Dissertations / Theses on the topic '3D cell'

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2007.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references (p. 135-152).
Cellular behavior is not dictated solely from within; it is also guided by a myriad of external cues. If cells are removed from their natural environment, apart from the microenvironment and social context they are accustomed to, it is difficult to study their behavior in any meaningful way. To that end, I describe a method for using optical trapping for positioning cells with submicron accuracy in three dimensions, then encapsulating them in hydrogel, in order to mimic the in vivo microenvironment. This process has been carefully optimized for cell viability, checking both prokaryotic and eukaryotic cells for membrane integrity and metabolic activity. To demonstrate the utility of this system, I have looked at a model "quorum sensing" system in Vibrio Fischeri, which operates by the emission and detection of a small chemical signal, an acyl-homoserine lactone. Through synthetic biology, I have engineered plasmids which express "sending" and "receiving" genes. Bacteria containing these plasmids were formed into complex 3D patterns, designed to assay signaling response. The gene expression of the bacteria was tracked over time using fluorescent proteins as reporters. A model for this system was composed using a finite element method to simulate signal transport through the hydrogel, and simple mass-action kinetic equations to simulate the resulting protein expression over time.
by Winston Timp.
Ph.D.

The thesis explains the development process of a production cell simulation in 3D implemented using Unity3D. The developed simulation communicates with existing control software and aims to test this control software in a 3D environment with physics simulation. The final result includes 3D models and also works as a visualization since it allows us to present the control system, and this visualization can be viewed using most web browsers. The thesis also includes a brief study and comparison between currently popular game engines to choose an appropriate option for this project.This is a project in collaboration with a local company (ARiSA) and has a high practical relevance.

Abstract The epithelial cells form barriers that compartmentalize the organs. Carcinomas are cancers stemming from epithelial cells and are the most common cancer type. The aim of this study was to understand the differentiation and malignant transformation of epithelial Madin-Darby canine kidney (MDCK) cells and to analyse the electrophysiological parameters which regulate their transport capacity. Emphasis was placed on comparing different culture environments, both in 2D and 3D. First, the effects of drugs or basal extracellular fluid composition on MDCK cell, cyst and lumen volumes were analysed using time-lapse microscopy. The results showed that MDCK cells were capable of both water secretion and reabsorption. The cells were able to perform these functions in a hyperpolarizing or depolarizing environment; change in osmolality of basal fluid was not required. Taken together, these results validate MDCK cells as a good basic model for studying kidney function. Next, the aim was to analyse the effect of 2D and 3D culture environments on the gene expression of untransformed MDCK and temperature sensitive ts-Src -transformed MDCK cells and the changes a single oncogene can induce. Microarray analysis revealed a decrease in the expression of survivin, an inhibitor of apoptosis protein, when switching the untransformed cells from 2D environment to 3D. This downregulation of survivin occurs in adult tissues as well, indicating that the cells grown in 3D are closer to the in vivo state than 2D cells. Src oncogene induced disintegration of cell junctions, but did not downregulate E-cadherin expression. The last part was to study further the factors controlling survivin expression and its significance to cell survival. MDCK cells grown in 3D did not suffer apoptosis if the cells remained in contact with the extracellular matrix. If MDCK cells were denied of ECM contacts they were more susceptible to apoptosis than survivin-expressing ts-Src MDCK cells. Finally, if cells were denied of cell-cell junctions, cells lacking survivin suffered apoptosis even though they had proper cell-matrix contacts. Taken together, these results highlighted the importance of cellular contacts to the cells: MDCK cells needed ECM contacts to differentiate and cell-cell contacts to avoid apoptosis
Tiivistelmä Epiteelisolut ovat erikoistuneet toimimaan rajapintana elimen ja ympäristön välillä. Ihmisten yleisin syöpä on epiteelisoluista alkunsa saanut karsinooma. Tämän tutkimuksen tarkoituksena oli ymmärtää Madin-Darby-koiran munuaisen solujen (MDCK) erilaistumista ja pahanlaatuistumista sekä analysoida sähköfysiologisia tekijöitä, jotka säätelevät näiden solujen kuljetustoimintaa. Erityisenä kiinnostuksen kohteena oli erilaisten kasvuympäristöjen vertailu. Farmakologisten aineiden tai basaalisen, solunulkopuolisen nesteen koostumuksen vaikutusta MDCK-solujen, -kystan sekä luumenin kokoon tutkittiin valomikroskooppisten aikasarjojen avulla. Tulokset osoittivat MDCK-solujen olevan kykeneviä sekä veden eritykseen että absorptioon, niin hyperpolarisoivassa kuin depolarisoivassakin ympäristössä. Basaalisen nesteen osmolaliteetin muutosta ei tarvittu. Nämä tulokset osoittavat MDCK-solujen olevan hyvä munuaisen tutkimuksen perusmalli. Seuraavaksi analysoitiin kaksi- ja kolmiulotteisten (2D ja 3D) viljely-ympäristöjen vaikutusta ei-transformoitujen MDCK-solujen ja lämpötilaherkkien ts-Src-transformoitujen MDCK-solujen geenien ilmentymiseen sekä yhden onkogeenin aktivoimisen aikaansaamia muutoksia. Microarray-analyysi osoitti apoptoosin estäjän, surviviinin, ilmentymisen vähenemisen, kun kasvuympäristö vaihdettiin 2D-ympäristöstä 3D-ympäristöön. Koska surviviinin väheneminen on normaali tapahtuma aikuisissa kudoksissa, voitiin todeta, että 3D-ympäristössä kasvatetut solut ovat lähempänä luonnonmukaista olotilaa kuin 2D-ympäristössä kasvaneet. Src-onkogeeni sai aikaan soluliitosten hajoamisen, mutta ei vähentänyt E-kadheriinin ilmentymistä. Tutkimuksen viimeinen osa keskittyi surviviinin ilmentymistä säätelevien tekijöiden analysoimiseen ja surviviinin merkitykseen solujen eloonjäämiselle. 3D-ympäristössä kasvaneet MDCK-solut eivät kärsineet apoptoosista edellyttäen, että solut pysyivät kosketuksissa soluväliaineeseen. Jos solut irtautuivat soluväliaineesta, ne päätyivät herkemmin apoptoosiin kuin surviviinia ilmentävät ts-Src MDCK-solut. Mikäli solujen väliset liitokset pakotettiin avautumaan, solut joutuivat apoptoosiin, vaikka ne olivat kosketuksissa soluväliaineeseen. Yhteenvetona nämä tulokset korostavat solujen kontaktien merkitystä: MDCK-solut tarvitsevat soluväliainekontakteja erilaistumiseen ja solujen välisiä kontakteja välttyäkseen apoptoosilta

La motilité des cellules est un phénomène fondamental en biologie souvent étudié sur des surfaces planes, conditions peu physiologiques. Nous avons analysé la migration cellulaire dans une matrice cellulaire 3D contenant de la fibronectine fluorescente. Nous démontrons que les cellules y sont confinées, et déforment leur environnement de manière cyclique avec une période de ~14 min avec deux centres de contractions à l’avant et à l’arrière de la cellule qui contractent avec un déphasage de ~3.5 min. Une perturbation de ces cycles entraîne une réduction de la motilité. Par l’utilisation d’inhibiteurs spécifiques, nous avons identifié l’acto-myosine comme étant l’acteur principal de ce phénomène. En imposant des contractions-relaxations locales par ablations laser, nous avons déclenché la motilité cellulaire ce qui confirme notre hypothèse. L’ensemble de cette étude met en évidence un nouveau mécanisme fondamental de dynamique cellulaire impliqué dans le mouvement des cellules
Cell motility is an important process in Biology. It is mainly studied on 2D planar surfaces, whereas cells experience a confining 3D environment in vivo. We prepared a 3D Cell Derived Matrix (CDM) labeled with fluorescently labeled fibronectin, and strikingly cells managed to deform the matrix with specific patterns : contractions occur cyclically with two contraction centers at the front and at the back of the cell, with a period of ~14 min and a phase shift of ~3.5 min. These cycles enable cells to optimally migrate through the CDM, as perturbation of cycles led to reduced motility. Acto-myosin was established to be the driving actor of these cycles, by using specific inhibitors. We were able to trigger cell motility externally with local laser ablations, which supports this framework of two alternating contractions involved in motion. Altogether, this study reveals a new mechanism of dynamic cellular behaviour linked to cell motility

Thesis (M.S.)--University of Cincinnati, 2009.
Advisors: Carla Purdy PhD (Committee Chair), Daria Narmoneva PhD (Committee Member), Ali Minai PhD (Committee Member). Title from electronic thesis title page (viewed April 30, 2009). Includes abstract. Keywords: Agent based modeling; cell growth; three dimensional. Includes bibliographical references.

In recent years, cells studies are often carried out with the help of three-dimensional (3D) nanostructured substrates. These kind of substrates can be useful in assisting the access in to the intracellular compartment and the delivery of molecules through holes or nanochannels, as well as in increasing the contact area of a cell with an electrode during electrical recordings.

Biofabrication has been receiving a great deal of attention in tissue engineering and regenerative medicine either by manual or automated processes. Different automated biofabrication techniques have been used to produce cell-laden alginate hydrogel structures, especially bioprinting approaches. , These approaches have been limited to 2D or simple 3D structures, however. In this thesis, a new extrusion-based bioprinting technique and a new simple, manual 3D biofabrication method are presented to culture cells in 3D. These methods do not rely on any complex fabrication methods. The bioprinting technique was developed to produce more complex alginate hydrogel structures. This was achieved by dividing the alginate hydrogel cross-linking process into 3 stages: primary calcium ion cross-linking for printability of the gel, secondary calcium cross-linking for rigidity of the alginate hydrogel immediately after printing and tertiary barium ion cross-linking for the long-term stability of the alginate hydrogel in the culture medium. Simple 3D structures including tubes were first printed to ensure the feasibility of the bioprinting technique. Complex 3D structures, such as branched vascular structures, were subsequently printed successfully. The static stiffness of the alginate hydrogel after printing was 20.18 ± 1.62 kPa which was rigid enough to sustain the integrity of the complex 3D alginate hydrogel structure during the printing. The addition of 60 mM barium chloride was found to significantly extend the stability of the cross-linked alginate hydrogel from 3 days to beyond 11 days without compromising the cellular viability. The results based on cell bioprinting suggested that the viability of U87-MG cells was 92.94 ± 0.91 % immediately after bioprinting. Cell viability was maintained above 88 ± 4.3 % in the alginate hydrogel over a period of 11 days. On the other hand, the manual biofabrication approach developed in this thesis enabled the fabrication of scalable 3D cell-laden hydrogel structures easily, without complex machinery. The technique could be carried out using only apparatus available in a typical cell biology laboratory. The fabrication method would involve micro coating cell-laden hydrogels covering the surface of a metal bar by dipping into cross-linking reagent CaCl2 or BaCl2, to form hollow tubular structures. This method could be used to form single- or multi-layered tubular structures. This fabrication method has incorporated the use alginate hydrogel as the primary biomaterial and secondary biomaterial could be added depending on the desired application. The feasibility of this method has been demonstrated by showing the cell survival rate and normal responsiveness of cells within these tubular structures using mouse dermal embryonic fibroblast cells and human embryonic kidney 293 cells containing a tetracycline responsive red fluorescence protein (tHEK cells). By adjusting the fabrication protocol, complex hollow alginate hydrogel structures could be generated.

L’ingénierie d’hydrogels ; leur structuration et fonctionnalisation à l’échelle cellulaire, est une étape clé pour aboutir à de modèles in-vitro plus physiologiques. À ce jour, elle reste difficile car ces matériaux polymères, mous et riches en eau, sont souvent trop fragiles pour la micro-fabrication traditionnelle. Pour pallier à ce fait, nous avons combinée illumination ultraviolette structurée et chambres de réaction perméables au gaz nous offrant la maitrise sur la distribution de photons, les réactifs et les gaz présents à chaque instant et en tout point d’un champ d’illumination. Nous pouvons ainsi contrôler une photochimie adaptée aux hydrogels les plus répandus et structurer, décorer ou liquéfier ces matériaux. Ensemble ces trois opérations forment une boite à outil complète adaptée aux substrats les plus communs que sont Matrigel, Agar, Poly(acrylamide) et Poly(éthylène-glycol). Nous avons par la suite fabriqué des micro-niches en hydrogel permettant la culture standardisée de lignées cellulaire et de neurones primaires soit par adhésion sur des topographies ou par auto-organisation en sphéroïdes. Ceci démontre que la plateforme est à même de répondre à des enjeux importants de culture cellulaire tridimensionnelle
Tailoring hydrogels into biomimetic templates represents a crucial step to build better in-vitro models but it is to date still challenging. Indeed, these synthetic or natural polymeric networks are often so frail they can’t be processed through standard micro-fabrication. Here, we combine a ultra-violet pattern projector with gas permeable microreactors to control gas, reagents and photon distribution and in fine, the reaction kinetics in space and time. Doing so, enabled a generic chemistry that can structure, liquefy or decorate (locally functionalize) common hydrogels. Altogether these three hydrogel engineering operations form a flexible toolbox that supports the most commonly used hydrogels: i.e. Matrigel, Agar-agar, poly(ethylene-glycol) and poly(acryl-amide). We successfully applied this solution to grow cells into standardized micro-niches demonstrating that it can readily address cell culture challenges such has controlled adhesion on topographical structures, standardization of spheroids or culture on shaped Matrigel

Conventionally, adherent cells are cultured in vitro using flat 2D cell culture trays. However the 2D cell culture method is tedious, unreliable and does not replicate the complexity of the 3D dynamic environment of native tissue. Nowadays 3D scaffolds can be used to culture cells. However a number of challenges still exist, including the need for destructive enzymes to release confluent cells. Poly(Nisopropylacrylamide) (PNIPAAm), a temperature responsive polymer, has revolutionised the cell culture fraternity by providing a non-invasive means of harvesting adherent cells, whereby confluent cells can be spontaneously released by simply cooling the cell culture medium and without requiring enzymes. While PNIPAAm monolayer cell culturing is a promising tool for engineering cell sheets, the current technology is largely limited to the use of flat 2D substrates, which lacks structural and organisational cues for cells. The aim of this project was to develop a 3D PNIPAAm scaffold which could be used efficiently for non-invasive 3D culture of adherent cells. This project was divided into three phases: Phase 1 (preliminary phase) involved development and characterisation of cross-linked PNIPAAm hydrogels; Phase 2 involved development and characterisation of PNIPAAm grafted 3D non-woven scaffolds, while Phase 3 focused on showing proof of concept for non-invasive temperature-induced cell culture from the 3D PNIPAAm grafted scaffolds. In Phase 1, PNIPAAm was cross-linked with N,N’-methylene-bis-acrylamide (MBA) using solution free-radical polymerisation to form P(PNIPAAm-co-MBA) hydrogels. A broad cross-link density (i.e. 1.1 - 9.1 Mol% MBA) was investigated, and the effect of using mixed solvents as the co-polymerisation medium. The P(PNIPAAm-co-MBA) gels proved unsuitable as a robust cell culture matrix, due to poor porosity, slow swelling/deswelling and poor mechanical properties. Subsequently, in Phase 2, polypropylene (PP), polyethylene terephthalate (PET), and nylon fibers were processed into highly porous non-woven fabric (NWF) scaffolds using a needle-punching technology. The NWF scaffolds were grafted with PNIPAAm using oxyfluorination-assisted graft polymerisation (OAGP). The OAGP method involved a 2 step process whereby the NWF was first fluorinated (direct fluorination or oxyfluorination) to introduce new functional groups on the fibre surface. The functionalised NWF scaffolds were then graft-polymerised with NIPAAm in an aqueous medium using ammonium persulphate as the initiator. Following oxyfluorination, new functional groups were detected on the surface of the NWF scaffolds, which included C-OH; C=O; CH2-CHF, and CHF-CHF. PP and nylon were both easily modified by oxyfluorination, while PET displayed very little changes to its surface groups. Improved wetting and swelling in water was observed for the oxyfluorinated polymers compared to pure NWF scaffolds. PP NWF showed the highest graft yield followed by nylon and then PET. PNIPAAm graft yield on the PP NWF was ~24 ±6 μg/cm2 on grafted pre-oxyfluorinated NWF when APS was used; which was found to be significantly higher compared to when pre-oxyfluorinated NWF was used without initiator (9 ±6 μg/cm2, p= 1.7x10-7); or when grafting was on pure PP with APS (2 ±0.3 μg/cm2, p = 8.4x10-12). This corresponded to an average PNIPAAm layer thickness of ~220 ±54 nm; 92 ± 60 nm; and 19 ± 3 nm respectively. Scanning electron microscopy (SEM) revealed a rough surface morphology and confinement of the PNIPAAm graft layer to the surface of the fibers when oxyfluorinated NWF scaffolds were used, however when pure NWF scaffolds were used during grafting, homopolymerisation was observed as a loosely bound layer on the NWF surface. The OAGP method did not affect the crystalline phase of bulk PP as was determined by X-ray diffraction (XRD), however, twin-melting thermal peaks were detected from DSC for the oxyfluorinated PP and PP-g-PNIPAAm NWF which possibly indicated crystal defects. Contact angle studies and microcalorimetric DSC showed that the PP-g-PNIPAAm NWF scaffolds exhibited thermoresponsive behaviour. Using the 2,2-Diphenyl-1-1-picrylhydrazyl (DPPH) radical method and electron-spin resonance (ESR), peroxides, as well as trapped long-lived peroxy radicals were identified on the surface of the oxyfluorinated PP NWF, which are believed to be instrumental in initiating graft polymerisation from the NWF. A free radical mechanism which is diffusion controlled was proposed for the OAGP method with initiation via peroxy radicals (RO•), as well as SO4•- and OH• radicals, whereby the latter result from decomposition of APS. In Phase 3 of this study, proof-of-concept is demonstrated for use of the PNIPAAm grafted NWF scaffolds in non-invasive culture of hepatocytes. Studies demonstrated that hepatocyte cells attached onto the 3D PNIPAAm scaffolds and remained viable in culture over long periods. The cells were released spontaneously and non-destructively as 3D multi-cellular constructs by simply cooling the cell culture medium from 37°C to 20°C, without requiring destructive enzymes. The PP-g- PNIPAAm NWF scaffolds performed the best in 3D cell culture. Additionally the CSIR is developing a thermo responsive 3D (T3D) cell culturing device, whereby the 3D thermo responsive NWF scaffolds are used in the bioreactor for cell culture. Temperature-induced cell release was also verified from the 3D Thermo responsive scaffolds in the bioreactor. This technology could lead to significant advances in improving the reliability of the in vitro cell culture model. Please cite as follows: Chetty, AS 2012, Thermoresponsive 3D scaffolds for non-invasive cell culture, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-06112013-151344/ > D13/4/713/ag
Thesis (PhD)--University of Pretoria, 2012.
Chemical Engineering
unrestricted

Under specific conditions short peptides modified with an N-terminal fluorenyl-9-methoxycarbonyl (Fmoc) group can self-assemble into hydrogel scaffolds similar in properties to the natural extracellular matrix. Fmoc-diphenylalanine (Fmoc-FF) for instance, has been shown to form hydrogels at physiological pH that have the ability to support 2D and 3D cell culture. The aim of this investigation is to provide further understanding of the self-assembly mechanism of such systems in order to progress towards the establishment of design rules for the preparation of scaffolds with tuneable properties.First, Fmoc-dipeptides composed of a combination of hydrophobic aromatic residues phenylalanine (F) and glycine (G) were studied with a particular emphasis on the effect of pH variations. The systems were investigated in order to assess what influence the position of such residues in the peptide sequence had on the physical properties of the molecules, and what impact the chemical structure had on the self-assembly behaviour and the gelation properties of the materials. Subsequently, phenylalanine was replaced by leucine (L), a non-aromatic amino acid that had the same relative hydrophobicity in order to determine whether the self-assembly of such molecules is driven by aromatic interactions or hydrophobic effects.Using potentiometry, the behaviour of the systems in solution has been investigated, revealing that they were all characterised by pKa shifts of up to six units above the theoretical values. Fmoc-FF exhibited two transitions whereas the other Fmoc-dipeptides only displayed one. These transitions were found to coincide with the formation of distinct self-assembled structures with differing molecular conformations and properties that were characterised using transmission electron microscopy, infrared and fluorescence spectroscopy, X-ray scattering and shear rheometry.π-stacking of the aromatic moieties was thought to be the driving force of the self-assembly mechanism, generating dimers that corresponded to the building blocks of the supramolecular structures formed. On the other hand, the peptide components were stabilised via hydrogen bonding and could form antiparallel β-sheets depending on the amino acid sequence and the associated influence on the rigidity of the molecules. Below their (first) apparent pKa transition, Fmoc-FF, Fmoc-LL, Fmoc-FG, Fmoc-LG and Fmoc-GG formed hydrogels, with the mechanical properties and stability varying depending on the amino acid sequence. Fmoc-FF and Fmoc-LL exhibited the lowest storage modulus values (G′ ~ 0.5–5 Pa) of the studied systems while Fmoc-LG displayed the highest (G′ ~ 1000–2100 Pa). Fmoc-FG and Fmoc-LG had the peculiarity of being obtained upon heating and where found to be particularly stable, as opposed to Fmoc-GG gels which showed a tendency to crystallise. On the microscopic scale, these gels were all associated with the presence of entangled fibrillar networks of different size and morphology, which in some cases could self-assemble further through a lamellar organisation. Again, Fmoc-FG and Fmoc-LG distinguished from the other systems as they were the only Fmoc-dipeptides to show a supramolecular chirality in the form of twisted ribbons under specific pH conditions. In contrast, Fmoc-GF and Fmoc-GL did not form hydrogels below their apparent pKa due to the formation of sheet-like and spherical structures respectively.

Current culture methods to generate large quantities of cells destined for tissue engineering and regenerative medicine commonly use enzymatic digestion. However, this method is not desirable for subsequent cell transfer to the body due to the destruction of important cell-surface proteins and the risk of enzymatic contamination [1]. Therefore, research has led to the development of thermo-responsive surfaces for the continued culture of mammalian cells, with passaging achieved via a drop in the culture temperature. Recognising that the three-dimensional (3D) culture environment influences the cell phenotype, our aim was to generate a thermo-responsive 3D fibre-based scaffold, using electrospinning, to create an enzyme-free 3D culture surface for mammalian cell expansion that would be suitable for cells destined for the clinic. Thermo-responsive poly (poly (ethylene glycol) methacrylate), poly (PEGMA188), with lower critical solution temperature (LCST) of 26°C has been proposed for use within this thesis. It was used in combination with poly (lactic-co-glycolic acid) (PLGA) and poly (ethylene terephthalate) (PET) polymers in order to create 3D thermo-responsive non-woven electrospun fibrous scaffolds, on which different cell types could be cultured and passaged. Poly (PEGMA188) was prepared by free radical polymerization, and then incorporated with PLGA and PET polymers via four different methods: (i) surface adsorption, (ii) NaOH surface treatment, (iii) surface entrapment and (iv) blend-electrospinning. Blend-electrospinning was chosen over the other methods as it produced nano-PET and micro-PLGA bead-less fibres with responsive behaviour. The biocompatibility was assessed via the adhesion and proliferation of different mammalian cell types, including (i) red fluorescent protein (RFP)-expressing 3T3 fibroblasts, (ii) green fluorescent protein (GFP)-expressing primary immortalized human mesenchymal stem cells (ihMSCs), (iii) human colon adenocarcinoma cells (Caco2) and (iv) primary human corneal stromal stem cells (hCSSCs). The cell viability (Alamar Blue assay) was determined to measure the difference in cell populations adherent to the scaffolds while changing the culture temperature. These thermo-responsive scaffolds were able to support cell adhesion and proliferation at 37°C (hydrophobic surface). Furthermore, it was possible to detach the cells from the scaffolds by decreasing the temperature to 17°C (hydrophilic surface). Irrespective of the concentration of poly (PEGMA188) used, all scaffolds exhibited thermo-responsive proprieties; the cells were viable and proliferated in a similar manner to those cultured on control surfaces (PLGA or PET scaffolds). Finally, the effects of the thermo-responsive polymer and 3D culture environment on the hCSSC phenotype were assessed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunocytochemistry. The application of 3D environments can promote the reversion of activated corneal stromal cells’ ‘fibroblastic phenotype’ to a desirable quiescent keratocyte phenotype. Therefore, seven thermal and enzymatic passages on responsive 3D scaffolds and 2D TCPS, respectively, were performed. Cell culture on the 3D scaffolds promoted the quiescent keratocyte phenotype, with the increased expression of the keratocyte markers, CD34 and ALDH, and decreased expression of the myofibroblast marker, ACTA2, when compared with cells cultured on the 2D culture flasks. In this thesis, the preparation and application of first generation, biocompatible thermo-responsive fibrous scaffolds are described. The combination of ease of preparation, positive cell response and the expansion of a desirable cell phenotype make the thermo-responsive fibres promising as a new class of materials for application in cell culture. The materials developed and studied in this thesis are believed to represent a significant contribution to the fields of biomaterials and tissue engineering.

It has been well established that the fundamental behaviors of mammalian cells are influenced by the physical cues that they experience from their surrounding environment. With respect to cells in our bodies, mechanically-driven morphological and phenotypic changes to our cells have been linked to responses critical to both normal development and disease progression, including lung, heart, muscle and bone disorders, and cancer. Although significant advancements to our understanding of cell behavior have been made using 2D cell culture methods, questions regarding how physical stretch guides cell behavior in more complex 3D biological systems remain unanswered. To address these questions, we used microfabrication techniques to develop vacuum-actuated stretchers for high throughput stretching and dynamic mechanical screening of 3D microtissue cultures. This thesis contains five research chapters that have utilized these devices to advance our understanding of how cells feel stretch and how it influences their behavior in a 3D matrix. In the first research chapter (chapter 2), we characterized how stretch is transferred from the tissue-level to the single-cell level and we investigated the cytoskeletal reinforcement response to long-term mechanical conditioning. In the second research chapter (chapter 3), we examined the effects of an acute dynamic stretch and found that 3D cultures soften through actin depolymerization to homeostatically maintain a mean tension. This softening response to stretch may lengthen tissues in our body, and thus may be an important mechanism by which airway resistance and arterial blood pressure are controlled. In the third and forth research chapters (chapter 4-5), we investigated the time dependencies of microtissues cultures and we found that their behavior differed from our knowledge of the rheological behavior of cells in 2D culture. Microtissues instead followed a stretched exponential model that seemed to be set by a dynamic equilibrium between cytoskeletal assembly and disassembly rates. The difference in the behavior from cells in 2D may reflect the profound changes to the structure and distribution of the cytoskeleton that occur when cells are grown on flat surfaces vs. within a 3D environment. In the fifth and final research chapter (chapter 6), we examined how mechanical forces may contribute to the progression of tissue fibrosis through activating latent TGF-β1. Our results suggest that mechanical stretch contributes to a feed forward loop that preserves a myofibroblastic phenotype. Together these investigations further our understanding of how cells respond to mechanical stimuli within 3D environments, and thus, mark a significant contribution to the fields of mechanobiology and cell mechanics.

This thesis established an innovative cell culture system to enable cells to grow by mimicking the environment of cells in living tissues and organs. The system can be used in different pharmaceutical industry fields, such as large-scale stem cell expansion, cell-based therapy, drug screening, and manufacturing of protein drugs and vaccines.

Articular cartilage damage associated with joint trauma seldom heals and often leads to osteoarthritis (OA). Current treatment often fails to regenerated functional cartilage close to native tissue. We previously identified a migratory chondrogenic progenitor cell (CPC) population that responded chemotactically to cell death and rapidly repopulated the injured cartilage matrix, which suggested their potential for cartilage repair. To test that potential we filled experimental full thickness chondral defects with an acellular hydrogel containing SDF-1α. We expect that SDF-1α can increase the recruitment of CPCs, and then promote the formation of a functional cartilage matrix with chondrogenic factors. Full-thickness bovine chondral defects were filled with hydrogel comprised of fibrin and hyaluronic acid and containing SDF-1α. Cell migration was monitored, followed by chondrogenic induction. Regenerated tissue was evaluated by histology, immunohistochemistry, and scanning electron microscopy. Push-out tests were performed to assess the strength of integration between regenerated tissue and host cartilage. Significant numbers of progenitor cells were recruited by SDF-1α within 12 days. By 5 weeks chondrogenesis, repair tissue cell morphology, proteoglycan density and surface ultrastructure were similar to native cartilage. SDF-1α treated defects had significantly greater interfacial strength than untreated controls. However, regenerated neocartilage had relatively inferior mechanical properties compared with native cartilage. In addition to that, we developed a 3D bioprinting platform, which can directly print chondrocytes as well as CPCs to fabricated articular cartilage tissue in vitro. We successfully implanted the printed tissue into an osteochondral defect, and observed tissue repair after implantation. The regerated tissue has biochemical and mechanical properties within the physiological range of native articular cartilage. This study showed that, when CPC chemotaxis and chondrogenesis are stimulated sequentially, in situ full thickness cartilage regeneration and bonding of repair tissue to surrounding cartilage could occur without the need for cell transplantation from exogenous sources. This study also demonstrated the potential of using 3D bioprinting to engineer articular cartilage implants for repairing cartilage defect.

Embryonic stem cells (ESCs) are promising as therapeutic material since they are pluripotent (potentially differentiate into any mature cell) and have “limitless” self-renewal capacity. To achieve widespread clinical utility, ESC cultures have to be designed to meet specific process requirements (e.g. quantity, quality etc.). Currently, most pluripotent stem cell (PSC) cultures are fragmented protocols relying on operator–intensive processing, as 2D monolayers on tissue culture plastic, at ambient O2 conditions. Incidentally, such culture conditions are sub-optimal, often leading to unscheduled stem cell behaviour. This thesis examines how ESC bioprocesses can be improved. Culture environment effects on ESCs are investigated, as well as computational tools for in silico design. I demonstrate how critical culture parameters and mathematical modelling can be exploited to improve the undifferentiated expansion of ESCs. Beginning with 3D murine ESCs (mESCs) cultures, 1) dynamic rotary cultures were demonstrated to improve self-renewal signalling activity, yielding improved proliferation of mESCs with higher “stemness” levels. 2) Culture metabolism was another critical factor. During batch feeding, metabolites accumulate within the culture environment especially at later stages in culture, causing stresses that impair ESC proliferation and “stemness”, independent of growth factor levels. In contrast, perfusion feeding maintained well-regulated culture environments that promoted the expansion of highly “naïve” mESCs. 3) Computational approaches can complement bioprocess design. Mathematical models identified novel multi-scale interactions within the bioprocess and effectively simulated bioreactor fluid dynamics. 4) As a means to further optimize the bioprocess, alternative signalling factors were combined with dynamic perfusion cultures in reduced (5%) O2 conditions, which generated increased cell yields having high “stemness” levels at half the costs. In conclusion, numerous ‘standard’ culture conditions were found to be sub-optimal for mESC culture, emphasizing the need for improved bioprocesses using rational design based on stem cell bioscience. It is anticipated that these integrated stem cell bioprocesses, can improve product yield and quality at reduced costs. Such bioprocess strategies will facilitate the usage of PSCs as therapeutics.

Supercritical Emulsion Extraction technology (SEE-C) was proposed for the production of poly-lactic-co-glycolic acid microcarriers. SEE-C operating parameters as pressure, temperature and flow rate ratios were analyzed and the process performance was optimized in terms of size distribution and encapsulation efficiency. Microdevices loaded with bovine serum insulin were produced with different sizes (2 and 3 µm) or insulin charges (3 and 6 mg/g) and with an encapsulation efficiency of 60%. The microcarriers were characterized in terms of insulin release profile in two different media (PBS and DMEM) and the diffusion and degradation constants were also estimated by using a mathematical model. PLGA microdevices were also used in a cultivation of embryonic ventricular myoblasts (cell line H9c2 obtained from rat) in a FBS serum free medium to monitor cell viability and growth in dependence of insulin released. Good cell viability and growth were observed on 3 µm microdevices loaded with 3 mg/g of insulin. PLGA microspheres loaded with growth factors (GFs) were charged into alginate scaffold with human Mesenchimal Steam Cells (hMSC) for bone tissue engineering with the aim of monitoring the effect of the local release of these signals on cells differentiation. These “living” 3D scaffolds were incubated in a direct perfusion tubular bioreactor to enhance nutrient transport and exposing the cells to a given shear stress. Different GFs such as, h-VEGF, h-BMP2 and a mix of two (ratio 1:1) were loaded and alginate beads were recovered from dynamic (tubular perfusion system bioreactor) and static culture at different time points (1st, 7th, 21st days) for the analytical assays such as, live/dead; alkaline phosphatase; osteocalcin; osteopontin and Van Kossa Immunoassay. The immunoassay confirmed always a better cells differentiation in the bioreactor with respect to the static culture and revealed a great influence of the BMP-2 released in the scaffold on cell differentiation.

Supercritical Emulsion Extraction technology (SEE-C) was proposed for the production of poly-lactic-co-glycolic acid microcarriers. SEE-C operating parameters as pressure, temperature and flow rate ratios were analyzed and the process performance was optimized in terms of size distribution and encapsulation efficiency. Microdevices loaded with bovine serum insulin were produced with different sizes (2 and 3 µm) or insulin charges (3 and 6 mg/g) and with an encapsulation efficiency of 60%. The microcarriers were characterized in terms of insulin release profile in two different media (PBS and DMEM) and the diffusion and degradation constants were also estimated by using a mathematical model. PLGA microdevices were also used in a cultivation of embryonic ventricular myoblasts (cell line H9c2 obtained from rat) in a FBS serum free medium to monitor cell viability and growth in dependence of insulin released. Good cell viability and growth were observed on 3 µm microdevices loaded with 3 mg/g of insulin. PLGA microspheres loaded with growth factors (GFs) were charged into alginate scaffold with human Mesenchimal Steam Cells (hMSC) for bone tissue engineering with the aim of monitoring the effect of the local release of these signals on cells differentiation. These “living” 3D scaffolds were incubated in a direct perfusion tubular bioreactor to enhance nutrient transport and exposing the cells to a given shear stress. Different GFs such as, h-VEGF, h-BMP2 and a mix of two (ratio 1:1) were loaded and alginate beads were recovered from dynamic (tubular perfusion system bioreactor) and static culture at different time points (1st, 7th, 21st days) for the analytical assays such as, live/dead; alkaline phosphatase; osteocalcin; osteopontin and Van Kossa Immunoassay. The immunoassay confirmed always a better cells differentiation in the bioreactor with respect to the static culture and revealed a great influence of the BMP-2 released in the scaffold on cell differentiation.

In recent years the study of three-dimensional (3D) cell culture has undergone a progressive development. The 3D models have shown characteristics more similar to the overall conditions in vivo with respect to 2D cultures (Hutmacher DW., 2001). The 3D approach decreases the gap between cell culture system and the cellular physiology, helping to understand better various cell functions such as proliferation, adhesion, viability, morphology, microenvironment, and response to drugs. Compared to 2D cultivation, the cells in 3D are completely different in terms of morphology, signaling, and microenvironmental metabolism (Astashkina A et al., 2014). The scaffold provides the support necessary for the cells to attach, proliferate, and maintain their differentiated function. As a drawback, the structural characteristics of 3D scaffolds make their characterization quite complex. In my project a new hydrogel scaffold (HS) has been produced and several methods have been modified and/or developed for characterizing its cell compatibility, porosity, mechanical and optical properties and molecular diffusion. The first condition that has been verified was that the absence of toxicity for the cell types used and the capacity of the scaffolds to promote cell adhesion and growth for a period suitable for biological tests. Different cell lines have been evaluated, like human endothelial vascular cell line (HECV), human keratinocyte cell line (NCTC), human fibroblast cell line and human liver carcinoma cell line. HECV and NCTC have been cultured in 3D for more than 30 days. The porosity of the lyophilized scaffold has been calculated by liquid displacement method, using acetonitrile as the displacement liquid, and resulted to be higher than 95%. The mechanical properties of the HS have been analysed and Young’s Modulus (YM) and yield strength (YS) were evaluated in dried state and in liquid immersion. The YM of HS ranges from 15 to 120 kPa, and the YS ranges from 3 to 21 kPa. The transparency of HS has been easily evaluated by UV-VIS transmittance study and by observing letters printed on a paper sheet placed under the scaffold completely immersed in PBS, measuring the sharpness of the typographical sign, with respect to a reference read through a PBS solution. The molecular diffusion through the scaffold has been evaluated using Franz diffusion cells at 37 °C, with metformin hydrochloride as diffusing molecule. The diffusion coefficient D was calculated, its value being approx. 1.2×10-9 m²/s.

Stem cell therapies and tissue engineering strategies are required for the clinical treatment of respiratory diseases. Previous studies have established protocols for the differentiation of airway epithelium from stem cells but have involved costly and laborious culture methods. The aim of this thesis was to achieve efficient and reproducible maintenance and differentiation of embryonic stem cells to airway epithelium, in 2D and 3D culture, by developing appropriate bioprocessing technology. Firstly, the 2D differentiation process of human and murine ES cells into pulmonary epithelial cells was addressed. The main finding in was that the proportion of type II pneumocytes, the major epithelial component of the gas-exchange area of lung, differentiated with this method was higher than that obtained in previous sudies, 33% of resultant cell expressed the specific marker surfactant protein C (SPC) compared with up to 10%. Secondly, the maintenance and differentiation was carried out in 3D. A protocol was devised that maintained undifferentiated human ES cells in culture for more than 200 days encapsulated in alginate without any feeder layer or growth factors. For ES cell differentiation in 3D, a method was devised to provide a relatively cheap and simple means of culture and use medium conditioned by a human pneumocyte tumour cell line (A549). The differentiation of human and murine ES cells into pulmonary epithelial cells, particularly type II pneumocytes, was found to be upregulated by culture in this conditioned medium, with or without embryoid body formation. The third step was to test whether this differentiation protocol was amenable to scale-up and automation in a bioreactor using cell encapsulation. It was possible to show that encapsulated murine ES cells cultured in static, co-culture or rotating wall bioreactor (HARV) systems, differentiate into endoderm and, predominantly, type I and II pneumocytes. Flow cytometry revealed that the mean yield of differentiated type II pneumocytes was around 50% at day 10 of cultivation. The final stage of the work was to design and produce a perfusion system airlift bioreactor to mimic the pulmonary microenvironment in order to achieve large scale production of biologically functional tissue. The results of these studies thus provide new protocols for the maintenance of ES cells and their differentiation towards pulmonary phenotypes that are relatively simple and cheap and can be applied in bioreactor systems that provide for the kind of scale up of differentiated cell production needed for future clinical applications.

Abstract While most of the in vitro cultures are carried out on bi-dimensional (2D) substrates, most of the in vivo extracellular matrices are threedimensional (3D). Consequently cells behave differently on 2D substrates as a way to self-adaptation to a non-physiological environment. This fact has encouraged the development of more relevant culture conditions seeking to provide more representative models for biomedicine (e.g. cancer, drug discovery and tissue engineering) and further insights into any dimension-dependent biological mechanism. Different 3D culture systems have been established though their variability and complexity hinder their standardisation in common cell culture procedures. So, this thesis deals with the dimensionality issue in cell/material interactions and introduces sandwich-like microenvironments as a versatile tool to study cell behaviour. Cells cultured within this system use both dorsal and ventral receptors to adhere and spread, undergoing important changes with respect to the 2D cultures and approaching to 3D conditions. Stimulation of dorsal receptors has been previously addressed by overlaying a protein gel on cells already attached on a 2D surface. Here we propose a sandwich-like system that consists of two 2D surfaces so that wider spectra of conditions can be investigated by changing the nature of the substrate (material, topography…) and the protein coatings of both ventral and dorsal sides. Since sandwich culture provides an altered cellular adhesion compared to the traditional 2D substrates by the excitation of the dorsal receptors, changes in the intracellular signalling are expected, which might alter important processes such as proliferation, morphology, migration and differentiation. Hence this thesis evaluates the effect of different sandwich culture parameters in cell behaviour. First, cell fate upon adhesion was evaluated in terms of morphology, proliferation and adhesion. Different conditions were studied such as materials with different properties or protein coatings (dorsal and ventral substrates), as well as the effect of sandwiching cells just after seeding or after been allowed to adhere to the ventral substrate. Interesting results were obtained such as the relationship between the ability of cells to reorganise the ECM with cell morphology, proliferation and adhesion, similarly as observed in 3D hydrogels (degradable vs nondegradable systems). Then, cell migration within sandwich culture was studied by live imaging of a wound healing assay. Results revealed the key effect of both ventral and dorsal substrates in determining the migration rate as well as the migration mode used by cells. Moreover cells within the sandwich culture migrating in the wound healing assay adopted an elongated cell morphology that resembled cells migrating in other 3D systems. Beyond differences in cell morphology and migration, dorsal stimulation promoted cell remodelling of the extra-cellular matrix (ECM) over simple ventral receptor activation in traditional 2D cultures. Finally the effect of sandwich culture on cell differentiation was evaluated. First we showed an increase in C2C12 myogenic differentiation when cultured within the sandwich system. This enhancement was shown to be dorsal stimulation dependent and related to an alteration of the signalling pathway and the growth factor release. To determine if sandwich culture leads only to myogenic differentiation or whether it allows differentiation to other lineages, 4 different human mesenchymal stem cells (hMSCs) lines were cultured under the same conditions. Results showed the same sandwich environment triggered different cell differentiation. This points out the importance of the microenvironment cell niche in vivo, which highly influence cell fate, and thus the need of mimicking it properly in vitro. Overall, sandwich-like microenvironments switch cell behaviour towards 3D-like patterns, demonstrating the importance of this versatile, simple and robust approach to mimic cell microenvironments in vivo.
Ballester Beltrán, J. (2014). Sandwich-like systems to engineer the cellular microenvironment [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48166
TESIS

In recent years, due to the public concern on global warming, both increasing energy efficiency and developing green energy become crucially important. Fuel cells can be one of the most suitable clean energy solutions for the environment because of its high energy conversion efficiency and near zero emissions of criteria air pollutants at the use stage. To increase the energy efficiency of fuel cells, effectively utilize the Pt catalyst and increase the fuel cell durability, the uniform distribution of the reactants over the fuel cell active area is of great importance. Over the last decade, many researchers have focused on developing flow field design to homogenously distribute the reactant and to decrease the pressure drop in the bipolar plates. However, most of the previous studies are in the stage of numerical simulation, and the few experimental studies have used very simple flow field geometries. Not to mention that complex transport phenomena inside a fuel cell make even the numerical simulation challenging and time consuming, which hinders the quick screening of proposed modifications and new designs. While the conventional fabrication techniques are expensive and time consuming, 3D printing is a very good rapid prototyping method that can be used both to validate the simulation results and to supplement the tedious simulation work. The question is whether the results from 3D printed flow fields could be as accurate and reliable as flow fields fabricated with conventional methods. In the present research, we investigated the applicability of 3D printing in validating the simulation results and as a fast screening method. State of the art designs for anode, cathode and water cooling BPPs proposed and fabricated using Polyjet 3D printing, SLA 3D printing and laser-cutter technologies and the pressure drop and velocity profiles were measured for each plate. The results demonstrated that SLA 3D printing has great promises to serve as a screening tool in modifying the flow field design, as well as in validating the simulation results.
Applied Science, Faculty of
Chemical and Biological Engineering, Department of
Graduate

Cell separation is an important component of modern medicine, with both clinical and research applications. Clinically, it is often desirable to isolate cell subpopulations providing focused treatment; on the research side, cell isolation is necessary for studies underpinning many discoveries in cell biology, further enabling research in areas such as regenerative medicine and cancer therapy. Cell separation requirements include high throughput, purity and recovery. Three cell separators dominate: fluorescence and magnetic-activated cell sorting and density-gradient centrifugation. Despite gold-standard establishing performances, they can be improved in affordability, throughput, and label-free cell separation implementation. A technology with potential to offer the next rotation of gold-standard cell separators is Dielectrophoresis, DEP. Two DEP cell separators are presented. The first, the Syringe Separator (SS), uses 3D-electrodes on a low-cost, disposable chip and a DEP field perpendicular to fluid flow; one cell type is passed through whilst the other is retained and subsequently recovered. Two-pass protocols achieved a 96.4% recovery at over 200,000 cells/second with <7% loss. Additionally, a three-step protocol removed 99.1% of RBCs spiked with cancer cells (100:1). Other SS implementations include hitherto unachieved separation of high and low quality nanowires and T-cell isolation. The second employs a novel electrode geometry termed the Canyon. Using a novel electrode fabrication method (Plotter-Canyon printing), Canyons were built of alternating layers of metal and non-metal. Cellular solutions flow through the Canyon directed to one of two outlets, one for each of the negative or positive DEP cell subpopulations. The Canyon cell separator achieved an 84% recovery and 10% loss at ~2,000 cells/second. We have demonstrated that DEP cell separators can be built to perform cell separations with high purity, rivalling established separators, at significantly higher throughput and recovery. The SS and Canyons are cheap, easy-to-operate and offer a stepwise improvement in conventional cell separation capabilities.

The derivation of embryonic stem cells (ESCs) has created an invaluable resource for scientific study and discovery. Further improvement in differentiation protocols is necessary to generate the large number of cells needed for clinical relevance. The goal of this work was to develop a method to incorporate biomaterial microparticles (MPs) within stem cell aggregates and to evaluate their use for local control of the cellular microenvironment for directed differentiation. The effects of unloaded MPs on ESC differentiation were first determined by controlled incorporation of poly(lactic-co-glycolic acid) (PLGA), agarose and gelatin MPs. Embryoid body (EB) formation, cell viability, and gross morphology were not affected by the presence of the MPs. Further analysis of gene expression and patterns of phenotypic marker expression revealed alterations in the differentiation profile in response to material incorporation. The ability of MPs to direct ESC differentiation was investigated by incorporation of growth factor loaded MPs within EBs. MPs were loaded with bone morphogenetic protein-4 (BMP-4). BMP-4 loaded MPs incorporated within EBs induced mesoderm gene expression while inhibiting expression of an ectoderm marker compared to untreated EBs. Finally, magnetic MPs (magMPs) were incorporated within EBs to induce magnetic sensitivity. The responsiveness of EBs to applied magnetic fields was controlled by the number of magMPs incorporated within the aggregates. Magnetic guidance was then used to control the precise location of single EBs or populations of EBs for bioreactor culture and for construction of heterogeneous cell constructs. Overall, the results indicated that PSC differentiation within spheroids is sensitive to various types of biomaterials. Incorporation of MPs within EBs can be used to direct ESC differentiation by control of the cellular environment from microscale interactions, by delivery of soluble factors, to macroscale interactions, by control of EB position in static and suspension cultures.

The interplay between cells and biomaterials constitutes a fertile ground to probe specific cellular functions and cues for therapeutic and research purposes. “Smart” materials encompass an extensive library that can lead to the design of dynamic multi-responsive constructs with great importance in the biomedical field. This work aims to describe diverse strategies on the modification of biological interfaces with synthetic polymers to promote the assembly of living cells and the design of multi-responsive healable cell-encapsulating constructs with interest in 3D in vitro modelling, drug delivery, cell-based therapies and tissue engineering. In the first part, cell membrane engineering approaches are introduced to create a responsive platform for the accelerated and simple formation of cellular aggregates/spheroids, and to study polymer-cell interactions by exploring biorthogonal ligand-receptor multivalent interactions under different conditions. Specifically, boronic acid- and succinimide-based copolymers were first synthesised and fully characterised by physicochemical methods, and found to bind covalently to natural moieties present on the membrane of several cell lines, which can regulate the development of cell spheroids and act as self-supporting “cellular glues”. The second part of the project is dedicated to the development of multi-responsive self-healing hydrogel nanocomposites for biomedical applications, where we further expanded the dynamic crosslinking nature of boronate ester bonds. The proposed gels could be prepared almost instantly, exhibited photo- and thermoreversible transient sol-gel type of transition with excellent healing properties, and no toxicity, which allows the system to be used as a versatile biologic delivery matrix. In summary, the results highlight novel and straightforward approaches that may pave the way to implement a biomaterial-cell platform with broad biotechnological applications.

Tissue engineering (TE) is a rapidly evolving interdisciplinary field that joins together materials science, biomedical engineering and cellular biology, in a quest to reconstruct living tissues upon injury or loss. For this reason TE has the potential to have a large impact in clinical implantations, expanding tissue supply for transplantation therapies. The scaffold is a centrepiece in TE, since it aims to mimic the extracellular matrix (ECM) that is found in natural tissue. Nonetheless, a major constraint in achieving larger constructs has been the lack of means to transport oxygen and waste produced by the cells. The construction of complex structures with an integrated vasculature, with high spatial resolution, is now a reality that opens the door for more complex and larger engineered tissues and organs. This thesis presents the results of a study on the impact on oxygen diffusion and cell viability in stem cell seeded constructs, after biomaterial (hydrogel) mechanical reinforcement with a laponite clay, considered to be of great potential for regenerative medicine. The impact on oxygen and nutrient diffusion and cell viability in stem cell seeded constructs after hydrogel mechanical reinforcement through polymer concentration is also presented and discussed. The impact on oxygen diffusion and cell viability after the creation of a microchannel network inside stem cell constructs, through a bioprinting technique, was quantified and constitutes the last part of the present work.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, February 2001.
Includes bibliographical references (leaves 121-129).
Approaches to achieve three dimensional (3D) reconstruction from 2D images can be grouped into two categories: computer-vision-based reconstruction and tomographic reconstruction. By exploring both the differences and connections between these two types of reconstruction, the thesis attempts to develop a new technique that can be applied to 3D reconstruction of biological structures. Specific attention is given to the reconstruction of the cell cytoskeleton from electron microscope images. The thesis is composed of two parts. The first part studies computer-vision-based reconstruction methods that extract 3D information from geometric relationship among images. First, a multiple-feature-based stereo reconstruction algorithm that recovers the 3D structure of an object from two images is presented. A volumetric reconstruction method is then developed by extending the algorithm to multiple images. The method integrates a sequence of 3D reconstruction from different stereo pairs. It achieves a globally optimized reconstruction by evaluating certainty values of each stereo reconstruction. This method is tuned and applied to 3D reconstruction of the cell cytoskeleton. Feasibility, reliability and flexibility of the method are explored.
(cont.) The second part of the thesis focuses on a special tomographic reconstruction, discrete tomography, where the object to be reconstructed is composed of a discrete set of materials each with uniform values. A Bayesian labeling process is proposed as a framework for discrete tomography. The process uses an expectation-maximization (EM) algorithm with which the reconstruction is obtained efficiently. Results demonstrate that the proposed algorithm achieves high reconstruction quality even with a small number of projections. An interesting relationship between discrete tomography and conventional tomography is also derived, showing that discrete tomography is a more generalized form of tomography and conventional tomography is only a special case of such generalization.
by Yuan Cheng.
Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 110-127).
For many cancers, dissemination of tumor cells to form metastases is not only a hallmark of the disease but an essential step to mortality. Migration and dissemination are complex, multistep processes, and study of their regulation has been challenging. Metastases need only be driven by a rare subpopulation of tumor cells, and a portion of dissemination is necessarily interaction with the cell's environment and thus cell extrinsic. Experimentally, there is additional uncertainty as exactly how to best assess migration outside of the complex in vivo environment. To develop a systems perspective of invasive disease, we first examine some of the experimental models used to study cell migration. We then apply this knowledge to examine regulation by proteases of endometrial cell invasion, and the pro-migratory effects of receptor crosstalk in breast carcinoma cells. Finally, extending from clear limitations in our knowledge of signaling regulation specifically within the invasive subpopulation of cells, we develop a model of ligand-mediated signaling for a receptor often expressed specifically during the process of dissemination. In total, this thesis extends systems biology techniques to the study of cell migration within the extracellular environment, with focus on that subpopulation of cells most directly implicated in the formation of metastatic disease.
by Aaron Samuel Meyer.
Ph. D.

[ES] El mieloma múltiple es una neoplasia hematológica caracterizada por una expansión descontrolada de células plasmáticas monoclonales (mPCs) en medula ósea que producen, en la mayoría de los casos, un componente monoclonal secretado en el suero y/o en orina. En la actualidad, se sigue considerando una enfermedad incurable con la constante aparición de recaídas en los pacientes. Una de las causas que condicionan esta situación, radica en la generación de resistencia frente a fármacos por parte de las mPCs. Este mecanismo de resistencia a fármacos (DR) se ha visto que no solo depende de factores intracelulares, sino que la propia interacción de las mPCs con el microambiente medular juega un papel fundamental para su supervivencia, crecimiento y desarrollo de DR. Entre los componentes del microambiente tumoral, destaca la adhesión de las mPCs a componentes de la matriz extracelular (ECM) que se ha visto relacionada con la generación de DR. Por este motivo el desarrollo de esta tesis doctoral consistió en la elaboración y validación de una plataforma de cultivo 3D basado en la síntesis de un microgel. Este sistema estará constituido por microesferas funcionalizadas con componentes de la ECM como son la fibronectina (FN), colágeno tipo I (COL), heparina (Hep), heparan sulfato (HS) y ácido hialurónico (HA), generando un entorno 3D biomimético con la capacidad de poder analizar la respuesta celular desencadenada por la interacción de las mPCs con los componentes de la ECM, así como la DR generada por la adhesión de las mPCs a estas biomoléculas. El primer estudio consistió en la realización y puesta a punto de varios protocolos para la síntesis de distintos microgeles; un primer sistema se produjo mediante la polimerización por vía radical en bloque de co-polímeros de poliacrilato de etilo (EA) y polimetacrilato de etilo (EMA) o bien por EA, EMA y ácido acrílico (AAc). Mediante una emulsión del tipo aceite en agua se consiguió producir con estos copolímeros, microesferas de un tamaño próximo al de las mPCs. Un segundo sistema se basó en microesferas de alginato. Estas microesferas se obtuvieron en un dispositivo de microfluidica produciéndose la gelificación externa de las micro-gotas con la incorporación de iones de calcio consiguiendo microesferas de un tamaño medio de 177 µm. Debido a la gran variedad de microesferas sintetizadas con diferentes grupos químicos en sus superficies, se consiguió establecer protocolos de funcionalización similares a los establecidos en la literatura, teniendo en cuenta la estabilidad de la biomolécula a lo largo del tiempo del cultivo celular. Este enfoque, permitió la funcionalización con una gran variedad de biomoléculas disponiendo así de microgeles funcionalizados con FN, COL, Hep, HS y HA. Una vez desarrollados los microgeles, en un segundo estudio se procedió a evaluar la respuesta celular en un entorno 3D basado en microgel, valorando la interacción con los componentes de la ECM. Entre los resultados observados se pudo determinar como el tamaño de las microesferas afecta al crecimiento celular incluso en ausencia de cualquier funcionalización. Con los microgeles constituidos por microesferas de un tamaño próximo al de las mPCs se obtuvo un mayor crecimiento celular que con los microgeles formados por partículas de mayor tamaño, y en ambos el crecimiento fue superior al del cultivo en suspensión. Se plantea la hipótesis de que la presencia de las microesferas favorece en gran medida que se produzca un mayor contacto célula-célula que se ve incrementado cuanto mayor es la superficie específica del microgel. Entre los componentes de la ECM estudiados, mientras que el COL no genera ninguna respuesta celular diferente al control (microgel no funcionalizado), el HA favorece la proliferación celular. La adhesión de las mPCs a la FN condiciona el bloqueo de las células en la fase G0-G1 del ciclo celular. Esta adhesión está mediada
[CA] El mieloma múltiple és una neoplàsia hematològica caracteritzada per una expansió descontrolada de cèl·lules plasmàtiques monoclonals (mPCs) en medul·la òssia que produeixen, en la majoria dels casos, un component monoclonal secretat en el sèrum i/o en orina. En l'actualitat, es continua considerant una malaltia incurable, amb la constant aparició de recaigudes en els pacients. Una de les causes que condicionen aquesta situació, radica en la generació de resistència enfront de fàrmacs per part de les mPCs. Aquest mecanisme de resistència a fàrmacs (DR) s'ha vist que no sols depèn de factors intracel·lulars, sinó que la mateixa interacció de les mPCs amb el microambient medul·lar juga un paper fonamental per a la seua supervivència, creixement i desenvolupament de DR. Entre els components del microambient tumoral, destaca l'adhesió de les mPCs a components de la matriu extracel·lular (ECM) que s'ha vist relacionada amb la generació de DR. Per aquest motiu, el desenvolupament d'aquesta tesi doctoral va consistir en l'elaboració i validació d'una plataforma de cultiu 3D basada en la síntesi d'un microgel. Aquest sistema estarà constituït per microesferes funcionalitzades amb components de l'ECM com són la fibronectina (FN), col·lagen tipus I (COL), heparina (Hep), heparan sulfat (HS) i àcid hialurònic (HA), generant un entorn 3D biomimètic amb la capacitat de poder analitzar la resposta cel·lular desencadenada per la interacció de les mPCs amb els components de la ECM, així com la DR generada per l'adhesió de les mPCs a aquestes biomolècules. El primer estudi va consistir en la realització i posada a punt de diversos protocols per a la síntesi de diferents microgels; un primer sistema es va produir mitjançant la polimerització per via radical en bloc de copolímers de poliacrilat d'etil (EA) i polimetacrilat d'etil (EMA), o bé per EA, EMA i àcid acrílic (AAc). Mitjançant una emulsió del tipus oli en aigua es va aconseguir produir amb aquests copolímers, microesferes d'una grandària pròxima al de les mPCs. Un segon sistema es va basar en microesferes d'alginat. Aquestes microesferes es van obtenir en un dispositiu de microfluidica produint-se la gelificació externa de les microgotes amb la incorporació d'ions de calci aconseguint microesferes d'una grandària mitjana de 177 ¿m. A causa de la gran varietat de microesferes sintetitzades amb diferents grups químics en les seues superfícies, es va aconseguir establir protocols de funcionalització similars als establerts en la literatura, tenint en compte l'estabilitat de la biomolècula al llarg del temps del cultiu cel·lular. Aquest enfocament va permetre la funcionalització amb una gran varietat de biomolècules disposant així de microgels funcionalitzats amb FN, COL, Hep, HS y HA. Una vegada desenvolupats els microgels, en un segon estudi es va procedir a avaluar la resposta cel·lular en un entorn 3D basat en microgel, valorant la interacció amb els components de l'ECM. Entre els resultats observats es va poder determinar com la grandària de les microesferes afecta el creixement cel·lular fins i tot en absència de qualsevol funcionalització. Amb els microgels constituïts per microesferes d'una grandària pròxima al de les mPCs es va obtenir un major creixement cel·lular que amb els microgels formats per partícules de major grandària, i en tots dos el creixement va ser superior al del cultiu en suspensió. Es planteja la hipòtesi que la presència de les microesferes afavoreix en gran manera que es produïsca un major contacte cèl·lula-cèl·lula que es veu incrementat com més gran és la superfície específica del microgel. Entre els components de l'ECM estudiats, mentre que el COL no genera cap resposta cel·lular diferent del control (microgel no funcionalitzat), l'HA afavoreix la proliferació cel·lular. L'adhesió de les mPCs a la FN condiciona el bloqueig de les cèl·lules en la fase G0-G1 del cic
[EN] Multiple myeloma is a haematological neoplasm characterized by an uncontrolled expansion of monoclonal plasma cells (mPCs) in bone marrow that produce, in most cases, a monoclonal component secreted in serum and/or urine. At present, it is still considered an incurable disease with the constant appearance of relapses in patients. One of the causes that condition this situation lies in the generation of drug resistance by the mPCs. This mechanism of drug resistance (DR) has been seen to depend not only on intracellular factors, but the very interaction of mPCs with the medullary microenvironment plays a fundamental role in their survival, growth and development of DR. Among the components of the tumor microenvironment, the adhesion of the mPCs to components of the extracellular matrix (ECM) stands out, which has been related to the generation of DR. For this reason, the development of this doctoral thesis consisted in the elaboration and validation of a 3D culture platform based on the synthesis of a microgel. This system will be made up of micropsheres functionalized with the components of the ECM such as fibronectin (FN), collagen type I (COL), heparin (Hep), heparan sulphate (HS) and hyaluronic acid (HA), generating a 3D biomimetic environment with the ability to analyse the cellular response triggered by the interaction of mPCs with the ECM components, as well as the DR generated by the adhesion of the mPCs to these biomolecules. The first study consisted in the realization and development of several protocols for the synthesis of different microgels. A first system was produced by the radical block polymerization of polyethylene acrylate (EA) and polymethacrylate (EMA) co-polymers or by EA, EMA and acrylic acid (AAc). By means of an oil-in-water emulsion technique, it was possible to produce, with these copolymers, microspheres of a size close to that of the mPCs. A second system was based on alginate microspheres. These microspheres were obtained in a microfluidic device producing the external gelification of the micro-drops with the incorporation of calcium ions, obtaining microspheres with an average size of 177 µm. Due to the great variety of microspheres synthesized with different chemical groups on their surfaces, it was possible to establish functionalization protocols similar to those established in the literature, taking into account the stability of the biomolecule along with the time of cell culture. This approach allowed for functionalization with a great variety of biomolecules, having in this way functionalized microgels with FN, COL, Hep, HS and HA. Once the microgels were developed, a second study was carried out to evaluate the cell response in a 3D microgel-based environment, assessing the interaction with the components of the ECM. Among the results observed, it was possible to determine how the size of the microspheres affects cell growth even in the absence of any functionalization. With the microgels constituted by microspheres close to the size of the mPCs, a greater cellular growth was obtained than with the microgels formed by larger particles, and in both the growth was higher than in suspended culture. It is hypothesized that the presence of microspheres greatly favours a greater cell-cell contact, which is increased the larger the specific surface area of the microgel. Among the components of the ECM studied, while the COL does not generate any cellular response different from the control (non-functionalized microgel), HA favours cell proliferation. The adhesion of mPCs to FN conditions the blocking of cells in the G0-G1 phase of the cell cycle. This adhesion is mediated by the integrin ¿4ß1.
La presente tesis doctoral no se podría haber realizado sin la financiación del proyecto PROMETEO/2016/063, trabajo que también estuvo parcialmente financiado con fondos FEDER (CIBERONC (CB16/12/00284)). La iniciativa CIBER-BBN está financiada por el proyecto VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. Las acciones CIBER están financiadas por el Instituto de Salud Carlos III con ayuda del Fondo Europeo de Desarrollo Regional.
Marin Paya, JC. (2021). 3D Culture o Multiple Myeloma Cell Line Using Microgel Environments [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/167427
TESIS

Vascularization is essential for living tissue and remains a major challenge in the field of tissue engineering. A lack of a perfusable channel network within a large and densely populated tissue engineered construct leads to necrotic core formation, preventing fabrication of functional tissues and organs. While many approaches have been reported for forming vascular networks, including materials processing techniques, such those involving lithography, bioprinting, and sacrificial templating; and cell-based approaches, in which cellular self-organization processes form vessels; all are deficient in their ability to form a vessel system of sufficient complexity for supporting a large cellular construct. What is missing from the literature is a method for forming a fully three-dimensional vascular network over the full range of length-scales found in native vessel systems, which can be used alongside cells and perfused with fluids to support their function. A large number of research groups are thus pursuing novel methods for fabricating vascular systems in order that new tissues and organs can be fabricated in the lab. In this project, a 3D printing-based approach was used to form vascular networks which are hierarchical, three-dimensional, and perfusable. This was performed in thick, cellularized hydrogels similar in composition to native tissue; these being collagen (ECM-like) and fibrin (woundlike), both of which are highly capable of supporting cellular activities, such as cell seeding, cell spreading, and capillary morphogenesis. In order to make use of 3D printed network templates in cellularized hydrogel environments, it was necessary to develop a new approach in which standard 3D printed materials were converted into a gelatin template, via an alginate intermediary, which can be removed quickly in physiologic conditions and which does not reduce cell viability. This multi-casting approach enables a hierarchical channel network to be formed in three-dimensions, capable of being perfused with cell medium to maintain the viability of a cell population, thereby addressing the fundamental problem. Using standard cell staining and immuno-histochemistry techniques, we showed good endothelial cell seeding and the presence of tight junctions between the channel endothelial cells. When fibroblasts were seeded into the bulk of the hydrogel, a high degree of cell viability and cell spreading was observed when a threshold flow rate is met. By counting the number of live and dead cells in a sample regions of the gel, we were able to show a dependency of cell viability upon the perfusion flow rate and further determine a regime in which the vast majority of cells are alive and spreading. This data informs future cellular experiments using this platform technology. The limits of existing 3D printing technology meant that the micro-scale vasculature needed to be formed by other means. Cellular co-culture of endothelial and stromal cell types has been shown to be capable of forming capillary-like structures in vitro. For inclusion with the 3D printed channel system, we investigated the use of an angiogenic method for capillary formation, using multi-cellular spheroids, and a vasculogenic approach, using individual cells, in order that the full vascular system could be constructed. Endothelial and mesenchymal stromal cells were encapsulated in small fibrin and collagen gels and maintained under static culture conditions in order to form capillaries by the above approaches. The aim here was to find a particular gel composition and cell concentration which would support capillary morphogenesis while being suitably robust to handle the mechanical stresses associated with perfusion. As future work, the next step will be to incorporate the vasculogenic co-culture technique, used to form capillary-sized vessels, into a perfusable gel containing the large templated channels, formed via the multi-casting approach. The challenge here is to anastomose the capillary-sized vessels to the large templated channels and thereby enable perfusion of the capillary vessels. This step would be a highly significant development in the field as it would mean large constructs could be fabricated with physiological densities of cells, which could lead to a range of potential therapeutic applications.

Les conditions du corps humain ne sont pas reproduites fidèlement par la culture cellulaire traditionnelle en 2D. Dans cette thèse, des cultures cellulaires 3D sont réalisées dans une plateforme microfluidique hautement intégrée. Des cellules mammifères adhérentes sont encapsulées dans des gouttes immobilisées dans un tableau de pièges capillaires à haute densité. Dans chaque goutte, les cellules se réorganisent pour former un unique microtissu 3D et fonctionnel appelé sphéroïde. L'utilisation d'un hydrogel permet d'alonger le temps de culture et de perfuser le tableau avec des solutions aqueuses, par exemple pour de l'immuno-cyto-chimie. Un unique sphéroïde, viable, peut aussi être extrait de cette puce microfluidique. Des données quantitatives sont extraites à haut débit au niveau de la population, du sphéroïde (dizaines de miliers de sphéroïdes) et au niveau cellulaire emph{in situ} (centaines de miliers de cellules) grâce à de l'imagerie de fluorescence et au dévelopement d'un code d'analyse d'image. Une première preuve de concept a été obtenue en démontrant la viabilité, la prolifération et la fonctionalité de sphéroïdes d'hépatocytes et en les corrélant à des paramètres morphologiques. Ensuite, des aggrégats de cellules souches mésenchymales ont été produits et les hétérogénéités spatiales dans l'expression de protéines impliquées dans leurs propriétés thérapeutiques ont été étudiées. Enfin, cette technologie a été encore dévelopée pour permettre d'appliquer des conditions biochimiques différentes dans chaque goutte. La production et la culture de sphéroïdes dans cette plateforme microfluidique peut mener à des dévelopements importants dans beaucoup de domaines tels que l'analyse de la toxicité des médicaments, le criblage de médicaments à haut débit, le traitement personnalisé du cancer, l'ingénierie tissulaire ou la modélisation de maladies
Conventional 2D cell culture fails to reproduce emph{in vivo} conditions. In this PhD thesis, 3D cell culture is implemented into a highly integrated microfluidic platform. Adherent mammalian cells are encapsulated in droplets immobilized on a high density array of capillary traps called anchors. In each droplet, the cells reorganize into a single functional 3D microtissue called spheroid. The use of an hydrogel allows to extend the culturing time in microdroplets and to perfuse the array with aqueous solutions, for instance for immuno-cyto-chemistry. A single and viable spheroid can also be selectively retrieved from the microfluidic chip. High throughput and quantitative data is extracted at the population, spheroid (tens of thousands of spheroids) and cellular level emph{in situ} (hundreds of thousands of cells) thanks to fluorescent imaging and a custom image analysis software. As a first proof of concept, the viability, proliferation and functionality of hp sh s were demonstrated and correlated with morphological parameters. Drug toxicity experiments were also performed on this liver model. Then, human mesenchymal stem cell aggregates were produced and the spatial heterogeneities of the expression of proteins involved in their therapeutic properties were investigated. Finally, this technology was further developed to enable applying different biochemical conditions in each droplet. The production and culture of spheroids in this microfluidic platform could lead to major advances in many fields such as drug toxicity, high throughput drug screening, personalized cancer treatment, tissue engineering or disease modeling

Pancreatic cancer continues to have one of the poorest prognoses amongst all cancers, with a 95% mortality rate. Standard of care chemotherapy has failed to provide significant clinical benefits, which has led to the development of targeted agents against validated signalling pathways. However, to date the approach of targeted agents alone, or in combination with traditional chemotherapeutics, has failed to significantly improve the prognosis for pancreatic cancer patients. The current standard of care chemotherapy for advanced pancreatic cancer provides only a modest increase in survival of several months. Models that improve the predictive potential of drug discovery programs and gain greater insights into the complexity of tumour biology are therefore urgently required. To better understand the mechanisms influencing the anti-cancer activities of current and novel therapies, we have developed a 3D in vitro micro-tumour cell culture model. Current in vitro models utilising cell monolayer cultures are unable to recapitulate the biological and physiological complexities of the in vivo pancreatic tumour microenvironment and may be poor predictors of drug efficacy. Pancreatic adenocarcinomas are characterised as having a highly dense and poorly vascularised stroma that is made up of extracellular matrix (ECM) components and host cells. This complex tumour microenvironment has been implicated in the chemoresistance profiles observed in pancreatic tumours.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Eskitis Institute for Cell and Molecular Therapies
Science, Environment, Engineering and Technology
Full Text

The field of biomaterials has seen huge development over the past decade with enormous efforts invested in discovering materials with improved biocompatibility, application and versatility. Polymers can display many properties that make them ideal biomaterials, such as their potential flexibility, low weight, low cost and biodegradability. Moreover, they can be prepared in a wide variety of compositions and forms and be readily fabricated into various shapes and structures. Polymer microarrays represent an efficient high-throughput platform for the screening and discovery of new materials compared to conventional assays with advantages such as high-density screening, internal consistency of assays and the requirement for only small quantities of material. The first part of this thesis describes work in the area of diabetes research with a focus on how dysfunctional β-cells could be replaced by the transplantation of β-cells obtained from pluripotent stem cells. To achieve this aim, high numbers of β-cells are required. A polymer microarray screening approach was used to identify a number of polymers that promoted the attachment of pancreatic progenitor cells and enhanced cell proliferation. Multiple scale-up fabrication techniques were assessed to establish the most suitable approach and surface for long term cell culture leading to the obtainment of reproducible in situ polymerised polymer layers with enhanced binding properties toward pancreatic progenitor cells. These surfaces have the potential to support cell adhesion and proliferation and could find potential use in the industrial sector to increase the production of pancreatic progenitor cells in vitro. In the second part, efforts were made to gain a better understanding of the maturation of β-cells and their behaviour, with the development of 3D hydrogels based on the previously identified polymers. In this scenario, parameters such as stiffness and porosity were evaluated to identify the best environmental conditions to support 3D cell culturing of pancreatic progenitor cells. Several approaches were tested to generate scaffolds with suitable stiffness and porosity leading to the obtainment of scaffolds based on the previously identified polymer composition and with controlled porosity and stiffness. These scaffolds could represent a suitable environment to allow a better understanding of cell organisation and regulation. In a third avenue of work, arrays of 3D biocompatible materials, which were tailored for varying elasticity, hardness, and porosity (to provide the necessary physical cues to control cellular functions) were fabricated. In this chapter, details of the development of an array of eighty 3D double-network hydrogel features are reported. The array features can be produced as single or double networks and modulated in terms of stiffness, viscoelasticity and porosity to assess cell response to materials with a wide range of properties. The final part of the thesis describes the development and screening of polymeric materials to allow a better understanding of cell–surface interactions with various cell types. To investigate the correlation between cell attachment and the nature of the polymer, a series of random and block copolymers were synthesised and examined for their abilities to attach and support the growth of human cervical cancer cells (HeLa) and human embryonic kidney cells (HEK293T), with attachment modelled on monomer ratios, arrangement, and polymer chain length. The results of this screening showed differences between block copolymers and random copolymers in cell adhesion and provide interesting insight into the improvement of polymer coatings for cell culture.

Ce travail de thèse se situe à l’interface de deux domaines : la culture cellulaire en trois dimensions et l’imagerie sans lentille.Fournissant un protocole de culture cellulaire plus réaliste sur le plan physiologique, le passage des cultures monocouches (2D) à des cultures tridimensionnelles (3D) - via l’utilisation de gels extracellulaires dans lesquels les cellules peuvent se développer dans les trois dimensions - permet de faire de grandes avancées dans de nombreux domaines en biologie tels que l’organogénèse, l’oncologie et la médecine régénérative. Ces nouveaux objets à étudier crée un besoin en matière d’imagerie 3D.De son côté, l’imagerie sans lentille 2D fournit un moyen robuste, peu cher, sans marquage et non toxique, d’étudier les cultures cellulaires en deux dimensions sur de grandes échelles et sur de longues périodes. Ce type de microscopie enregistre l’image des interférences produites par l’échantillon biologique traversé par une lumière cohérente. Connaissant la physique de la propagation de la lumière, ces hologrammes sont rétro-propagés numériquement pour reconstruire l’objet recherché. L’algorithme de reconstruction remplace les lentilles absentes dans le rôle de la formation de l’image.Le but de cette thèse est de montrer la possibilité d’adapter cette technologie sans lentille à l’imagerie des cultures cellulaires en 3D. De nouveaux prototypes de microscopes sans lentille sont conçus en parallèle du développement d’algorithmes de reconstructions tomographiques dédiés.Concernant les prototypes, plusieurs solutions sont testées pour converger vers un schéma alliant deux conditions. La première est le choix de la simplicité d’utilisation avec une culture cellulaire en boîte de Petri standard et ne nécessitant aucune préparation spécifique ou aucun changement de contenant. Cette condition entraînant de fortes contraintes géométriques sur l’architecture, la deuxième est de trouver la meilleure couverture angulaire possible des angles d’éclairage. Enfin, une version adaptée aux conditions en incubateur est développée et testée avec succès.Concernant les algorithmes, quatre types de solutions sont proposés, basées sur le théorème de diffraction de Fourier classiquement utilisé en tomographie diffractive optique. Toutes cherchent à corriger deux problèmes inhérents au microscope sans lentille : l’absence de l’information de phase, le capteur n’étant sensible qu’à l’intensité de l’onde reçue, et la couverture angulaire limitée. Le premier algorithme se limite à remplacer la phase inconnue par celle d’une onde incidente plane. Rapide, cette méthode est néanmoins source de nombreux artefacts. La deuxième solution, en approximant l’objet 3D inconnu par un plan moyen, utilise les outils de la microscopie sans lentille 2D pour retrouver cette phase manquante via une approche inverse. La troisième solution consiste à implémenter une approche inverse régularisée sur l’objet 3D à reconstruire. C’est la méthode la plus efficace pour compenser les deux problèmes mentionnés, mais elle est très lente. La quatrième et dernière solution est basée sur un algorithme de type Gerchberg-Saxton modifié avec une étape de régularisation sur l’objet.Toutes ces méthodes sont comparées et testées avec succès sur des simulations numériques et des données expérimentales. Des comparaisons avec des acquisitions au microscope classique montrent la validité des reconstructions en matière de tailles et de formes des objets reconstruits ainsi que la précision de leur positionnement tridimensionnel. Elles permettent de reconstruire des volumes de plusieurs dizaines de millimètres cubes de cultures cellulaires 3D, inaccessibles en microscopie standard.Par ailleurs, les données spatio-temporelles obtenues avec succès en incubateur montrent aussi la pertinence de ce type d’imagerie en mettant en évidence des interactions dynamiques sur de grandes échelles des cellules entres elles ainsi qu’avec leur environnement tridimensionnel
This PhD work is at the interface of two fields: 3D cell culture and lens-free imaging.Providing a more realistic cell culture protocol on the physiological level, switching from single-layer (2D) cultures to three-dimensional (3D) cultures - via the use of extracellular gel in which cells can grow in three dimensions - is at the origin of several breakthroughs in several fields such as developmental biology, oncology and regenerative medicine. The study of these new 3D structures creates a need in terms of 3D imaging.On another side, 2D lens-free imaging provides a robust, inexpensive, non-labeling and non-toxic tool to study cell cultures in two dimensions over large scales and over long periods of time. This type of microscopy records the interferences produced by a coherent light scattered by the biological sample. Knowing the physics of the light propagation, these holograms are retro-propagated numerically to reconstruct the unknown object. The reconstruction algorithm replaces the absent lenses in the role of image formation.The aim of this PhD is to show the possibility of adapting this lens-free technology for imaging 3D cell culture. New lens-free microscopes are designed and built along with the development of dedicated tomographic reconstruction algorithms.Concerning the prototypes, several solutions are tested to finally converge to a scheme combining two conditions. The first requirement is the choice of simplicity of use with a cell culture in standard Petri dish and requiring no specific preparation or change of container. The second condition is to find the best possible angular coverage of lighting angles in regards of the geometric constraint imposed by the first requirement. Finally, an incubator-proof version is successfully built and tested.Regarding the algorithms, four major types of solutions are implemented, all based on the Fourier diffraction theorem, conventionally used in optical diffractive tomography. All methods aim to correct two inherent problems of a lens-free microscope: the absence of phase information, the sensor being sensitive only to the intensity of the incident wave, and the limited angular coverage. The first algorithm simply replaces the unknown phase with that of an incident plane wave. However, this method is fast but it is the source of many artifacts. The second solution tries to estimate the missing phase by approximating the unknown object by an average plane and uses the tools of the 2D lens-free microscopy to recover the missing phase in an inverse problem approach. The third solution consists in implementing a regularized inverse problem approach on the 3D object to reconstruct. This is the most effective method to deal with the two problems mentioned above but it is very slow. The fourth and last solution is based on a modified Gerchberg-Saxton algorithm with a regularization step on the object.All these methods are compared and tested successfully on numerical simulations and experimental data. Comparisons with conventional microscope acquisitions show the validity of the reconstructions in terms of shape and positioning of the retrieved objects as well as the accuracy of their three-dimensional positioning. Biological samples are reconstructed with volumes of several tens of cubic millimeters, inaccessible in standard microscopy.Moreover, 3D time-lapse data successfully obtained in incubators show the relevance of this type of imaging by highlighting large-scale interactions between cells or between cells and their three-dimensional environment

Natural Killer cells are a type of lymphocyte belonging to the innate immune system and they operate cell-mediated cytotoxicity and release of pro-inflammatory cytokines against cancerous cells. However, in vivo testings have shown a reduced activity of NK cells against solid tumors probably due to the negative influence of the immunosuppressive tumor microenvironment. Multicellular tumor spheroids may constitute an advantageous model in cancer biology for studying the mechanisms behind cancer immune editing since it more closely mimics the complexity of the human body compared with the 2D model counterpart. This study investigated the interaction between NK cells isolated from blood and tumor spheroids obtained from A498 renal carcinoma cells, using light-sheet microscopy imaging which allows satisfactory cell tracking in the inner layers of the spheroids. NK cells not only indeed interact with tumor spheroids, but many of them were able to penetrate the spheroids inducing some changes in the structure of the latter. NK cells were also tracked over time, displaying the migration path and calculating the speed. The fluorescence intensity of the NK cells was found reduced as soon as they penetrate the spheroid but, conversely, the speed seems to increase inside the spheroid, a possible sign of the fallibility of the tracking algorithm in this specific case. We propose solutions for more sophisticated future implementations, involving the use of marks during the experimental phase and drift corrections at the data analysis level.

This study aimed to develop a 3-Dimensional (D) hydrogel system for the co-culture of autologous human renal and immune cells. Previous studies have shown that human renal epithelial cells are able to modulate autologous immune cell responses. However, these studies were undertaken in a standard 2D culture system. The 3D model was developed to re-capitulate these observations within a more physiological relevant in vivo like environment.

Mesenchymal stem cells (MSCs) have great potential to improve clinical outcomes for many inflammatory and degenerative diseases through delivery of exogenous MSCs via injection or cell-laden scaffolds and through mobilization and migration of endogenous MSCs to injury sites. MSC fate and function is determined by microenvironmental cues, specifically dimensionality, topography, and cell-cell interactions. MSC responses of migration and differentiation are the focus of this dissertation. Cell migration occurs in several physiological and pathological processes; migration mode and cell signaling are determined by the environment and type of confinement in three-dimensional (3D) models. Tendon injury is a common musculoskeletal disorder that occurs through cumulative damage to the extracellular matrix (ECM). Studies combining nanofibrous scaffolds and MSCs to determine an optimal topographical environment have promoted tenogenic differentiation under various conditions. We investigated cellular response of MSCs on specifically designed nanofiber matrices fabricated using a novel spinneret-based tunable engineered parameters production method (STEP). We designed suspended and aligned nanofiber scaffolds to study cellular morphology, tendon marker gene expression, and matrix deposition as determinants for tendon differentiation. The delivery and maintenance of MSCs at sites of inflammation or injury are major challenges in stem cell therapies. Enhancing stem cell homing could improve their therapeutic effects. Homing is a process that involves cell migration through the vasculature to target organs. This process is defined in leukocyte transendothelial migration (TEM); however, far less is known about MSC homing. We investigated two population subsets of MSCs in a Transwell system mimicking the vasculature; migrated cells that initiated transmigration on the endothelium and nonmigrated cells in the apical chamber that failed to transmigrate. Gene and protein expression changes were observed between these subsets and evidence suggests that multiple signaling pathways regulate TEM. The results of these experiments have demonstrated that microenvironmental cues are critical to understanding the cellular and molecular mechanisms of MSC response, specifically in homing and differentiation. This knowledge has identified scaffold parameters required to stimulate tenogenesis and signaling pathways controlling MSC homing. These findings will allow us to target key regulatory molecules and cell signaling pathways involved in MSC response towards development of regenerative therapies.
Ph. D.

La formation et la consolidation de souvenirs est l’une des caractéristiques fondamentales du cerveau, responsable de l’apprentissage et de comportements cognitifs élevés. Malgré son importance, ce processus n’est pas entièrement compris à ce jour et fait l’objet de nombreux travaux de de recherche, allant de l’analyse de l’activité des synapses individuelles à la reconstruction de cartes de connectivité du cerveau. Dans ce travail, nous proposons une approche intégrée pour mesurer in vivo l’activité de chaque neurone du corps pédonculé (Mushroom body, MB) de la Drosophila melanogaster dans une procédure entièrement automatisée. Il s’agit d’imager en 3D et dans le temps le MB dans sa totalité par microscopie confocale et d’opérer un suivi temporel de la position de chaque neurone afin de relever leur niveau individuel d’activité. En utilisant cette approche, nous avons découvert que pendant la formation de la mémoire à long terme, de nouveaux neurones sont recrutés au sein du corps pédonculés, tandis que l’intensité de la réponse des neurones individuels reste inchangée. Au delà de l’apport méthodologique qui permet à présent de quantifier automatiquement l’activité d’un grand nombre de neurones, ce travail a contribué à une meilleure compréhension de la formation de la mémoire à long terme
Formation and consolidation of new memories is one of the fundamental characteristics of the brain, responsible for learning and high cognitive behavior. While important, the process isn’t fully understood to the present day and is the subject of various studies, spanning from the activity analysis of individual synapses to the reconstruction of brain connectivity maps. In this work, we propose a bold approach, on which we aim to measure in vivo the activity of every single neuron from the whole Mushroom body (MB) of the Drosophila melanogaster, in a fully automated procedure. After a 3D image acquisition over time of the MB by means of confocal microscopy, an automated detection and tracking of the neurons is performed. The whole process takes place while the fly is awake and subjected to different odor stimulations, so that it is possible to associate the activity patterns at the single cell level to the stimulus that is being received. By comparing the response patterns from flies that were trained and flies that were not trained to associate an odor with an electric shock we identified changes in neuronal activity, providing information on how memory is formed. Beyond the methodological innovation that brought the possibility to track the activity of a large set of single neurons, this work contributed to the current understanding of long term memory formation

The development of different organs and tissues along the gastrointestinal tract, including the pancreas, depends on signalling between the endoderm and the adjacent mesenchyme. The Nkx gene Bapx1 is involved in spatial control of organ-positioning in the spleno-pancreatic region, and deficiency in this gene results in unacceptable proximity of the splenic mesenchyme to the pancreas. This permits agitating signals from the splenic mesenchyme to induce an in vivo (and in vitro) transformation of pancreatic epithelium to a cystic structure with gut like features. Also, wild type splenic mesenchyme is competent to induce a similar transformation. These findings illustrate the importance for strict control of organ positioning during spleno-pancreatic development. Several growth factors and receptors involved in pancreatic development are activated by protease processing. Some of these growth factors have been implicated as substrates for members of the A Disintegrin And Metalloprotease (ADAM) family. The ADAMs 9, 10, and 17 are expressed during pancreatic development and in the adult pancreas, suggesting a possible role for these ADAMs in pancreatic development and function. Animal model systems are widely used to investigate gene function during development and disease. However, spatial, molecular, and quantitative phenotype screening in animals is a time consuming effort. Optical Projection Tomography is a 3-dimensional imaging technique that, in combination with improvements in sample preparation and computer processing, can be used to visualize and quantify characteristics of intact adult mouse organs such as the total β-cell content in the pancreas.