Dissertations / Theses on the topic 'Cell culture'
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1
Liu, Mengfei, and 刘梦菲. "Epithelial morphogenesis in three-dimensional cell culture system." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/208611.
Full textAbstract:
In human body, the most common structures formed by epithelial cells are hollow cysts or tubules. The key feature of the cysts and tubules is the central lumen, which is lined by epithelial cell sheets. The central lumen allows material exchange, thus it is indispensable for the proper function of the epithelial tissue.
In order to understand the way that the epithelial cells form highly specialized structure, an in vitro three-dimensional (3D) culture system was established. The Caco-2 cells were embedded in reconstituted basement membrane termed matrigel, whose biochemical constitution and physical properties were similar with the in vivo environment. The Caco-2 cells in matrigel spontaneously formed spherical multi-cell cysts, which could continuously expand. The confocal imaging and reconstruction technique helped understand the cyst structure and its formation process. The cysts developed central lumen surrounded by a layer of polarized cells. The apical domain of the cells faced the lumen, while the basal domain attached to the extracellular matrix.
In the mature cysts, fluid was secreted by the cells around the lumen at the apical domain, and accumulated in the central lumen. The laser burning experiment showed that the intraluminal pressure was higher than the outer environment. The intact cell sheet was required to keep the engorged morphology of the cysts. The tension of the cell layer balanced with the intraluminal pressure.
To investigate the effect of pressure on cyst development, the cysts were treated with cholera toxin, which could increase intraluminal pressure through promoting apical secretion. The time-lapse images showed that under cholera toxin treatment, the expansion of the cysts was accelerated. The high intraluminal pressure led to shape change of thecells, followed by increase in cell proliferation rate. Cholera toxin itself could not promote cell growth. In the3D cultured cysts, it was the increased intraluminal pressure that directly induced the acceleration of cell proliferation. It indicated that not only biochemical signals, but also mechanical force, contributed to epithelial morphogenesis.
The mechanical stimulation could be converted into biochemical signals, further affect cell behavior. In response to mechanical stimulation, the focal adhesion kinase was activated in the cells around the cyst lumen. Furthermore, the microarray analysis suggested that multiple signaling pathways were altered under intraluminal pressure stimulation, including the pathways related to cytoskeleton organization, cell cycle and cell adhesion.
Taken together, comparing with the conventional two-dimensional cell culture on rigid surface, the three-dimensional culture system provided the cells a more physiological environment. The 3D culture system allows the epithelial cells to form well-organized hollow structure. It is a convenient model for investigating the process and mechanism of epithelial morphogenesis.published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy
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Theil, Ian. "Anchorage-dependent mammalian cell culture." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=56768.
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Genetically engineered anchorage-dependent human embryonic kidney (293) cells were cultured at 37$ sp circ$C on 1 mm thick sheets of a fibrous polymeric matrix having an average fibre diameter of 10.2 $ mu$m and a void fraction of 0.81 using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2.5 mM glutamine. Immobilization efficiencies above 70% were observed when cells were added to 100 mL spinner flasks (operating at 60 rpm) containing 70 mL of medium and two 1 x 1 cm squares of matrix (total gross area of 2 cm$ sp2$) fastened to the base of the stirrer shaft. Loadings in excess of 2.4 $ times 10 sp7$ cells per cm$ sp2$ of matrix were measured after 2 h.The state of the cultures was followed by measuring the consumption of glucose and glutamine and the production of lactate and ammonium.
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Machado, Roque Ana Isabel. "Protein scaffolds for cell culture." Thesis, University of Newcastle Upon Tyne, 2013. http://hdl.handle.net/10443/1843.
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We report here the design, purification and structural characterization of a new protein scaffold for cell culture. Prior studies in our group revealed the structure of the bacterial protein Caf1 to be flexible protein nanofibres, up to 1.5 μm. The existing Cafl expression system was cumbersome and difficult to mutate, we have now produced a system containing the caf operon which allows for the incorporation of specific peptide motifs. The small peptide, RGDS from fibronectin was inserted into 5 different surface loops of Caf1. The Caf1 mutants were expressed and purified and a structural characterization by biophysical methods was conducted. This revealed permissive sites into which new motifs can be inserted. The characterised proteins were sterilised and used to coat 96 well plates for cell culture. In this study we used mammalian cell lines such as 3T3 fibroblasts, PC12 neuronal cells and primary osteoblasts to understand how they behave in the presence of this biomaterial, in particular the formation of focal adhesions, changes in cytoskeleton rearrangement and nuclear and cell morphology. The controlled engineering of sites within the polymer allowed us to study their implication in cell attachment, survival and proliferation. Our preliminary results have shown that cells interact poorly with the unmodified protein e.g. without any motif associated. This reveals that the polymer is inert and does not influence cell growth by itself. In contrast, the incorporation of RGDS, can invert the scenario of cell growth; promoting cell attachment, survival and proliferation. In a second stage of the project we designed a separate compatible plasmid encoding caf1 gene and used it with the previous plasmid to co-express hybrid Caf1 polymers. The long fibres can also be crosslinked with a non-toxic and non-immunogenic chemical compound – NHS-PEG. Thus a protein hydrogel composed of interchangeable folding units which can be used to incorporate different cell interacting peptide motifs. It is robust and, in the unmodified state highly protease resistant. Future studies will elucidate the versatility and potentiality for this peptide hydrogel in stem cell differentiation.
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Lee, Kevin Shao-Kwan. "Microscale controlled continuous cell culture." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/64579.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2011.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 489-500).
Measurements of metabolic and cellular activity through substrate and product interactions are highly dependent on environmental conditions and cellular metabolic state. For such experiments to be feasible, continuous cultures are utilized to ensure consistent conditions. However, since medium must be replenished every cell doubling time, costs can be prohibitive in large reactors. An integrated microscale bioreactor with built-in fluid metering and environmental control will enable programmed experiments capable of generating reproducible data routinely. This work develops an instrument capable of supporting automated microscale continuous culture experiments. The instrument consists of a plastic-PDMS device capable of continuous flow reactions without volume drift. A novel bonding process is invented to fabricate devices with chemically stable interfaces against water, acids, and bases. We introduce a direct CNC machining and chemical bonding fabrication process for production of fluidic devices with a 1 mL working volume, high oxygen transfer rate (kLa ~ 0.025 s-1), fast mixing (2 s), accurate flow control (± 18 nL), and closed loop control over temperature, cell density, oxygen, and pH. Providing control over environmental parameters allows the system to perform different types of cell culture on a single device, such as batch, fed-batch, chemostat, and turbidostat continuous culture. Validation experiments demonstrate that cells can be grown to high optical densities (OD = 50) and production of commercially relevant chemicals such as DNA vaccines are comparable to large scale bench fermentations. Continuous cultures are also demonstrated without contamination for 3 weeks in a single device and both steady state and dynamically controlled conditions are possible, allowing observations of cell metabolic dynamics.
by Kevin Shao-Kwan Lee.
Ph.D.
5
Schley, Jeremiah P. "Single Cell Culture Wells (SiCCWells)." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1406292709.
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Balagopal, Tulika C., Xiao Lucy Cheng, Jessica Gamboa, John Harper, Sheridan McPheeters, and Bryce Notheis. "A Dynamic Cell Culture System." Thesis, The University of Arizona, 2010. http://hdl.handle.net/10150/146852.
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The following includes information regarding the design, construction, and initial testing of A Dynamic Cell Culture System. This project has been undertaken in order to provide the Wu Lab with a tool that will allow a method to more accurately simulate human vasculature for research purposes by applying pulsatile shear, tensile, and normal pressures. This is accomplished by utilizing upper and lower Chambers between which reside tissue scaffolds (composed of silk and elastin) which are seeded with cells. A high flow-rate pump is used to pass cell culture media over the scaffolds in order to create shear. Solenoid valves are used in conjunction with pressure sensors to cause pressure to build up in one Chamber, causing both normal and tensile stresses in the scaffolds. At this time, the system has been fully constructed and has undergone only initial testing, the results of which showed that more work on sealing the Chamber and tube connections is necessary.
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Reiland, Joanne Elizabeth Donovan Maureen D. "Analysis of cell culture models of mammary drug transport." Iowa City : University of Iowa, 2009. http://ir.uiowa.edu/etd/316.
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Ekström, Jens-Ola. "Ljungan Virus Replication in Cell Culture." Doctoral thesis, Högskolan i Kalmar, Naturvetenskapliga institutionen, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:hik:diva-10.
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Ljungan virus (LV) is a recently identified picornavirus of the genus Parechovirus. LV has been isolated from voles trapped in Sweden and also in the United States. LV infected small rodents may suffer from diabetes type 1 and type 2 like symptoms, myocarditis and encephalitis. LV has been proposed as a human pathogen, with indications of causing diabetes type 1, myocarditis and intrauterine fetal deaths. In this thesis, cell culture adapted LV strains were utilised for development and adaptation of several basic methodological protocols to study the LV biology, e.g. real time PCR, highly specific antibodies and a reverse genetics system. These methods allowed detailed studies of this virus and how it interacts with the host cell. The genomic 5'-end was identified and modelling showed unique secondary structure folding of this region. The LV encodes an aphthovirus-like 2A protein with a DvExNPGP motif. This motif was found to mediate primary cleavage of the LV polyprotein in vitro and is proposed to constitute the carboxy terminus of the structural protein VP1 in LV. Rabbit polyclonal antibodies generated against recombinant structural proteins were used to verify that the LV virion is composed of the structural proteins VP0, VP1 and VP3. Cell culture studies showed that LV replicates to low titer with an absent or delayed cell lysis. LV is proposed to be able to spread by a, for picornaviruses, not previously demonstrated direct cell-to-cell transmission. All results taken together suggest a maintenance strategy of LV including low amounts of the LV genome and persistently infected hosts. Stability studies showed that the LV virion not only maintain activity in acidic and alkaline environments but also exhibit resistance to the commonly used disinfectant Virkon®.The results presented in this thesis show that LV has several unique properties, not previously observed for a picornavirus.
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Ekström, Jens-Ola. "Ljungan virus replication in cell culture /." Kalmar : University of Kalmar, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:hik:diva-10.
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Jayawarna, Vineetha. "Fmoc-peptide gels for cell culture." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.489519.
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Spontaneous formation of macroscopic hydrogels from small molecule building blocks via self-assembly is a powerful tool for the preparation of novel materials with well defined properties. Peptides are particularly interesting as building blocks for these materials. Self-assembled nanowires, fibres, sheets and tubes from peptide systems have all been described.
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Johnstone, Alex. "Microfluidic systems for neuronal cell culture." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/37822/.
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At a high level of abstraction, the brain is a system for analysing sensory information, and responding appropriately. That information is encoded and stored in the millions of neural circuits that comprise the brain. Deciphering this code is essential to understanding how memories are implemented in physiologically normal brain tissue, and to inferring the nature of some neurological disorders affecting memory such as Alzheimer’s disease, in which the neural encoding is aberrant or unsuccessful. One approach to this problem is to reduce the complexity of the brain functionality to three elements: stimuli, response, and reinforcement. The electrical activity of individual neurons can be recorded with electrodes, capturing the pathways of signal propagation in a network of cells. Individual neurons can be also induced to reliably respond to electrical or optical stimuli, so that they initiate, relay, or even block a signal. If the stimuli to a finite network of cells can be made heterogeneous so that only a sub-population of cells is targeted, then the network can be trained to react in a repeatable way to a given stimulus, testing the concept that the higher order functions of the brain can emerge from a simple set of underlying computational rules. Training however requires a mechanism for reinforcing only some of the possible pathways, in synchrony with stimuli and in response to the recorded network activity. In the intact brain, this mechanism is pharmacological: a neuromodulator such as dopamine is released throughout the brain, but as it only coincides with some but not all neuronal activity, the reinforcement is temporally selective. The key task of this project is to emulate this selective neuromodulator reinforcement in vitro in a finite neuronal network. The project must also provide capacity for heterogeneous stimulation and individual cell recording, which can be coordinated with the reinforcement under computer control. The strategy used was to develop microscale chambers to house a small network of cultured neurons. The chambers were integrated with existing cell recording and stimulating technologies, so that specific connections between neurons could be both monitored and induced. Neuronal cultures of a few hundred cells were successfully grown in microchannels, on substrates capable of recording their electrical activity. Thus it was possible to create a small cultured network in which complete network activity could be detected, subject to a sufficiently precise recording technique. Additionally, a fluid-handling system was developed in order to emulate the continual replenishment of nutrients and soluble gases that are essential to cell survival. The system is intended to deliver soluble chemicals that modulate neuronal activity, on a timescale that is consistent with neuromodulator delivery in the body. The fluid handling system comprises a set of pressure driven pumps under automated computer control. This system has the capacity to deliver neuromodulator in solution with high spatiotemporal precision. The ability to reliably deliver and wash off precise volumes of drugs in a matter of seconds, with no dilution of the intended concentration, will be of great benefit to researchers investigating the response of various cell types to different agonists.
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Murphy, Eric James. "Cell culture models for neural trauma /." The Ohio State University, 1989. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487672245901403.
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Reichen, M. "Multiplexed microfabricated cell culture device for stem cell process development." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1349012/.
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In this thesis, a multiplexed micro-fabricated cell culture device is presented to probe the soluble micro-environment of stem cells. A large number of biological, physical and chemical variables determine the micro-environment of stem cells and affect therefore their fate. The soluble micro-environment is believed to play a pivotal role in controlling stem cell fate and its optimisation may therefore allow exploitation of applications such as regenerative medicine or drug discovery. However, novel tools are required to probe and optimise the soluble micro-environment. The ability to move small volumes of liquid and the minimal use of resources are important characteristics of microfluidic systems and thus, they are perfectly suited to study the micro-environment of stem cells. A microfluidic cell culture device must ful fil three important requirements to be of utility in stem cell bioprocess development. The first aspect addresses the need for adaptability and flexibility to implement changes in microfluidic designs to take account of the rapid progress in stem cell research. To this end, a packaging solution specifi cally designed for a microfluidic cell culture device has been developed. The packaging system has thus been complemented with a rapid fabrication method using a micro-milling machine to quickly fabricate disposable and easily reconfi gurable microfluidic chips. The packaging solution has a maximum burst pressure of approximately 7.5 bar and the fabrication method has a dimensional fidelity with less than 10% deviation from the nominal value. The second aspect focuses on the scalability and comparability of results and the feasibility of continuous culture of stem cells - both critical elements for the success of a microfluidic cell culture system for stem cell bioprocess development. A novel cell seeding method has been developed using a pipette to directly and carefully seed cells into a culture chamber within a microfluidic cell culture device. To prevent a wash-out of viable cells during continuous culture, a low hydrodynamic shear stress microfluidic chip has been developed. The cell culture device has been successfully tested using human embryonic stem cells (hESC) on feeder cells. The third aspect concerns the automated monitoring of stem cell bioprocesses in the cell culture device. A multiplexed micro fluidic bioreactor platform with time-lapse imaging has been developed to obtain data-rich experimental sets. The platform consisting of a cooled media reservoir and a pumping mechanism has been characterised and tested using mouse embryonic stem cells (mESC) as a proof of concept. The combination of these three aspects provides a basis towards a multiplexed microfabricated cell culture device, which allows data-rich experimentation and comparability with current benchscale culture vessels for stem cell expansion and di fferentiation.
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Berdugo, Claudia. "Cell Damage Mechanisms and Stress Response in Animal Cell Culture." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1269467441.
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Kim, Narae. "External pH in culture on somatic cell reprogramming and cell differentiation in mouse and chicken cells." Kyoto University, 2017. http://hdl.handle.net/2433/218018.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第20092号
農博第2199号
新制||農||1046(附属図書館)
学位論文||H29||N5026(農学部図書室)
33208
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 今井 裕, 教授 松井 徹, 教授 久米 新一
学位規則第4条第1項該当
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Capra, J. (Janne). "Differentiation and malignant transformation of epithelial cells:3D cell culture models." Doctoral thesis, Oulun yliopisto, 2018. http://urn.fi/urn:isbn:9789526218236.
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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 apoptosisTiivistelmä 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
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Huynh, Minh Diem. "Reduction in apparent stromal cell culture density through transient fusions with osteosarcoma cells." Thesis, The University of Sydney, 2007. http://hdl.handle.net/2123/4465.
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Benign tumours grow by expanding and displacing the surrounding tissues, while malignant tumours replace and destroy the surrounding tissues by invasion. Although there is extensive literature on mechanisms of tumour invasion and metastasis, with an emphasis on angiogenesis, adhesion, degradation of the extracellular matrix and migration, an important question not clearly addressed by the literature, but nonetheless approached in this thesis, is that of the fate of normal cells during tissue replacement by migrating invasive malignant cells. Earlier work in the laboratory where this PhD candidature was carried out, investigated the effect of osteosarcoma cells on endothelium. In contrast to the expected angiogenic effect of malignant cells for endothelium, it was found that the human osteosarcoma cell line (SAOS-2) induced apoptosis in human umbilical vein endothelial cells (HUVEC) in contact dependent manner (McEwen et al., 2003). It was suggested that apoptosis of endothelium by malignant tumour cells may facilitate tumour invasion and metastasis (McEwen et al., 2003), and one of the aims of the current study was to extend these findings to include human gingival fibroblasts (HGF) and human umbilical artery smooth muscle cells (HUASMC). The major finding of this thesis was that SAOS-2 induced a reduction in the apparent cell culture density of HGF and HUASMC in a contact-dependent manner. The SW480 colorectal carcinoma cell line did not have any clear effect upon the apparent stromal cell culture density of either HGF or HUASMC, suggesting that the effect under investigation was tumour cell line specific. Surprisingly and in contrast to the similar effect reported for endothelium (Chen et al., 2005; McEwen et al., 2003), the effect of SAOS-2 upon HGF and HUASMC was not due to stromal cell apoptosis. Apoptosis was ruled out as a possible mechanism for the reduced apparent culture density under study, by using widely accepted methods which are dependent upon intermucleosomal fragmentation of DNA, the permeability of plasma membranes to dyes in advanced apoptosis and necrosis, phosphatidylserine translocation as well as inhibitor studies blocking both caspase dependent and independent pathways. While apoptosis was not demonstrated, the possibility emerged that reduced apparent stromal cell culture density reflected fusion events rather than the simple removal of cells as had been earlier reported for HUVEC (McEwen et al., 2003). This idea was supported by reduced SAOS-2 circularity in co-culture. Confocal microscopy of cells pre-labelled with fluorescent dyes further supported this idea, with dual-labelling as evidence of cell fusion. Although occasional homotypic fusion of stromal cells was seen, heterotypic fusion of stromal cells with SAOS-2 was much more prevalent. Time lapse microscopy was performed to further characteristic cell fusion in co-cultures, and revealed multiple transient fusions between SAOS-2 and HGF. To work towards determining the biological relevance of the key observation, two stable SAOS-2 GFP clones were generated for future planned studies using human gingival explants and nude mice. Importantly, the clones were similar to native SAOS-2 with regard to alkaline phosphatise expression and reducing apparent stromal cell culture density. Transient fusions between HGF and SAOS-2, may be a mechanism for cooption of stromal cells into the malignant process, facilitating tumour invasion. Additionally, heterocellular fusion of SAOS-2 with stromal cells may facilitate immune evasion, while it seems likely that despite the absence of an identical activity in SW480 cells, other malignant tumour cells may also express similar activity.
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Huynh, Minh Diem. "Reduction in apparent stromal cell culture density through transient fusions with osteosarcoma cells." University of Sydney, 2007. http://hdl.handle.net/2123/4465.
Full textAbstract:
Doctor of PhilosophyBenign tumours grow by expanding and displacing the surrounding tissues, while malignant tumours replace and destroy the surrounding tissues by invasion. Although there is extensive literature on mechanisms of tumour invasion and metastasis, with an emphasis on angiogenesis, adhesion, degradation of the extracellular matrix and migration, an important question not clearly addressed by the literature, but nonetheless approached in this thesis, is that of the fate of normal cells during tissue replacement by migrating invasive malignant cells. Earlier work in the laboratory where this PhD candidature was carried out, investigated the effect of osteosarcoma cells on endothelium. In contrast to the expected angiogenic effect of malignant cells for endothelium, it was found that the human osteosarcoma cell line (SAOS-2) induced apoptosis in human umbilical vein endothelial cells (HUVEC) in contact dependent manner (McEwen et al., 2003). It was suggested that apoptosis of endothelium by malignant tumour cells may facilitate tumour invasion and metastasis (McEwen et al., 2003), and one of the aims of the current study was to extend these findings to include human gingival fibroblasts (HGF) and human umbilical artery smooth muscle cells (HUASMC). The major finding of this thesis was that SAOS-2 induced a reduction in the apparent cell culture density of HGF and HUASMC in a contact-dependent manner. The SW480 colorectal carcinoma cell line did not have any clear effect upon the apparent stromal cell culture density of either HGF or HUASMC, suggesting that the effect under investigation was tumour cell line specific. Surprisingly and in contrast to the similar effect reported for endothelium (Chen et al., 2005; McEwen et al., 2003), the effect of SAOS-2 upon HGF and HUASMC was not due to stromal cell apoptosis. Apoptosis was ruled out as a possible mechanism for the reduced apparent culture density under study, by using widely accepted methods which are dependent upon intermucleosomal fragmentation of DNA, the permeability of plasma membranes to dyes in advanced apoptosis and necrosis, phosphatidylserine translocation as well as inhibitor studies blocking both caspase dependent and independent pathways. While apoptosis was not demonstrated, the possibility emerged that reduced apparent stromal cell culture density reflected fusion events rather than the simple removal of cells as had been earlier reported for HUVEC (McEwen et al., 2003). This idea was supported by reduced SAOS-2 circularity in co-culture. Confocal microscopy of cells pre-labelled with fluorescent dyes further supported this idea, with dual-labelling as evidence of cell fusion. Although occasional homotypic fusion of stromal cells was seen, heterotypic fusion of stromal cells with SAOS-2 was much more prevalent. Time lapse microscopy was performed to further characteristic cell fusion in co-cultures, and revealed multiple transient fusions between SAOS-2 and HGF. To work towards determining the biological relevance of the key observation, two stable SAOS-2 GFP clones were generated for future planned studies using human gingival explants and nude mice. Importantly, the clones were similar to native SAOS-2 with regard to alkaline phosphatise expression and reducing apparent stromal cell culture density. Transient fusions between HGF and SAOS-2, may be a mechanism for cooption of stromal cells into the malignant process, facilitating tumour invasion. Additionally, heterocellular fusion of SAOS-2 with stromal cells may facilitate immune evasion, while it seems likely that despite the absence of an identical activity in SW480 cells, other malignant tumour cells may also express similar activity.
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Oliver, Nicole Ann. "Developing a robust harvest for high cell density CHO cell culture." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/130614.
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Thesis: S.M., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020Thesis: M.B.A., Massachusetts Institute of Technology, Sloan School of Management, in conjunction with the Leaders for Manufacturing Program at MIT, May, 2020
Cataloged from the official PDF version of thesis.
Includes bibliographical references (pages 96-98).
by Nicole Ann Oliver.
S.M.
M.B.A.
S.M. Massachusetts Institute of Technology, Department of Biological Engineering
M.B.A. Massachusetts Institute of Technology, Sloan School of Management
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Sutton, Amy. "A New Active Cell Culture Material for Controlled Cell Micro-Manipulation." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493600.
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Mechanical forces in the cell’s natural environment have a crucial impact on growth and behavior, from single-cell gene expression and cell division to the spatial patterning of complex organ architectures. Few areas of biology can be understood without taking into account how both individual cells and networks of cells sense and transduce physical stresses as part of a tightly-controlled and highly integrated, hierarchical collective. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here, we present a new active cell culture material that allows highly localized, directional, and reversible deformation of the cell growth substrate, with control at scales ranging from the entire surface to the subcellular, and response times on the order of a few seconds. This material has a hybrid architecture composed of a polymeric array of microstructure actuators that serve as anchorage points for adherent cells, embedded in a temperature-responsive poly(N-isopropylacrylamide) hydrogel layer. By incorporating gold nanorods as a phototransducer component into the hydrogel, we are able to remotely control with focused near-infrared light the localized and highly directional actuation of the supported microstructures over a range of scales down to 10 um, with response times under 5 s. Additionally, the surface cues provided by this hybrid material provide opportunities for controlling the cell attachment and alignment patterns. Thus, by triggering surface deformations at selected locations, we applied uniaxial mechanical stresses directly on defined portions of single cells within a population, with resolution over the cell distortion close to ~3 um, cell strains between 0 and ~45% reached locally in different regions within the same cell, cycling frequencies on the order of ~0.1 Hz, and no losses in cell viability. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques.Chemistry and Chemical Biology
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Vajrala, Sucheta Gowthami. "Mechanism of CO2 inhibition in insect cell culture." Thesis, University of Iowa, 2010. https://ir.uiowa.edu/etd/611.
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The prominence of insect cell culture has grown rapidly due to its ability to produce baculovirus biopesticides and recombinant proteins using the Baculovirus Expression Vector System. A critical problem in the mass production of these products is CO2 accumulation to inhibitory levels within the bioreactor. The current research investigated the effect of elevated CO2 concentrations on insect cell growth and metabolism and the roles of oxidative stress and intracellular pH (pHi) in CO2 inhibition.
Spodoptera frugiperda Sf-9 insect cells were cultured in a 3 L bioreactor (1.2 L working volume) controlled at 20% air saturation, 27oC and a pH of 6.2. The cells were exposed to a constant CO2 concentration by purging the medium with CO2 and the headspace with air. The experiments were repeated for different CO2 concentrations and samples were taken every 24 h to determine cell density, viability, metabolism and oxidative stress.
The population doubling time (PDT) of Sf-9 cells increased with increasing CO2 concentration. Specifically, the PDT for 0-37, 73, 147, 183 and 220 mm Hg CO2 concentrations were 23.2 ± 6.7, 32.4 ± 7.2, 38.1 ± 13.3, 42.9 ± 5.4 and 69.3 ± 35.9 h (n = 3 or 4; 95% confidence level), respectively. An 80 mL working volume shaker flask was maintained as a control and had an average PDT of 24.9 ± 3.1 h (n = 7; 95% confidence level). The viability of cells in all experiments was above 90%. The osmolality for all bioreactor experiments was observed to be 300 - 360 mOsm/kg, a range that is known to have a negligible effect on insect cell culture. Elevated CO2 concentration did not alter the cell specific glucose consumption rate (2.5 to 3.2 x 10-17 mol/cell-s), but slightly increased the specific lactate production rate from -3.0 x 10-19 mol/cell-s to 10.2 x 10-19mol/cell-s. Oxidative stress did not contribute to CO2 inhibition in uninfected Sf-9 cells as no significant increase in the levels of lipid hydroperoxide and protein carbonyl concentrations was discovered at elevated CO2 concentration. The experiments conducted to determine the effect of CO2 on pHi were not successful and different experimental methods tested were well documented.
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Sjögren, Frida. "Microstructuring of Hyaluronic acid cell culture scaffolds." Licentiate thesis, Uppsala universitet, Mikrosystemteknik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-334653.
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Chen, Michael C. W. "Hydrogel-based microfluidic system for cell culture." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/7209.
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Traditionally, cell culture has been done in culture flasks or well plates where the volumes and length scales involved in the culture environment are many orders of magnitude larger than the size scale of individual cells. To better tailor medical care to an individual patient, it may be necessary to carry out genetic, physiological, and biochemical analyses on very small cell samples and to have an in vitro cell culture environment that more closely approximates the in vivo conditions.
A microfluidic device that integrates both cell handling and long-term 3-D cell culture techniques is presented. The designed microdevice traps cells with alginate, an ionically cross-linking hydrogel, which mimics the extra cellular matrix within our body. To encapsulate the cells, a solution of calcium ions is introduced in parallel with the alginate precursor cell suspension. Alginate hydrogel forms at the interface and as the region of gel grows it traps cells inside. This is a reversible process; the gel matrix can be dissolved and the cells can be released by the addition of ethylene-diaminetetraacetic acid (EDTA), a calcium chelator.
To show that the microfluidic device is reliable for long term mammalian cell culture, hepatocytes and breast tumor cells were cultured within the alginate gel layer inside the microfluidic device for more than two weeks. Hepatocytes were able to form three-dimensional aggregates within the microfluidic hydrogel environment. We further demonstrate the possibility of performing anticancer agent screening within this device. Breast tumor cells seeded in the microchannel were treated with doxorubicin, a common chemotherapy drug. Compared to controls, the doxorubicin inhibited cell proliferation.
In future, this system will have applications in cell-based testing and in studies involving small cell populations, such as cancer cells obtained from needle biopsies of tumors.
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Lamb, Keith A. "Cell culture models for transdermal drug delivery." Thesis, University of Nottingham, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334854.
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Zupke, Craig Allen. "Metabolic flux analysis in mammalian cell culture." Thesis, Massachusetts Institute of Technology, 1993. http://hdl.handle.net/1721.1/12661.
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Macown, R. J. "Microfabricated devices for adherent stem cell culture." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1465828/.
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This thesis details the development of a system of microfabricated devices for the adherent culture of stem cells. The multipotency and self renewal of stem cells make them a potentially abundant source of valuable human cells, for both drug screening and regenerative medicine. However, processing stem cells is challenging due to the complexity of whole cell products, the number of process parameters, and the typical use of adherent culture. It is hypothesised that a microfabricated adherent culture system could facilitate process development with minimal use of resources. Furthermore, microfluidic systems offer advantages in spatial and temporal control over the microenvironment that would benefit process development. An existing prototype culture system is critically evaluated by: assessing the design, modelling fluid flow and dissolved oxygen, and successfully co-culturing human embryonic stem cells, on inactivated mouse embryonic fibroblasts, under perfused conditions. The utilisation of reversible seals facilitates the use of standard tissue-culture polystyrene culture surfaces and manual seeding techniques. The evaluation of the prototype system is used to inform improvements to the design, making it easier to use, increasing the robustness, allowing monitoring of whole culture chambers by microscopy, and improving control over mean pericellular dissolved oxygen. Modelling shows the improved culture system also achieves more uniform distribution of both pericellular dissolved oxygen and fluid velocity. The improved culture system shows similar mouse embryonic stem cell seeding behaviour to tissue culture flasks, but, with medium perfused at 300 μl.h 1, mouse embryonic stem cells reach full confluency in less than 48 h, compared with 72 hours for cells maintained statically in flasks. There is also inconclusive data suggesting that the growth rate is limited by pericellular dissolved oxygen and is, therefore, increased and made more uniform by the inclusion of a gas permeable lid system. The reliability, ease of use, comparability with traditional culture systems, and control over process parameters of the improved system should make it a useful tool for stem cell process development.
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Atefi, Ehsan. "Aqueous Biphasic 3D Cell Culture Micro-Technology." University of Akron / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=akron1443112692.
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Chattopadhyay, Devamita. "Studies on protective additives in cell culture /." The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487861796818776.
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Ambury, Rachael. "Bioactive sugar surfaces for hepatocyte cell culture." Thesis, University of Manchester, 2010. https://www.research.manchester.ac.uk/portal/en/theses/bioactive-sugar-surfaces-for-hepatocyte-cell-culture(122af33a-35b1-47c1-9579-4568fef47543).html.
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The primary objective of this study was to identify, develop and characterise a novel bioactive surface capable of binding hepatocytes and enabling the retention of hepatocyte-specific cell function during in-vitro culture. The materials were designed to exploit a unique characteristic of hepatocyte biology, with β-galactose moieties displayed to allow cellular adhesion via the specific asialoglycoprotein receptors (ASGP-R) found on hepatocytes. Hydrogels were created by modifying a commercially available block co-polymer of polyethylene glycol (PEG) and acrylamide, (PEGA) with galactose moieties contained within lactobionic acid (LA), producing a unique bioactive sugar-based gel. A control sugar, D-glucuronic acid (GA), was used as a non-ASGP-R binding control. Monomers used were mono- and bis-acryloamido PEG (Mw=1900 gmol-1), and dimethylacrylamide. The pendant PEGA amine groups were used as ligands to bind to the sugars. The resultant gels were characterised using Fourier Transform Infrared Spectroscopy (FT-IR), protein adsorption, Fmoc-Phe and dansyl chloride labelling. The biocompatibility of the gel surfaces was evaluated using a hepatocyte cell line and the degree of attachment, proliferation, and morphology was characterised using light microscopy, live/dead assays, DNA assays, immunochemical staining, flow cytometry and reverse-transcription polymerase chain reaction (RT-PCR).FT-IR analysis of LA revealed a distinctive band at approximately 1740cm-1 corresponding to carbonyl stretching (C=O) of carboxylic acid. This unique peak disappeared as the galactose moieties within the LA were incorporated into the PEGA gel. A similar trend was also observed with the control GA sugar within the PEGA gel, confirming that the sugars had been integrated into the material. Protein adsorption assays confirmed the non-fouling nature of PEGA. Cell culture experiments showed that hepatocytes attached preferentially to the sugar surfaces, with few cells seen on the PEGA surfaces. It was observed that cells on the PEGA with LA surface were more metabolically active, than the controls and proliferated to a monolayer by day 7 in culture. Immunocytochemical staining of the cells for actin, vinculin and phosphorylated focal adhesion kinase illustrated differences in cell morphology between cells grown on different surfaces. It was determined that the sugar PEGA surfaces maintained some characteristics of hepatocyte functionality e.g. urea synthesis over the course of 7 days. To improve the reproducibility of the surfaces generated, a preliminary investigation of two-dimensional PEG monolayer surfaces as a well defined platform for surface reactions was conducted. These were chemically functionalised in a stepwise manner with the sugars. The number of coupling steps and the choice of solvent were shown to affect the efficiency of the reaction. Further more, the need for careful sample preparation was highlighted as contamination could potentially inhibit the interpretation of the surface chemistry.The overall conclusion of this work is that saccharides within non-fouling surfaces composed of thin layers of PEG-acrylamide hydrogels are able to support hepatocyte attachment and the retention of cell type specific functions in culture. However, this preliminary work has shown that much further research is necessary to elucidate the role that the surface chemistry plays in the attachment of hepatocytes.
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Isomura, Akihiro. "Spatiotemporal pattern formation in cardiac cell culture." 京都大学 (Kyoto University), 2009. http://hdl.handle.net/2433/124393.
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Barry, Megan M. Crockett Robert S. "Three-dimensional scaffolds for mammary epithelial cell growth : a thesis /." [San Luis Obispo, Calif. : California Polytechnic State University], 2008. http://digitalcommons.calpoly.edu/theses/12/.
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Thesis (M.S.)--California Polytechnic State University, 2008.Major professor: Robert S. Crockett, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Engineering." "May 2008." Includes bibliographical references (leaves 38-45). Also available on microfiche (1 sheet).
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Ouyang, Anli. "Embryonic stem cell culture in fibrous bed bioreactor." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1149001795.
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Kittler, Ralf. "Functional genomic analysis of cell cycle progression in human tissue culture cells." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1161253856455-48321.
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The eukaryotic cell cycle orchestrates the precise duplication and distribution of the genetic material, cytoplasm and membranes to daughter cells. In multicellular eukaryotes, cell cycle regulation also governs various organisatorial processes ranging from gametogenesis over multicellular development to tissue formation and repair. Consequently, defects in cell cycle regulation provoke a variety of human cancers. A global view of genes and pathways governing the human cell cycle would advance many research areas and may also deliver novel cancer targets. Therefore this work aimed on the genome-wide identification and systematic characterisation of genes required for cell cycle progression in human cells. I developed a highly specific and efficient RNA interference (RNAi) technology to realize the potential of RNAi for genome-wide screening of the genes essential for cell cycle progression in human tissue culture cells. This approach is based on the large-scale enzymatic digestion of long dsRNAs for the rapid and cost-efficient generation of libraries of highly complex pools of endoribonuclease-prepared siRNAs (esiRNAs). The analysis of the silencing efficiency and specificity of esiRNAs and siRNAs revealed that esiRNAs are as efficient for mRNA degradation as chemically synthesized siRNA designed with state-of-the-art design algorithms, while exhibiting a markedly reduced number of off-target effects. After demonstrating the effectiveness of this approach in a proof-of-concept study, I screened a genome-wide esiRNA library and used three assays to generate a quantitative and reproducible multi-parameter profile for the 1389 identified genes. The resulting phenotypic signatures were used to assign novel cell cycle functions to genes by combining hierarchical clustering, bioinformatics and proteomic data mining. This global perspective on gene functions in the human cell cycle presents a framework for the systematic documentation necessary for the understanding of cell cycle progression and its misregulation in diseases. The identification of novel genes with a role in human cell cycle progression is a starting point for an in-depth analysis of their specific functions, which requires the validation of the observed RNAi phenotype by genetic rescue, the study of the subcellular localisation and the identification of interaction partners of the expressed protein. One strategy to achieve these experimental goals is the expression of RNAi resistant and/or tagged transgenes. A major obstacle for transgenesis in mammalian tissue culture cells is the lack of efficient homologous recombination limiting the use of cultured mammalian cells as a real genetic system like yeast. I developed a technology circumventing this problem by expressing an orthologous gene from a closely related species including its regulatory sequences carried on a bacterial artificial chromosome (BAC). This technology allows physiological expression of the transgene, which cannot be achieved with conventional cDNA expression constructs. The use of the orthologous gene from a closely related species confers RNAi resistance to the transgene allowing the depletion of the endogenous gene by RNAi. Thus, this technology mimics homologous recombination by replacing an endogenous gene with a transgene while maintaining normal gene expression. In combination with recombineering strategies this technology is useful for RNAi rescue experiments, protein localisation and the identification of protein interaction partners in mammalian tissue culture cells. In summary, this thesis presents a major technical advance for large-scale functional genomic studies in mammalian tissue culture cells and provides novel insights into various aspects of cell cycle progression. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 217 MB: Movies, Rohdaten - Nutzung: Referat Informationsvermittlung der SLUB)
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Konagaya, Shuhei. "Design of cell culture substrates for large-scale preparation of neural cells." 京都大学 (Kyoto University), 2013. http://hdl.handle.net/2433/174963.
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Lecault, Véronique. "Microfluidic cell culture arrays for clonal expansion and characterization of mammalian cells." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43667.
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Sage, Elizabeth Ann. "The functional competence of animal cells in culture : the NSO cell proteome." Thesis, University of Kent, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399618.
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McKilligin, Elaine. "Characterisation and comparison of cell culture models of intimal smooth muscle cells." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624223.
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Katalinich, Micheal. "Characterization of Chitosan Films for Cell Culture Applications." Fogler Library, University of Maine, 2001. http://www.library.umaine.edu/theses/pdf/KatalinichM2001.pdf.
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Pasturel, Aurélien. "Tailoring common hydrogels into 3D cell culture templates." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0302.
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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 tridimensionnelleTailoring 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
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Huynh, Minh Diem. "Reduction in apparent stromal cell culture density through transient fusions with osteosarcoma cells." Thesis, The University of Sydney, 2007. http://hdl.handle.net/2123/4675.
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Shrader, Carl D. "Insulin-induced endothelial cell proliferation and viability in stretched murine skin and cell culture." Morgantown, W. Va. : [West Virginia University Libraries], 2007. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=5260.
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Thesis (Ph. D.)--West Virginia University, 2007.Title from document title page. Document formatted into pages; contains xiii, 127 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.
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Najm, Nour Addeen. "In vitro culture studies of tick cell lines." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-146638.
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Afshar-Sterle, Shoukat. "Cell, tissue culture and transformation of Triticum tauschii /." Title page, abstract and contents only, 2000. http://web4.library.adelaide.edu.au/theses/09APSP/09apspa258.pdf.
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Brown, Stephen Alan. "Cell response to physical stimuli in dynamic culture." Thesis, University of Ulster, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.558780.
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Endothelial cells are known to respond directly to their physical environment, with morphological changes in response to fluid shear stress being associated with a number of key cellular functions and disease processes such as angiogenesis, cell migration and atherosclerosis. Alignment and elongation of endothelial cells can result from culturing them on substrates with specific micro-features. If simple, cost-effective physical cues can be used to direct endothelial cell function, significant advances could be made in a number of fields, including Tissue Engineering and Regenerative Medicine (TERM), vascular biomaterials and drug development. This research considers two distinct forms of physical stimuli of endothelial cells, namely Laminar Shear Stress (LSS) and hot embossed surface micro-features, created on Tissue Culture Treated Polystyrene (TCPS). Using both experimental and computational methods the nature of the effects of these forms of stimuli have been investigated separately and in combination. The resulting information enables a number of key experimental outcomes to be presented. Firstly, while hot embossing is able to produce consistent and repeatable micro-scale features, it has a subtle but significant effect on the TCPS surface, changing both surface chemistry and roughness in a manner that can result in changes in cell adhesion and proliferation. Secondly, Bovine Aortic Endothelial Cells (BAECs) align and elongate in response to culture on hot embossed ridge features and to LSS stimulus, with notable cytoskeletal differences observed between cells aligned as a result of the different forms of stimuli. Application of these stimuli in combination suggests that the nature of the cell adhesion to the substrate is a key factor in determining the response to LSS. Thirdly, the specific topography of an adhered endothelial monolayer changes the resulting shear stress and so must be considered if the responses to LSS are to be correctly understood. Computational Fluid Dynamics (CFD) modelling has determined that spatial gradients of the shear stress may be more important than its magnitude when considering the adhesion of endothelial cells. Finally, it has been observed that a combination of specific surface chemistry, roughness and micro-features is able to promote a pronounced form of directional tubule formation in BAECs.
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Bailey, Christopher Michael. "Modelling and control of a plant cell culture." Thesis, University of Sheffield, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270201.
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Sivathanu, Vivek. "In vitro models for airway epithelial cell culture." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/81726.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2013.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 40-41).
This work is about the development of a physiologically relevant model of the human airway. Various factors such as the cell model, physiochemical factors such as the cell substrate properties including its stiffness, shear stress, stretch, the air-liquid interface and the biochemical factors in the medium influence the biology of the cells. The aim of this work is to closely approximate conditions in an in vivo situation by engineering the above conditions in to the in vitro platform. An assay to introduce the cell substrate properties was developed in a glass bottomed petri dish type culture as well as a microfluidic device culture. The influence of the cell substrate on airway epithelial cell monolayer formation was investigated in detail by changing the stiffness of the substrate independently by changing the gel concentration, the gel formation pH and the height of the gel from a hard substrate. Further, we found that biochemical growth factors have a huge role in cell monolayer formation. A real-time measurement of monolayer integrity using electrical resistance measurements was developed. A shear stress application platform was developed and a stretch application platform was designed. The applications of such a platform with the inclusion of various physiologically relevant factors include the study of physiologic evolution of microbes such as the influenza virus.
by Vivek Sivathanu.
S.M.
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Hill, C. J. "Regulating stem cell behaviour using bioengineered culture substrates." Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3006120/.
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Stem cells hold enormous potential for the treatment of injuries and degenerative diseases. In the pursuit of stem cell therapies, a plethora of biomaterials have been developed to induce lineage-specific differentiation or support cell propagation for research and clinical applications. However, the use of stem cells is hindered by the cost of scale-up and risk of zoonotic transmissions from animalderived culture components. Here, we utilise a recombinant protein scaffold composed of self-assembling nanofibres, termed ZT, and assess the systems adaptability for in vitro applications. Protein-based scaffolds offer distinct advantages over conventional materials such as the display of peptidic motifs with near-native stoichiometries and control of the spatial density and nanotopographical distribution of genetically-encoded bioactivities. Herein, the functionalisation potential of the ZT system is explored via the generation of chimeric protein building blocks that exhibit the integrin-binding RGD motif. Specific sites within the building blocks were found to tolerate diversification, in the form of exogenous peptides or a fused protein domain, without structural perturbation or inhibition of assembly. The bioactivity of functionalised ZT nanofibres was assessed using murine mesenchymal stem cells (mMSCs), which recognised the integrin-binding moieties. The ability of one ZT nanofibre variant to induce mMSC chondrogenesis was investigated, which proved unsuccessful in the current context. A second generation of ZT variants were generated by the incorporation of chondroinductive motifs for future applications in cartilage tissue engineering. Additionally, the capacity of functionalised ZT nanofibres to act as culture substrates for human embryonic stem cell (hESC) self-renewal was explored.
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Ge, Cheng. "Novel technologies for cell culture and tissue engineering." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:ab1014cf-80a4-4675-b607-96dc52c39b17.
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Cell culture has been a fundamental tool for the study of cell biology, tissue engineering, stem cell technology and biotechnology in general. It becomes more and more important to have a well-defined physiochemical microenvironment during cell culture. Conventional cell cultures employ expensive, manually controlled incubation equipment, making it difficult to maximize a cultures yield. Furthermore, previous studies use qualitative methods to assess cell culture proliferation that are inherently inaccurate and labour intensive, thereby increasing the cost of production. In addition, three dimensional cell culture, in scaffold, has been shown to provide more physiological relevant information as it mimic more accurate conditions that are similar to the physiological conditions of the human body compared with two dimension, which has special interest to regenerative medicine. Therefore, a portable and automated total-analysis-system (μTAS) was proposed with microenvironment control and quantitative analysis techniques to monitor cell proliferation and metabolic activity. The automated portable heating system was validated to be capable to maintain a stable physiochemical microenvironment, with little margin of error, for cellular substrate outside of conventional incubation. A standalone platform system was designed and fabricated with accurate temperature control by employing an optically transparent ITO-film with a large heating area. The transparency of the film is critical for continuous in-situ microscopic observation over long-term cell culture process. Previous studies have attempted to use ITO-film as a heating element, but were unable to distribute the heat evenly onto the microbioreactor platform. This nagging problem in the literature was improved through a novel film design. As a result, the ITO-film based heating system was evaluated and constructed successfully to serve as a heating element for long-term static cell culture with facilitated proliferation rate in gas-permeable PDMS microbioreactor outside of conventional incubation. In addition to maintaining a stable microenvironment, a non-invasive in-situ technology for monitoring cell viability and proliferation rate was constructed and developed based on bioimpedance spectroscopy (BIS). It was primarily focused on making decisions for structure and specification of proposed system-on a chip BIS measurement. The miniaturization of BIS system on microbioreactor platform was achieved by utilizing and integrating switching matrix array, impedance analyzer chip with reliable analogue-front-end circuitry. The realized system was verified with the DLD-1 cells and its monitored data were validated with conventional bioassays. Three dimensional cell cultures with scaffold is a key to the success of tissue engineering. Engineered cornea collagen scaffold may be feasible using re-seeding proper human cells onto a decellularized corneal scaffold. The quality of the scaffold and the interaction of the cells are critical to the key function (i.e transparency, haze and total transmittance) of final products. An integrated corneal collagen scaffold quality assessment system, via optical property inspection unit, was innovatively designed and realized with non-invasive and non-destructive characteristics. The H1299 cells were seeded onto inspected corneal scaffold and BIS system, which were realized in the previous chapter, were used to validate its applicability for 3D cell culture. The cell adhesion as an outcome at different scaffolds with different optical properties has revealed the importance of the microstructure of scaffold on the cell functions. The results showed the developed technologies can be used for the quality control of corneal scaffold and the fabricated μTAS not only enabled environmental control but, with BIS-based in-situ assay, it also facilitate the function (i.e adhesion) and viability monitoring with quantitative and qualitative analysis in 3D-alike cell culture. Additionally, by considering its low decontamination and cost-effective nature with compatibility for high-throughput screening applications, the fabricated and integrated systems has significant applications in tissue engineering.
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Makene, Vedastus Wilfred. "Cell culture biomarkers for monitoring of wastewater pollutants." University of the Western Cape, 2021. http://hdl.handle.net/11394/8148.
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Philosophiae Doctor - PhDWastewater is normally composed of a mixture of pollutants. The type and composition of pollutants in a particular wastewater depend on the source of origin. The source and characteristics of a particular wastewater determine the ideal method of sewage treatment. Specific treatment techniques are effective in the removal of certain types of pollutants and may have no impact on the levels of other types of pollutants. Therefore, a combination of treatments and assessment of the quality of effluent before release into the environment is normally recommended. The assessment of effluent can be achieved by various techniques including chemical analysis and biological assays. Chemical analyses are commonly employed; however, they often pose detection problems and are considered to be uneconomical.
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Chetty, Avashnee Shamparkesh. "Thermoresponsive 3D scaffolds for non-invasive cell culture." Thesis, University of Pretoria, 2012. http://hdl.handle.net/2263/25463.
Full textAbstract:
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/agThesis (PhD)--University of Pretoria, 2012.
Chemical Engineering
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