Literatura científica selecionada sobre o tema "4D cell culture"

(1) Background: We describe a 4D cell culture platform with which we tried to detect and to characterize migration dynamics of single hematopoietic stem cells in polymer film microcavity arrays integrated into a microtiter plate. (2) Methods: The system was set up with CD34-expressing KG-1a cells as a surrogate for hematopoietic stem cells. We then evaluated the system as an artificial hematopoietic stem cell niche model comprised of a co-culture of human hematopoietic stem cells from cord blood (cord blood CD34+ cells, hHSCs) and human mesenchymal stromal cells (hMSCs) from bone marrow over a period of 21 days. We used a software-based cell detection method to count single hematopoietic stem cells (HSCs) in microcavities. (3) Results: It was possible to detect single HSCs and their migration behavior within single microcavities. The HSCs displayed a pronounced migration behavior with one population of CD34-expressing cells located at the bottom of the microcavities and one population located in the middle of the microcavities at day 14. However, at day 21 the two populations seemed to unite again so that no clear distinction between the two was possible anymore. (4) Conclusions: Single cell migration detection was possible but microscopy and flow cytometry delivered non-uniform data sets. Further optimization is currently being developed.

Abstract Multiple myeloma (MM) bone disease (MMBD) is characterized by activation of osteoclasts and suppression of osteoblastic differentiation, with these changes in the bone microenvironment supporting MM cell growth and drug resistance. These complex interactions between MM cells and bone cells are incompletely understood. Current bone targeted therapy with bisphosphonates or Denosumab only blocks bone resorption but has no effect on osteoblast activity and only modest effects on MM growth. Therefore, new MMBD treatments are needed. Semaphorin-4D (Sema4D; CD100), is made by osteoclasts and inhibits osteoblasts by binding to the Plexin B receptor. Breast cancers also express Sema4d, and silencing sema4D in MDA-MB-231 breast cancer cells suppresses bone metastasis (Yang Y et al, PLoS One 2016). Since breast cancers and MM both cause osteolytic bone destruction and soluble Sema4D and Plexin B levels are increased in sera of MM patients (Terpos et al, 2012), we tested if sema4D contributed to MMBD. qPCR analysis of human MM cell lines and primary CD138+ cells showed MM cells express high levels of sema4D mRNA, comparing to the MDA-MB-231 breast cancer cells. Analysis of previously reported gene expression array data confirmed that MM cells express sema4D at a higher level compared to bone marrow plasma cells of MGUS and healthy donors (GenomicScape.com; Zhan F et al, Blood 2007; Mattiolo M et al, Oncogene, 2005). These results plus those of Terpos et al suggest that MM cells commonly express Sema4D. We next asked if the bone microenvironment increases MM expression of Sema4D. We co-cultured human MM cell lines RPMI8226 and JJN3 with mouse bones. Species -specific changes in tumor and bone were evaluated by quantitative RT-PCR. MM cells engrafted onto mouse bones, increasing markers of osteolysis similar to those seen in MM bone disease. After a week of co-culture, Sema4D expression was increased in MM cells (mean ±SD; 4.2±0.4; p=0.023), compared to MM cells grown alone. In addition, bones co-cultured with MM cells expressed higher Sema4D mRNA than bones alone (mean ±SD; 3.6±0.21; p=0.03). While co-culture increased both MM and bone Sema4D, markers of osteoblast activity, Col1a1, alkaline phosphatase and osteocalcin were suppressed. Preliminary experiments suggest that osteocytes are a major source of Sema4D expression in bone, in addition to active osteoclasts, which are much rarer cells than osteocytes. The induction of Sema4D in bone was only partially inhibited by 100nM zoledronic acid to inhibit osteoclast activity. Since osteocytes can physically interact with MM cells in vivo (Delgado Calle, Cancer Res 2016), we then tested the effect of MM cells on osteocyte sema4D expression in co-cultures of RPMI 8226 and JJN3 MM cells with MOL-Y4 osteocytic cells, separated by transwells. Both MM cell lines increased the Sema4D mRNA content of MLO-Y4 cells (mean ±SD; 3.1±0.4; p=0.036), suggesting that myeloma-secreted factors regulate osteocyte Sema4D expression. Since Sema4D is a potent osteoblast inhibitor, our data suggest that osteocyte -derived Sema4D may be a major contributor to MMBD, and that neutralization of Sema4D activity should improve the suppressed bone formation in MM. Disclosures Roodman: Amgen: Consultancy.

During the last decades, the study of cell behavior was largely accomplished in uncoated or extracellular matrix (ECM)-coated plastic dishes. To date, considerable cell biological efforts have tried to model in vitro the natural microenvironment found in vivo. For the lung, explants cultured ex vivo as lung tissue cultures (LTCs) provide a three-dimensional (3D) tissue model containing all cells in their natural microenvironment. Techniques for assessing the dynamic live interaction between ECM and cellular tissue components, however, are still missing. Here, we describe specific multidimensional immunolabeling of living 3D-LTCs, derived from healthy and fibrotic mouse lungs, as well as patient-derived 3D-LTCs, and concomitant real-time four-dimensional multichannel imaging thereof. This approach allowed the evaluation of dynamic interactions between mesenchymal cells and macrophages with their ECM. Furthermore, fibroblasts transiently expressing focal adhesions markers incorporated into the 3D-LTCs, paving new ways for studying the dynamic interaction between cellular adhesions and their natural-derived ECM. A novel protein transfer technology (FuseIt/Ibidi) shuttled fluorescently labeled α-smooth muscle actin antibodies into the native cells of living 3D-LTCs, enabling live monitoring of α-smooth muscle actin-positive stress fibers in native tissue myofibroblasts residing in fibrotic lesions of 3D-LTCs. Finally, this technique can be applied to healthy and diseased human lung tissue, as well as to adherent cells in conventional two-dimensional cell culture. This novel method will provide valuable new insights into the dynamics of ECM (patho)biology, studying in detail the interaction between ECM and cellular tissue components in their natural microenvironment.

The classic cell culture involves the use of support in two dimensions, such as a well plate or a Petri dish, that allows the culture of different types of cells. However, this technique does not mimic the natural microenvironment where the cells are exposed to. To solve that, three-dimensional bioprinting techniques were implemented, which involves the use of biopolymers and/or synthetic materials and cells. Because of a lack of information between data sources, the objective of this review paper is, to sum up, all the available information on the topic of bioprinting and to help researchers with the problematics with 3D bioprinters, such as the 3D-Bioplotter™. The 3D-Bioplotter™ has been used in the pre-clinical field since 2000 and could allow the printing of more than one material at the same time, and therefore to increase the complexity of the 3D structure manufactured. It is also very precise with maximum flexibility and a user-friendly and stable software that allows the optimization of the bioprinting process on the technological point of view. Different applications have resulted from the research on this field, mainly focused on regenerative medicine, but the lack of information and/or the possible misunderstandings between papers makes the reproducibility of the tests difficult. Nowadays, the 3D Bioprinting is evolving into another technology called 4D Bioprinting, which promises to be the next step in the bioprinting field and might promote great applications in the future.

The rates of synthesis and turnover of the rare amino acid hypusine [N6-(4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid] in protein were studied in relationship to polyamine metabolism and growth rates in rat hepatoma tissue-culture (HTC) cells. Hypusine is selectively formed in the eukaryotic translation initiation factor eIF-4D, by a post-translational mechanism involving spermidine [Cooper, Park, Folk, Safer & Braverman (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1854-1857]. The half-life of the hypusine-containing protein was longer than 24 h. In cells whose intracellular spermidine pools had been initially depleted, by using DL-alpha-difluoromethylornithine (DFMO), maximum synthesis rates of hypusine in protein were 5-10 times higher, on restoration of endogenous spermidine contents by exogenous addition, than those observed in untreated exponential-phase cultures. In cells pretreated with DFMO, the rate of hypusine synthesis was constant for up to 1 h after the addition of 5 microM-spermidine, whereas endogenous spermidine contents varied from less than 1 to more than 10 nmol/mg of protein. However, the overall amount of hypusine formed, during the first 1 h after the addition of various concentrations of spermidine (0.05-10 microM) to the culture medium, was markedly dependent on the final endogenous spermidine content achieved at the end of the 1 h measurement interval. Early in exponential-phase growth, protein-bound hypusine was synthesized at a rate of 1-2 pmol/h per mg of protein. This rate decreased to less than 0.5 pmol/h per mg of protein when cell growth rates decreased as cultures reached high cell densities. Analysis of the polyamine substrate specificity for hypusine formation showed that N1-acetylspermidine did not compete with spermidine in the reaction, nor did N1-(buta-2,3-dienyl)-N2-methylbutane-1,4-diamine, and irreversible inhibitor of polyamine oxidase, block the reaction. On the basis of comparative radiolabelling experiments, spermine was either a poor substrate, or not a substrate, for hypusine formation. These results confirm that spermidine is the likely precursor of the aminohydroxybutyl moiety of hypusine, and show that overall hypusine formation, but not necessarily the synthesis rate, is dependent on the endogenous spermidine concentration, especially under conditions where spermidine concentrations are initially low, as is the case after DFMO treatment, and then increase.

In vivo, les cellules sont situées dans un microenvironnement poreux et dynamique en 3D qui fournit des signaux biochimiques et biophysiques ainsi que des signaux dynamiques influençant le comportement des cellules dans des contextes physiologiques et pathologiques. Afin de mieux reproduire ces conditions in vitro pour des applications en biologie cellulaire fondamentale, en ingénierie tissulaire et en dépistage de médicaments, cette thèse présente le développement d'échafaudages 4D électro-actifs, combinant une architecture 3D passive et microporeuse nommée polyHIPE et un polymère électroactif, PEDOT. Ces échafaudages servent de plateforme de culture cellulaire dynamique capable de délivrer une stimulation électromécanique. L'étude s'est d'abord concentrée sur la synthèse et la caractérisation des échafaudages polyHIPE-PEDOT électroactifs, qui ont présenté une structure hautement poreuse (10 à 100 µm) et interconnectée, bénéfique pour une colonisation cellulaire rapide. Notamment, ces échafaudages peuvent subir des changements volumétriques en réponse à une stimulation électrique. La deuxième partie de ce travail a porté sur l’évaluation de la compatibilité des échafaudages polyHIPE-PEDOT avec les exigences de la culture cellulaire. Les échafaudages se sont révélés cytocompatibles, favorisant l'adhésion, la migration et la prolifération des cellules. Les cellules à l'intérieur de l'échafaudage ont adopté une morphologie cellulaire fusiforme typique des micro-environnements cellulaires 3D et ont synthétisé de la fibronectine, une protéine de la matrice extracellulaire essentielle pour les interactions cellule-matrice. Dans la troisième partie de cette thèse, un dispositif de stimulation électromécanique adapté aux études de culture cellulaire in vitro (plaque à 6 puits) et à l'imagerie des cellules vivantes (boîte de Petri à fond de verre) a été développé. Un protocole de stimulation a été déterminé et n'a pas induit d'effets cytotoxiques aigus. Après stimulation, les cellules présentent une morphologie hétérogène, cependant, elles sont restées attacher dans la structure poreuse de l'échafaudage. Différentes sondes employées pour marquer des cellules vivantes ont permis de suivre en temps réel la dynamique cellulaire pendant la stimulation électromécanique. En outre, les cellules stimulées présentaient un profil de cytokines différent de celui des cellules non stimulées. Ainsi, cette thèse a démontré la preuve de concept de l'échafaudage polyHIPE-PEDOT électroactif en tant qu'outil pour la culture cellulaire 4D et pour de futures études de mécanobiologie
In vivo, cells are situated within a 3D porous and dynamic microenvironment that provides biochemical and biophysical cues as well as dynamic signals influencing cell behavior across physiological and pathological contexts. To better replicate these conditions in vitro for applications in fundamental cell biology, tissue engineering, and drug screening this thesis presents the development of 4D electroactive scaffolds, combining a 3D passive microporous polyHIPE architecture and an electroactive polymer, PEDOT. These scaffolds serve as a dynamic cell culture platform capable to deliver electromechanical stimulation. The study first focused on the synthesis and characterization of electroactive polyHIPE-PEDOT scaffolds, which demonstrated a highly porous (10 to 100 µm) and interconnective structure beneficial for rapid cell colonization. Notably, these scaffolds could undergo volumetric changes in response to electrical stimulation. The second part of this work focused the polyHIPE-PEDOT scaffolds were found to be suitable for cell culture applications. The scaffolds were found to be cytocompatible, supporting cell adhesion, migration and proliferation. Cells within the scaffold adopted a spindle-like cell morphology typical of 3D cell microenvironments and synthesized fibronectin, an extracellular matrix protein essential for cell-matrix interactions. In the third part of this thesis, an electromechanical stimulation device suitable for in vitro cell culture studies (6-well cell culture plate) and live cell imaging (glass bottomed petri dish) was developed. A stimulation protocol was established and did not induce acute cytotoxic effects. After stimulation, cells exhibited heterogenic cell morphology, however, remained spread within the porous structure of the scaffold. Different live cell probes allowed the real-time monitoring of the cell dynamics during electromechanical stimulation. Furthermore, the stimulated cells exhibited different cytokine profile compared to non-stimulated cells. Thus, this thesis demonstrated the proof of concept of the electroactive polyHIPE-PEDOT scaffold as a tool for 4D cell culture and for future mechanobiological studies

AbstractCell migration is a very dynamic process involving several chemical as well as biological interactions with other cells and the environment. Several models exist to study cell migration ranging from simple 2D in vitro cultures to more demanding 3D multicellular assays, to complex evaluation in animals. High-resolution 4D (XYZ, spatial + T, time dimension) intravital imaging using transgenic animals with a fluorescent label in cells of interest is a powerful tool to study cell migration in the correct environment. Here we describe an advanced dorsal skinfold chamber model to study endothelial cell and pericyte migration and association.

Hydrogels crosslinked by homopolymerization of single component acrylate/methacrylate terminated polymers (e.g., poly(ethylene glycol) diacrylate, or PEGDA) were once the dominant biomaterials in biomedical applications, including the encapsulation of therapeutic agents and biological molecules. However, accumulating evidence has revealed many disadvantages of homopolymerized hydrogels, including heterogeneity of the crosslinking that adversely impacted the bioactivity of the encapsulated molecules. As such, recent years have witnessed the expansive use of modular click chemistry for the crosslinking of multicomponent hydrogels, typically consisting of two or more functionally distinct macromolecular building blocks. This chapter provides an overview of the crosslinking and applications of multicomponent hydrogels, focusing on those crosslinked by strain-promoted alkyne–azide cycloaddition (SPAAC), Michael-type addition, Diels–Alder (DA) reactions, inverse electron-demand Diels–Alder (iEDDA), thiol–ene polymerizations, and imine/hydrazone/oxime click reactions. This chapter also summarizes information regarding the characteristics, advantages, and limitations of commonly used synthetic (e.g., PEG, poly(acrylate), poly(vinyl alcohol), etc.) and naturally-derived macromers (e.g., gelatin, hyaluronic acid, etc.) for forming multicomponent hydrogels. Finally, an overview is given on the applications of multicomponent hydrogels in drug delivery, biofabrication, and 3D/4D cell culture.