Literatura científica selecionada sobre o tema "Culture cellulaire 4D"

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.

Fishing cat (Prionailurus viverrinus) and clouded leopard (Neofelis nebulosa) are wild felids, currently in vulnerable status according to the International Union for Conservation of Nature red list (2017). Several measures in assisted reproductive technology (e.g. AI, embryo transfer) have been used by the Zoological Park Organization of Thailand (ZPO) to increase their offspring in captivity. Recently, the generation of induced pluripotent stem cell (iPS cells) becomes popular and provides alternative way to preserve good genetics in the form of cell with diverse capacities. This great potential of iPS cells is unlimited self-renewal and pluripotency, similar to embryonic stem cells (ESC). Under the right cell culture conditions, pluripotent stem cells can differentiate into all cell types of the body. Here, we aimed to find the optimal condition to generate integration-free iPS cells from fishing cat and clouded leopard. At first, to obtain somatic cells for cellular reprogramming, adult dermal fibroblast cell lines from both species were established from belly skin tissues. Subsequently, several nucleofection programs of AmaxaTM 4D-nucleofectorTM (Lonza, Basel, Switzerland) were examined to introduce integration-free DNA vectors carrying reprogramming factors into the felid fibroblasts. The transfected cells were cultured under numerous conditions: (1) matrix/defined surface including irradiated mouse embryonic fibroblast, gelatin, vitronectin, and Geltrex® (Thermo Fisher Scientific, Waltham, MA, USA); (2) ESC/iPS cell medium including Essential 8TM (Thermo Fisher Scientific) DMEM containing KnockOutTM Serum Replacement (KOSR; Thermo Fisher Scientific) and/or fetal bovine serum (FBS); and (3) supplement including basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), l-ascorbic acid, nicotinamide, ALK5 inhibitor (A83-01) and RevitaCellTM (Thermo Fisher Scientific). We found that optimal nucleofection programs for human dermal fibroblast including FF-135 and EN-150 were able to transfer episomal vectors and excisable piggyBAC transposon carrying reprogramming factors into fishing cat and clouded leopard fibroblasts, respectively. The iPS-like colonies appeared around 26 to 30 days post-nucleofection. The culture of transfected cells on either Geltrex® or Vitronectin-coated surface supports the formation of iPS-like colonies with different derivation efficiency (0.01 and 0.005%, respectively). In addition, all colonies were formed under medium containing FBS, together with both bFGF and LIF supplements. Taken together, we have developed a platform to generate iPS cells from tissue collection to the establishment of iPS cell culture. This will further enable us to apply the technique to obtain iPS cells from other endangered and vulnerable felid species.

Biomimetic scaffolds imitate native tissue and can take a multidimensional form. They are biocompatible and can influence cellular metabolism, making them attractive bioengineering platforms. The use of biomimetic scaffolds adds complexity to traditional cell cultivation methods. The most commonly used technique involves cultivating cells on a flat surface in a two-dimensional format due to its simplicity. A three-dimensional (3D) format can provide a microenvironment for surrounding cells. There are two main techniques for obtaining 3D structures based on the presence of scaffolding. Scaffold-free techniques consist of spheroid technologies. Meanwhile, scaffold techniques contain organoids and all constructs that use various types of scaffolds, ranging from decellularized extracellular matrix (dECM) through hydrogels that are one of the most extensively studied forms of potential scaffolds for 3D culture up to 4D bioprinted biomaterials. 3D bioprinting is one of the most important techniques used to create biomimetic scaffolds. The versatility of this technique allows the use of many different types of inks, mainly hydrogels, as well as cells and inorganic substances. Increasing amounts of data provide evidence of vast potential of biomimetic scaffolds usage in tissue engineering and personalized medicine, with the main area of potential application being the regeneration of skin and musculoskeletal systems. Recent papers also indicate increasing amounts of in vivo tests of products based on biomimetic scaffolds, which further strengthen the importance of this branch of tissue engineering and emphasize the need for extensive research to provide safe for humansbiomimetic tissues and organs. In this review article, we provide a review of the recent advancements in the field of biomimetic scaffolds preceded by an overview of cell culture technologies that led to the development of biomimetic scaffold techniques as the most complex type of cell culture.

Abstract OBJECTIVE: Intraoperative detection of residual tumor tissue in glioma surgery remains an important challenge because the extent of tumor removal is related to the prognosis of the disease. Multiphoton excited fluorescence tomography of living tissues provides high-resolution structural and photochemical imaging at a subcellular level. In this conceptual study, we have used multiphoton microscopy and fluorescence lifetime imaging (4D microscopy) to image cultured glioma cell lines, solid tumor, and invasive tumor cells in an experimental mouse glioma model and human glioma biopsy specimens. MATERIAL AND METHODS: A laser imaging system containing a mode-locked 80 MHz titanium:sapphire laser with a tuning range of 710 to 920 nm, a scan unit, and a time correlated single photon counting board was used to generate autofluorescence intensity images and fluorescence lifetime images of cultured cell lines, experimental intracranial gliomas in mouse brain, and biopsies of human gliomas. RESULTS: Multiphoton microscopy of native tumor bearing brain provided structural images of the normal brain anatomy at a subcellular resolution. Solid tumor, the tumor-brain interface, and single invasive tumor cells could be visualized. Fluorescence lifetime imaging demonstrated significantly different decay of the fluorescent signal in tumor versus normal brain, allowing a clear definition of the tumor-brain interface based on this parameter. Distinct fluorescence lifetimes of endogenous fluorophores were found in different cellular compartments in cultured glioma cells. The analysis of the relationship between the laser excitation wavelength and the lifetime of excitable fluorophores demonstrated distinct profiles for cells of different histotypes. CONCLUSION: Multiphoton excited fluorescence of endogenous fluorophores allows structural imaging of tumor and central nervous system histo-architecture at a subcellular level. The analysis of the decay of the fluorescent signal within specific excitation volumes by fluorescent lifetime imaging discriminates glioma cells and normal brain, and the excitation/lifetime profiles may further allow differentiation of cellular histotypes. This technology provides a noninvasive optical tissue analysis that may potentially be applied to an intraoperative analysis of resection plains in tumor surgery.

Abstract The subventricular zone (SVZ) of lateral ventricles in brain contains neuronal stem cells (NSC) that form neurospheres when cultured with EGF and/or bFGF. Progeny from transplanted NSC migrate throughout the brain and replace multiple differentiated cells. Our goal is to develop this phenomenon into a cellular/gene therapeutic approach for treatment of neurological disease. Progeny of normal or gene transduced NSC can replace defective host cells or act as enzyme delivery vehicles. One model for testing this approach is the Mucopolysaccharidosis Type VII (MPS VII) mouse that is deficient in β-glucuronidase (GUS) expression, causing lysosomal storage disease (Sly Syndrome). Undegraded substrates accumulate in brain, causing cognitive dysfunction. A second model is deficient in palmitoyl-protein thioesterase 1 (PPT1), causing neurodegeneration in humans and mice known as infantile neuronal ceroid lipofuscinosis, or Battens Disease. By 24–30wks of age, mice develop motor abnormalities and seizures. Obstacles to NSC therapy include suitable source of donor cells and immunological rejection. Since mesenchymal stem cells (MSC) derived from bone marrow and cord blood have neuronal differentiation capacity, we believe these tissues can generate NSC. If so, self bone marrow or cord blood derived NSC could be transduced with a lentivirus that restores enzyme expression, solving both obstacles. We cultured 15d fetal liver (the murine equivalent of cord blood) from eGFP transgenic mice in neurosphere medium containing EGF and bFGF +/− Noggin/Fc. Noggin is an antagonist for bone morphogenic protein, is expressed in ependymal cells of the SVZ, upregulates neurogenesis, and inhibits bone formation. Growth factors were added days 1–5, and alternating days thereafter. Half the media was replaced every 4d. At 14d, the +Noggin culture had 2–3x the confluency of the -Noggin culture and contained spindle-shaped cells resembling MSC. After another 14d with EGF and bFGF alone, the +Noggin cells formed neurospheres. Neurospheres reformed after trituration, indicating self-renewal capacity. The neurospheres were Nestin+, a marker for NSC, and upon differentiation with serum, β-NGF, BDNF, and NT-3, stained positive for neurons (MAP2) and astrocytes (GFAP). Next, we transplanted 250,000 normal eGFP fetal liver derived NSC (FL NSC) into the lateral ventricles of PPT1−/ − neonatal recipients. Brains 18–54 days post transplant revealed cells in the SVZ of the lateral, dorsolateral, and third ventricles. Donor cells migrated away from the ventricles, into the hippocampal fimbria, under the corpus callosum, and into the rostral migratory stream. Later time points revealed increased migration away from ventricles. We also tested transduction of NSC from MPS VII fetal brain using a lentiviral SIN vector driving human GUS from a PGK promoter. Transduction efficiency was 88–90%. Transplantation of 250,000 transduced NSC into neonatal MPS VII recipients revealed a similar pattern of transplantation as described above for FL NSC. Donor GUS+ cells were detected 4mo post transplant, indicating long-term gene expression and donor cell survival. In conclusion, NSC are efficiently transduced with lentivirus, can be cultured from hematopoietic tissue, and engraft long-term following neonatal injection. These results demonstrate a method to circumvent both donor cell availability and immune barriers to transplantation, providing hope for patients with devastating neurological disorders.

Background:Autophagy is catabolic process allowing cells to degrade unnecessary or dysfunctional cellular organelles. Failure of appropriate regulation of autophagy, however, can severely perturb tissue homeostasis. Recent studies demonstrate that autophagy is activated in several fibrotic diseases such as liver fibrosis, renal interstitial fibrosis, cardiac fibrosis.Objectives:The objective of this work was to characterize the activation of autophagy in systemic sclerosis (SSc) and to decipher its role in the pathogenesis of SSc.Methods:Activation of autophagy in skin samples of patients and murine models of SSc was assessed by co-staining of LC3B and P62 with the lysosomal marker LAMP2. The role of the autophagy was investigated in the model of bleomycin-induced dermal fibrosis. Beclin1 was overexpressed using adenovirus encoding for Beclin1. To knockdown Atg7 in vivo was achieved by subcutaneous injections of Atg7 siRNA or non-targeting siRNA. In vivo, 3-methyladenine (3-MA) was administered i.p. in a concentration of 15 mg/kg ones daily. Protein expression was measured by Western blot. Target genes were analyzed by qPCR. To monitor the autophagic flux, we generated adenoviral vectors encoding for tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3).Results:In the present study, we demonstrate that autophagy is activated in fibroblasts in SSc skin and also in experimental fibrosis models as compared to respective non-fibrotic control tissue with enhanced activity in in vivo and in vitro autophagy reporter studies. The aberrant activation of autophagy had profound stimulatory effects on fibroblasts. Activation of autophagy by forced expression of BECLIN1 promoted fibroblast-to-myofibroblast transition and stimulated the collagen release by cultured human fibroblasts and induced fibrosis in murine model. Nevertheless, inhibition of autophagy can deactivate myofibroblasts and induce regression of tissue fibrosis. Knockdown of ATG7 or BECLIN1 in human fibroblasts reduced the expression of αSMA and the number of stress fibers in myofibroblasts, indicating re-differentiation of myofibroblasts into resting fibroblasts upon inhibition of autophagy. Similar results were obtained with the autophagy inhibitors CQ and 3-MA. In vivo, siRNA mediated knockdown of Atg7 effectively prevented progression of fibrosis in a model of established bleomycin-induced skin fibrosis. Inactivation of autophagy decreased dermal thickness, myofibroblast counts and hydroxyproline content to below pretreatment levels, indicating regression of bleomycin-induced skin fibrosis. In addition, treatment of mice with the autophagy inhibitor 3-MA ameliorated bleomycin-induced skin fibrosis.Conclusion:We demonstrate that autophagy activity is enhanced in fibroblasts of SSc patients and in murine models of SSc. The increased activation of autophagy induces fibroblast-to-myofibroblast transition and promotes fibrotic tissue remodeling. However, inhibition of autophagy can deactivate myofibroblasts and induce regression of tissue fibrosis.References:[1]Wynn, T. Cellular and molecular mechanisms of fibrosis. J Pathol 214, 199-210 (2008).[2]Klionsky DJ, Abeliovich H, Agostinis P, et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4, 151-175 (2008).[3]Wang, CW & Klionsky, DJ. The molecular mechanism of autophagy. Mol Med 9, 65-76 (2003).[4]Hernández-Gea V, Ghiassi-Nejad Z, Rozenfeld R, et al. Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues. Gastroenterology 142, 938-946 (2012).Disclosure of Interests:Ariella Zehender: None declared, Yi-Nan Li: None declared, Neng-Yu Lin: None declared, Andrea-Hermina Györfi: None declared, Alina Soare: None declared, Christina Bergmann: None declared, Andreas Ramming: None declared, Georg Schett: None declared, Jörg H.W. Distler Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB., Grant/research support from: Anamar, Active Biotech, Array Biopharma, aTyr, BMS, Bayer Pharma, Boehringer Ingel-heim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB., Employee of: stock owner of 4D Science and Scientific head of FibroCure

Abstract Abstract 2987 Background. We have recently shown HIF1A to be expressed in 95.4% of CD138-purified myeloma cell samples from previously untreated patients (n= 329), with significantly higher [lower] expression in case of presence of t(4,14) [hyperdiploidy] vs. patients without the respective aberration. This makes HIF1A an interesting target in myeloma treatment. Additionally, we have shown about 40% of myeloma cell samples to have a proliferation-index above the median plus three standard-deviations of normal bone-marrow plasma cells, and we and others have proven proliferation to be associated with adverse prognosis in myeloma. Here, we report on 2 members of a novel class of sulfonanilides, their preclinical activity and pharmacology, and their dual mechanism of action, targeting HIF1A-signaling and inducing apoptosis via cell cycle arrest and tubulin depolymerization. Patients and Methods. The effect of the novel sulfonanilides ELR510444 and ELR510552 on the proliferation of 20 human myeloma cell lines and the survival of 5 primary myeloma cell-samples cultured within their microenvironment were tested. The results of efficacy studies in in two murine models (RPMI8226-xenograft-model and 5T33-model) are also presented. The mechanism of action was investigated using a variety of in-vitro assays (see below). Results. Preclinical activity in Myeloma. i) The sulfonanilides ELR510444 and ELR510552 completely inhibit proliferation of 20/20 tested myeloma cell lines at low nM concentrations and ii) induce apoptosis in 5/5 primary myeloma cell-samples at 6.4 – 32 nM concentration, without major effect on the bone marrow microenvironment. iii) They significantly inhibit tumor growth (xenograft; RPMI8226 mouse model, 6 mg p.o. bid for ELR510444, 15 mg p.o. bid for ELR510552) and bone marrow infiltration (5T33-model; ELR510444, 6 mg/kg p.o. bid × 4d, rest 3d (cycle)). Mechanism of action. Apoptosis induction and G2/M-block. i) Both compounds lead to caspase-3/7 activation and subsequent apoptosis with cellular EC50 values of 50–100 nM. ii) The compounds induce an initial cellular arrest in G2/M and a significant tubulin depolymerizing effect, followed by an increase in a sub-G1 (apoptotic) population after 24h. HIF1A-inhibition. i) Both compounds show a potent inhibition of HIF1A signaling in a cell based reporter assay (HRE-bla HCT-116) at EC50s of 1–25nM, whereas ii) at concentrations of 1 μ M, neither of the compounds shows an effect in assay systems monitoring the JAK/STAT, NFκB, PI3K/AKT/FOXO or Wnt/β-catenin-signaling pathways. iii) Kinase inhibition profiling showed no significant inhibition at 1μ M in two assays assessing 100 (Invitrogen) and 442 (Ambit) kinases, respectively. Pre-clinical pharmacology. Single dose exposure of 25 mg p.o. yields a maximum concentration of 1.1 μ M with a half life time of 3.6 hours (ELR510444) and 2.7 μ M and 6.6 h (ELR510552) in mice, respectively. The compounds are well-tolerated at levels that are significantly above the in vitro EC50 in all myeloma cell lines and primary samples tested. Conclusion. ELR510444 and ELR510552 are very active on all tested myeloma cell lines and primary myeloma cells without major impact on the bone marrow microenvironment, and show activity in two different mouse models. The compounds inhibit HIF1A-signaling and induce apoptosis via cell cycle arrest and tubulin depolymerization. Preclinical pharmacology data show favorable in vivo profiles with exposure levels in mice significantly higher than concentrations required for in vitro activity. Therefore, this novel class of compounds represents a promising weapon in the therapeutic arsenal against multiple myeloma entering a phase I/II trial within the next year. Disclosures: Leber: ELARA Pharmaceuticals GmbH: Employment. Janssen:ELARA Pharmaceuticals GmbH: Employment. Lewis:ELARA Pharmaceuticals GmbH: Employment. Schultes:ELARA Pharmaceuticals GmbH: Employment.

BackgroundNuclear receptors (NR) are a family of transcription factors. Several members of the NR family are candidates for targeted intervention in inflammatory and fibrotic diseases (1-2). Testicular receptor 4 (TR4), also known as Nr2c2, has been shown to regulate fundamental cellular processes such as differentiation, proliferation and growth factor signaling (3-4). However, its role in fibrotic diseases has not been investigated so far.ObjectivesThe aim of the present study was to characterize the role of TR4 in the pathogenesis of fibrotic tissue remodeling in SSc and to interrogate its underlying mechanism.MethodsExpression of TR4 was quantified by RT-PCR, Western blot and immunofluorescence. The effects of TR4 knockdown on collagen production and myofibroblast differentiation were analyzed in cultured human fibroblasts and in three mouse models with fibroblast-specific knockout of TR4. RNA sequencing was performed in TGFβ-stimulated human dermal fibroblasts transfected with TR4 siRNA or non-targeting siRNA. The implication of TR4 in cytoskeleton regulation was analyzed by ROCK activity assays, stress fiber formation and quantification of the ratio of filamentous (F)-actin/ globular (G)-actin. The functional role of ROCK and Gα12 was analyzed using small molecule inhibitors and siRNA, respectively.ResultsTR4 expression was upregulated in fibroblasts in the skin of SSc patients and fibrotic mouse models. The expression of TR4 was upregulated in a TGFβ- and SMAD3-dependent manner. TR4 knockdown inhibited TGFβ-induced myofibroblast differentiation and collagen release, whereas overexpression of TR4 promoted fibroblast activation. Fibroblastspecific knockout of TR4 ameliorated experimental dermal fibrosis induced by bleomycin, adTBR and in sclGVHD with decreases in dermal thickening, myofibroblast counts and hydroxyproline content. The RNASeq of TR4 knockdown fibroblasts stimulated with TGFβ identified 651 differentially expressed genes as compared to control fibroblasts. Differentially expressed genes included central profibrotic genes such as ACTA2, COL3A1, CCL12. gProfiler enrichment analysis of the TR4-DEGs revealed enrichment of multiple GO, GSEA and Reactome terms related to ECM release, cytoskeleton organization and Rho GTPases activity. Indeed, knockdown of TR4 ameliorated the induction of ROCK activity by TGFβ stimulation and reduced the shift in the ratio from globular (G) actin to filamentous (F) actin. Knockdown of TR4 strongly reduced the expression of the G-protein alpha-12 (Gα12). ROCK inhibition by Y27632 or knockdown of Gα12 inhibited the induction of αSMA and stress fiber formation induced by overexpression of TR4.ConclusionTR4 is upregulated in SSc in a TGFβ-dependent manner to promote fibroblast activation. Inhibition of TR4 interferes with TGFβ-induced activation of ROCK, prevents cytoskeletal remodeling and fibroblast-to-myofibroblast transition and ameliorates experimental fibrosis. As nuclear receptors are common targets for therapeutic intervention, TR4 may offer potential for antifibrotic therapies.References[1]Avouac J, Palumbo-Zerr K, et al. The nuclear receptor constitutive androstane receptor/NR1I3 enhances the profibrotic effects of transforming growth factor beta and contributes to the development of experimental dermal fibrosis. Arthritis Rheumatol. 2014;66(11):3140-50.[2]Palumbo-Zerr K, Zerr P, et al. Orphan nuclear receptor NR4A1 regulates transforming growth factor-beta signaling and fibrosis. Nat Med. 2015;21(2):150-8.[3]Bookout AL, Jeong Y, Downes M, Yu RT, Evans RM, Mangelsdorf DJ. Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network. Cell. 2006;126(4):789-99.[4]Simandi Z, Cuaranta-Monroy I, Nagy L. Nuclear receptors as regulators of stem cell and cancer stem cell metabolism. Seminars in cell & developmental biology. 2013;24(10-12):716-23.Disclosure of InterestsLichong Shen: None declared, Yun Zhang: None declared, ZHU Honglin: None declared, Jörg H.W. Distler Shareholder of: stock owner of 4D Science GmbH, Consultant of: JHWD has consultancy relationships and/or has received research funding from AbbVie, Actelion, BMS, Celgene, Bayer Pharma, Boehringer Ingelheim, JB Therapeutics, Sanofi-Aventis, Novartis, UCB, GSK, Array Biopharma and Active Biotech in the area of potential treatments of SSc.

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