Cell therapy of the liver : interest of a cell sheet approach

Le foie est l'organe endocrinien le plus volumineux du corps humain. Il présente de nombreuses fonctions telles que la détoxification, la synthèse des protéines et de la bile, le stockage du sucre et du fer. Le foie est également caractérisé par sa grande capacité de régénération. Toutefois, du fait d'agressions chroniques ou de certaines maladies, il perd cette capacité et une transplantation hépatique est alors nécessaire. Dans un contexte d'augmentation des besoins de greffons hépatiques, et de la baisse du nombre de donneurs, les nouvelles approches d'ingénierie tissulaire (IT) peuvent représenter un intérêt majeur. Les hépatocytes sont des cellules polarisées, interagissant entre elles et avec des cellules endothéliales. Cette polarisation est importante pour leur viabilité et leur fonctionnalité. Des méthodes de culture in-vitro permettant de préserver cette polarisation sont nécessaires pour conserver les capacités de ces cellules. Nos travaux de recherches ont ainsi porté sur deux nouvelles méthodes de culture : la technologie des feuillets cellulaires et la culture cellulaire par lévitation acoustique. La technologie des feuillets cellulaires consiste à cultiver des cellules sur un polymère thermosensible, le poly NIPAM. En dessous de 32°C, le polymère devient hydrophile et permet de détacher les cellules en feuillets. Pour la fabrication des feuillets hépatiques, nous avons cultivé les cellules HepaRG sur un polymère thermosensible. Les feuillets ont ensuite été recouverts d'une couche de gel de fibrine afin de faciliter leur transfert et leur manipulation. Nous avons pu montrer que les feuillets cellulaires obtenus conservent une très bonne viabilité, que les cellules restent polarisées et fonctionnelles, et ce jusqu'à un mois après les avoir détachées du polymère. Une deuxième méthode de culture cellulaire a été développée, basée sur la lévitation acoustique. La preuve de concept de cette technologie a été réalisée sur des cellules stromales mésenchymateuses (CSM). Cette méthode a permis de démontrer que les cellules s'organisent en feuillets, puis vont former des sphéroïdes du fait des forces mécaniques des cellules. Cette technique de lévitation acoustique ne modifie pas la viabilité cellulaire, ni l'expression des marqueurs de surface. Elle permet de surcroît de meilleures capacités de différenciation dans les voies de l'adipogenèse et de l'ostéogenèse. Enfin, dans l'optique d'évaluer les capacités de vascularisation de feuillets cellulaires, ou d'organoïdes hépatiques, nous avons étudié les HUVEC comme modèle de cellules endothéliales. In-vitro, deux sous-populations ont été identifiées, sur la base de l'expression du marqueur CD34. La comparaison des cellules CD34+ et CD34- a montré que l'expression du CD34 est réversible, et qu'elle augmente avec la confluence. L'étude transcriptomique a révélé dans les cellules CD34+ une sous-expression des gènes de prolifération, ainsi qu'une sur-expression de l'IL-33, par rapport aux cellules CD34- . Le profil immunologique de ces cellules en co-culture avec des cellules mononucléées sanguines a montré une prolifération plus importante des lymphocytes T régulateurs en présence d'HUVEC CD34+. Pour conclure, nous avons mis au point deux méthodes de culture cellulaire qui pourraient être utilisées pour de futures applications cliniques de thérapie cellulaire hépatiques. Nous avons également élucidé un profil immunologique distinct des cellules endothéliales en fonction de l'expression du marqueur CD34 qui pourrait représenter un biomarqueur des mécanismes de la revascularisation ou des pathologies hépatiques, impliquant les cellules endothéliales.The liver is the largest endocrine organ in the human body. It has many functions such as detoxification, protein and bile synthesis, sugar and iron storage. The liver is also characterized by its great capacity for regeneration. However, due to certain diseases, it loses this ability and a liver transplant is then necessary. In a context of increasing needs for liver transplantation, and the decline in the number of donors, new tissue engineering (TI) approaches may represent a major interest. Hepatocytes are polarized cells, interacting with each other and with endothelial cells. This polarization is important for their viability and functionality. In-vitro culture methods allowing to preserve this polarization are necessary to preserve the capacities of these cells. Our research work has thus focused on two new culture methods: cell sheet technology and cell culture by acoustic levitation. Cell sheet technology involves growing cells on a thermoresponsive polymer, poly NIPAM. Below 32°C, the polymer becomes hydrophilic and makes it possible to detach the cells into sheets. For the production of liver sheets, we cultured HepaRG cells on a thermoresponsive polymer. The sheets were then covered with a layer of fibrin gel to facilitate their transfer and manipulation. We were able to show that the cell sheets obtained retain very good viability, that the cells remain polarized and functional, up to one month after having detached them from the polymer. A second cell culture method has been developed, based on acoustic levitation. The proof of concept of this technology was carried out on mesenchymal stromal cells (MSCs). This method has made it possible to demonstrate that the cells organize themselves into sheets, then will form spheroids due to the mechanical forces of the cells. This acoustic levitation technique does not modify cell viability or the expression of surface markers. It also allows better differentiation capacities in the pathways of adipogenesis and osteogenesis. Finally, in order to evaluate the vascularization of cell sheets, or hepatic organoids, we have studied HUVECs as a model of endothelial cells. In-vitro, two subpopulations have been identified, based on the expression of the CD34 marker. Comparison of CD34+ and CD34- cells showed that CD34 expression is reversible, and that it increases with confluence. The transcriptomic study revealed in the CD34+ cells an under-expression of proliferation genes, as well as an over-expression of IL-33, compared to the CD34- cells. The immunological profile of these cells in co-culture with blood mononuclear cells showed a greater proliferation of regulatory T lymphocytes in the presence of HUVEC CD34+. To conclude, we have developed two cell culture methods that could be used for future clinical applications of hepatic cell therapy. We also elucidated a distinct immunological profile of endothelial cells according to the expression of the CD34 marker which could represent a biomarker of the mechanisms of revascularization or liver pathologies, involving endothelial cells

Trends in 3D bioprinting for esophageal tissue repair and reconstruction

International audienc

Self-organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation

International audienceIn recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells

Author Correction: Self‑organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Toward the creation of 2D or 3D clusters of cells in acoustic levitation

International audienceIntroduction Today, three-dimensional (3D) cell cultures tend to replace 2D conventional method because of their more relevant tissue-mimicking characteristics. Indeed, the 3D cell architecture (spheroïd, organoïd, etc) and the microenvironment is closer to In Vivo physiological behaviour [1, 2]. The main difficulties remain in creating a scaffold compatible with the targeted cells and tissues. Bioprinting is one the great objective for tissue engineering. For instance, stereolithography is a 3D printing technology where the freestanding object is built layer by layer with a photosensitive polymer resin through the projection of a UV image in the top plane. In recent promising work, stereolithography has been applied to create 3D hydrogel structures to guide cells like hepatocytes [3]. Nevertheless, ideally, scaffold-free methods are needed. We propose a new method combining microfluidic channels and the acoustic radiation force (ARF) to structure and to control the shape of stem-cells aggregates

Biodistribution of allogenic umbilical cord-derived mesenchymal stromal cells after fetal repair of myelomeningocele in an ovine model

Abstract Background Myelomeningocele (MMC) is a spinal cord congenital defect that leads to paraplegia, sphincter disorders and potential neurocognitive disabilities. Prenatal surgery of MMC provides a significant benefit compared to surgery at birth. Mesenchymal stromal cell (MSC) therapy as an adjuvant treatment for prenatal surgery showed promising results in animal experiments which could be considered for clinical use in human fetuses. Despite numerous reassuring studies on the safety of MSCs administration in humans, no study focused on MSCs biodistribution after a local MSCs graft on the fetal spinal cord. Aim The purpose of our study was to assess the biodistribution of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) at birth in lambs who had a prenatal myelomeningocele repair using a fibrin patch seeded with allogenic UC-MSCs. Methods After isolation, UC-MSCs were tagged using a green fluorescent protein (GFP)-containing lentiviral vector. MMC defects were surgically created at 75 days of gestation and repaired 15 days later using UC-MSCs patch. Lambs were delivered at 142 days and sacrificed. DNA extraction was performed among biopsies of the different organs and q-PCR analysis was used to detect the expression of GFP (GFP DNA coding sequence). Results In our 6 surviving lambs grafted with UC-MSCs, GFP lentivirus genomic DNA was not detected in the organs. Conclusion These reassuring data will support translational application in humans, especially since the first human clinical trial using mesenchymal stromal cells for in-utero treatment of MMC started recently in U.S.A

Toward the creation of 2D or 3D clusters of cells in acoustic levitation

International audienceIntroduction Today, three-dimensional (3D) cell cultures tend to replace 2D conventional method because of their more relevant tissue-mimicking characteristics. Indeed, the 3D cell architecture (spheroïd, organoïd, etc) and the microenvironment is closer to In Vivo physiological behaviour [1, 2]. The main difficulties remain in creating a scaffold compatible with the targeted cells and tissues. Bioprinting is one the great objective for tissue engineering. For instance, stereolithography is a 3D printing technology where the freestanding object is built layer by layer with a photosensitive polymer resin through the projection of a UV image in the top plane. In recent promising work, stereolithography has been applied to create 3D hydrogel structures to guide cells like hepatocytes [3]. Nevertheless, ideally, scaffold-free methods are needed. We propose a new method combining microfluidic channels and the acoustic radiation force (ARF) to structure and to control the shape of stem-cells aggregates

Self-organization of human mesenchymal stem cells into spheroids trapped in acoustic levitation

International audienc

Endothelial CD34 expression and regulation of immune cell response in-vitro

Abstract Endothelial cells cover the lining of different blood vessels and lymph nodes, and have major functions including the transport of blood, vessel homeostasis, inflammatory responses, control of transendothelial migration of circulating cells into the tissues, and formation of new blood vessels. Therefore, understanding these cells is of major interest. The morphological features, phenotype and function of endothelial cells varies according to the vascular bed examined. The sialomucin, CD34, is widely used as an endothelial marker. However, CD34 is differentially expressed on endothelial cells in different organs and in pathological conditions. Little is known about regulation of endothelial CD34 expression or function. Expression of CD34 is also strongly regulated in-vitro in endothelial cell models, including human umbilical vein endothelial cells (HUVEC) and endothelial colony forming cells (ECFC). We have therefore analysed the expression and function of CD34 by comparing CD34high and CD34low endothelial cell subpopulations. Transcriptomic analysis showed that CD34 gene and protein expressions are highly correlated, that CD34high cells proliferate less but express higher levels of IL-33 and Angiopoietin 2, compared with CD34low cells. Higher secretion levels of IL-33 and Angiopoietin 2 by CD34high HUVECs was confirmed by ELISA. Finally, when endothelial cells were allowed to interact with peripheral blood mononuclear cells, CD34high endothelial cells activated stronger proliferation of regulatory T lymphocytes (Tregs) compared to CD34low cells whereas expansion of other CD4+-T cell subsets was equivalent. These results suggest that CD34 expression by endothelial cells in-vitro associates with their ability to proliferate and with an immunogenic ability that favours the tolerogenic response

A clinical‐grade acellular matrix for esophageal replacement

International audienc