Stabiliser hépatocellulaire phénotype en utilisant des surfaces synthétiques optimisés
Summary
This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.
Abstract
Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.
Introduction
Les matières biologiques ont été largement utilisés dans l'entretien et la différenciation des cellules souches pluripotentes 1. Tout en permettant, ces substrats biologiques contiennent souvent une multitude de composants définis. Matrigel est un substrat communément utilisé pour la culture de cellules souches et de la différenciation. Malheureusement, sa composition variable influe sur la fonction des cellules et le phénotype. Bien qu'une grande variété de matrices biologiques alternatives, mieux définis ont été utilisés 2-7, leur origine animale ou pauvres évolutivité en fait des candidats inappropriés pour la production industrielle. Par conséquent, l'identification des alternatives synthétiques, à composition définie et des performances fiables, sont des objectifs clés de la recherche sur les cellules souches.
Dans une tentative pour surmonter les limitations des indéfinis substrats de culture de cellules, des collaborations interdisciplinaires entre la chimie et de la biologie ont identifié des matériaux synthétiques ayant la capacité de supporter le phénotype cellulaire. Synthsubstrats étiques sont évolutives, rentables, et peuvent être fabriqués dans des structures 3D complexes, imitant l'environnement in vivo. En raison de ces propriétés des substrats synthétiques ont été largement utilisés pour supporter et entraîner la différenciation de nombreux types de cellules 8-10.
Essais de débit de pointe et haute ont facilité la sélection rapide de matériaux synthétiques, de grandes bibliothèques, et livré de nouveaux matériaux aux propriétés flexibles avec de larges applications dans la recherche et le développement 11-13 biomédicale. Utilisant à haut débit, la technologie de dépistage micro-réseau polymère, nous avons identifié rapidement d'une simple polyuréthane (PU134), adapté pour l'entretien des hépatocytes dérivés de cellules souches humaines. Ce polymère a été jugée supérieure à des substrats provenant des animaux en ce qui concerne la différenciation et la fonction des hépatocytes 14-16. Nous avons par la suite optimisé le processus conditions de revêtement, de la topographie et de stérilisation pour accéder à effetssur le rendement en polymère stabilisant fonction des hépatocytes et la durée de vie. Ceci a des implications importantes en ce qui concerne la compréhension des fondamentaux de la biologie des hépatocytes pour la modélisation à base de cellules et des applications en médecine régénérative.
La technologie décrite ici est un exemple de la façon dont la surface d'un polymère synthétique peut être optimisée pour conserver le phénotype cellulaire. Nous croyons que la combinaison de cette technologie avec un hépatocyte libre protocole de différenciation de sérum efficace a le potentiel de fournir une production évolutive des hépatocytes pour l'utilisation de la modélisation in vitro et la médecine régénérative.
Protocol
Representative Results
Discussion
Many of the current methods used to generate hepatocytes from stem cells rely on undefined matrices of animal origin. These substrates can be costly and highly variable, affecting cell function and stability, representing a significant barrier to application. Therefore, we performed a screen for synthetic materials which support the culture of stem cell derived hepatocytes. We have identified, a simple polyurethane (PU134), formed by polymerizing PHNGAD, MDI and an extender, that in combination with a robust hepatocyte d…
Declarações
The authors have nothing to disclose.
Acknowledgements
D.C.H., M.B. and F.K. were supported by an EPSRC Follow on Fund. B.L-V and D.S. were each supported by MRC PhD studentships. K.C. was supported by funding from the UK Regenerative Medicine Platform.
Materials
| Synthesis, preparation, coating and characterization of polymer PU134 coated coverslips | |||
| Shaker | Edmun Bühler | KS-15 | |
| Irradiator | CIS Biointernational | IBL 637 | |
| Spin coater | Specialty Coating System | P-6708 | |
| Scanning Electron Microscope | Philips | XL30CPSEM | |
| Atomic Force Microscope | DimensionV Nanoscope, VEECO | ||
| p4-GLO CYP3A4 | Promega | V8902 | |
| UV bulb | ESCO | ||
| NanoScope analysis software | VEECO | version 1.20 | |
| Fluorescence microscope | Olympus | TH45200 | Use Volocity 4 Software |
| Tissue culture plates | Corning, UK | 3527 | |
| glass slides | Scientific Laboratory Supplies | MIC3308 | |
| Diethylene glycol | Sigma–Aldrich | 93171 | |
| 1,6-hexanediol | Sigma–Aldrich | 240117 | |
| Neopentyl glycol | Sigma–Aldrich | 408255 | |
| Adipic acid | Sigma–Aldrich | 9582 | |
| anhydrous N,N-Dimethylformamide | Sigma–Aldrich | 227056 | |
| Diethyl ether | Sigma–Aldrich | 676845 | |
| titanium (IV) butoxide | Sigma–Aldrich | 244112 | |
| 1,4-butanediol | Sigma–Aldrich | 493732 | |
| Vacuum oven | Thermoscientific | ||
| 4,4’-Methylenebis(phenyl isocyanate) | Sigma–Aldrich | 101688 | |
| Tetrahydrofurane | Sigma–Aldrich | 401757 | |
| Sputter coater | Bal-Tec SCD 050 | ||
| Inmunostaining | |||
| Phosphate buffer saline (-MgCl2, -CaCl2) | Gibco | 10010031 | Store at room temperature |
| PBST, PBS made up with 0.1% TWEEN 20 | Scientific Laboratory Supplies Ltd | EC607 | |
| Methanol | Scientific Laboratory Supplies Ltd | CHE5010 | |
| Bovine Serum Albumin | Sigma-Aldrich, UK | A7906 | |
| MOWIOL 488 DAPI | Calbiochem | 475904 | Made up in Tris HCL and glycerol as per manufacturers instructions |
| Cell culture and Functional assay | |||
| CYP3A activity pGLO kit | Promega | V8902 | |
| Hepatozyme | Gibco | 17705021 | |
| TryLE express | Life Technologies | 12604013 |
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