Estabilizar hepatocelular Fenotipo Usando superficies sintéticas optimizadas
Summary
Este artículo se centrará en el desarrollo de superficies de polímero recubierto de largo plazo, la cultura estable de células madre derivadas de hepatocitos humanos.
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
Los materiales biológicos han sido ampliamente utilizados en el mantenimiento y diferenciación de células madre pluripotentes 1. Al tiempo que permite, estos sustratos biológicos a menudo contienen una gran cantidad de componentes no definidos. Matrigel es un sustrato comúnmente utilizado para el cultivo de células madre y la diferenciación. Desafortunadamente, su composición variable influye en la función celular y el fenotipo. Aunque una variedad de matrices biológicas alternativas, más definidos se han utilizado 2-7, su origen animal o pobre escalabilidad les hace candidatos inadecuados para la fabricación industrial. Por lo tanto, la identificación de alternativas sintéticas, con composición definida y un rendimiento fiable, son objetivos clave en la investigación de células madre.
En un intento de superar las limitaciones de los sustratos de cultivo de células definidos, colaboraciones interdisciplinarios entre la química y la biología han identificado materiales sintéticos con la capacidad de soportar el fenotipo celular. Synthsustratos etic son escalables, rentables, y se pueden fabricar en complejas estructuras 3D, imitando el entorno en vivo. Debido a estas propiedades sustratos sintéticos han sido ampliamente utilizados para apoyar y conducir la diferenciación de muchos tipos de células 8-10.
Ensayos avanzados y de alto rendimiento han facilitado la rápida detección de los materiales sintéticos, de grandes bibliotecas, y entregado nuevos materiales con propiedades flexibles con amplias aplicaciones en la investigación y el desarrollo 11-13 biomédica. La utilización de un alto rendimiento, tecnología de tramado polímero micro-array, identificamos rápidamente en un sencillo de poliuretano (PU134), adecuado para el mantenimiento de los hepatocitos derivados de células madre humanas. Este polímero se encontró que era superior a sustratos derivados de animales con respecto a la diferenciación y la función de los hepatocitos 14-16. Hemos optimizado posteriormente el proceso de condiciones de recubrimiento, la topografía y la esterilización para acceder a efectosen el rendimiento del polímero en la estabilización de función de los hepatocitos y la vida útil. Esto tiene implicaciones significativas en cuanto a la comprensión de los fundamentos de la biología de los hepatocitos para el modelado basado en células y aplicaciones de medicina regenerativa.
La tecnología aquí descrita representa un ejemplo de cómo la superficie de un polímero sintético puede ser optimizado para preservar el fenotipo celular. Creemos que la combinación de esta tecnología con un protocolo de diferenciación de hepatocitos sin suero eficiente tiene el potencial de proporcionar una producción escalable de hepatocitos para su uso en modelado in vitro y la medicina regenerativa.
Protocol
Representative Results
Discussion
Muchos de los métodos actuales utilizados para generar hepatocitos a partir de células madre se basan en matrices indefinidos de origen animal. Estos sustratos pueden ser costosos y altamente variable, que afecta a la función celular y la estabilidad, lo que representa una barrera significativa a la aplicación. Por lo tanto, se realizó una pantalla para materiales sintéticos que apoyan la cultura de células madre de hepatocitos derivados. Hemos identificado, un poliuretano sencilla (PU134), formado por la polimer…
Disclosures
The authors have nothing to disclose.
Acknowledgements
DCH, MB y FK fueron apoyados por un seguimiento EPSRC el Fondo. BL-V y DS fueron cada apoyados por becas de doctorado MRC. KC fue apoyado por la financiación de la Plataforma de Medicina Regenerativa del Reino Unido.
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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