Dried human skin fibroblasts as a new substratum for functional culture of hepatic cells

• Authors: Agnieszka Wencel , Karolina Zakrzewska , Anna Samluk , Bartłomiej Noszczyk , Dorota Pijanowska , Krzysztof Pluta
• Languages of publication: EN

Abstracts

EN

The primary hepatocytes culture is still one of the main challenges in toxicology studies in the drug discovery process, development of in vitro models to study liver function, and cell-based therapies. Isolated hepatocytes display a rapid decline in viability and liver-specific functions including albumin production, conversion of ammonia to urea, and activity of the drug metabolizing enzymes. A number of methods have been developed in order to maintain hepatocytes in their highly differentiated state in vitro. Optimization of culture conditions includes a variety of media formulations and supplements, growth surface coating with the components of extracellular matrix or with synthetic polymers, three-dimensional growth scaffolds and decellularized tissues, and coculture with other cell types required for the normal cell-cell interactions. Here we propose a new substratum for hepatic cells made by drying confluent human skin fibroblasts' culture. This growth surface coating, prepared using maximally simplified procedure, combines the advantages of the use of extracellular matrices and growth factors/cytokines secreted by the feeder layer cells. In comparison to the hepatoma cells grown on a regular tissue culture plastic, cells cultured on the dried fibroblasts were able to synthesize albumin in larger quantities and to form greater number of apical vacuoles. Unlike the coculture with the living feeder layer cells, the number of cells grown on the new substratum was not reduced after fourteen days of culture. This fact could make the dried fibroblasts coating an ideal candidate for the substrate for non-dividing human hepatocytes.

Keywords

EN

cocultures; culture substratum; dried fibroblasts; human skin fibroblasts; C3A cells

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2017
• Volume: 64
• Issue: 2
• Pages: 357-363

Dates

published

2017

received

2016-11-23

revised

2017-03-15

accepted

2017-03-15

(unknown)

2017-06-09

Contributors

author

Agnieszka Wencel

• Department of Hybrid Microbiosystems Engineering, Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland

author

Karolina Zakrzewska

• Department of Hybrid Microbiosystems Engineering, Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland

author

Anna Samluk

• Department of Hybrid Microbiosystems Engineering, Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland

author

Bartłomiej Noszczyk

• Department of Plastic Surgery, Medical Centre of Postgraduate Education, Orlowski Memorial Hospital, Warsaw, Poland

author

Dorota Pijanowska

• Department of Hybrid Microbiosystems Engineering, Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland

author

Krzysztof Pluta

• Department of Hybrid Microbiosystems Engineering, Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland

References

• Baker TK, Carfagna MA, Gao H, Dow ER, Li Q, Searfoss GH et al., (2001) Temporal gene expression analysis of monolayer cultured rat hepatocytes. Chem Res Toxicol 14: 1218-1231.
• Bartolo L De, Morelli S, Lopez LC, Giorno L, Campana C, Salerno S, et al., (2005) Biotransformation and liver-specific functions of human hepatocytes in culture on RGD-immobilized plasma-processed membranes. Biomaterials 26: 4432-4441. doi: 10.1016/j.biomaterials.2004.11.009.
• Bhatia SN, Balis UJ, Yarmush ML, Toner M (1999) Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. FASEB J 13: 1883-1900.
• Bhatia SN, Underhill GH, Zaret KS, Fox IJ (2014) Cell and tissue engineering for liver disease. Sci Transl Med 6: 245sr2. doi: 10.1126/scitranslmed.3005975.
• Blachier M, Leleu H, Peck-Radosavljevic M, Valla D, Roudot-Thoraval F (2013) The burden of liver disease in Europe: A review of available epidemiological data. J Hepatol 58: 593-608. doi: 10.1016/j.jhep.2012.12.005.
• Cheng Y, Wang Y, Kang YZ, Hu PY, Gao Y, Pan MX (2013) In vitro culture of tumour-derived hepatocytes in decellularised whole-liver biological scaffolds. Digestion 87: 189-195. doi: 10.1159/000349949.
• Cho CH, Berthiaume F, Tilles AW, Yarmush ML (2008) A new technique for primary hepatocyte expansion in vitro. Biotechnol Bioeng 101: 345-356. doi: 10.1002/bit.21911.
• Evenou F, Hamon M, Fujii T, Takeuchi S, Sakai Y (2011) Gas-permeable membranes and co-culture with fibroblasts enable high-density hepatocyte culture as multilayered liver tissues. Biotechnol Prog 27: 1146-1153. doi: 10.1002/btpr.626.
• Faulk DM, Wildemann JD, Badylak SF (2015) Decellularization and cell seeding of whole liver biologic scaffolds composed of extracellular matrix. J Clin Exp Hepatol 5: 69-80. doi: 10.1016/j.jceh.2014.03.043.
• Forbes SJ, Gupta S, Dhawan A (2015) Cell therapy for liver disease: From liver transplantation to cell factory. J Hepatol 62 (Suppl 1): S157-S169. doi: 10.1016/j.jhep.2015.02.040.
• Gatmaitan Z, Jefferson DM, Ruiz-Opazo N, Biempica L, Arias IM, Dudas G et al., (1983) Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata. J Cell Biol 97: 1179-1190.
• Hui EE, Bhatia SN (2007) Micromechanical control of cell - cell interactions. Proc Natl Acad Sci USA 104: 5722–5726. doi: 10.1073/pnas.0608660104.
• Jeong D, Han C, Kang I, Park HT, Kim J et al., (2016) Effect of concentrated fibroblast - conditioned media on in vitro maintenance of rat primary hepatocyte. PLoS ONE 11: e0148846. doi: 10.1371/journal.pone.0148846.
• Kenneth D et al., Deaths: Final Data for 2014. National Vital Statistics Reports. 65(4) [cited 2016 Sep 22]. Available from: http://www.cdc.gov/nchs/data/nvsr/nvsr65/nvsr65_04.pdf
• Khetani SR, Szulgit G, Del Rio JA, Barlow C, Bhatia SN (2004) Exploring interactions between rat hepatocytes and nonparenchymal cells using gene expression profiling. Hepatology 40: 545-554. doi: 10.1002/hep.20351.
• Kono Y, Roberts EA (1996) Modulation of the expression of liver-specific functions in novel human hepatocyte lines cultured in a collagen gel sandwich configuration. Biochem Biophys Res Commun 220: 628-632. doi: 10.1006/bbrc.1996.0454.
• Lee DH, Lee KW (2014) Hepatocyte isolation, culture, and its clinical applications. Hanyang Med Rev 34: 165-172. doi: 10.7599/hmr.2014.34.4.165
• Nelson LJ, Navarro M, Treskes P, Samuel K, Tura-Ceide O, Morley SD, et al., (2015) Acetaminophen cytotoxicity is ameliorated in a human liver organotypic co-culture model. Sci Rep 5: 17455. doi: 10.1038/srep17455.
• OPTN Database. National Data. [cited 2016 Sep 22]. Available from:. http://optn.transplant.hrsa.gov/data/view-data-reports/national-data/
• Palakkan AA, Hay DC, Pr AK, Tv K, Ross JA (2013) Liver tissue engineering and cell sources: Issues and challenges. Liver Int 33: 666-676. doi: 10.1111/liv.12134.
• Panda S, Bisht S, Malakar D, Mohanty AK, Kaushik JK (2015) In vitro culture of functionally active buffalo hepatocytes Isolated by Using a Simplified Manual Perfusion Method. PLoS ONE 10: e0118841. doi: 10.1371/journal.pone.0118841.
• Rowe C, Gerrard DT, Jenkins R, Berry A, Durkin K, Sundstrom L, et al., (2013) Proteome-wide analyses of human hepatocytes during differentiation and dedifferentiation. Hepatology 58: 799-809. doi: 10.1002/hep.26414.
• Samluk A, Zakrzewska KE, Pluta KD (2013) Generation of fluorescently labeled cell lines, C3A hepatoma cells, and human adult skin fibroblasts to study coculture models. Artif Organs 37: E123-E130. doi: 10.1111/aor.12064.
• Sellaro TL, Ranade A, Faulk DM, Mccabe GP, et al., (2010) Maintenance of human hepatocyte function in vitro by liver-derived extracellular matrix gels. Tissue Eng Part A 16: 1075-1082. doi: 10.1089/ten.tea.2008.0587.
• Shoji R, Sakai Y, Sakoda A, Suzuki M (2000) Preservation of microplate-attached human hepatoma cells and their use in cytotoxicity tests. Cytotechnology 32: 147-155. doi: 10.1023/A:1008124228377.
• Soto-Gutierrez A, Zhang L, Medberry C, Fukumitsu K, Faulk D, Jiang H, et al., (2011) A whole-organ regenerative medicine approach for liver replacement. Tissue Eng Part C Methods 17: 677-686. doi: 10.1089/ten.TEC.2010.0698.
• Yang Y, Li J, Pan X, Zhou P, Yu X, Cao H, et al., (2013) Co-culture with mesenchymal stem cells enhances metabolic functions of liver cells in bioartificial liver system. Biotechnol Bioeng 110: 958-968. doi: 10.1002/bit.24752.

Identifiers

DOI

10.18388/abp.2016_1481