title: Constrained spheroids for prolonged hepatocyte culture.
abstract: Liver-specific functions in primary hepatocytes can be maintained over extended duration in vitro using spheroid culture. However, the undesired loss of cells over time is still a major unaddressed problem, which consequently generates large variations in downstream assays such as drug screening. In static culture, the turbulence generated by medium change can cause spheroids to detach from the culture substrate. Under perfusion, the momentum generated by Stokes force similarly results in spheroid detachment. To overcome this problem, we developed a Constrained Spheroids (CS) culture system that immobilizes spheroids between a glass coverslip and an ultra-thin porous Parylene C membrane, both surface-modified with poly(ethylene glycol) and galactose ligands for optimum spheroid formation and maintenance. In this configuration, cell loss was minimized even when perfusion was introduced. When compared to the standard collagen sandwich model, hepatocytes cultured as CS under perfusion exhibited significantly enhanced hepatocyte functions such as urea secretion, and CYP1A1 and CYP3A2 metabolic activity. We propose the use of the CS culture as an improved culture platform to current hepatocyte spheroid-based culture systems.
authors.affiliation: Institute of Biotechnology and Nanotechnology, A*STAR, The Nanos, #04-01, 31 Biopolis Way, Singapore 138669, Singapore; NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences (CeLS), #05-01, 28 Medical Drive, Singapore 117456, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, MD9-03-03, 2 Medical Drive, Singapore 117597, Singapore.
authors.forename: Wen Hao
authors.initials: WH
authors.lastname: Tong
authors.affiliation: Institute of Biotechnology and Nanotechnology, A*STAR, The Nanos, #04-01, 31 Biopolis Way, Singapore 138669, Singapore; NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences (CeLS), #05-01, 28 Medical Drive, Singapore 117456, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, MD9-03-03, 2 Medical Drive, Singapore 117597, Singapore.
authors.forename: Yu
authors.initials: Y
authors.lastname: Fang
authors.affiliation: Institute of Biotechnology and Nanotechnology, A*STAR, The Nanos, #04-01, 31 Biopolis Way, Singapore 138669, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, MD9-03-03, 2 Medical Drive, Singapore 117597, Singapore; Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #10-01 CREATE Tower, Singapore 138602, Singapore.
authors.forename: Jie
authors.initials: J
authors.lastname: Yan
authors.affiliation: Department of Physiology, Yong Loo Lin School of Medicine, MD9-03-03, 2 Medical Drive, Singapore 117597, Singapore.
authors.forename: Xin
authors.initials: X
authors.lastname: Hong
authors.affiliation: Institute of Biotechnology and Nanotechnology, A*STAR, The Nanos, #04-01, 31 Biopolis Way, Singapore 138669, Singapore.
authors.forename: Nisha
authors.initials: N
authors.lastname: Hari Singh
authors.affiliation: Bruker Singapore Pte Ltd., 11 Biopolis Way #10-10, The Helios, Singapore 138667, Singapore.
authors.forename: Shu Rui
authors.initials: SR
authors.lastname: Wang
authors.affiliation: Institute of Biotechnology and Nanotechnology, A*STAR, The Nanos, #04-01, 31 Biopolis Way, Singapore 138669, Singapore; NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences (CeLS), #05-01, 28 Medical Drive, Singapore 117456, Singapore; ETH Zürich, Department of Biosystem Science and Engineering (D-BSSE), Mattenstrasse 26, CH-4058 Basel, Switzerland; Roche Pharmaceutical Research and Early Development, Department of Discovery Technology, Roche Innovation Center Basel, Grenzacherstrasse 124, CH-4070 Basel, Switzerland.
authors.forename: Bramasta
authors.initials: B
authors.lastname: Nugraha
authors.affiliation: Department of Physiology, Yong Loo Lin School of Medicine, MD9-03-03, 2 Medical Drive, Singapore 117597, Singapore.
authors.forename: Lei
authors.initials: L
authors.lastname: Xia
authors.affiliation: Department of Physiology, Yong Loo Lin School of Medicine, MD9-03-03, 2 Medical Drive, Singapore 117597, Singapore.
authors.forename: Eliza Li Shan
authors.initials: ELS
authors.lastname: Fong
authors.affiliation: Institute of Biotechnology and Nanotechnology, A*STAR, The Nanos, #04-01, 31 Biopolis Way, Singapore 138669, Singapore. Electronic address: ciliescu@ibn.a-star.edu.sg.
authors.forename: Ciprian
authors.initials: C
authors.lastname: Iliescu
authors.affiliation: Institute of Biotechnology and Nanotechnology, A*STAR, The Nanos, #04-01, 31 Biopolis Way, Singapore 138669, Singapore; NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences (CeLS), #05-01, 28 Medical Drive, Singapore 117456, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, MD9-03-03, 2 Medical Drive, Singapore 117597, Singapore; Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #10-01 CREATE Tower, Singapore 138602, Singapore; Mechanobiology Institute, National University of Singapore, T-Lab, #05-01, 5A Engineering Drive 1, Singapore 117411, Singapore; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: hanry_yu@nuhs.edu.sg.
authors.forename: Hanry
authors.initials: H
authors.lastname: Yu
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation*; D016764:Cell Polarity; D002470:Cell Survival; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation; D004867:Equipment Design; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D008297:Male; D008567:Membranes, Artificial; D010477:Perfusion / Q000295:instrumentation; D011108:Polymers / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D018874:Spheroids, Cellular / Q000166:cytology* / Q000378:metabolism; D014992:Xylenes / Q000737:chemistry
chemical_list: D008567:Membranes, Artificial; D011108:Polymers; D014992:Xylenes; C011055:parylene; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2
keywords: Drug testing; Hepatocytes; Liver; Perfusion; Spheroids