Intensive promotion of spheroid formation by soluble factors in a hepatocyte-conditioned medium.
We developed a hybrid artificial liver and a drug metabolism simulator using polyurethane foam (PUF) in which primary hepatocytes spontaneously form functional spheroids. Gel filtration liquid chromatography analysis of a hepatocyte-conditioned medium during spheroid formation showed that some substances secreted by primary rat hepatocytes accumulated advantageously inside the pores of PUF compared with outside. Similar substances were detected in a hepatocyte-conditioned medium from a positively-charged surface by concentrating the substances using an ultrafiltration membrane of a molecular weight-cutoff of 50 kD. These substances were shown to act as soluble factors on freshly isolated primary rat hepatocytes to promote spontaneous and rapid spheroid formation, depending on their concentration by preventing them from initially attaching and spreading on a positively-charged surface. In particular, using 50-fold concentrated substances, about 80% of total hepatocytes formed the floating spheroids within 72 h of culture. The resulting spheroids had a diameter distribution mainly ranging from 40 to 70 microm and expressed high-level liver-specific functions compared with a conventional monolayer.
Fujii
Y
Y
Department of Chemical Systems and Engineering, Graduate School of Engineering, Kyushu University, Fukuoka, Japan.
Nakazawa
K
K
Funatsu
K
K
D000641:Ammonia / Q000032:analysis; D000818:Animals; D001672:Biocompatible Materials; D018929:Cell Culture Techniques / Q000379:methods; D002467:Cell Nucleus / Q000648:ultrastructure; D048429:Cell Size; D002478:Cells, Cultured; D002850:Chromatography, Gel; D017077:Culture Media, Conditioned / Q000032:analysis*; D022781:Hepatocytes / Q000166:cytology* / Q000502:physiology*; D019164:Liver, Artificial; D008297:Male; D011140:Polyurethanes; D051381:Rats; D017208:Rats, Wistar; D012709:Serum Albumin / Q000032:analysis; D014462:Ultrafiltration
D001672:Biocompatible Materials; D017077:Culture Media, Conditioned; D011140:Polyurethanes; D012709:Serum Albumin; D000641:Ammonia