1. Establishment of large canine hepatocyte spheroids by mixing vascular endothelial cells and canine adipose-derived mesenchymal stem cells
- Author
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Akito Ichikawa, Sakurako Neo, Ryouhei Nukui, Yoko Yamada, Suguru Nitta, Hidetoshi Iwaki, Yusuke Yanagi, Koichi Nakayama, Shoichi Sato, Satoko Tateishi, and Masaharu Hisasue
- Subjects
hFGF, human fibroblast growth factor ,Medicine (General) ,Three-dimensions ,TAT, tyrosine aminotransferase ,CD, cluster of differentiation ,Biomedical Engineering ,PBS, phosphate-buffered saline ,CDH1, cadherin-1 (epithelital-cadherin) ,Canine ,Biomaterials ,SF-HGM, hepatocyte growth medium for spheroid formation ,iPSC, induced pluripotent stem cells ,R5-920 ,CYP, cytochrome ,FBS, fetal bovine serum ,α1-AT, α1-antitrypsin ,HUVEC, human umbilical vein endothelial cells ,rf-HGF, recombinant ferine hepatocyte growth factor ,Hepatocyte ,DILI, drug induced liver injury ,ALB, albumin ,3D, three-dimensions ,HTM, hepatocyte thawing medium ,HGM, hepatocyte growth medium ,QH573-671 ,AFP, α-fetoprotein ,EGM, endothelial cell growth medium ,LcHS, large canine hepatocyte spheroids ,cASC, canine adipose-derived mesenchymal stem cells ,MSCGM, mesenchymal stem cell growth medium ,embryonic structures ,Mesenchymal stem cells ,Original Article ,Spheroids ,Cytology ,cHep, canine primary-cultured hepatocytes ,Developmental Biology - Abstract
Introduction Differentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs). Methods Spheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes (ALB, AFP, α1-AT, CDH1, CYP2E1, CYP3A12, and TAT) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes. Results The ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P, Graphical abstract Image 1, Highlights • cHep spheroids was co-cultured using canine hepatocytes, mesenchymal stem cells, and human umbilical vein endothelial cells. • The ratio of spheroid formation was improved and expression of liver related genes and protein was maintained. • The three-mix culture could promote large spheroids without impairing liver function in canine hepatocyte.
- Published
- 2021