日本語フィールド
著者：*Akito Ichikawa, Sakurako Neo, Ryouhei Nukui, Yoko Yamada, Suguru Nitta, Hidetoshi Iwaki, Yusuke Yanagi, Koichi Nakayama, Shoichi Sato, Satoko Tateishi, Masaharu Hisasue題名：Establishment of large canine hepatocyte spheroids by mixing vascular endothelial cells and canine adipose-derived mesenchymal stem cells発表情報：Regen Ther 巻: 19 ページ: 1-8キーワード：3D, three-dimensions; AFP, α-fetoprotein; ALB, albumin; CD, cluster of differentiation; CDH1, cadherin-1 (epithelital-cadherin); CYP, cytochrome; Canine; DILI, drug induced liver injury; EGM, endothelial cell growth medium; FBS, fetal bovine serum; HGM, hepatocyte growth medium; HTM, hepatocyte thawing medium; HUVEC, human umbilical vein endothelial cells; Hepatocyte; LcHS, large canine hepatocyte spheroids; MSCGM, mesenchymal stem cell growth medium; Mesenchymal stem cells; PBS, phosphate-buffered saline; SF-HGM, hepatocyte growth medium for spheroid formation; Spheroids; TAT, tyrosine aminotransferase; Three-dimensions; cASC, canine adipose-derived mesenchymal stem cells; cHep, canine primary-cultured hepatocytes; hFGF, human fibroblast growth factor; iPSC, induced pluripotent stem cells; rf-HGF, recombinant ferine hepatocyte growth factor; α1-AT, α1-antitrypsin. 概要：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 < 0.001). Histological evaluation revealed that the three-mix spheroids formed large canine hepatocyte spheroids (LcHS), and hepatocytes were distributed in the center of the spheroids. Quantitative gene expression analysis of LcHS showed that liver-related genes expression were maintained the same levels with that of a culture of cHep alone from days 4-7.
Conclusion: These results revealed that the three-mix culture method using cHep, HUVECs, and cASCs was capable of promoting LcHS without impairing liver function in cHep, suggesting that LcHS could be used for the application of high-volume culture techniques in dogs.抄録：英語フィールド
Author：*Akito Ichikawa, Sakurako Neo, Ryouhei Nukui, Yoko Yamada, Suguru Nitta, Hidetoshi Iwaki, Yusuke Yanagi, Koichi Nakayama, Shoichi Sato, Satoko Tateishi, Masaharu HisasueTitle：Establishment of large canine hepatocyte spheroids by mixing vascular endothelial cells and canine adipose-derived mesenchymal stem cellsAnnouncement information:Regen Ther Vol: 19 Page: 1-8Keyword：3D, three-dimensions; AFP, α-fetoprotein; ALB, albumin; CD, cluster of differentiation; CDH1, cadherin-1 (epithelital-cadherin); CYP, cytochrome; Canine; DILI, drug induced liver injury; EGM, endothelial cell growth medium; FBS, fetal bovine serum; HGM, hepatocyte growth medium; HTM, hepatocyte thawing medium; HUVEC, human umbilical vein endothelial cells; Hepatocyte; LcHS, large canine hepatocyte spheroids; MSCGM, mesenchymal stem cell growth medium; Mesenchymal stem cells; PBS, phosphate-buffered saline; SF-HGM, hepatocyte growth medium for spheroid formation; Spheroids; TAT, tyrosine aminotransferase; Three-dimensions; cASC, canine adipose-derived mesenchymal stem cells; cHep, canine primary-cultured hepatocytes; hFGF, human fibroblast growth factor; iPSC, induced pluripotent stem cells; rf-HGF, recombinant ferine hepatocyte growth factor; α1-AT, α1-antitrypsin. An 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 < 0.001). Histological evaluation revealed that the three-mix spheroids formed large canine hepatocyte spheroids (LcHS), and hepatocytes were distributed in the center of the spheroids. Quantitative gene expression analysis of LcHS showed that liver-related genes expression were maintained the same levels with that of a culture of cHep alone from days 4-7.
Conclusion: These results revealed that the three-mix culture method using cHep, HUVECs, and cASCs was capable of promoting LcHS without impairing liver function in cHep, suggesting that LcHS could be used for the application of high-volume culture techniques in dogs.