源自人脂肪组织间充质基质细胞的条件培养基可改善原代肝细胞的维持。
Conditioned Media Derived from Human Adipose Tissue Mesenchymal Stromal Cells Improves Primary Hepatocyte Maintenance.
作者信息
Azhdari Tafti Zahra, Mahmoodi Mehdi, Hajizadeh Mohamad Reza, Ezzatizadeh Vahid, Baharvand Hossein, Vosough Massoud, Piryaei Abbas
机构信息
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Department of Clinical Biochemistry, School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
出版信息
Cell J. 2018 Oct;20(3):377-387. doi: 10.22074/cellj.2018.5288. Epub 2018 May 15.
OBJECTIVE
Recent advances in cell therapy have encouraged researchers to provide an alternative for treatment and restoration of damaged liver through using hepatocytes. However, these cells quickly lose their functional capabilities in vitro. Here, we aim to use the secretome of mesenchymal stromal cells (MSCs) to improve in vitro maintenance conditions for hepatocytes.
MATERIALS AND METHODS
In this experimental study, following serum deprivation, human adipose tissue-derived MSCs (hAT-MSCs) were cultured for 24 hours under normoxic (N) and hypoxic (H) conditions. Their conditioned media (CM) were subsequently collected and labeled as N-CM (normoxia) and H-CM (hypoxia). Murine hepatocytes were isolated by perfusion of mouse liver with collagenase, and were cultured in hepatocyte basal (William's) medium supplemented with 4% N-CM or H-CM. Untreated William's and hepatocyte-specific media (HepZYM) were used as controls. Finally, we evaluated the survival and proliferation rates, as well as functionality and hepatocyte-specific gene expressions of the cells.
RESULTS
We observed a significant increase in viability of hepatocytes in the presence of N-CM and H-CM compared to HepZYM on day 5, as indicated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) assay. Indocyanine green (ICG) uptake of hepatocytes in the H-CM and HepZYM groups on days 3 and 5 also suggested that H-CM maintained the hepatocytes at about the same level as the hepatocyte-specific medium. The HepZYM group had significantly higher levels of albumin (Alb) and urea secretion compared to the other groups (P<0.0001). However, there were no significant differences in cytochrome activity and cytochrome gene expression profiles among these groups. Finally, we found a slightly, but not significantly higher concentration of vascular endothelial growth factor (VEGF) in the H-CM group compared to the N-CM group (P=0.063).
CONCLUSION
The enrichment of William's basal medium with 4% hAT-MSC-H-CM improved some physiologic parameters in a primary hepatocyte culture.
目的
细胞治疗的最新进展促使研究人员尝试通过使用肝细胞为受损肝脏的治疗和修复提供一种替代方法。然而，这些细胞在体外会迅速丧失其功能。在此，我们旨在利用间充质基质细胞（MSC）的分泌组来改善肝细胞的体外培养条件。
材料与方法
在本实验研究中，血清饥饿处理后，将人脂肪组织来源的间充质干细胞（hAT-MSC）在常氧（N）和低氧（H）条件下培养24小时。随后收集其条件培养基（CM），分别标记为N-CM（常氧）和H-CM（低氧）。通过用胶原酶灌注小鼠肝脏分离小鼠肝细胞，并在补充有4% N-CM或H-CM的肝细胞基础培养基（威廉姆斯培养基）中培养。未处理的威廉姆斯培养基和肝细胞特异性培养基（HepZYM）用作对照。最后，我们评估了细胞的存活率、增殖率以及功能和肝细胞特异性基因表达。
结果
MTS（3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲氧基苯基)-2-(4-磺基苯基)-2H-四唑）检测表明，与HepZYM相比，在第5天，N-CM和H-CM存在时肝细胞活力显著增加。第3天和第5天，H-CM组和HepZYM组肝细胞对吲哚菁绿（ICG）的摄取也表明，H-CM维持肝细胞的水平与肝细胞特异性培养基大致相同。与其他组相比，HepZYM组白蛋白（Alb）和尿素分泌水平显著更高（P<0.0001）。然而，这些组之间细胞色素活性和细胞色素基因表达谱没有显著差异。最后，我们发现H-CM组血管内皮生长因子（VEGF）浓度略高于N-CM组，但差异不显著（P=0.063）。
结论
用4% hAT-MSC-H-CM富集威廉姆斯基础培养基可改善原代肝细胞培养中的一些生理参数。
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