Mohajerani S A, Nourbakhsh M, Cadili A, Lakey J R, Kneteman N M
Department of Surgery, University of Alberta , Edmonton, Alberta , Canada.
† Division of Surgical Research, Department of Surgery, University of California , Irvine, CA , USA.
Cell Med. 2010 Nov 5;1(2):81-92. doi: 10.3727/215517910X536627. eCollection 2010.
Hepatocytes are vulnerable to loss of function and viability in culture. Modified culture methods have been applied to maintain their functional status. Heterotypic interactions between hepatocytes and nonparenchymal neighbors in liver milieu are thought to modulate cell differentiation. Cocultivation of hepatocyte with various cell types has been applied to mimic the hepatic environment. Bone marrow stromal cells (BMSC) are plastic cell lines capable of transforming to other cell types. In this study hepatocyte coculture with BMSCs achieved long-term function of human hepatocytes in culture for 4 weeks. In vitro functional status of human hepatocytes in BMSC coculture was compared with fibroblast coculture and collagen culture by measuring albumin, human-α-1-antitrypsin (hAAT), urea secretion, CYP450 activity, and staining for intracellular albumin and glycogen. After 2 weeks in culture hepatocytes were retrieved and transplanted to severe combined immunodeficiency/albumin linked-urokinase type plasminogen activator (SCID Alb-uPA) mice and engraft-ment capacity was analyzed by human hepatic-specific function measured by hAAT levels in mouse serum, and Alu staining of mouse liver for human hepatocytes. Hepatocytes from BMSC coculture had significantly higher albumin, hAAT secretion, urea production, and cytochrome P450 (CYP450) activity than other culture groups. Staining confirmed the higher functional status in BMSC coculture. Transplantation of hepatocytes detached from BMSC cocultures showed significantly higher engraftment function than hepatocytes from other culture groups measured by hAAT levels in mouse serum. In conclusion, BMSC coculture has excellent potential for hepatocyte function preservation in vitro and in vivo after transplant. It is possible to use BMSC hepatocyte coculture as a supply of cell therapy in liver disease.
肝细胞在培养过程中容易出现功能丧失和活力下降的情况。人们已应用改良的培养方法来维持其功能状态。肝脏微环境中肝细胞与非实质细胞邻域之间的异型相互作用被认为可调节细胞分化。肝细胞与各种细胞类型共培养已被用于模拟肝脏环境。骨髓基质细胞（BMSC）是能够转变为其他细胞类型的可塑性细胞系。在本研究中，肝细胞与BMSC共培养实现了人肝细胞在培养4周后的长期功能。通过测量白蛋白、人α-1-抗胰蛋白酶（hAAT）、尿素分泌、CYP450活性以及细胞内白蛋白和糖原染色，将BMSC共培养体系中人肝细胞的体外功能状态与成纤维细胞共培养体系和胶原蛋白培养体系进行了比较。培养2周后，取出肝细胞并移植到严重联合免疫缺陷/白蛋白连接尿激酶型纤溶酶原激活剂（SCID Alb-uPA）小鼠体内，通过测量小鼠血清中hAAT水平所反映的人肝脏特异性功能以及对小鼠肝脏进行人肝细胞的Alu染色来分析植入能力。与其他培养组相比，BMSC共培养体系中的肝细胞具有显著更高的白蛋白、hAAT分泌、尿素生成和细胞色素P450（CYP450）活性。染色证实了BMSC共培养体系中肝细胞具有更高的功能状态。通过测量小鼠血清中hAAT水平发现，从BMSC共培养体系中分离的肝细胞移植后的植入功能明显高于其他培养组的肝细胞。总之，BMSC共培养在体外保存肝细胞功能以及移植后在体内保存肝细胞功能方面具有出色的潜力。将BMSC与肝细胞共培养有可能作为肝病细胞治疗的一种细胞来源。
