HEPATOLOGY SERIES - ISOLATED HEPATOCYTES - USE IN EXPERIMENTAL AND CLINICAL HEPATOLOGY
HEPATOLOGY SERIES - ISOLATED HEPATOCYTES - USE IN EXPERIMENTAL AND CLINICAL HEPATOLOGY
复制标题
DOI：
10.1136/gut.35.4.433
复制
发表时间：
1994-04-01
期刊：
影响因子：
24.5
通讯作者：
STRAIN, AJ
中科院分区：
文献类型：
--
作者：
STRAIN, AJ
自从Howard和Pesch首次使用胶原酶分解成人肝组织，并成功分离出具有活性和功能性的肝细胞以来，已经有25年多的时间了。在此之前，人们使用了许多机械和酶方法来破坏肝组织，仅取得了有限的成功。替代方法，如使用组织切片或外植体器官培养也不令人满意，仅有有限的用途。然后，Berry和Friend在描述第一个肝脏灌流程序时介绍了一个主要的发展。2这大大提高了肝细胞的活力，产量估计高达原始组织的50%。尽管许多研究人员后来报告了修改，但在一系列细致的研究中，塞格伦在很大程度上被认为是大幅改进了这项技术并建立了所谓的两步胶原酶灌注技术，这已经成为成千上万研究人员的“标准”方案。这项技术包括用二价无阳离子缓冲液灌流肝脏，有效地松开相邻细胞和细胞外基质之间的桥粒和半桥粒连接。然后用胶原酶对基质进行蛋白质分解，并仔细保持温度、pH和氧合。这一过程导致实质组织完全解体，而不会发生机械破坏，这对肝细胞的生存是非常不利的。然后，可以移除包膜，将肝细胞简单地摇出到培养液中，产生具有始终如一的高活力和纯度的细胞。这篇评论的目的不是回顾方法学，因为这是在一本优秀的综合性专著中实现的，4而是讨论有趣的最新发展及其对实验和临床肝病的影响。这些进展现在使研究人员能够从包括人类在内的许多物种中分离肝细胞，从小的活组织切片中分离细胞，并在体外将它们在各种条件下长期保存，而不会失去分化的表型。这些改进为广泛的肝脏生长、代谢和毒理学的体外研究以及与临床肝病相关的令人兴奋的新研究开辟了道路，例如人工肝支持、肝细胞移植和基因治疗。所引用的已发表报告并不是一个完全全面的清单，在适当的情况下，包括了最近的审查。
It is now over 25 years since Howard and Pesch first used collagenase to disaggregate adult liver tissue, with the resultant successful isolation of viable and functional hepa-tocytes.'Before that, numerous mechanical and enzymatic ways of disrupting liver tissue had been used, with a limited degree ofsuccess only. Alternatives such as the use of tissue slices or explant organ cultures were also unsatisfactory and of only limited use. Then Berry and Friend introduced a major development when they described the first liver perfusion procedure. 2 This led to a considerably improved viability of hepatocytes and a yield that was estimated to comprise up to 50% of the original tissue. Although many investigators subsequently reported modi-fications, it is Seglen, in a series of meticulous studies, who is largely credited with substantially refining the technique and establishing theso called two step collagenase perfusion technique3 which has become the'standard'protocol for many thousands of investigators. This technique involves perfusing the liver with a divalent cation free buffer, effectively loosening the desmosome and hemi-desmosome junctions between the adjacent cells and extracellular matrix. This is followed by proteolytic digestion of the matrix with collagenase, and careful maintenance of the temperature, pH, and oxygenation. This procedure results in complete disaggregation ofthe parenchymal tissue without the mechanical disruption which is so detrimental to the viability of hepatocytes. The capsule can then be removed and the hepatocytes simply shaken out into the medium, yielding cells with consistently high viability and purity. This commentary does not aim to review the methodology, since this has been achieved in an excellent comprehensive monograph, 4 but to discuss interesting recent developments and their impact on experimental and clinical hepatology. These developments now enable investigators to isolate hepatocytes from numerous species, including man, to isolate cells from small biopsy sized fragments of liver, and to maintain them under various conditions in vitro for pro-longed periods without loss of differentiated phenotype. These improvements have opened the way for extensive in vitro studies of liver growth, metabolism, and toxicology and exciting novel investigations relating to clinical liver disease, for example artificial liver support, hepatocyte transplanta-tion, and gene therapy. The published reports cited are not intended to be a fully comprehensive list, and where appro-priate, recent reviews are included.