Collagen gel immobilization: a useful cell culture technique for long-term metabolic studies on human hepatocytes.
Collagen gel immobilization: a useful cell culture technique for long-term metabolic studies on human hepatocytes.
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DOI：
10.3109/00498259409043224
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发表时间：
1994-02
期刊：
影响因子：
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通讯作者：
H. Koebe;S. Pahernik;P. Eyer;F. Schildberg
中科院分区：
文献类型：
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作者：
H. Koebe;S. Pahernik;P. Eyer;F. Schildberg
1.人肝细胞原代培养已被广泛应用于异种代谢的研究。这些方法中的大多数都是在新鲜分离的细胞或短期原代培养上进行的。标准培养技术在体外1周内不能维持P450酶的功能稳定性。2.本研究的目的是证明一种易于应用的细胞外基质构型对培养的人肝细胞的长期性能的有利影响。光镜检查表明，培养的细胞在1个月以上仍能存活。电子显微镜下，肝细胞内可见胆小管和桥粒，线粒体和内质网丰富，提示代谢活动。3.培养早期(分离后3天)，培养液DNA含量下降，丙氨酸氨基转移酶(ALAT)峰值升高，白蛋白合成增加。在这个适应期过后，DNA含量和白蛋白合成水平保持稳定；ALAT恢复到低值。4.通过测量依赖于P450 1A1的对硝基苯甲醚O-脱甲基化为对硝基苯酚来监测功能活性，这一过程在3周内似乎是恒定的，1 mM苯巴比妥诱导作用较弱。另一种装置研究了对乙酰氨基酚在亚毒性浓度下的偶联反应：对乙酰氨基酚被代谢成其葡萄糖醛酸苷和硫酸盐；未检测到3-(谷胱甘肽-S基)-对乙酰氨基酚。比较培养的第3天和第16天，发现代谢几乎相同。5.胶原蛋白凝胶固定化不仅为培养的肝细胞提供了机械支持，而且为代谢研究提供了细胞的长期分化功能。
1. Primary cultures of human hepatocytes have already been employed in various applications for the study of xenobiotic metabolism. Most of these approaches were performed either on freshly isolated cells or on short-term primary cultures. Standard culture techniques do not maintain functional stability of P450 enzymes for > 1 week in vitro. 2. The aim of this study was to demonstrate the beneficial effect of an easy to apply, extracellular matrix configuration on the long-term performance of cultured human liver cells. Light microscopical examination of the cultures indicated that the cells remained viable over 1 month. As revealed by electron microscopy, hepatocytes exhibited bile canaliculi and desmosomes and were rich in mitochondria and endoplasmatic reticulum, indicating metabolic activity. 3. An early culture phase (3 days after isolation) could be described with decreasing DNA content of the cultures, peak values of alanine-amino-transferase (ALAT), and increasing albumin synthesis. After this adaptive period stable levels for DNA content and albumin synthesis were noted; ALAT returned to low values. 4. Functional activity was monitored by measurements of P450 1A1-dependent O-demethylation of p-nitroanisole to p-nitrophenol, which appeared to be constant over 3 weeks and weakly inducible by 1 mM phenobarbital. Another set-up examined conjugation of acetaminophen at subtoxic concentrations: acetaminophen was metabolized to its glucuronide and sulphate; 3-(glutathione-S-yl)-acetaminophen was not detected. Almost identical metabolism was found, comparing day 3 with 16 of culture. 5. We concluded that collagen gel immobilization not only provides mechanical support to cultured hepatocytes, but also supports long-term differentiated function of the cells for metabolic studies.