Guan Shuwen, Gao Botao, Xiao Jiangwei
Biomedical Materials Research Office, Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou 510000, P. R. China.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2022 Aug 25;39(4):776-783. doi: 10.7507/1001-5515.202108056.
Primary human hepatocytes (PHH) are the gold standard of human liver model for drug screening. However, a problem of culturing PHH is the rapid decline of cytochrome P450 (CYP450) activity, which plays an important role in drug metabolism. In this study, thermo-responsive culture dishes were used to explore the conditions for murine embryonic 3T3-J2 fibroblasts to form cell sheet. Based on the cell sheet engineering technology, a three-dimensional (3D) "sandwich" co-culture system of 3T3-J2 cell sheet/PHH/collagen gel was constructed. The tissue structure and protein expression of the model section were observed by hematoxylin eosin staining and immunofluorescence staining respectively. Phenacetin and bupropion were used as substrates to determine the activity of CYP450. The contents of albumin and urea in the system were determined by enzyme linked immunosorbent assay (ELISA). The results showed that the complete 3T3-J2 cell sheet could be obtained when the cell seeding density was 1.5×10 /dish (35 mm dish) and the incubation time at low temperature was 60 min. Through cell sheet stacking, a 3D liver model was developed. Compared with the two-dimensional (2D) model, in the 3D model, the cell-cell and cell-matrix connections were tighter, the activities of cytochrome P450 CYP1A2 and cytochrome P450 CYP2B6 were significantly increased, and the secretion levels of albumin and urea were increased. These indexes could be maintained stably for 21 d. Therefore, cell sheet stacking is helpful to improve the level of liver function of 3D liver model. This model is expected to be used to predict the metabolism of low-clearance drugs in preclinical, which is of great significance for drug evaluation and other studies.
原代人肝细胞（PHH）是药物筛选的人肝模型的金标准。然而，培养PHH的一个问题是细胞色素P450（CYP450）活性迅速下降，而CYP450在药物代谢中起重要作用。在本研究中，使用热响应培养皿探索小鼠胚胎3T3-J2成纤维细胞形成细胞片的条件。基于细胞片工程技术，构建了3T3-J2细胞片/PHH/胶原凝胶的三维（3D）“三明治”共培养系统。分别通过苏木精伊红染色和免疫荧光染色观察模型切片的组织结构和蛋白表达。以非那西丁和安非他酮为底物测定CYP450的活性。采用酶联免疫吸附测定（ELISA）法测定系统中白蛋白和尿素的含量。结果表明，当细胞接种密度为1.5×10⁵/皿（35 mm培养皿）且低温孵育时间为60 min时，可获得完整的3T3-J2细胞片。通过细胞片堆叠，构建了一种3D肝脏模型。与二维（2D）模型相比，在3D模型中，细胞-细胞和细胞-基质连接更紧密，细胞色素P450 CYP1A2和细胞色素P450 CYP2B6的活性显著增加，白蛋白和尿素的分泌水平升高。这些指标可稳定维持21 d。因此，细胞片堆叠有助于提高3D肝脏模型的肝功能水平。该模型有望用于临床前预测低清除率药物的代谢，对药物评价等研究具有重要意义。
