Long term maintenance of cytochrome P450 activity in a cell sheet-based three-dimensional human hepatic model_Journal of Biomedical Engineering

Authors：

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;

Corresponding author：

GAO Botao, Email: gaobotao1984@outlook.com

Keywords：

Primary human hepatocyte; Co-culture; Fibroblast; Cell sheet; Cytochrome P450

DOI：

10.7507/1001-5515.202108056

Video：

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Primary human hepatocytes (PHH) are the gold standard of in vitro human liver model for drug screening. However, a problem of culturing PHH in vitro 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 1.5×10⁶ /dish (35 mm dish) and the incubation time at low temperature was 60 min. Through cell sheet stacking, a 3D in vitro 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.

Citation： GUAN Shuwen, GAO Botao, XIAO Jiangwei. Long term maintenance of cytochrome P450 activity in a cell sheet-based three-dimensional human hepatic model. Journal of Biomedical Engineering, 2022, 39(4): 776-783. doi: 10.7507/1001-5515.202108056 Copy

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2.	Obach R S. Predicting clearance in humans from in vitro data. Curr Top Med Chem, 2011, 11(4): 334-339.
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4.	Smith C M, Nolan C K, Edwards M A, et al. A comprehensive evaluation of metabolic activity and intrinsic clearance in suspensions and monolayer cultures of cryopreserved primary human hepatocytes. J Pharm Sci, 2012, 101(10): 3989-4002.
5.	孙英辉, 慈小燕, 李薇, 等. 应用人肝细胞研究人参皂苷Rb_1和Rg_1、丹参素钠、冰片对CYP450药物代谢酶1A2、2B6和3A4的体外诱导作用. 药物评价研究, 2021, 44(1): 38-44.
6.	董航, 张海晖, 窦桂芳, 等. γ射线辐射对大鼠肝药物代谢酶CYP1A2,CYP2C9和CYP2D6蛋白表达及药物代谢活性的影响. 中国药理学与毒理学杂志, 2021, 35(4): 290-296.
7.	徐聪, 徐思云, 胡海红, 等. CYP2B6体外诱导活性评价模型的建立及其在中药活性成分筛选的应用. 药学学报, 2013, 48(1): 119-124.
8.	Di L, Obach R S. Addressing the challenges of low clearance in drug research. AAPS J, 2015, 17(2): 352-357.
9.	阮婷婷, 鞠武建, 熊海伟, 等. 低清除率药物的代谢稳定性预测模型研究进展. 中国药科大学学报, 2019, 50(2): 152-160.
10.	Khetani S R, Szulgit G, Del Rio J A, et al. Exploring interactions between rat hepatocytes and nonparenchymal cells using gene expression profiling. Hepatology, 2004, 40(3): 545-554.
11.	Gu J, Shi X, Zhang Y, et al. Establishment of a three-dimensional co-culture system by porcine hepatocytes and bone marrow mesenchymal stem cells in vitro. Hepatol Res, 2009, 39(4): 398-407.
12.	Thomas R J, Bhandari R, Barrett D A, et al. The effect of three-dimensional co-culture of hepatocytes and hepatic stellate cells on key hepatocyte functions in vitro. Cells Tissues Organs, 2005, 181(2): 67-79.
13.	Haraguchi Y, Shimizu T, Yamato M, et al. Scaffold-free tissue engineering using cell sheet technology. Chem Inform, 2012, 2(6): 2184-2190.
14.	Kobayashi J, Kikuchi A, Aoyagi T, et al. Cell sheet tissue engineering: cell sheet preparation, harvesting/manipulation, and transplantation. J Biomed Mater Res A, 2019, 107(5): 955-967.
15.	Yang J, Yamato M, Kohno C, et al. Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials, 2005, 26(33): 6415-6422.
16.	Yang J, Yamato M, Shimizu T, et al. Reconstruction of functional tissues with cell sheet engineering. Biomaterials, 2007, 28(34): 5033-5043.
17.	Kim K, Ohashi Kazuo, Utoh R, et al. Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets. Biomaterials, 2012, 33(5): 1406-1413.
18.	Gao B, Sakaguchi K, Ogawa T, et al. Functional analysis of induced human ballooned hepatocytes in a cell sheet-based three dimensional mode. Tissue Eng Regen Med, 2021, 18(2): 217-224.
19.	Hynds R E, Gowers K, Nigro E, et al. Cross-talk between human airway epithelial cells and 3T3-J2 feeder cells involves partial activation of human MET by murine HGF. PLoS One, 2018, 13(5): e0197129.
20.	Barreca A, de Luca M, Del Monte P, et al. In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors. J Cell Physiol, 1992, 151(2): 262-268.
21.	Ruoß M, Kieber V, Rebholz S, et al. Cell-type-specific quantification of a scaffold-based 3D liver co-culture. Methods Protoc, 2019, 3(1): 1-19.
22.	Ware B R, Liu J S, Monckton C P, et al. Micropatterned coculture with 3T3-J2 fibroblasts enhances hepatic functions and drug screening utility of HepaRG cells. Toxicol Sci, 2021, 181(1): 90-104.
23.	Haraguchi Y, Shimizu T, Sasagawa T, et al. Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nat Protoc, 2012, 7(5): 850-858.
24.	Kukla D, Crampton A L, Wood D K, et al. Microscale collagen and fibroblast interactions enhance primary human hepatocyte functions in three-dimensional models. Gene Expr, 2020, 20(1): 1-18.

1. Wienkers L C, Heath T G. Predicting in vivo drug interactions from in vitro drug discovery data. Nat Rev Drug Discov, 2005, 4(10): 825-833.
2. Obach R S. Predicting clearance in humans from in vitro data. Curr Top Med Chem, 2011, 11(4): 334-339.
3. Houston J B. Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem Pharmacol, 1994, 47(9): 1469-1479.
4. Smith C M, Nolan C K, Edwards M A, et al. A comprehensive evaluation of metabolic activity and intrinsic clearance in suspensions and monolayer cultures of cryopreserved primary human hepatocytes. J Pharm Sci, 2012, 101(10): 3989-4002.
5. 孙英辉, 慈小燕, 李薇, 等. 应用人肝细胞研究人参皂苷Rb_1和Rg_1、丹参素钠、冰片对CYP450药物代谢酶1A2、2B6和3A4的体外诱导作用. 药物评价研究, 2021, 44(1): 38-44.
6. 董航, 张海晖, 窦桂芳, 等. γ射线辐射对大鼠肝药物代谢酶CYP1A2,CYP2C9和CYP2D6蛋白表达及药物代谢活性的影响. 中国药理学与毒理学杂志, 2021, 35(4): 290-296.
7. 徐聪, 徐思云, 胡海红, 等. CYP2B6体外诱导活性评价模型的建立及其在中药活性成分筛选的应用. 药学学报, 2013, 48(1): 119-124.
8. Di L, Obach R S. Addressing the challenges of low clearance in drug research. AAPS J, 2015, 17(2): 352-357.
9. 阮婷婷, 鞠武建, 熊海伟, 等. 低清除率药物的代谢稳定性预测模型研究进展. 中国药科大学学报, 2019, 50(2): 152-160.
10. Khetani S R, Szulgit G, Del Rio J A, et al. Exploring interactions between rat hepatocytes and nonparenchymal cells using gene expression profiling. Hepatology, 2004, 40(3): 545-554.
11. Gu J, Shi X, Zhang Y, et al. Establishment of a three-dimensional co-culture system by porcine hepatocytes and bone marrow mesenchymal stem cells in vitro. Hepatol Res, 2009, 39(4): 398-407.
12. Thomas R J, Bhandari R, Barrett D A, et al. The effect of three-dimensional co-culture of hepatocytes and hepatic stellate cells on key hepatocyte functions in vitro. Cells Tissues Organs, 2005, 181(2): 67-79.
13. Haraguchi Y, Shimizu T, Yamato M, et al. Scaffold-free tissue engineering using cell sheet technology. Chem Inform, 2012, 2(6): 2184-2190.
14. Kobayashi J, Kikuchi A, Aoyagi T, et al. Cell sheet tissue engineering: cell sheet preparation, harvesting/manipulation, and transplantation. J Biomed Mater Res A, 2019, 107(5): 955-967.
15. Yang J, Yamato M, Kohno C, et al. Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials, 2005, 26(33): 6415-6422.
16. Yang J, Yamato M, Shimizu T, et al. Reconstruction of functional tissues with cell sheet engineering. Biomaterials, 2007, 28(34): 5033-5043.
17. Kim K, Ohashi Kazuo, Utoh R, et al. Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets. Biomaterials, 2012, 33(5): 1406-1413.
18. Gao B, Sakaguchi K, Ogawa T, et al. Functional analysis of induced human ballooned hepatocytes in a cell sheet-based three dimensional mode. Tissue Eng Regen Med, 2021, 18(2): 217-224.
19. Hynds R E, Gowers K, Nigro E, et al. Cross-talk between human airway epithelial cells and 3T3-J2 feeder cells involves partial activation of human MET by murine HGF. PLoS One, 2018, 13(5): e0197129.
20. Barreca A, de Luca M, Del Monte P, et al. In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors. J Cell Physiol, 1992, 151(2): 262-268.
21. Ruoß M, Kieber V, Rebholz S, et al. Cell-type-specific quantification of a scaffold-based 3D liver co-culture. Methods Protoc, 2019, 3(1): 1-19.
22. Ware B R, Liu J S, Monckton C P, et al. Micropatterned coculture with 3T3-J2 fibroblasts enhances hepatic functions and drug screening utility of HepaRG cells. Toxicol Sci, 2021, 181(1): 90-104.
23. Haraguchi Y, Shimizu T, Sasagawa T, et al. Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nat Protoc, 2012, 7(5): 850-858.
24. Kukla D, Crampton A L, Wood D K, et al. Microscale collagen and fibroblast interactions enhance primary human hepatocyte functions in three-dimensional models. Gene Expr, 2020, 20(1): 1-18.

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