Production of decellularized porcine, leporine and murine renal biological scaffold and co-culture exploration of renal scaffolds slices with cells in vitro
WANG Xin-wang,ZHOU Le-bin,MEI Jin,ZHANG De-ming,LIU Dan,DU Ao-ling,CHEN Sheng-hua
Chinese Journal of Clinical Anatomy ›› 2018, Vol. 36 ›› Issue (2) : 174-181.
Production of decellularized porcine, leporine and murine renal biological scaffold and co-culture exploration of renal scaffolds slices with cells in vitro
Objective To produce the decellularized biological renal scaffold of the pig, New Zealand white rabbit and SD rat, and then to explore the influence of porcine, leporine and murine renal biological scaffolds on the co-cultured seed cells HEK. Methods Adult healthy kidneys from 10 pigs, 28 New Zealand white rabbits, and 28 SD rats were harvested and divided into normal groups and scaffold groups randomly and equally. The scaffold group kidneys were perfused for decellularization with heparinized PBS, 1% Triton X-100 and 1% SDS through the renal artery successively. The kidneys in each group were characterized in histomorphology and detected for mechanical properties. And the co-culture experiments were conducted using the slices of each group and HEK cells, and the expression of PCNA in HEK cells was analyzed by immunofluorescence assay using the value of the gray level. Results In each scaffold group after decellularization, the HE staining of cell nucleus in the slices was negative, Masson's trichrome staining of collagen was positive, and immunofluorescence staining of Collagen I and Collagen IV was also positive. And with the scanning electron microscopy, the faveolate orifice structure and the typical glomerulus-like structure were observed. Characterized by the mechanical properties, the Young's modulus of each scaffold group is similar with that of the corresponding normal group. After the co-culture, the PCNA/DAPI value of every renal scaffold group is higher than that of the blank control. Furthermore, there was no significant difference on the PCNA/DAPI of HEK between the pig, New Zealand white rabbit and SD rat renal scaffolds. Conclusions The decellularization method of our study can eliminate the cells in the porcine, leporine and murine kidney, retain the extracellular matrices and preserve the spatial structure and similar mechanical strength. And it is an effective way to produce the decellularized pig, New Zealand white rabbit and SD rat renal biological scaffolds. Importantly, the produced decellularized porcine, leporine and murine kidney scaffold slices can improve the proliferation activity of heterogeneous HEK cells when co-cultured in vitro, and there is no species difference on the improvement between the pig, New Zealand white rabbit and SD rat.
/ Decellularization / Biological scaffold / Kidney regeneration / Co-cuture / Tissue engineering
[1] 肖月, 隋宾艳, 赵琨. 我国终末期肾病现状及透析技术的应用、费用及支付情况分析[J]. 中国卫生政策研究, 2011, 4(5):29-33.
[2] 宋康康, 赵德龙, 陈香美. 维持性血液透析患者死亡相关危险因素综述[J]. 解放军医学院学报, 2017, 38(5): 471-474.
[3] Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart[J]. Nat Med, 2008, 14(2):213-221.
[4] 史跃先, 黎磊石, 周虹, 等. IgA肾病肾组织内胶原蛋白Ⅳ的分布及意义[J]. 解放军医学杂志, 1993, 14(2):114-117.
[5] Peloso A, Petrosyan A, Da Sacco S, et al. Renal extracellular matrix scaffolds from discarded kidneys maintain glomerular morphometry and vascular resilience and retains critical growth factors[J]. Transplantation, 2015, 99(9):1807-1816.
[6] Huang J, Wang H, Fan ST, et al. The national program for deceased organ donation in China[J]. Transplantation, 2013, 96(1):5-9.
[7] Madariaga ML, Ott HC. Bioengineering kidneys for transplantation[J]. Semin Nephrol, 2014, 34(4):384-393.
[8] 韩雪峰, 杨大平, 郭铁芳. 曲拉通X-100对制备脱细胞血管基质影响的实验研究[J]. 中华外科杂志, 2002, 40(1):27-29.
[9] 秦雨萌, 周涛, 张璐, 等. 大鼠单叶肝去细胞生物支架的制备与鉴定[J]. 解剖学报, 2017, 48(4):477-481.
[10]赵丽娜, 余雅玲, 林刻智, 等. 在体制备大鼠全肾去细胞支架的程序性分析[J]. 解剖学报. 2014, (2):267-272.
[11]Yu YL, Shao YK, Ding YQ, et al. Decellularized kidney scaffold-mediated renal regeneration[J]. Biomaterials, 2014, 35(25):6822-6828.
[12]Guan Y, Liu S, Sun C, et al. The effective bioengineering method of implantation decellularized renal extracellular matrix scaffolds[J]. Oncotarget, 2015, 6(34):36126-36138.
[13]Du C, Narayanan K, Leong MF, et al. Functional Kidney Bioengineering with Pluripotent Stem-Cell-Derived Renal Progenitor Cells and Decellularized Kidney Scaffolds[J]. Adv Healthc Mater, 2016, 5(16):2080-2091.
[14]Abolbashari M, Agcaoili SM, Lee MK, et al. Repopulation of porcine kidney scaffold using porcine primary renal cells[J]. Acta Biomater, 2016, 29:52-61.
/
| 〈 |
|
〉 |
