Cryopreserved skin epithelial cell sheet combined with acellular amniotic membrane as an off-the-shelf scaffold for urethral regeneration

Dongliang Zhang; Jiangang Hou; Yubo Gu; Jialiang Shao; Shukui Zhou; Jingming Zhuang; Lujie Song; Xiang Wang

doi:10.1016/j.msec.2021.111926

Cryopreserved skin epithelial cell sheet combined with acellular amniotic membrane as an off-the-shelf scaffold for urethral regeneration

Mater Sci Eng C Mater Biol Appl. 2021 Mar:122:111926. doi: 10.1016/j.msec.2021.111926. Epub 2021 Feb 3.

Authors

Dongliang Zhang¹, Jiangang Hou², Yubo Gu³, Jialiang Shao¹, Shukui Zhou⁴, Jingming Zhuang², Lujie Song⁵, Xiang Wang⁶

Affiliations

¹ Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
² Department of Urology, Huashan Hospital Fudan University, Shanghai, China.
³ Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
⁴ Department of Urology, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China.
⁵ Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China. Electronic address: ljsong@sjtu.edu.cn.
⁶ Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Electronic address: xiang.wang1@shgh.cn.

PMID: 33641919
DOI: 10.1016/j.msec.2021.111926

Abstract

Background: Autologous tissue transplantation for urethral repair is often limited and causes donor site complications. Here, a cryopreserved rabbit skin epithelial cell sheet (SEC) combined with an acellular amniotic membrane (AM) was used to repair rabbit urethral defects.

Methods: Abdominal skin was collected from 4-week-old New Zealand rabbits, and primary epithelial cells were extracted and cultured to form a cell sheet. Fresh SEC-AMs were constructed and cryopreserved. A cryopreservation system including optimized medium, two-pump perfusion, a programmed freezer and liquid nitrogen storage was established. Cell viability, mechanical strength, electron microscopy, and histological staining were performed in vitro after 1 month. Next, the sheets were transplanted subcutaneously for 2 weeks, and the graft was used to repair the rabbit urethral defect. Urinary function was measured and samples were collected for histological staining after 1 month.

Results: We confirmed that cryopreservation damage of SECs was reduced by composition with acellular AMs in terms of high cell activity. The SEC mechanical strength was also enhanced by AMs, which was convenient for the operation. In in vivo experiments, we transplanted sheets into the groin area for two weeks and found that cryopreservation reduced inflammatory cell infiltration and significantly improved vascular density. In the urethral repair experiment, the near-normal passive urine flow rate, smooth mucosa of the gross specimen, intact epithelialization and abundant neovascularization were confirmed in the cryopreserved-SEC-AM group compared with the other groups.

Conclusions: Cryopreserved SEC-AMs demonstrated similar outcomes of rabbit urethral defect repair as fresh SEC-AMs, showing good clinical application prospects.

Keywords: Amniotic Membrane; Cryopreservation; Macrophage polarization; Skin epithelial cell sheet; Urethra regeneration.

MeSH terms

Amnion*
Animals
Cryopreservation
Epithelial Cells
Male
Rabbits
Regeneration
Urethra*