Abstract
BackgroundHuman epithelial cell sheets (ECSs) are used to clinically treat epithelial conditions such as burns, corneal blindness, middle ear cholesteatoma and vitiligo. As a widely used material in clinic, there is little information on the biobanking of ECSs and its repair effect after storage.ResultsTwo methods for biobanking foreskin ECSs were compared in a short term (7 days): 4-degree storage and programmed cryopreservation. Cell sheet integrity, viability, apoptosis, immunogenicity, mechanical properties and function were evaluated. In vivo, ECSs were directly transplanted to skin defect models and histological examination was performed at 1 week postoperatively. We successfully extracted human foreskin-derived primary epithelial cells and fabricated them into ECSs. Compared with 4-degree storage, programmed cryopreservation preserved the ECS structural integrity, enhanced the mechanical properties, decreased HLA-I expression, and increased cell viability and survival. An increased proportion of melanocytes with proliferative capacity remained in the cryopreserved sheets, and the undifferentiated epithelial cells were comparable to those of the fresh sheets. In vivo, cryopreserved ECSs could reduce inflammatory cell infiltration and promote connective tissue remodeling, epithelial cell proliferation and vascular regeneration.ConclusionsProgrammed cryopreservation of ECSs was superior and more feasible than 4-degree storage and the cryopreserved ECSs achieved satisfying skin wound healing in vivo. We anticipate that the off-the-shelf ECSs could be quickly used, such as, to repair human epithelial defect in future.Graphical abstract
Highlights
Human epithelial cell sheets (ECSs) are used to clinically treat epithelial conditions such as burns, corneal blindness, middle ear cholesteatoma and vitiligo
Human foreskin keratinocytes (HFKs) originating from foreskin show typical characteristics, including colony formation and a cobblestone-like shape
By the two-pump perfusion system (Fig. 1d), cryoprotectant Dimethyl sulfoxide (DMSO) was introduced with a linear gradient (Fig. 1e)
Summary
Human epithelial cell sheets (ECSs) are used to clinically treat epithelial conditions such as burns, corneal blindness, middle ear cholesteatoma and vitiligo. Epithelial cell sheets (ECSs), as a scaffold-less material, are widely used to repair various epithelial tissue defects, including those in the skin [1, 2], cornea [3], middle ear mucosa [4], urethra and bladder mucosa [5, 6]. It is necessary to stock enough engineered cell sheets to ensure that a supply is available to deal with unpredictable factors [7]. This would require an efficient preservation technique that can store engineered grafts for several weeks or months and be ready for use immediately. The cell viability would decrease rapidly over time that ECSs couldn’t meet the quality control requirements of transplantation
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