Development of a vitrification method for preserving human myoblast cell sheets for myocardial regeneration therapy

Abstract

BackgroundTissue-engineered cardiac constructs have potential in the functional recovery of heart failure; however, the preservation of these constructs is crucial for the development and widespread application of this treatment. We hypothesized that tissue-engineered skeletal myoblast (SMB) constructs may be preserved by vitrification to conserve biological function and structure.ResultsScaffold-free cardiac cell-sheet constructs were prepared from SMBs and immersed in a vitrification solution containing ethylene glycol, sucrose, and carboxyl poly-l-lysine. The cell sheet was wrapped in a thin film and frozen rapidly above liquid nitrogen to achieve vitrification (vitrification group, n = 8); fresh, untreated SMB sheets (fresh group, n = 8) were used as the control. The cryopreserved SMB sheets were thawed at 2 days, 1 week, 1 month, and 3 months after cryopreservation for assessment. Thawed, cryopreserved SMB sheets were transplanted into rat hearts in a myocardial infarction nude rat model, and their effects on cardiac function were evaluated. Cell viability in the cardiac constructs of the vitrification group was comparable to that of the fresh group, independent of the period of cryopreservation (p > 0.05). The structures of the cell-sheet constructs, including cell-cell junctions such as desmosomes, extracellular matrix, and cell membranes, were maintained in the vitrification group for 3 months. The expression of cytokine genes and extracellular matrix proteins (fibronectin, collagen I, N-cadherin, and integrin α5) showed similar levels in the vitrification and fresh groups. Moreover, in an in vivo experiment, the ejection fraction was significantly improved in animals treated with the fresh or cryopreserved constructs as compared to that in the sham-treated group (p < 0.05).ConclusionsOverall, these results show that the vitrification method proposed here preserves the functionality and structure of scaffold-free cardiac cell-sheet constructs using human SMBs after thawing, suggesting the potential clinical application of this method in cell-sheet therapy.

High-mobility group box 1 fragment suppresses adverse post-infarction remodeling by recruiting PDGFRα-positive bone marrow cells

“…To date, CBRT has been the mainstream approach, and numerous studies have reported its usefulness with different types of stem cells [30,31], including MSCs [8,9]. CBRT requires an in-house cell-processing center with an aseptic environment in the hospital [34]. Because drug-induced regenerative therapy does not require any cell culture, the quality of those cells is more easily maintained than with CBRT.…”

Biobanked human foreskin epithelial cell sheets reduce inflammation and promote wound healing in a nude mouse model

“…More studies have tried to preserve cell sheet by cryopreservation and have successfully cryopreserved limbal stem cell sheets [ 14 ], chondrocyte sheets [ 15 ], myoblast cell sheets [ 16 ], and mesenchymal stem cell sheets [ 17 ], in which 48–97% cell viability was reported. However, cryopreservation of ECSs remains a challenge because the ability to tolerate cryopreservation varies by cell type.…”

Vitrification and storage of oral mucosa epithelial cell sheets

“…Cell sheet technology has been developing for the past decade to target the delivery of cell sheets onto damaged organs. Success of both cell sheet engineering and transplantation have been reported in numerous articles; however, only three recent publications have reported the vitrification of cell sheets (Maehara et al, ; Ohkawara et al, ; Tani et al, ). Little is known about cell sheet vitrification; more studies are needed to increase our knowledge and to improve the vitrification methodology of cell sheets.…”

“…For this reason, we focused on analyzing CAOMECS stored in the absence of vitrification solutions. In addition, Ohkawara et al showed that human myoblasts cell sheets vitrified from 2 days to 3 months had the same curative effect on the heart after transplantation, indicating that the time of storage has no effect on the curative properties of the cell sheets (Ohkawara et al, ). These authors also mentioned that the cryopreserved cell sheets by slow freezing were not capable of rebuilding the extra cellular matrix upon thawing, confirming the need for a fast freezing of cell sheets.…”

“…A recent publication reported that slow freezing destroys the extracellular matrix (ECM) and was unable to be repaired confirming the need for our approach for fast freezing in absence of vitrification solutions (dry condition). During the vitrification step, ECM was maintained and preserved the cell sheet structure (Ohkawara et al, ). CAOMECS stored in absence of vitrification solution did not break during thawing, and the cell–cell connection was preserved as shown in the immunohistostaining.…”