Cryopreserved human adipogenic-differentiated pre-adipocytes: A potential new source for adipose tissue regeneration

Background: Previously, we have shown that in vitro adipogenic differentiation of pre-adipocytes before implantation can enhance in vivo adipose tissue formation. For large-scale adipose tissue engineering or repeat procedures, cryopreservation of fat grafts has been commonly used in recent years. However, the feasibility of cryopreservation of adipogenic differentiated pre-adipocytes has not been investigated. Methods: To examine the impact of cryopreservation on the adipogenic functions of adipogenic-differentiated pre-adipocytes, freeze-thawed adipocytes were compared with fresh differentiated adipocytes in vitro and in vivo. Adipogenic function was assessed by Oil red O staining, ELISA analysis of leptin secretion and RT-PCR of adipogenic-related genes. After transplantation, adipose tissue formation was assessed by histomorphologic and volumetric analysis. Results: Freeze-thawed adipocytes constantly showed typical adipogenic functions in terms of lipid content, leptin secretion and adipogenic gene expression, as well as good viability. Importantly, implants derived from freeze-thawed adipocytes were successfully developed to adipose tissue and newly formed adipose tissues were similar to those developed from fresh differentiated adipocytes, based on histomorphologic and volumetric analysis. In addition, CD34-positive endothelial cells were detected in implants. These results demonstrate that the specific characters of adipogenic-differentiated pre-adipocytes are successfully conserved after cryopreservation without any significant alteration. Discussion: Cryopreservation of adipogenic-differentiated pre-adipocytes is a feasible method and extends their clinical use in adipose tissue-engineering applications and transplantation.