Optimization of Adipose Tissue Cryopreservation Techniques in a Murine Model

Abstract

Background:. The aim of this study was to develop an adipose tissue (AT) cryopreservation protocol that is effective, simple, and maintains the functionality and viability of AT after thawing and transplantation. Methods:. Two cryopreservation temperatures (T°), −20°C and −80°C, and two cryoprotective agents (CPAs), trehalose and hydroxyethyl starch (HES), were compared first in an experimental study, using a slowfreezing protocol. The five experimental groups were the following: (a) Fresh AT (control group), (b) T = −20°C, 10%HES, (c) T = −80°C, 10%HES, (d) T = −20°C, 0.35M trehalose, (e) T = −80°C, 0.35M trehalose. We evaluated the morphology (histological studies) and tissue viability by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genic expression. Based on the results of the preliminary study, an in vivo study was performed, choosing as cryopreservation T° −20°C. HES and trehalose were compared as cryoprotective agents and with a control group (fresh AT). AT grafts were transplanted into immunodeficient mice. After 1 month of inoculation, animals were euthanized and samples were recovered. Samples were weighted and processed for histological study, viability study (GAPDH genic expression), and vascularization study (VEGF genic expression). Results:. The initial histological study demonstrated that all AT cryopreserved group samples showed typical histological features of AT, similar to that of the control group. Statistically significant differences were not observed (P > 0.05) in GAPDH expression between different groups related to temperature or CPA. Referring to the in vivo studies, cryopreserved groups showed good take of the graft and normal AT architectural preservation, as well as a clear vascular network. Statistically significant differences were not found (P > 0.05) with regard to graft take (%), GAPDH, or VEGF expression. Conclusion:. Slow freezing at −20°C using trehalose, and −20°C using HES as cryoprotective agents are both straightforward and easy AT cryopreservation procedures, with results similar to those of fresh AT in terms of tissue viability and morphohistological characteristics.