Cryopreservation of hMSCs seeded silk nanofibers based tissue engineered constructs

Abstract

Long term cryopreservation of tissue engineering constructs is of paramount importance to meet off-the shelf requirements for medical applications. In the present study, the effect of cryopreservation using natural osmolytes such as trehalose and ectoin with and without conventional Me2SO on the cryopreservation of tissue engineered constructs (TECs) was evaluated. MSCs derived from umbilical cord were seeded on electrospun nanofibrous silk fibroin scaffolds and cultured to develop TECs. TECs were subjected to controlled rate freezing using nine different freezing solutions. Among these, freezing medium consisting of natural osmolytes like trehalose (40mM), ectoin (40mM), catalase (100μg) as antioxidant and Me2SO (2.5%) was found to be the most effective. Optimality of the chosen cryoprotectants was confirmed by cell viability (PI live/dead staining), cell proliferation (MTT assay), microstructure analysis (SEM), membrane integrity (confocal microscopy) and in vitro osteogenic differentiation (ALP assay, RT-PCR and histology) study carried out with post-thaw cryopreserved TECs. The mechanical integrity of the cryopreserved scaffold was found to be unaltered. The performance of the freezing medium towards cryopreservation of TEC was superior than the performance achieved using conventional Me2SO and similar to the non cryopreserved TEC. Overall we have formulated an efficient freezing medium that may pave the way of long term preservation of TECs with maintaining its integrity, MSCs viability and differentiation potentiality. It was observed that the performance of freezing medium for cryopreservation of TECs was better than the Me2SO.

Keywords: Cryopreservation; Natural osmolytes; Silk nanofibrous scaffolds; Tissue engineering constructs.