Cryopreservation of mesenchymal stem cell and tissue engineered constructs using non-toxic cryoprotective agents

Abstract

The thesis work deals with the development of cryopreservation strategy for long term storage of MSCs and MSCs seeded tissue engineered constructs using non toxic cryoprotective agents as freezing medium. In the first phase, different freezing medium consisting of a combination of natural extracellular cryoprotectants namely trehalose, hydroxyl ethyl starch, polyvinyl pyrolidine and intracellular CPAs like erythritol, taurine and ectoin were used for cryopreservation of MNCs following the Taguchi Orthogonal Array method. Among the various combinations, freezing medium consisting of trehalose (0.05mM), ectoin (0.10mM) and catalase (100μg/ml) has shown maximum MNCs viability. These CPAs were further investigated individually as well as in combination to see their effectiveness towards long term preservation of MSCs. Among the freezing solutions, solution prepared using trehalose (0.3mM), ectoin (0.3mM), and catalase (100μg/ml) was found to be the most effective in preserving MSCs in long term basis. The viability of MSCs (73%) is found to be higher than the viability achieved with 10% (v/v) Me2SO (61%) used as control. The apoptotic study has indicated that the addition of general caspase and calpain inhibitors can reduce the apoptosis rate upto 10-15% thereby achieving increased cell viability of 80%. The optimum condition for the controlled rate freezing of MSCs was established as prenucleation cooling rate -1oC/min, nucleation temperature -7.5oC, cold spike -80oC/min, post nucleation holding time 5min, post nucleation cooling rate -1oC/min, cell density (3×106/ml/cell) and storage temperature (-150°C ) using the most effective freezing medium achieving cell viability of 85%. The developed freezing medium has also shown its ability to preserve MSCs seeded tissue engineered construct. The maximum viability of 80% achieved at optimum controlled rate freezing of TECs was established as cooling rate -1oC/min, nucleation temperature -7.5OC and freezing medium consisting of trehalose (0.3mM), ectoin (0.3mM), catalase (100μg/ml) in presence of caspase (50μg) and calpain (50μg) inhibitors. Overall, it is demonstrated that the developed freezing medium may pave the way for long term preservation of MSCs and also MSCs seeded scaffold.