Investigating ice nucleation temperature effects on mesenchymal stem cell recovery from cryostorage [Abstract

Alexandra Stolzing (1251348)

Nick Wragg (1260240)

English

Loughborough University Institutional Repository

Page | 60Investigating ice nucleation temperature effects on mesenchymal stem cellrecovery from cryostorageNicholas M. Wragg1, Alexandra Stolzing11 Centre for Biological Engineering, Loughborough University, Loughborough, LE113TUCorresponding email: n.m.wragg2@lboro.ac.ukPoster Number: A47Keywords: Cryostorage, Expansion, Cell Engineering, MusculoskeletalIntroductionDuring the cryopreservation process, cells are frozen gradually before transfer to cryogenic temperaturesfor long term storage. During this process, ice crystals form at nucleation sites within the storage solution.As a stochastic process, the structure of crystallisation is governed by chaos, so each time the freezingprocess takes place, the temperature of crystal formation and the structure changes. During crystalformulation, temperature spikes occur, linked to a change in phase, which can result in supercoolingevents that damage cells due to osmotic gradients and intracellular ice formation.By controlling the ice nucleation temperature, the randomised formation effects could be curtailedpotentially increasing the recovery of the cell product from storage.MethodsMesenchymal stem cells (MSCs) were seeded at 4500 cells/well into a 96-well plate and cultured inDMEM+10%FBS overnight. The culture medium was then replaced with growth medium+10% DMSO. Acommercially available ice nucleation array (IceStart, Asymptote) was then placed into half of the 96-wellplate and then the whole plate placed into a controlled rate freezing device (ViaFreeze, Asymptote). Onceat -80oC the plate was removed to liquid nitrogen storage (vapour phase). Each condition was analysedusing MTT, one and four days after the implementation of slow (30 minute) and fast (5 minute) thawingconditions and removal of the IceStart.ResultsThe addition of IceStart moves the nucleation temperature closer to the melting point of the cryomedium.Furthermore, MTT analysis shows that IceStart Arrays enhance the viability of the MSCs significantly(p≤0.05) after just one day of culture in comparison to no-IceStart controls.ConclusionsThe addition of a known nucleation site reduces the damaging variation observed during a standardfreezing process without optimisation of cryomedium and in a high throughput setting.

Investigating ice nucleation temperature effects on mesenchymal stem cell recovery from cryostorage [Abstract]

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Investigating ice nucleation temperature effects on mesenchymal stem cell recovery from cryostorage [Abstract]

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