The effect of Me 2 SO overexposure during cryopreservation on HOS TE85 and hMSC viability, growth and quality

Abstract

With the cell therapy industry continuing to grow, the ability to preserve clinical grade cells, including mesenchymal stem cells (MSCs), whilst retaining cell viability and function remains critical for the generation of off-the-shelf therapies. Cryopreservation of MSCs, using slow freezing, is an established process at lab scale. However, the cytotoxicity of cryoprotectants, like MeSO, raises questions about the impact of prolonged cell exposure to cryoprotectant at temperatures >0 °C during processing of large cell batches for allogenic therapies prior to rapid cooling in a controlled rate freezer or in the clinic prior to administration. Here we show that exposure of human bone marrow derived MSCs to MeSO for ≥1 h before freezing, or after thawing, degrades membrane integrity, short-term cell attachment efficiency and alters cell immunophenotype. After 2 h's exposure to MeSO at 37 °C post-thaw, membrane integrity dropped to ∼70% and only ∼50% of cells retained the ability to adhere to tissue culture plastic. Furthermore, only 70% of the recovered MSCs retained an immunophenotype consistent with the ISCT minimal criteria after exposure. We also saw a similar loss of membrane integrity and attachment efficiency after exposing osteoblast (HOS TE85) cells to MeSO before, and after, cryopreservation. Overall, these results show that freezing medium exposure is a critical determinant of product quality as process scale increases. Defining and reporting cell sensitivity to freezing medium exposure, both before and after cryopreservation, enables a fair judgement of how scalable a particular cryopreservation process can be, and consequently whether the therapy has commercial feasibility.

The Impact of Varying Cooling and Thawing Rates on the Quality of Cryopreserved Human Peripheral Blood T Cells

“… Assumption that data from sperm and embryos translated to somatic cells. Slow thawing – confounding of post thaw DMSO toxicity and slow thawing 36 , 37 . …”

Icariin Improves the Viability and Function of Cryopreserved Human Nucleus Pulposus‐Derived Mesenchymal Stem Cells

“…Currently, the existing cryopreservation methods for MSCs can be classified into two main categories, slow freezing and vitrification (rapid freezing) [ 10 ]. But both methods have some notable limitations [ 11 ], and there is an increasing amount of evidence showing the adverse effects of conventional cryopreservation methods for MSCs, such as the cytotoxicity of the standard cryoprotectant dimethyl sulfoxide (DMSO), cell apoptosis, cellular structure damages, and oxidative stress [ 12 – 14 ]. Recently, many approaches have been proposed for a more efficient application of cryopreserved MSCs, among which addition of selected agents in cryopreservation solution has shown good application prospects [ 15 , 16 ].…”

Cryopreservation of midbrain dopaminergic neural cells differentiated from human embryonic stem cells

“…Recent studies with HepG2 cells suggested slow thawing up to -10°C, and then rapid thawing from -10°C to 4°C is beneficial due to the reduction of microfluctuations [39]. Finally, rapid thawing may not be beneficial due to the potential for increased toxicity of DMSO, especially if temperatures approach 37°C before wash-out [40]. An ideal thawing protocol for some cell types may consist of slow thawing to 4°C, followed by immediate dilution of cells in cold medium to reduce the toxic effects of DMSO prior to centrifugation.…”