139 Low temperature cell pausing: A short-term hypothermic preservation method for cells

Encouraging advancements in stem cell therapies have produced a requirement for an effective short-term cell preservation method, enabling time for quality assurance testing and transport to their clinical destination. Low temperature pausing of cells (>0 °C) effectively bypasses cell damage caused by extreme temperature shifts during cryopreservation and using ambient temperatures in particular, will greatly reduce costs and reliability of specialist machinery. Although initial work is with an osteoblast cell line, this research aims to apply the process to therapeutically relevant cells and optimize methodologies to enhance cell viability and maintain quality post-preservation. Successful pausing of stem cells will increase the possibility of many novel cellular therapies becoming approved marketed products. An adherent osteosarcoma cell line (HOS TE85) was used for initial investigations. Cells were seeded at 2 × 10 5 cells/ml into 6-well plates and incubated for 24 h at 37 °C, 5% CO 2 . Cells were paused at 80% confluence at ambient temperatures (no atmospheric control) for up to 48 h. Cell assessments were performed immediately after cell pausing and following 24 h recovery at 37 °C, 5% CO 2 . The effect of cell pausing was assessed qualitatively using optical light microscopy and quantitatively using a panel of viability assays. Viability was evaluated by membrane integrity (Trypan Blue exclusion) and ATP-dependent metabolic activity (PrestoBlue). Alkaline phosphatase activity was measured as a secondary indicator of osteoblast-like phenotype. Immediately after hypothermic exposure, cells appear smaller and more spherical in shape when compared to the fibroblastic, adherent structure of untreated controls. After the recovery period, morphology returned to normal. The percentage of viable cells (Trypan Blue negative) retrieved immediately after 24 and 48 h of storage at ambient temperature was 81% and 76% ( n  = 3), respectively. After a recovery period, viable cell number increased to 170% (24 h hypothermia) and 88% (48 h hypothermia) of that measured prior to cell pausing. PrestoBlue fluorescence intensity decreased following 24 and 48 hours storage at ambient temperatures compared to untreated controls by 53% and 64% ( n  = 3), respectively. Following recovery, fluorescence intensity increased to 151% and 144% after 24 and 48 h of low temperature storage compared to values before hypothermic exposure. We show that HOS TE85 cells effectively recover in terms of morphology, membrane integrity and ATP-dependent enzyme activity from up to 48 h of cell pausing. Additionally, recovered cell yield was lower with increased cell pausing time. These promising results mean we can begin to look at cell types relevant to cell therapy such as mesenchymal stromal cells and pausing in suspension. Work so far is ‘proof of concept’ that HOS TE85 cells can be preserved for 48 h in standard media by entering a phase of suspended animation during pausing at ambient temperatures. Whilst low temperature preservation in standard media holds great potential for HOS TE85 cells, more complex stem cells may require specialised media formulations compatible with cell pausing. Successful cell pausing will create a simpler, cost effective short-term preservation method, which once applied to therapeutically relevant cells, will accelerate the progression of cellular therapies from bench to bedside. Source of funding: Loughborough University Graduate School Studentship. Conflict of interest: None declared. n.robinson@lboro.ac.uk