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Manufacturing processes for autologous cell therapy need to reproducibly generate in specification (quality and quantity) clinical product. However, patient variability prevents the level of control of cell input material that could be achieved in a cell line or allogeneic-based process. We have applied literature data on bone marrow-derived mesenchymal stromal cells variability to estimate probability distributions for stem cell yields given underlying truncated normal distributions in total nucleated cell concentration, stem cell percentage and plausible aspirate volumes. Monte Carlo simulation identified potential variability in harvested stem cell number in excess of an order of magnitude. The source material variability was used to identify the proportion of donor manufacturing runs that would achieve a target yield specification of 2E7 cells in a fixed time window with given proliferative rates and different aspirate volumes. A rapid, screening, development approach was undertaken to assess culture materials and process parameters (T-flask surface, medium, feed schedule) to specify a protocol with identified proliferative rate and a consequent model-based target aspirate volume. Finally, four engineering runs of the candidate process were conducted and a range of relevant quality parameters measured including expression of markers CD105, CD73, CD44, CD45, CD34, CD11b, CD19, HLA-DR, CD146 (melanoma cell adhesion molecule), CD106 (vascular cell adhesion molecule) and SSEA-4, specific metabolic activity and vascular endothelial growth factor secretion, and osteogenic differentiation potential. Our approach of using estimated distributions from publicly available information provides a route for data-poor earl- stage developers to plan manufacture with defined risk based on rational assumptions; furthermore, the models produced by such assumptions can be used to evaluate candidate processes, and can be incrementally improved with accumulating distribution understanding or subdivision by new process variables.

Background & Aim The commercialization of cells for therapeutic use drives a critical need for the refinement of cell storage and handling procedures, ensuring the integrity of cell-based products is maintained throughout the product life cycle. One area for refinement is the transfer of cells from a sealed container system after they have been cryopreserved, stored, shipped and thawed. There are many parameters that may affect either the total volume or the viability of the cells recovered. These include the type of transfer equipment used, the properties of the cryo-formulations that may have different viscosities or rheology, and the proficiency of the manual operator performing the task. This study investigates the comparability of a vial adaptor when compared to a traditional needle and syringe for recovering cells from a sealed cyclic olefin polymer (COP) vials after cryopreservation and storage for one week at -180°C. Methods, Results & Conclusion One experiment was designed to evaluate the effect of operator variability (n = 4) when transferring 1 ml fill volumes containing 1 × 106 cells/ml stored in Cryostor CS10 using a vial adaptor or needle and syringe. Bone marrow-derived human mesenchymal stem cells (hMSCs) and cord blood hematopoietic stem cells (HSCs) were used as exemplar adherent and suspension cell types. Total volume recovery from each system was measured, as was morphology, cell yield and viability after a 4-day outgrowth to determine cell and volume losses from the transfer process. A second experiment looked at the effect different cell densities and cryo-formulations may have during transfer. 1ml vials of hMSCs at different densities (5 e5/ml and 4 e6/ml) were prepared and stored in 4 different formulations with differing colloidal components. The cell yield and viability were measured immediately post-transfer as was total recovered volume. The results showed that recovery of both intact cells and product volume were >80% for recovered volume with a suitable cryo-formulation regardless of transfer method (Figure 1). The vial adaptor was comparable in performance to a needle and syringe, thus providing a safe needle-free alternative for operators during manual transfer. Furthermore, the data highlighted the how cryo-formulation choice and operator variability can impact upon overall product recovery in a cell therapy context.

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 Me$_{2}$SO, 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 Me$_{2}$SO 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 Me$_{2}$SO 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 Me$_{2}$SO 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.

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

Regenerative medicine is an emerging technology and with over 1000 cell therapies currently in clinical trials there is a need to expand the limited knowledge related to the storage, shipment and preservation of cell therapies. An accepted gold standard of human cell cryopreservation is 10% (v/v) dimethyl sulfoxide (Me 2 SO) in serum. Concerns mean safer and optimized protocols are needed, including Me 2 SO toxicity and thawing. Given its potential toxicity, Me 2 SO usage is a key issue in cryopreservation. Methods specify the need to reduce cell exposure time to Me 2 SO above 0 °C as much as possible. The maximum amount of time cells can be exposed to Me 2 SO to prevent a detrimental effect needs to be clarified. Thawing is poorly understood in cryopreservation. It is as vital as freezing to prevent injury to cells. Protocols are currently too vague for cell therapy regulation, and need improvement. The time dependent Me 2 SO cytotoxicity was evaluated by overexposing human mesenchymal stem cells (hMSCs) to Me 2 SO during cryopreservation. The effect on several metrics has been investigated and it has been determined that after 1 h overexposure after thawing viability is reduced from 96.3 ± 0.6% to 89.1 ± 2.6%, the co-expression of four characteristic markers is changed from 91.1 ± 1.7% to 82.1 ± 0.6%. This significant change could cause adverse effects on product efficacy and patient health, to prevent this, Me 2 SO exposure must be kept to below 1 h. A heat transfer model has been developed and determined that 655 J is the required energy to thaw a vial, associated heat transfer rates have also been calculated. Using these values, and additional factors such as pre-thaw stabilization, and fresh-media composition, a multi-parameter experiment will help determine an improved thawing process.

Background: Advancements in cell therapies have produced a requirement for an effective short-term cell preservation method, enabling time for quality testing and transport to their clinical destination. We aim to ‘pause’ cells at ambient temperature whilst maintaining cell viability and quality post preservation. Storage at ambient temperature effectively bypasses cell damage from freezing and will greatly reduce costs and reliability of specialist machinery. Methodology: Initially, a human osteosarcoma cell line (HOS TE85) was paused 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 ,95% humidity. Results: Viability (propidium iodide) remained above 98% and 91% for cells paused at 24 and 48 h respectively and significantly lower reductive metabolism (PrestoBlue) was observed during pausing compared with non-paused controls ( p ⩽ 0.01). Following recovery, metabolic activity became similar to the control and alkaline phosphatase activity (typically secreted by osteoblasts) remained unchanged. Conclusion: HOS TE85 cells effectively recover following 48 h pausing in standard media and show evidence of ‘pausing’ with regards to lower reductive metabolism at ambient temperature exposure. Current experiments are investigating the effects of prolonged pausing (144 h), pausing in suspension and application to mesenchymal stem cells. Successful cell pausing will create a simpler, cost effective short-term preservation method for cell therapies.