A Monte Carlo framework for managing biological variability in manufacture of autologous cell therapy from mesenchymal stromal cells therapies.

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.

The effect of Me2SO overexposure during cryopreservation on HOS TE85 and hMSC viability, growth and quality.

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 Me2SO, 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 Me2SO 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 Me2SO 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 Me2SO 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.

Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process.

Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 10(5) cells.mL(-1) in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal-free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post-harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony-forming potential. The hMSCs were held in suspension post-harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum-free vehicle solution using a controlled-rate freezing process. Post-thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component-free hMSC production process from expansion through to cryopreservation.

Low temperature cell pausing: an alternative short-term preservation method for use in cell therapies including stem cell applications.

Encouraging advances in 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 offers many advantages over cryopreservation, including the ability to store cells at scale, reduced cost and a simplified procedure with increased reliability. This review will focus on the importance of developing a short-term cell preservation platform as well highlighting the major successes of cell pausing and the key challenges which need addressing, to enable application of the process to therapeutically relevant cells.

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation.

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me2SO). Me2SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me2SO. Although reduced (by 22 ± 2%, p=0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me2SO, where the doubling time was significantly reduced (by 41 ± 4%, p=0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.