Scale-out of massively parallel patient-specific cell cultures with a modified transportable conditioned cell culture chamber

Barrier Isolators, which separate the cell culture processing atmosphere from the bioburden of personnel, are the best means to reduce contamination risks. These isolators are currently being used for cGMP-compliant clinical trials1, 2. Scaling cell production processes presents non-obvious restrictions to most people. Compared to open processing, modular Cytocentric isolators can be replicated to scale proportionately with each stage in cell processing until all steps are accommodated maximally. This allows a process to efficiently and quickly scale with operations from pre-clinical through clinical studies3. However, for processing of massively parallel patient-specific cell cultures, incubation capacity in a barrier isolator, unlike in the open room, can be a bottleneck. Inexpensive and infinitely elastic incubation capacity can be provided by existing external incubators if cultures can be safely transported to and from the isolator for processing. We tested a modified transportable conditioned cell culture chamber (TC4) designed to enclose cell cultures inside the exterior incubator and fit through the airlocks of the barrier isolator to safely deliver cells to the interior for processing. We have previously published on good cell growth using this processing system to expand K562 cells, a hematopoietic stem cell-like cell line that has been used as a surrogate for CAR-T cell processing. In this study, we addressed sterility concerns by running mock production runs with a highly permissive color-changing bacterial broth. We ran three production runs, moving mock cultures between the barrier isolator and the external incubator with the TC4 transport chamber. We took samples of the final mock cell product, sealed them into sterile vials, and incubated them long-term, monitoring for bacterial growth. We also performed environmental monitoring of the barrier isolator processing chamber with an air sampler and contact plates. Positive control samples were all yellow and turbid. Negative samples and all test materials were negative for microbial growth. We concluded that this transport chamber could help safely alleviate the bottleneck in cell production presented by the unique needs of massively-parallel patient specific cell incubation. References: Mei, S.H., et al., Isolation and large-scale expansion of bone marrow-derived mesenchymal stem cells with serum-free media under GMP-compliance. mortality, 2014. 40: p. 1. Marathe, C.S., et al., Islet cell transplantation in Australia: screening, remote transplantation, and incretin hormone secretion in insulin independent patients. Horm Metab Res, 2015. 47(1): p. 16-23. Yufit, T., P. Carson, and V. Falanga, Topical Delivery of Cultured Stem Cells to Human Non-Healing Wounds: GMP Facility Development in an Academic Setting and FDA Requirements for an IND and Human Testing. Current drug delivery, 2014. 11(5): p. 572-581

Reducing variability in conditions for cell handling improves MSC yields

Efficient cell expansion in vitro is essential to commercialization of human MSC as a cellular therapy. The cost of goods sold (COGS) is dramatically affected by how long it takes to expand the cells in vitro and the cell yield determines the number of doses generated for profit. Therefore, maximizing MSC growth in culture is critical for the success of MSC-based cellular therapies. Studies by others have shown that temperature differences in cell production can adversely affect cell yields. Here we study the effects of variability in temperature and CO2, like changes seen during routine cell handling in a room air BSC, on human MSC yield. We cultured human bone marrow mesenchymal stromal/stem cells for 8 biweekly subpassages (P4-P12) with conventional room air CO2 incubator conditions (37 degrees C/ 5% CO2). The culture was divided into separate cultures for routine cell handling in two different conditions (1) room air BSC conditions (RT/ 0.1% CO2) (variable) or (2) the same conditions as incubation (constant). At each passage, cells were plated in 96-well plates which were assayed over time for cell growth kinetics. Consistently, MSC incubated and handled in constant conditions recovered more quickly after subpassage and were more likely to continue to divide, improving final cell yields. We conclude that constant conditions for cell handling are critical for maximum MSC cell yield

Cytocentric measurement for regenerative medicine

Any Regenerative Medicine (RM) business requires reliably predictable cell and tissue products. Regulatory agencies expect control and documentation. However, laboratory tissue production is currently not predictable or well-controlled. Before conditions can be controlled to meet the needs of cells and tissues in culture for RM, we have to know what those needs are and be able to quantify them. Therefore, identification and measurement of critical cell quality attributes at a cellular or pericellular level is essential to generating reproducible cell and tissue products. Here, we identify some of the critical cell and process parameters for cell and tissue products as well as technologies available for sensing them. We also discuss available and needed technologies for monitoring both 2D and 3D cultures to manufacture reliable cell and tissue products for clinical and non-clinical use. As any industry matures, it improves and standardizes the quality of its products. Cytocentric measurement of cell and tissue quality attributes are needed for RM