Multiple Uses for CryoStor in Cell Manufacturing Journal articles recently added to BioLife Solutions’ Evidence Library illustrate several stages during the cell manufacturing process for which CryoStor CS10® cryopreservation solution is applicable. The three articles that follow include several different cell types cryopreserved in CryoStor CS10. An expanded list of cell types biopreserved in BioLife media, “BioPreservation Media Products in Customer Clinical Trials,” is available here. In the first article cited, Thirlwell et al. employ CryoStor in the beginning stage of manufacturing, to establish a cell bank following outgrowth of pancreatic-derived mesenchymal stromal cells. 1. Thirlwell, K. L., Colligan, D., Mountford, J. C., Samuel, K., Bailey, L., Cuesta-Gomez, N., Hewit, K. D., Kelly, C. J., West, C. C., McGowan, N. W., Casey, J. J., Graham, G. J., Turner, M. L., Forbes, S. and Campbell, J. D. Pancreas-derived mesenchymal stromal cells share immune response-modulating and angiogenic potential with bone marrow mesenchymal stromal cells and can be grown to therapeutic scale under Good Manufacturing Practice conditions. Cytotherapy. 2020. doi: 10.1016/j.jcyt.2020.07.010. The article by Thirlwell et al. points out that mesenchymal stromal cells (MSCs) isolated from a number of sources are being considered for therapeutic purposes for a variety of diseases. Pancreatic-derived MSCs have been described. Enzymatic dissociation of pancreata under Good Manufacturing Practices (GMP) conditions produces highly pure pancreatic islets that can be transplanted into individuals to treat type 1 diabetes mellitus. The authors show that MSCs can be isolated from the exocrine tissue and low-purity islet fraction left over from the production of high-purity islets. When cells resulting from explant outgrowth in cell culture reached 80 – 90% confluent, the cells were recovered, and debris was removed. The cells, designated passage “0”, were counted, and cryopreserved at a density of 1 x 10∧6 per 2 mL cryovial in CryoStor CS10, or re-cultured. The authors compared LPI MSCs expanded under GMP conditions to bone marrow-derived MSCs in vitro and in vivo for pro-regenerative and inflammation-modulating functions. In the second article cited, Jones et al. discuss manufacturing strategies they have worked out for regulatory T cells (Tregs). Tregs were harvested and cryopreserved after 9 days in culture. 2. Jones, M., Nankervis, B., Roballo, K. S., Pham, H., Bushman, J., and Coeshott, C. (2020). A Comparison of Automated Perfusion- and Manual Diffusion-Based Human Regulatory T Cell Expansion and Functionality Using a Soluble Activator Complex. Cell Transplantation. 2020; 29: 1-15. doi: 10.1177/0963689720923578. Given the goal to manufacture billions of Tregs at scale, authors Jones et al. compared expansion of Tregs in the Quantum® Cell Expansion System versus growth in T225 flasks, starting from the same inoculum of cells (3 x 10∧7 Tregs in 124 mL of medium, originating from a three donor pool), using identical culture media, identical media volumes, and identical time in culture (9 days), in triplicate. Cell cultures were monitored to measure cell metabolism as well as to determine cell and population doubling times. Cells were harvested on Day 9 and counted to determine cell harvest numbers and viability. For later analysis (phenotyping, cytokine secretion and in vitro rat splenocyte suppression assay), cells were cryopreserved at 2.0 x 10∧7 cells/ml in CryoStor CS10, stored at -80℃ overnight, then transferred to vapor-phase liquid nitrogen for long-term storage. Briefly, the Quantum Cell Expression System produced a 17.7-fold increase in Treg yield compared to expansion in T225 flasks. The Quantum Cell Expression System produced Tregs, of which 97.7% exhibited a memory phenotype compared to 93.7% for T225 flask culture, demonstrated an 8-fold greater IL-10 cytokine stimulation index than cells grown in T225 flasks, and that suppressed effector cells, following cryopreservation. In the third article cited, Makkar et al. manufactured allogeneic cardiosphere-derived cells (CDCs) for evaluation of safety and efficacy, by intracoronary administration, to patients with myocardial infarction, in clinical trial NCT01458405 (ALLSTAR). 3. Makkar, R. R., Kereiakes, D. J., Aguirre, F., Kowalchuk, G., Chakravarty, T., Malliaras, K., Francis, G. S., Povsic, T. J., Schatz, R., Traverse, J. H., Pogoda, J. M., Smith, R. R., Marbán, L., Ascheim, D. D., Ostovaneh, M. R., Lima, J. A. C., DeMaria, A., Marbán, E. and Henry, T. D. Intracoronary ALLogeneic heart STem cells to Achieve myocardial Regeneration (ALLSTAR): a randomized, placebo-controlled, double-blinded trial. European Heart Journal. 2020. doi: 10.1093/eurheartj/ehaa541. Initial pre-clinical and early phase trials examined the use of autologous cardiosphere-derived cells to treat study patients ill with post-myocardial infarction, hypoplastic left heart syndrome, dilated cardiomyopathy or Duchenne muscular dystrophy. Treatment with autologous cells is limited by constraints including post-biopsy processing time, donor age, and co-morbidities. These constraints could be mitigated by the use of allogeneic CDCs. The manufacture of CAP-1002 is outlined in the journal article by Chakravarty, T., Makkar, R., Ascheim, D., Traverse, J., Schatz, R., & Demaria, A. et al. titled, “ALLogeneic Heart STem Cells to Achieve Myocardial Regeneration (ALLSTAR) Trial: Rationale and Design,” Transplantation (2017); 26(2), 205-214. doi: 10.3727/096368916×692933. In brief, explant-derived cells from donor hearts were cryopreserved to create a Master Cell Bank. Cardiospheres were generated from cells from the MCB and CDCs were derived from cardiospheres. According to the authors in the current journal article, “a dose unit of the CAP-1002 formulation consisted of 25 million allogeneic CDCs suspended in 11.5 mL of cryopreservation solution (CryoStor CS10; BioLife Solutions Inc.) containing 10% Dimethylsulfoxide, heparin (1800 U total), and nitroglycerine (450 micrograms total).” The authors explained that heparin and nitroglycerine were added to resolve the inherent thrombogenicity of CDCs, initially observed during studies in large animals. BioLife Solutions has class-defining solutions for your cell manufacturing workflow. BioLife Solutions’ best-in-class CryoStor® and HypoThermosol® biopreservation media are headliners in our media lineup that includes BloodStor® and Cell Thawing Media® product lines. Cryopreserved cells need process and storage you can count on. Take charge of your freezing process with BioLife Solutions’ Custom Biogenic Systems Series 2101 Controlled Rate Freezers. For storing cells frozen in vapor-phase liquid nitrogen, CBS’ line of Isothermal Liquid Nitrogen Freezers (patented) maximizes storage space while minimizing chances of cross-contamination. Thaw precious frozen cells with precision! BioLife’s ThawSTAR CB delivers first-of-its-kind automated ThawSTAR technology for water-free thawing of cells frozen in 25 mL to 1000 mL cryobags. View the ThawSTAR CB video here! The ThawSTAR product lineup includes ThawSTAR systems for cells frozen in vials: CFT1.5 (1.5 mL cryovials), CFT2 (1.8 – 2.0 mL cryovials), and AT6 (6.0 mL AT-Closed Vial®). How do you handle temperature-sensitive frozen biosamples? Check out the BioT line of transporters (ULT, LN2) and the ULT Mobile Workstation. Ship informed! Ship your cell and gene therapies in evo® DV-10 liquid nitrogen dry vapor smart shippers developed by SAVSU Technologies. The evo system features the most advanced smart containers and Software-as-a-Service technologies that enable specialty couriers to offer a higher level of service to you and those you serve. Need more information? Ask the Scientist to help you optimize your cell manufacturing workflow. Visit our website at www.biolifesolutions.com. Search our Evidence Library. To place an order or request more information, call us today! 1.866.6543 (toll-free in North America) or direct: 1.425.402.1400.