Large-scale expansion and characterization of CD3+ T-cells in the Quantum® Cell Expansion System.

BACKGROUND: The rapid evolution of cell-based immunotherapies such as chimeric antigen receptor T-cells for treatment of hematological cancers has precipitated the need for a platform to expand these cells ex vivo in a safe, efficient, and reproducible manner. In the Quantum® Cell Expansion System (Quantum system) we evaluated the expansion of T-cells from healthy donors in a functionally-closed environment that reduces time and resources needed to produce a therapeutic dose. METHODS: Mononuclear cells from leukapheresis products from 5 healthy donors were activated with anti-CD3/CD28 Dynabeads® and expanded in the Quantum system for 8-9 days using xeno-free, serum-free medium and IL-2. Harvested cells were phenotyped by flow cytometry and evaluated for cytokine secretion by multiplex assays. RESULTS: From starting products of 30 or 85 × 106 mononuclear cells, CD3+ T-cell populations expanded over 500-fold following stimulation to provide yields up to 25 × 109 cells within 8 days. T-cell yields from all donors were similar in terms of harvest numbers, viability and doubling times. Functionality (secretion of IFN-γ, IL-2 and TNF-α) was retained in harvested T-cells upon restimulation in vitro and T-cells displayed therapeutically-relevant less-differentiated phenotypes of naïve and central memory T-cells, with low expression of exhaustion markers LAG-3 and PD-1. CONCLUSIONS: The Quantum system has been successfully used to produce large quantities of functional T-cells at clinical dosing scale and within a short timeframe. This platform could have wide applicability for autologous and allogeneic cellular immunotherapies for the treatment of cancer.

Genetic stability of bone marrow-derived human mesenchymal stromal cells in the Quantum System.

BACKGROUND AIMS: The Quantum® Cell Expansion System (Quantum; Terumo BCT, Inc, Lakewood, CO, USA) is a novel hollow fiber-based device that automates and closes the cell culture process, reducing labor intensive tasks such as manual cell culture feeding and harvesting. The manual cell selection and expansion processes for the production of clinical-scale quantities of bone marrow-derived human mesenchymal stromal cells (BM-hMSCs) have been successfully translated onto the Quantum platform previously. The formerly static, manual, in vitro process performed primarily on tissue culture polystyrene substrates may raise the question of whether BM-hMSCs cultured on a hollow fiber platform yields comparable cell quality. METHODS: A rigorous battery of assays was used to determine the genetic stability of BM-hMSCs selected and produced with the Quantum. In this study, genetic stability was determined by assessing spectral karyotype, micronucleus formation and tumorigenicity to resolve chromosomal aberrations in the stem cell population. Cell phenotype, adherent growth kinetics and tri-lineage differentiation were also evaluated. HMSC bone marrow aspirates, obtained from three approved donors, were expanded in parallel using T225 culture flasks and the Quantum. RESULTS: BM-hMSCs harvested from the Quantum demonstrated immunophenotype, morphology and tri-lineage differentiation capacity characteristics consistent with the International Society of Cell Therapy standard for hMSCs. Cell populations showed no malignant neoplastic formation in athymic mice 60 days post-transplant, no clonal chromosomal aberrations were observed and no DNA damage was found as measured by micronucleus formation. CONCLUSIONS: Quantum-produced BM-hMSCs are of comparable quality and demonstrate analogous genetic stability to BM-hMSCs cultured on tissue culture polystyrene substrates.

Evaluation of reagents used to coat the hollow-fiber bioreactor membrane of the Quantum® Cell Expansion System for the culture of human mesenchymal stem cells.

The addition of a coating reagent to promote cell adherence is necessary to prepare the membrane surface of the Quantum® Cell Expansion System hollow-fiber bioreactor for the culture of mesenchymal stem cells. In this study, the efficacy of 8 potential coating reagents has been compared in terms of the doubling times of their cell populations, cell morphology, characterization via flow cytometry, and capacity for trilineage differentiation. Human fibronectin (FN), pooled human cryoprecipitate (CPPT), and recombinant human vitronectin (VN) were successful as coating reagents, and each product has advantages in different cell culture contexts. Mesenchymal stem cells harvested from Quantum cultured with each of these 3 compounds as coating reagents all met International Society for Cellular Therapy standards for plastic adherence, surface marker expression, and successful trilineage differentiation. No significant differences were observed among the doubling times from Quantum harvests using FN, CPPT, or VN as coating reagents (P = 0.31). Coating with gelatin, human serum albumin, collagen I, poly­l­lysine, and poly­d­lysine resulted in significantly lower harvest yield; these agents are not recommended for use as coating reagents in the Quantum system.

Optimizing T Cell Expansion in a Hollow-Fiber Bioreactor.

PURPOSE OF REVIEW: Recent developments in regenerative medicine have precipitated the need to expand gene-modified human T cells to numbers that exceed the capacity of well-plate-based, and flask-based processes. This review discusses the changes in process development that are needed to meet the cell expansion requirements by utilizing hollow-fiber bioreactors. Maintenance of cell proliferation over long periods can become limited by unfilled demands for nutrients and oxygen and by the accumulation of waste products in the local environment. RECENT FINDINGS: Perfusion feeding, improved gas exchange, and the efficient removal of lactate can increase the yield of T cells from an average of 10.8E +09 to more than 28E +09 in only 10 days. SUMMARY: Aggressively feeding cells and actively keeping cells in the bioreactor improves gas exchange and metabolite management over semi-static methods. The ability to remove the environmental constraints that can limit cell expansion by using a two-chamber hollow-fiber bioreactor will be discussed.