- Use of Gas Permeable Rapid Expansion (G-Rex®) Culture ware with Chemically Defined Media for Simplistic and Robust T Lymphocyte Expansion
Use of Gas Permeable Rapid Expansion (G-Rex®) Culture ware with Chemically Defined Media for Simplistic and Robust T Lymphocyte Expansion
Published on 7 November 2021
G-Rex-based Expansion of T Lymphocytes
Randy Alfano, PhD, VP Product Development, InVitria
As immune cell therapies continue to show promising clinical efficacy, advancements in manufacturing platforms must continue in parallel in order to enhance process control, definition, and ultimately, process yield. The single use Gas Permeable Rapid Expansion (G-Rex) vessel offers a practical, robust, and cost-effective solution for immune cell expansion scale up and scale out. Published studies have demonstrated that processes established in early phases of product development easily translate to large scale production with minimal changes to the cell product.1 Furthermore, due the unique design of the G-Rex vessel, large media volumes can be employed to minimize vessel manipulations and reduce labor required. Finally, application of the G-Rex has been shown to be highly reproducible from donor to donor in both expansion kinetics and resulting expanding phenotype.2
However, the degree of process control and reproducibility also heavily depends not only on the expansion vessel used but also the growth media inside the vessel. Most cell culture media used for the expansion of immune cells for clinical use either contain a low percentage of human serum or components purified from human serum, such as albumin and transferrin. Serum albumin, for example, enables the significant enhancement of cell growth and viability of mammalian cells cultured ex vivo. Given the central functional role of albumin (both in vivo and invitro) and its high concentration in cell culture media, inconsistencies in the quality of the raw material can greatly affect the overall function of the protein.3
InVitria leverages an animal component free protein expression platform to produce recombinant proteins at large scale that are critical for cell culture applications, including human serum albumin and transferrin.4,5 These animal-free components were used to formulate a completely chemically defined formulation specific for human T Lymphocyte expansion. This blood-free formulation, known as OptiPEAK T Lymphocyte, was paired with the G-Rex and compared to the standard xeno-free formulation XViVo-15 supplemented with 5% human serum.
The data presented here demonstrate that the chemically defined formulation exhibited robust expansion kinetics of human T cells isolated form peripheral blood of healthy donors in the G-Rex. Furthermore, the expanded cells harvested for the G-Rex exhibited high viability and equivalent phenotype to the XViVo formulation. Taken together, these data provide proof of principal that the G-Rex vessels can be used with chemically defined and blood-free media to enhance the productivity of the cell culture process and the quality and reproducibility of the expanded cell product.
G-Rex-based Expansion of T Lymphocytes
Peripheral blood from healthy donors were obtained from a commercial source (ZenBio, Research Triangle Park, NC). All blood samples were processed at a maximum of 48 hours post collection. To isolate Lymphocytes, PBMC were isolated from whole blood using a Lymphoprep gradient with 1.077 ± 0.001 g/mL density (StemCell Technologies, Vancouver, BC) and subsequently washed with basal RPMI1640 (Life Technologies, Grand Island, NY) to remove all traces of donor serum.
PBMC were seeded in T-flasks at a density of 1 x 106 cells/mL in the presence of 100 ng/mL αCD3, 50 ng/mL αCD28 (Miltenyi Biotech, Bergisch Gladbach, Germany) and 10 ng/mL rhIL-2 (PeproTech, Rocky Hill, NJ) for three days in either OptiPEAK T Lymphocyte (InVitria, Junction City, KS) or XViVo-15 (Lonza, Walkersville, MD) with 5% Human Serum (Sigma Aldrich, St. Louis, MO) at 37 °C and 5% CO2.
Activated cells were subsequently harvested by centrifugation and resuspended in a minimum volume of each medium supplemented with 10 ng/mL rhIL-2 and counted with a pre-gated flow cytometer using Calcein AM-based flow cytometry for viable cell quantification (Accuri, BD Biosciences, East Rutherford, NJ). For seeding activated T cells into G-Rex10 vessels (Wilson Wolf, St Paul, MN), 4 mL media (4 mL/cm2) was equilibrated at 37 °C and 5% CO2 for at least two hours prior to seeding cells. Activated T cells were seeded at an initial cell density of 1-5 x 105 cells/cm2. At 24 hours post seed, G-Rex vessels were fed by adding the additional 36 mL of growth media.
Samples were pulled throughout the growth phase for glucose measurements as well as cell counting. Cell counting was performed with a pre-gated flow cytometer using Calcein AM-based flow cytometry for viable cell quantification.
The same process was followed for G-Rex100 vessels.
Post expansion, cells were collected from the G-Rex vessels and spun down to remove all traces of growth media. Cells were resuspended in DPBS supplemented with 10% FBS, 0.1% F-68 (Thermo Fisher, Waltham, MA). They were then stained with mouse APC α human CD3, mouse PE α human CD4 (BD Biosciences, East Rutherford, NJ), and mouse Pe-Cy7 α human CD8 (Biorbyt, St. Louis, MO) for 30 minutes at room temperature. Cells were analyzed on a pre-gated flow cytometer (Accuri, BD Biosciences, East Rutherford, NJ).
Difference of means was determined using a student’s unpaired t-test with a 95% confidence interval (CI).
G-Rex-based Expansion of T Cells in Chemically Defined Conditions Exhibit Robust Growth Kinetics
To establish expansion kinetics of T cells in serum free conditions using the G-Rex, T cells activated with αCD3/αCD28 soluble antibodies were seeded at 1-5 x 105 cells/cm2 in G-Rex10 vessels in OptiPEAK T Lymphocyte or the standard XViVo-15 + 5% human male AB serum from ten independent donors for seven days as described in the methods section.
T cells in both media exhibited robust expansion kinetics in the G-Rex with cell growth being detected within 48 hours post seed of the vessel (Figure 1). XViVo supplemented with human serum exhibited an average of 33.64 ± 13.02-fold expansion amongst the donors expanded. OptiPEAK T Lymphocyte exhibited similar expansion kinetics. Donors expanded for seven days exhibited a 36.90 ± 11.98-fold expansion (p ≥ 0.05). After seven days of expansion, donors exhibited variable expansion behaviors. Select donors continued to expand to upwards of 50-60-fold while other donors ceased growth (data not shown).
Viabilities were also determined via flow cytometry of the expanded cell product. Cells expanded in XViVo were found to be 85.81 ± 8.69% viable while OptiPEAK demonstrated a slight, but statistically insignificant, increase at 90.54 ± 8.05% viability.
To determine the scalability of the chemically defined media, the larger G-Rex100 was evaluated with two donors. The expansion kinetics were compared between OptiPEAK T Lymphocyte and XViVO-15 + 5% HS. The G-Rex100 vessels represent an order of magnitude increase in cell culture surface over the G-Rex10 vessels. The G-Rex100 also has a higher volume of growth media to surface area ratio (5 mL/cm2 compared to the G-Rex10’s 4 mL/cm2).
Donors were activated and seeded in the G-Rex100 in the same fashion as the G-Rex10. As expected, the donors exhibited robust expansion kinetics in the G-Rex100s. An increase in cell density was detected as early as 48 hours post vessel seeding with final expansion kinetics being slightly higher than what was observed in the G-Rex10s.
Donors expanded in XViVo-15 + 5% HS exhibited 50.64 ± 5.52 fold expansion. Cells expanded in OptiPEAK T Lymphocyte were slightly higher at 57.81 ± 9.10 fold expansion (Figure 2).
Phenotypes of the expanded cell products were obtained via flow cytometry. Total CD3, CD4, and CD8 was measured. CD4/CD8 ratios of expanded cell products between the two media were found to vary from donor, as expected. However, no significant differences were demonstrated in CD4/CD8 ratios of the expanded cell product between the two media (Table 1 and Figure 3).
The use of the G-Rex offers multiple benefits in a clinical manufacturing setting for the rapid and robust production of T Cells. These vessels offer large media holding capacity to minimize feedings during the expansion process. In addition, these vessels possess a small footprint and are compatible with standard tissue culture incubators which can simplify implementation. However, most T cell expansion media currently used in the G-Rex still rely on either human serum or serum-derived components, such as albumin and transferrin, to achieve commercially viable expansion kinetics.
Removal of serum or serum-derived components is essential in a manufacturing setting due to risk of contamination from pathogenic adventitious agents, supply chain constraints due to high demand and limited global supply, and unintended changes to the cell product due variable degree of composition observed in serum.6 Currently, regulatory agencies are encouraging the use of non-animal derived substitutes.
It is demonstrated that the chemically defined and blood-free OptiPEAK T Lymphocyte exhibits equivalent T cell expansion kinetics, viability, and phenotype compared to the popular XViVo-15 + 5% human serum using a simplified seeding and expansion protocol in the G-Rex vessel. There was no phenotypic change observed with the chemically defined medium, suggesting that recombinant proteins can achieve equivalent functionality with T cells.
These observations provide a proof of principal that chemically defined T cell expansion processes in the G-Rex can eliminate serum and blood-derived components from the process.
- Xiao L, et al. “Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor-modified effector cells.” Cytotherapy, 20(3): 420-435, 2018.
- Grau-Vorster M, et al. “Characterization of a Cytomegalovirus-Specific T Lymphocyte Product Obtained Through a Rapid and Scalable Production Process for Use in Adoptive Immunotherapy.” Front Immunol, 25;11:271, 2020.
- Miyamura M, et al. “Comparison of Posttranslational Modification and the Functional Impairment of Human Serum Albumin in Commerical Preparations.” J Pharm Sci, 105, 3: 1043-1049, 2016.
- Pennybaker A, A. R. “Utilization of Recombinant Albumins in the Expansion of Human T Lymphocytes.” Cell Gene Ther Insights, 4, 4:231-239, 2018.
- Zhang D., et al. “Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.).” BMC Biotechnol, 12: 92, 2012
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