Adoptive T cell therapy is a rapidly progressing field, and optimized protocols for the scalable manufacture of T cells are essential to maximize the therapeutic potential of patient-derived T cells. A single administration of adoptive T cell therapy can require billions of expanded T cells. Large-scale production of human T cells for cellular therapy is a complex, multi-step process that presents many opportunities for optimization in order to obtain maximum yield while retaining desired end phenotype and function. In this Technical Bulletin, we highlight the importance of cell density on T cell expansion. Our findings suggest that maintaining T cells at lower cell densities improves T cell growth and viability, especially at early stages of cell expansion. With optimized culture protocols, up to 800-fold expansion of human T cells with > 85% cell viability was achieved over 10 - 14 days in static culture. This culture strategy can also promote overall cell growth using the Xuri™ Cell Expansion System W25 (GE Healthcare). The protocol described here can be easily implemented in a T cell expansion workflow to ensure optimal viability and total fold expansion.

To investigate the effects of early cell dilution on T cell expansion, T cells were activated with ImmunoCult™ Human T Cell Activator in ImmunoCult™-XF T Cell Expansion Medium supplemented with rhIL-2 at a cell density of 1 x 10⁶ cells/mL. On day 3, T cell culture volume was increased by 2-, 4-, 8-, 16-, or 32-fold by the addition of fresh ImmunoCult™-XF T Cell Expansion Medium supplemented with rhIL-2. On days 5 and 7, T cell culture volume was increased by 4-fold. Where indicated, T cells were transferred into a 2L Xuri™ Cellbag™ Bioreactors, and maintained in the Xuri™ Cell Expansion System W25 on day 5. T cells were harvested for analysis on day 10 or 11 as indicated.

Results

Effect of Early Cell Dilution on Human T Cell Expansion

To investigate the effect of cell density on human T cell expansion, we activated T cells on day 0 using ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator or ImmunoCult™ Human CD3/28 T Cell Activator. On day 3, we increased the total culture volume by 2-, 4-, 8-, 16- or 32-fold, and determined the effects on total expansion. The 4- and 8-fold culture volume increases resulted in the highest total fold expansion and cell viability without any significant effects on the activation phenotype of expanded cells (Figure 2).

Comparison of T Cell Activators

The 8-fold volume increase resulted in significantly higher cell expansion compared to the 4-fold volume increase for cells activated using ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (Figure 3A). On the contrary, the 8-fold volume increase did not cause a statistically significant increase in expansion compared to 4-fold volume increase for cells activated using ImmunoCult™ Human CD3/CD28 T Cell Activator (Figure 3B).

Recommended Human T Cell Expansion Protocol

The findings above have led us to revise our human T cell expansion protocol with optimized cell dilutions (Figure 3C). It is optimal to dilute cells by increasing volume 8-fold or maintaining cell density at 1 - 2.5 x 10 ⁵ cells/mL at day 3 post-activation. We further dilute the expanding cells by increasing culture volume 4-fold at days 5 and 7 post-activation.

Human T Cell Expansion Using the Xuri™ Cell Expansion System

Obtaining billions of T cells for cell therapy requires large volumes of cells to be expanded in a controlled environment. The optimized T cell expansion protocol above can be adapted for use with the Xuri™ Cell Expansion System W25 for T cell expansion in a closed system for cell therapy manufacturing.

T cells were initially seeded in cell culture flasks as outlined in Figure 1. On day 3 of culture, fresh medium was added to increase the total culture volume by 2-, 4-, or 8-fold. Cells were then transferred into a 2 L Xuri™ Cellbag™ Bioreactors on day 5. We found that the 8-fold culture volume increase on day 3 resulted in the highest total fold expansion (Figure 4).

Phenotype of Expanded T Cells

The T cell phenotype plays a critical role in the efficacy of cellular therapy products in addition to cell number. For example, studies in mouse models suggest that minimally differentiated T cells (naïve T cells, stem cell memory T cells, or central memory T cells) exhibit superior antitumor activity compared to highly differentiated T cell subsets.¹

The majority of T cells expanded using ImmunoCult™ reagents in static culture alone or in the Xuri™ Cell Expansion System had a central memory phenotype as determined by their co-expression of CD62L and CD45RO (Figure 5).

Discussion

The implication of having an optimized method to produce T cells for cell therapy is significant. As described in this Technical Bulletin, we have identified cell density following T cell activation as a key factor in optimizing T cell expansion.

We show that maintaining T cells at lower cell densities during early T cell expansion improves T cell growth and viability. Our study identifies day 3 as a key timepoint for culture volume adjustment when optimizing T cell expansion protocols. At day 3 post-activation, cultured T cells display high levels of cell activation, as indicated by the expression of CD25 and PD-1, but have not significantly expanded (Figure 2). We speculate that lowering cell density at this time point improves T cell expansion by increasing the amount of available nutrients and space per cell after they have received their activation signals. On the other hand, lowering cell density too much may reduce the concentration of autocrine survival factors or cell-cell contact necessary to support optimal fold expansion and cell viability.

One variable that needs to be taken into account when researchers optimize their cell expansion protocol is genetic modification. Producing T cells for therapy often requires genetic modification of the T cells, which can be achieved using viral vectors ² or gene-editing nucleases.³ How these methods may affect optimal culture dilutions remain to be investigated.

The protocol described here provides a basic framework for testing the effects of other variables that can affect T cell expansion, including starting cell populations (T cell subsets), cytokines, and growth factors. The optimal culture volume increases will vary depending on these variables, so we recommend that researchers find the culture volume increases that work best with their specific protocols. Ultimately, having a well-validated, fully optimized protocol can contribute to solving some of the major challenges of T cell therapy, including lack of standardization.

Optimized T Cell Activation and Expansion Reagents

The T cell culture reagents used in this Technical Bulletin were developed to enable T cell therapy research and contribute to bringing T cell therapy research from bench to bedside. Learn more: