Manufacturing autologous and allogeneic cell therapies can be challenging for various reasons. One of the biggest challenges to successful cell therapy processing is the manufacturing process itself. Because cell therapy manufacturing involves several steps, processing can become cumbersome and affect the quality of the final cell therapy product. Thus, optimizing manufacturing processes and workflows that support efficient manufacturing, decreases manual hands-on handling and, ultimately, the cost of producing cell therapies is crucial.
The Gibco™ CTS™ DynaCellect™ Magnetic Separation system is specifically designed to allow for the isolation of T cells using the CTS Dynabeads™ technology. In this study, we were interested in learning if washing thawed leukopak cells before the magnetic separation of T cells on the CTS DynaCellect system would affect T cell isolation efficiency, purity, and cell viability.
We compared these results to cells that were not washed prior to T cell isolation on the CTS DynaCellect system.
Methods overview:
A quarter leukopak containing PBMCs was thawed using the BioLife Thawstar®. Following thawing, the cells were washed on the Gibco™ CTS Rotea™Counterflow Centrifugation system and calibrated to a 1×106/mL cell density. Another quarter of thawed leukopak cells was diluted at a 1:1 ratio with DPBS and calibrated to a 1×106/mL cell density using the CTS DynaCellect system.
CTS Dynabeads CD3/CD28 beads were added to each portion of leukopak cells at a 3:1 bead-to-cell ratio using the CTS DynaCellect system. CD3+/CD28+ cells were isolated from each of these leukopak bags using the CTS DynaCellect system. The isolated T cells were cultured in G-Rex®100 bioreactor vessels containing CTS OpTmizer™ Pro Serum Free Medium supplemented with IL-2 for three days.
All data shown is representative of three independent experiments.
Data summary:
While there was a slightly higher, but not statistically significant, isolation efficiency for CD3+ and CD3+/CD28+ T cells when the cells were washed on day 0 (figure 1), there was no difference in the proportion of T cells (figure 2) from Day 0 through Day 2 post-isolation and upon removal of the CTS Dynabeads CD3/CD28 beads.
Isolation efficiency was calculated with the formula below after T cell isolation on the CTS DynaCellect system:
There was also no difference in the viability of T cells isolated between cells that were first washed using the CTS Rotea system and cells that were directly isolated on the CTS DynaCellect system (figure 3). Cell viability remained high (>90%) all through cell processing.
Furthermore, there were no significant differences in CD25 and CD69 activation marker expression between the washed and unwashed (directly isolated) cells on days 1 and 2 following T cell isolation and expansion (figure 4).
Figure 1: T cell isolation efficiencies for CD3+ and CD3+/CD28+ T cells following isolation on the CTS DynaCellect system.
Figure 2: T cell composition from day 0 of cell processing through day 2 when the CTS Dynabeads CD3/CD28 beads were removed.
Figure 3: Cell viability from day 0 of cell processing through day 2 when the CTS Dynabeads CD3/CD28 beads were removed.
Figure 4: CD69 and CD25 activation marker expression on T cells from day 0 to day 2.
Discussion and conclusions
Herein, we investigated if washing leukopak cells before T cell isolation on the CTS DynaCellect system would have an impact on cell viability, T cell purity, T cell isolation efficiency, as well as the expression of T cell activation markers. Our studies show that while washing leukopak cells before isolating T cells on the CTS DynaCellect system can slightly improve T cell isolation efficiency, it did not significantly alter T cell purity, viability, or T cell activation markers when those cells were activated and cultured for up to two days.
These results suggest that for cell therapy manufacturers who may absolutely need to wash their cell inputs prior to T cell isolation, they can do so with little concern that prior washing will reduce or affect the quality of T cells for downstream processes.
Furthermore, our results confirm already published data that shows excellent isolation efficiencies and T cell quality for direct CAR T cell processing and isolation using the CTS DynaCellect system.
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