The efficiency and efficacy of the formulation of cell ** and the manufacturing process are affected by cryofreezing, so it is necessary to understand the impact of cryopreservation on cells for the successful commercial production of cell **. In this study, the investigators investigated the effects of some process parameters (CPPs) on the survival rate after thawing, post-activation expansion, and cytokine secretion of human PAN CD3+ T cells to evaluate the importance of these parameters during cryopreservation.
The CPPS parameters in this study included cryopreservation solution formulation, DMSO content, post-thaw (pre-dilution wash) stability, and activation timeline, and activity was assessed by membrane integrity, proliferation rate, and post-activation INFY secretion to characterize the cell and cryopreservation process.
· Cell culture
Cells are seeded in 6-well plates by thawing of human PAN CD3+ T cells, 1 h after thawing, cells are activated with anti-CD3 CD28 CD2 T cell activator, and the cell culture plates are placed in an incubator at 37 °C and 5% CO2 for 10 days, and the cells are removed from the incubator to collect cells for formulation and cryopreservation.
· Cryopreservation solution selection
Four different cryopreservation solutions, Cryostor CS5, Cryostor CS10 and 2 common lab-made cryopreservation solutions. For cryopreserved T cells. Two homemade formulations contain Normosol-R or Plasmalyte (PLA) and the same components, 5% W V recombinant human serum albumin and 10% V DMSO (BloodStor 100, BioLife Solutions).
· Cryopreservation
Remove the medium containing the cells from the incubator and collect in a 15 ml centrifuge tube. Remove each set of samples prior to freezing to assess count and viability. T cell pellets are spheroided and resuspended in cryopreservation solution. Distribute in 2 ml cryovials in a 1 ml volume and incubate the cells at 2-8C for 15 min. The vials are then moved to a programmed cooler to be cooled at -1 °C min and manually nucleated at 10 °C. After reaching -70 °C, transfer the vials to LN2 for storage for at least 24 h.
· Defrosting and evaluation
Samples are thawed in a 37 °C water bath and then resuspended 1:10 in pre-warmed XF T cell culture medium. Samples were collected for viability and counting measurements immediately after thawing. Each cell group is seeded in 2 wells of a conventional 6-well plate, one well for the resting control, and one well for assessing activation. After thawing, anti-CD3 CD28 CD2 activation reagents were added for activation. At this point, transfer the 6-well plate to an incubator with 37 and 5% CO2 for the following series of studies
1- To study the effect of resting prior to activation, a set of T cell samples were cryopreserved in Cryostor CS5 and activated after resting for 24 h in the post-thaw culture.
2- To investigate the effect of DMSO concentrations, two groups of T cell samples cryopreserved in CryoStor CS5 and CryoStor CS10 with 5% and 10% v DMSO, respectively, were compared.
3- To investigate the necessity of a cell resting period prior to cell treatment after thawing, a set of samples is left at room temperature for 1 h immediately after thawing prior to further processing.
For all samples, at 24 h and 72 h post-thaw, a small piece was removed from each well to assess viability, count (Chemometec NC-3000), and detect Infy secretion (ELISA).
1. Research on the influence of ready-to-use commercially available CS10 and self-made formula cryopreservation solution
Among the three different groups with 10% V V with similar DMSO content, the CS10 group recovered viability more quickly than the other two home-made cryopreservation solutions, which was close to that of the unfrozen T cell control group.
After T cell activation, the viability of T cells decreased in all groups, and the cell viability of the CS10 group at 72 h after thawing was closest to that of the unfrozen control group, and was also statistically higher than that of the other two homemade cryopreservation groups.
Overall, a comparison of the 3 groups of cryopreservation solutions using 10% DMSO showed little difference in viability between thawed and activated viability. However, at 72 h after thawing, there were significant differences in amplification and INFY secretion between the three groups. The number of cells cryopreserved in CryoStor CS10 increased to the same level as the unfrozen control over 72 h, which was higher than the number of cells cryopreserved in Normosol HSA and PLA HSA cryopreservation solution in the other two groups. The same was true for the INFY comparison: secretion levels were significantly higher in the CS10 group than in the other groups, and only slightly lower than in the unfrozen control group.
2. Research on the influence of DMSO concentration
The effect of increasing DMSO concentrations in cryopreservation solution formulations on cell cryopreservation was investigated by comparing the effects of Cryostor CS5 and CS10 formulations (5% and 10% V DMSO, respectively).
It can be seen from Figure 2 that the viability of the CS10 group at 72 h after thawing was lower than that of the activated CS5 group, but the cell expansion rate of the CS10 group was significantly higher than that of the CS5 group, and the secretion of INFY was also significantly higher than that of the CS5 group. In addition, it must be mentioned that in terms of cell viability and expansion potential, the performance of cryopreserved cells at lower DMSO concentrations (Cryostor CS5 at 5% V v) is comparable to that of lab-made cryopreservation with 10% V DMSO. CS5 cryopreserved cells secrete more INFY compared to a homemade formulation of PLA containing 10% v DMSO.
3. Research on the necessity of cell resting period before cell treatment after thawing
Comparison of 3 different scenarios of activation after T cell thawing:
1) Treat and activate immediately after thawing.
2) Wait (1 hour) after thawing for further processing.
3) Activated and incubated 24 h after treatment.
As can be seen from the figure, activation results in a loss of viability within a short period of time after the cells are thawed. Therefore, it is hypothesized that ensuring that there is a cell resting period after thawing (24 h in this case) prior to activation may be beneficial in reducing cell loss and increasing amplification rate.
However, post-thaw stability is a risk concern in cell manufacturing and practice, and it is advisable to know how long cells remain stable after thawing in advance before washing or patient administration. The results of this study suggest that waiting 1 h after thawing at room temperature (20-25 °C) has a greater effect on cell expansion capacity as well as INFY secretion compared to samples treated immediately after thawing, and interestingly, cell viability remained statistically unchanged in the 1 h waiting group, so viability alone is not sufficient to assess the effect of these process parameters on T cell quality. In addition, it was observed that 24 h of incubation in the culture medium after thawing and prior to activation did not necessarily reduce the loss of cell viability after activation (Figure 2).
Compared to traditional lab-made cryopreservation solutions, CryoStor is a more reliable formulation of human CD3+ T cell cryopreservation, i.e., human CD3+ T cells cryopreserved in serum-free, protein-free CryoStor CS10 cryopreservation solution compared to the other two test formulations, Normosol-R + 5% W V RhSA + 10% V V DMSO and Plasmalyte-A + 5% W V Rhsa + 10% V v DMSO, Shows enhanced or at least equivalent quality and functionality after thawing.
In addition, the performance of cells cryopreserved at lower DMSO concentrations (5% VV) was comparable to that of lab-made cryopreservation solutions using 10% VV DMSO, suggesting that the use of cryopreservation solutions such as cryostor formulations is a potential way to reduce DMSO content in products.
Higher concentrations of DMSO (10% Vv) appear to be more effective at preserving human T cells than lower concentrations of DMSO (5% Vv) compared to Cryostor's two cryopreservation products.
The expansion rate and INFY secretion of resting cultures were significantly lower than those in the immediate cell activation group at the same post-thaw time, suggesting that it may be more beneficial to activate T cells immediately after thawing in order to optimize manufacturing time.
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