Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Good manufacturing practice production of an off-the-shelf CD34 + progenitor-derived NK cell product with preserved anti-tumor functionality post-infusion in NOD/SCID/IL2Rg null mice

doi: 10.1007/s00018-025-05727-4

Figure Lengend Snippet: High post-thawing viability and proliferative capacity of G-rex expanded HSPC-NK cells cryopreserved using an optimized freezing and thawing protocol A Overview of the GMP-compliant generation of NK cells from HSPCs in a G-rex scalable bioreactor using six standardized medium additions. B Fold expansion at day 35. C Absolute number of CD34 + HSPCs at day 0 and CD56 + HSPC-NK cells at day 35. D Composition of the HSPC-NK cell product. E Trypan blue-based post-thawing viability of HSPC-NK cells frozen in cryobags. F Recovery of viable cells post-thawing compared to pre-freezing dosage of bag-frozen cells. G HSPC-NK cell dosage post-thawing. H Stability of thawed HSPC-NK cells in CryoStor CS10 for a 30-min timespan. I Post-thawing viability following twelve months of storage in the liquid nitrogen vapor phase, assessed using cryovials. Stability study ongoing till five years post-freezing. J Absolute number of viable fresh or cryopreserved HSPC-NK cells cultured up to seven days in 50 ng/ml IL-15. K Proliferation score, defined as the fold change reduction in MFI of Proliferation Dye eFluor450 compared to day zero, of fresh and cryopreserved HSPC-NK cells following seven days of culture in 50 ng/ml IL-15. Donor 4 (▼) involves a non-GMP compliant pre-validation run. For donor 3 (∎), t = 0 represents t = 15 ( E - G ) and t = 30 represents t = 45 ( G ) due to technical issues. Bars in B, D-G, K represent the mean. Significance was evaluated using the Wilcoxon test for H, Friedman test with Dunn’s multiple comparison correction for I, and a two-way ANOVA with Šídák multiple comparison correction for K; no statistical significances were found

Article Snippet: Afterwards, cells were cryopreserved in either room temperature (RT) or 4 °C pre-cooled CryoStor CS10 freeze medium (BioLife Solutions, 210102) at a concentration of 10-100 × 10 6 cells/ml in Cryotube vials (cryovials; ThermoFisher Scientific, 363401 or Greiner Bio-One, 126263) or CryoMACS freezing bags 50 (Miltenyi Biotech, 200-074-400).

Techniques: Cell Culture, Biomarker Discovery, Comparison