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Natural killer (NK) cells have recently shown renewed promise as therapeutic cells for use in treating hematologic cancer indications. Despite this promise, NK cell manufacturing workflows remain largely manual, open, and disconnected, and depend on feeders, as well as outdated unit operations or processes, often utilizing research-grade reagents. Successful scale-up of NK cells critically depends on the availability and performance of nutrient-rich expansion media and cryopreservation conditions that are conducive to high cell viability and recovery post-thaw. In this paper we used Cytiva hardware and media to expand the NK92 cell line in a model process that is suitable for GMP and clinical manufacturing of NK cells. We tested a range of cryopreservation factors including cooling rate, a range of DMSO-containing and DMSO-free cryoprotectants, ice nucleation, and cell density. Higher post-thaw recovery was seen in cryobags over cryovials cooled in identical conditions, and cooling rates of 1°C/min or 2°C/min optimal for cryopreservation in DMSO-containing and DMSO-free cryoprotectants respectively. Higher cell densities of 5x107 cells/ml gave higher post-thaw viability than those cryopreserved at either 1x106 or 5x106 cells/ml. This enabled us to automate, close and connect unit operations within the workflow while demonstrating superior expansion and cryopreservation of NK92 cells. Cellular outputs and performance were conducive to clinical dosing regimens, serving as a proof-of-concept for future clinical and commercial manufacturing. [ABSTRACT FROM AUTHOR]

Background: Cryopreserved peripheral blood mononuclear cells (PBMCs) are frequently collected and provide disease- and treatment-relevant data in clinical studies. Here, we developed combined protein (40 antibodies) and transcript single-cell (sc)RNA sequencing (scRNA-seq) in PBMCs. Results: Among 31 participants in the Women's Interagency HIV Study (WIHS), we sequenced 41,611 cells. Using Boolean gating followed by Seurat UMAPs (tool for visualizing high-dimensional data) and Louvain clustering, we identified 50 subsets among CD4+ T, CD8+ T, B, NK cells, and monocytes. This resolution was superior to flow cytometry, mass cytometry, or scRNA-seq without antibodies. Combined protein and transcript scRNA-seq allowed for the assessment of disease-related changes in transcriptomes and cell type proportions. As a proof-of-concept, we showed such differences between healthy and matched individuals living with HIV with and without cardiovascular disease. Conclusions: In conclusion, combined protein and transcript scRNA sequencing is a suitable and powerful method for clinical investigations using PBMCs. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation tests before clinical use, the cells are cryopreserved to bridge the necessary evaluation time. Standard degranulation and chromium release cytotoxicity assays confirm the ability of cryopreserved NK cells to kill target cells. Here, we report that tumor cells embedded in a 3-dimensional collagen gel, however, are killed by cryopreserved NK cells at a 5.6-fold lower rate compared to fresh NK cells. This difference is mainly caused by a 6-fold decrease in the fraction of motile NK cells after cryopreservation. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function. Cryopreservation is standard protocol prior to using NK cells in immunotherapy. Here the authors show that cryopreservation substantially reduces the clinical utility of these cells owing to a defect in their motility, an effect that might account for failure to treat some cancers with NK cell immunotherapy. [ABSTRACT FROM AUTHOR]

Cytokine-induced killer (CIK) cells are an immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (HSCT) in acute leukemia or myelodysplastic syndrome (MDS) patients. Prompt and sequential administration of escalating cell doses improves the efficacy of CIK cell therapy without exacerbating graft vs. host disease (GVHD). This study addresses manufacturing-related issues and aimed to develop a time-, personal- and cost-saving good manufacturing process (GMP)-compliant protocol for the generation of ready-for-use therapeutic CIK cell doses starting from one unstimulated donor-derived peripheral blood (PB) or leukocytapheresis (LP) products. Culture medium with or without the addition of either AB serum, fresh frozen plasma (FFP) or platelet lysate (PL) was used for culture. Fresh and cryopreserved CIK cells were compared regarding expansion rate, viability, phenotype, and ability to inhibit leukemia growth. Cell numbers increased by a median factor of 10-fold in the presence of FFP, PL, or AB serum, whereas cultivation in FFP/PL-free or AB serum-free medium failed to promote adequate CIK cell proliferation (p < 0.01) needed to provide clinical doses of 1 × 106 T cells/kG, 5 × 106 T cells/kG, 1 × 107 T cells/kG, and 1 × 108 T cells/kG recipient body weight. CIK cells consisting of T cells, T- natural killer (T-NK) cells and a minor fraction of NK cells were not significantly modified by different medium supplements. Moreover, neither cytotoxic potential against leukemic THP-1 cells nor cell activation shown by CD25 expression were significantly influenced. Moreover, overnight and long-term cryopreservation had no significant effect on the composition of CIK cells, their phenotype or cytotoxic potential. A viability of almost 93% (range: 89–96) and 89.3% (range: 84–94) was obtained after freeze-thawing procedure and long-term storage, respectively, whereas viability was 96% (range: 90-97) in fresh CIK cells. Altogether, GMP-complaint CIK cell generation from an unstimulated donor-derived PB or LP products was feasible. Introducing FFP, which is easily accessible, into CIK cell cultures was time- and cost-saving without loss of viability and potency in a 10-12 day batch culture. The feasibility of cryopreservation enabled storage and delivery of sequential highly effective ready-for-use CIK cell doses and therefore reduced the number of manufacturing cycles. [ABSTRACT FROM AUTHOR]

Background: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. Methods: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 106 cells/mL to 1.67 × 106 cells/mL. Results: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 106 cells/mL. Conclusion: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C. [ABSTRACT FROM AUTHOR]