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Effect of Cryopreservation on Autologous Chimeric Antigen Receptor T Cell Characteristics

ABSTRACT: As clinical applications for chimeric antigen receptor T cell (CART) therapy extend beyond early phase trials, commercial manufacture incorporating preservation steps becomes a logistical necessity. The effect of cryopreservation on CART characteristics is unclear. We retrospectively evaluated the effect of cryopreservation on product release criteria and in vivo characteristics in 158 autologous CART products from 6 single-center clinical trials. Further, from 3 healthy donor manufacturing runs, we prospectively identified differentially expressed cell surface markers and gene signatures among fresh versus cryopreserved CARTs. Within 2 days of culture initiation, cell viability of the starting fraction (peripheral blood mononuclear cells [PBMNCs]) decreased significantly in the cryo-thawed arm compared to the fresh arm. Despite this, PBMNC cryopreservation did not affect final CART fold expansion, transduction efficiency, CD3%, or CD4:CD8 ratios. In vivo CART persistence and clinical responses did not differ among fresh and cryopreserved final products. In healthy donors, compared to fresh CARTs, early apoptotic cell-surface markers were significantly elevated in cryo-thawed CARTs. Cryo-thawed CARTs also demonstrated significantly elevated expression of mitochondrial dysfunction, apoptosis signaling, and cell cycle damage pathways. Cryopreservation during CART manufacture is a viable strategy, based on standard product release parameters. The clinical impact of cryopreservationrelated subtle micro-cellular damage needs further study

ORGANISM(S): Homo sapiens

PROVIDER: GSE158929 | GEO | 2020/10/03

REPOSITORIES: GEO

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Project description:Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. The advent of a microfluidics-based multi-omics approaches to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. challenges to cell-based research include site to- site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh (Samples labeled as FRE) or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO, Samples labeled as FBS), FBS/DMSO after a DNase step (a step often included in epigenetic studies, samples labeled as DNA), and cryopreservation using commercially available Recovery©media (Samples labeled as REC). In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further, demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

Project description:Study question: Does storage time impact on transcriptome of slowly frozen cryopreserved human metaphase II (MII) oocytes? Summary answer: For the first time, we demonstrate that the length of cryostorage has no effect on the gene expression profile of human metaphase II oocytes. What is known already: Oocyte cryopreservation is a largely-used technique in IVF for storage of surplus oocytes, as well as for fertility preservation (i.e., women undergoing gonadotoxic therapies) and oocyte donation programs. Although it is known that cryopreservation negatively impacts on oocyte physiology and it is associated with decrease of transcripts, no experimental data about the effect of storage time on the oocyte molecular profile are available to date. Study design, size, duration: This study included 27 women undergoing IVF treatment, < 38 years aged, without any ovarian pathology. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed. Participants/materials, setting, methods: Pools of ?10 oocytes for each group were prepared. Total RNA was extracted from each pool, amplified, labeled and hybridized on 4x44K v2 microarrays (Agilent). Analyses were performed by R v3.1.3 software using the limma package v3.22.7. Main results and the role of chance: Comparison of gene expression profiles between surviving thawed oocytes after 3 and 6 years of storage in liquid nitrogen found no differently expressed genes. The expression profiles of cryopreserved MII oocytes significantly differed from those of non-cryopreserved oocytes in 107 probe sets corresponding to 73 down-regulated and 29 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics resource disclosed that cryopreservation deregulates genes involved in oocyte function and early embryo development, such as chromosome organization, RNA splicing and processing, cell cycle process, response to DNA damage stimulus, cellular response to stress and DNA repair, calcium ion binding, malate dehydrogenase activity, mitochondrial membrane respiratory chain. Among the probes significantly up-regulated in cryopreserved oocytes, 2 corresponded to ovary-specific expressed large intergenic noncoding (linc)RNAs. This study included 27 women undergoing IVF treatment, < 38 years aged, without any ovarian pathology. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed.

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Effect of Cryopreservation on Autologous Chimeric Antigen Receptor T Cell Characteristics

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