Purpose of Review Cryopreservation and its associated freezing and thawing procedures–short “freeze-thawing”–are among the final steps in economically viable manufacturing and clinical application of diverse cellular therapeutics. Translation from preclinical proof-of-concept studies to larger clinical trials has indicated that these processes may potentially present an Achilles heel to optimal cell product safety and particularly efficacy in clinical trials and routine use. Recent Findings We review the current state of the literature on how cryopreservation of cellular therapies has evolved and how the application of this technique to different cell types is interlinked with their ability to engraft and function upon transfer in vivo, in particular for hematopoietic stem and progenitor cells (HSPCs), their progeny, and therapeutic cell products derived thereof. We also discuss pros and cons how this may differ for non-hematopoietic mesenchymal stromal/stem cell (MSC) therapeutics. We present different avenues that may be crucial for cell therapy optimization, both, for hematopoietic (e.g., effector, regulatory, and chimeric antigen receptor (CAR)-modified T and NK cell based products) and for non-hematopoietic products, such as MSCs and induced pluripotent stem cells (iPSCs), to achieve optimal viability, recovery, effective cell dose, and functionality of the cryorecovered cells. Summary Targeted research into optimizing the cryopreservation and freeze-thawing routines and the adjunct manufacturing process design may provide crucial advantages to increase both the safety and efficacy of cellular therapeutics in clinical use and to enable effective market deployment strategies to become economically viable and sustainable medicines.
Cell manufacturing facilities need to define the potency of Mesenchymal Stromal Cells (MSCs) as cellular therapeutics in advanced clinical trials or marketing approval. Since MSCs’ mechanism of actions in humans are not well defined, more than a single functional property of MSCs need to be captured as a surrogate measure of potency utilizing assay matrix technologies. However, the current limitation is the sole investigation of MSC mediated T cell suppression as a surrogate measure of potency. We investigated the effect of MSCs on B cell matrix responses to be incorporated into the assay matrix potency analytical system. Our results demonstrate that MSCs inhibit B cell differentiation and block pan-antibody secretion upon activation of B cells in the PBMCs. In contrast, MSCs are inferior in blocking B cell matrix responses when purified B cells are used. Mechanistic analysis has demonstrated that MSC mediated inhibition of B cell matrix responses are non-contact dependent and Tryptophan metabolic pathway plays a major role, akin to the mechanism of MSC mediated T cell suppression. MSCs also inhibit both T cell and B cell responses when both of these lymphoid populations are concurrently activated in the PBMCs. Secretome analysis of MSC and T/B cell activated PBMC cocultures identified direct and inverse correlative matrix signatures between humoral antibody isotypes and secretory molecules. The current analysis of the combined and concomitant investigation of T cell and B cell matrix responses fulfil the potency assay matrix strategy by incorporating MSCs’ interaction with more than a single inflammatory immune responder.
B7 family proteins serve as checkpoint molecules that protect tumors from T cell mediated lysis. Tryptophan degrading enzymes indoleamine 2,3 dioxygenase (IDO) and tryptophan 2,3 dioxygenase (TDO) also induce T cell immune tolerance. However, little is known about the relative contribution of B7 molecules, tryptophan degrading enzymes, as well as the impact of tumor and stromal cell interactions to the development of immunosuppressive tumor microenvironment. To investigate such interactions, we used a tripartite model of human hepatocellular carcinoma cell line (HepG2) and mesenchymal stromal cells (MSCs) co-cultured with peripheral blood mononuclear cells (PBMCs). Co-culture of HepG2 cells and activated PBMCs demonstrate that HepG2 cells undergo PBMC mediated cytolysis, despite constitutive expression of B7-H3 and upregulation of PD-L1 by IFNγ. Knockdown of B7-H3, PD-L1 or IDO does not modulate PBMC mediated lysis of HepG2 cells. However, TNFα preactivation enhances lysis of HepG2 cells, and blocking of TNFα production from PBMCs protects HepG2 cells. On the other hand, MSCs protect HepG2 cells from PBMC mediated lysis, even in the presence of TNFα. Further investigation showed that MSC mediated protection is associated with the unique secretome profile of upregulated and downregulated cytokines and chemokines. IFNγ activated MSCs are superior to TNFα activated or control MSCs in protecting HepG2 cells. Blockade of IFNγ driven IDO activity completely abolishes the ability of MSCs to protect HepG2 cells from cytolysis by PBMCs. These results suggest that inhibition of IFNγ activation of IDO induction in stromal cells, combined with usage of TNFα, could be a novel immunotherapeutic strategy to induce regression of hepatocellular carcinoma.
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