Hematology · Medical Literature
Impact of cryopreservation on PBMC-derived CAR-T cell manufacturing outcomes: a systematic review.
Abstract
Chimeric antigen receptor T-cell (CAR-T) therapy is an established treatment for several hematological malignancies, with peripheral blood mononuclear cells (PBMCs) serving as the starting material for manufacturing. Cryopreservation of PBMCs may offer logistical flexibility, although its influence on manufacturing outcomes remains incompletely defined. This review aimed to compare the effect of fresh versus cryopreserved PBMC starting material on CAR-T cell manufacturing outcomes, including viability, fold expansion, and transduction efficiency. A systematic review was conducted following PRISMA guidelines. PubMed and Google Scholar were searched from inception through March 2026 for original studies comparing fresh and cryopreserved PBMCs in human CAR-T cell manufacturing. Methodological quality was assessed using the design-appropriate quality-appraisal tools, and findings were synthesized narratively due to heterogeneity in study design and protocols. Five studies published between 2019 and 2025 met the inclusion criteria, comprising two clinical and three experimental analyses. Post-thaw viability and recovery of cryopreserved PBMCs ranged between 77% and 97%, slightly lower than fresh material. Fold expansion, transduction efficiency, and cytotoxic activity were generally comparable between groups, although some studies reported transient early differences including prolonged doubling times, mitochondrial dysfunction signals, and increased TIM-3 expression in cryopreserved-derived products. Cryopreservation can be considered a feasible approach in CAR-T manufacturing, with generally comparable outcomes despite early post-thaw cellular changes. These differences do not seem to consistently compromise the overall manufacturing performance. However, the current evidence remains limited and heterogeneous, and further studies are required to increase confidence in our initial findings.
Clinical relevance
Does the use of cryopreserved versus fresh peripheral blood mononuclear cells (PBMCs) as starting material adversely affect CAR-T cell manufacturing outcomes—specifically post-thaw viability, fold expansion, and transduction efficiency—in human CAR-T cell production? __SNAPSHOT__ {"studyDesign":"Systematic review (narrative synthesis); PRISMA-compliant; no meta-analysis performed due to study heterogeneity","setting":"Multinational; PubMed and Google Scholar searched from inception through March 2026; no single institutional setting","sampleSize":"5 included studies (2 clinical analyses, 3 experimental analyses); aggregate patient/sample numbers across studies not reported in abstract","population":"Studies involving human PBMC-derived CAR-T cell manufacturing comparing fresh versus cryopreserved PBMC starting material; published 2019–2025; hematological malignancy context (specific diagnoses not further detailed in abstract)","intervention":"Cryopreserved PBMCs used as CAR-T cell manufacturing starting material","comparator":"Fresh (non-cryopreserved) PBMCs used as CAR-T cell manufacturing starting material","primaryOutcome":"CAR-T cell manufacturing outcomes: post-thaw PBMC viability and recovery, fold expansion, and transduction efficiency","followUp":"Not applicable (manufacturing process endpoints); clinical follow-up duration of included studies not reported in abstract","fundingSource":"Not reported"}
Key findings
Across the 5 included studies, post-thaw viability and recovery of cryopreserved PBMCs ranged from 77% to 97%, described as slightly lower than fresh PBMC benchmarks (exact fresh PBMC viability comparator values not reported in the abstract). No absolute or relative effect sizes, confidence intervals, p-values, or NNT/NNH values are reported in the abstract for the primary manufacturing endpoints. Fold expansion and transduction efficiency outcomes are noted as investigated but specific quantitative results from the pooled evidence are not provided in the available abstract text. Findings were synthesized narratively rather than statistically, precluding calculation of pooled effect estimates. No clinically important adverse events are described in the abstract; safety endpoints were not a stated primary focus of the review. Methodological quality was assessed using design-appropriate tools, though quality scores for individual studies are not reported in the abstract.
Practice implications
This systematic review addresses a clinically meaningful manufacturing question: whether PBMC cryopreservation—which offers significant logistical flexibility for centralized CAR-T production, advance cell banking prior to intensive chemotherapy, and decoupling of leukapheresis timing from manufacturing—compromises the quality of the final cellular product. The abstract signals that post-thaw viability and recovery fall within a range (77–97%) that may be acceptable for manufacturing, though the lower bound (77%) approaches thresholds at which downstream manufacturing adequacy could be questioned. The review does not establish whether observed differences in starting material viability translate into clinically meaningful differences in final CAR-T product potency, in vivo expansion, or patient outcomes. No clinical response data (e.g., complete remission rates, overall survival) are reported in the abstract. The findings appear to apply to patients undergoing autologous CAR-T manufacturing for hematological malignancies, but disease-specific subgroup analyses, patient-level covariates (e.g., prior treatment burden, lymphocyte count), and long-term cryostorage duration effects cannot be assessed from the abstract alone. Patients with heavily pretreated disease—who may have the lowest baseline T-cell quantity and quality—are not separately characterized. Because only an abstract is available, the full quality appraisal scores, individual study-level data, and any sensitivity analyses remain inaccessible; conclusions must therefore be treated as preliminary pending full-text review. __LIMITATIONS__ Extremely small evidence base: only 5 studies published over a 6-year period (2019–2025) met inclusion criteria, severely limiting the power and generalizability of any synthesis.,Heterogeneity in study design (2 clinical, 3 experimental) and manufacturing protocols (vector types, activation methods, cryoprotectants, storage durations) precluded meta-analysis and formal quantitative pooling.,Narrative synthesis is inherently susceptible to qualitative bias; no statistical weighting of study quality or sample size is possible.,Post-thaw viability range (77–97%) spans a clinically meaningful range; the lower bound may not reliably support successful downstream manufacturing, yet subgroup analysis by viability threshold is not reported.,Clinical patient outcomes (response rates, progression-free survival, overall survival, cytokine release syndrome rates) are not reported as endpoints, limiting translation to bedside practice.,Variability in cryopreservation protocols (cryoprotectant type, cooling rate, storage duration, thaw methodology) across studies introduces major confounding that cannot be adjusted for in a narrative synthesis.,Selection bias inherent to included studies; experimental analyses may use healthy donor PBMCs rather than heavily pretreated patient-derived material, limiting applicability to the intended clinical population.,Surrogate manufacturing endpoints (viability, fold expansion, transduction efficiency) may not correlate with clinical CAR-T potency, persistence, or efficacy in vivo.,Funding source not reported; potential industry influence on included primary studies cannot be assessed.,Only an abstract was available for this analysis; full methodology, individual study data tables, quality appraisal scores, and sensitivity analyses were not accessible. __BOTTOM_LINE_CATEGORY__ promising_but_preliminary __BOTTOM_LINE__ This PRISMA-compliant systematic review of 5 studies suggests that cryopreserved PBMCs can serve as viable starting material for CAR-T manufacturing, with post-thaw viability ranging 77–97%, though this is slightly lower than fresh PBMC benchmarks. The review is limited by a small, heterogeneous evidence base, reliance on narrative synthesis, and the absence of clinical efficacy or safety outcome data. Current evidence is insufficient to recommend cryopreserved PBMCs as equivalent to fresh PBMCs for all manufacturing contexts; this question warrants prospective, protocol-standardized comparative studies with patient-level clinical endpoints before formal practice change.