Cryopreservation of mesenchymal stem/stromal cells using a DMSO-free solution is comparable to DMSO-containing cryoprotectants: results of an international multicenter PACT/BEST collaborative study.

BACKGROUND AND AIM: An essential aspect of ensuring availability and stability of mesenchymal stem/stromal cells (MSCs) products for clinical use is that these cells are cryopreserved before individual infusion into patients. Currently, cryopreservation of MSCs involves use of a cryoprotectant solution containing dimethyl sulfoxide (DMSO). However, it is recognized that DMSO may be toxic for both the patient and the MSC product. In this Production Assistance for Cellular Therapies (PACT) and Biomedical Excellence for Safer Transfusion (BEST) Collaborative study, we compared a novel DMSO-free solution with DMSO containing cryoprotectant solutions for freezing MSCs. METHODS: A DMSO-free cryoprotectant solution containing sucrose, glycerol, and isoleucine (SGI) in a base of Plasmalyte A was prepared at the University of Minnesota. Cryoprotectant solutions containing 5-10% DMSO (in-house) were prepared at seven participating centers (five from USA, one each from Australia and Germany). The MSCs were isolated from bone marrow or adipose tissue and cultured ex vivo per local protocols at each center. The cells in suspension were frozen by aliquoting into vials/bags. For six out of the seven centers, the vials/bags were placed in a controlled rate freezer (one center placed them at -80°C freezer overnight) before transferring to liquid nitrogen. The cells were kept frozen for at least one week before thawing and testing. Pre- and post-thaw assessment included cell viability and recovery, immunophenotype as well as transcriptional and gene expression profiles. Linear regression, mixed effects models and two-sided t-tests were applied for statistical analysis. RESULTS: MSCs had an average viability of 94.3% (95% CI: 87.2-100%) before cryopreservation, decreasing by 4.5% (95% CI: 0.03-9.0%; P: 0.049) and 11.4% (95% CI: 6.9-15.8%; P< 0.001), for MSCs cryopreserved in the in-house and SGI solutions, respectively. The average recovery of viable MSCs cryopreserved in the SGI was 92.9% (95% CI: 85.7-100.0%), and it was lower by 5.6% (95% CI: 1.3-9.8%, P < 0.013) for the in-house solution. Additionally, MSCs cryopreserved in the two solutions had expected level of expressions for CD45, CD73, CD90, and CD105 with no significant difference in global gene expression profiles. CONCLUSION: MSCs cryopreserved in a DMSO-free solution containing sucrose, glycerol, and isoleucine in a base of Plasmalyte A had slightly lower cell viability, better recovery, and comparable immunophenotype and global gene expression profiles compared to MSCs cryopreserved in DMSO containing solutions. The average viability of MSCs in the novel solution was above 80% and, thus, likely clinically acceptable. Future studies are suggested to test the post-thaw functions of MSCs cryopreserved in the novel DMSO-free solution.

Aberrant GPA expression and regulatory function of red blood cells in sickle cell disease.

ABSTRACT: Glycophorin A (GPA), a red blood cell (RBC) surface glycoprotein, can maintain peripheral blood leukocyte quiescence through interaction with a sialic acid-binding Ig-like lectin (Siglec-9). Under inflammatory conditions such as sickle cell disease (SCD), the GPA of RBCs undergo structural changes that affect this interaction. Peripheral blood samples from patients with SCD before and after RBC transfusions were probed for neutrophil and monocyte activation markers and analyzed by fluorescence-activated cell sorting (FACS). RBCs were purified and tested by FACS for Siglec-9 binding and GPA expression, and incubated with cultured endothelial cells to evaluate their effect on barrier function. Activated leukocytes from healthy subjects (HS) were coincubated with healthy RBCs (RBCH), GPA-altered RBCs, or GPA-overexpressing (OE) cells and analyzed using FACS. Monocyte CD63 and neutrophil CD66b from patients with SCD at baseline were increased 47% and 27%, respectively, as compared with HS (P = .0017, P = .0162). After transfusion, these markers were suppressed by 22% and 17% (P = .0084, P = .0633). GPA expression in RBCSCD was 38% higher (P = .0291) with decreased Siglec-9 binding compared with RBCH (0.0266). Monocyte CD63 and neutrophil CD66b were suppressed after incubation with RBCH and GPA-OE cells, but not with GPA-altered RBCs. Endothelial barrier dysfunction after lipopolysaccharide challenge was restored fully with exposure to RBCH, but not with RBCSCD, from patients in pain crisis, or with RBCH with altered GPA. Pretransfusion RBCSCD do not effectively maintain the quiescence of leukocytes and endothelium, but quiescence is restored through RBC transfusion, likely by reestablished GPA-Siglec-9 interactions.

Acute pulmonary injury in hematology patients supported with pathogen-reduced and conventional platelet components.

ABSTRACT: Patients treated with antineoplastic therapy often develop thrombocytopenia requiring platelet transfusion, which has potential to exacerbate pulmonary injury. This study tested the hypothesis that amotosalen-UVA pathogen-reduced platelet components (PRPCs) do not potentiate pulmonary dysfunction compared with conventional platelet components (CPCs). A prospective, multicenter, open-label, sequential cohort study evaluated the incidence of treatment-emergent assisted mechanical ventilation initiated for pulmonary dysfunction (TEAMV-PD). The first cohort received CPC. After the CPC cohort, each site enrolled a second cohort transfused with PRPC. Other outcomes included clinically significant pulmonary adverse events (CSPAE) and the incidence of treatment-emergent acute respiratory distress syndrome (TEARDS) diagnosed by blinded expert adjudication. The incidence of TEAMV-PD in all patients (1068 PRPC and 1223 CPC) was less for PRPC (1.7 %) than CPC (3.1%) with a treatment difference of -1.5% (95% confidence interval [CI], -2.7 to -0.2). In patients requiring ≥2 PCs, the incidence of TEAMV-PD was reduced for PRPC recipients compared with CPC recipients (treatment difference, -2.4%; 95% CI, -4.2 to -0.6). CSPAE increased with increasing PC exposure but were not significantly different between the cohorts. For patients receiving ≥2 platelet transfusions, TEARDS occurred in 1.3% PRPC and 2.6% CPC recipients (P = .086). Bayesian analysis demonstrated PRPC may be superior in reducing TEAMV-PD and TEARDS for platelet transfusion recipients compared with CPC recipients, with 99.2% and 88.8% probability, respectively. In this study, PRPC compared with CPC demonstrated high probability of reduced severe pulmonary injury requiring assisted mechanical ventilation in patients with hematology disorders dependent on platelet transfusion. This trial was registered at www.ClinicalTrials.gov as #NCT02549222.