Assessment of stored red blood cells through lab-on-a-chip technologies for precision transfusion medicine.

Transfusion of red blood cells (RBCs) is one of the most valuable and widespread treatments in modern medicine. Lifesaving RBC transfusions are facilitated by the cold storage of RBC units in blood banks worldwide. Currently, RBC storage and subsequent transfusion practices are performed using simplistic workflows. More specifically, most blood banks follow the "first-in-first-out" principle to avoid wastage, whereas most healthcare providers prefer the "last-in-first-out" approach simply favoring chronologically younger RBCs. Neither approach addresses recent advances through -omics showing that stored RBC quality is highly variable depending on donor-, time-, and processing-specific factors. Thus, it is time to rethink our workflows in transfusion medicine taking advantage of novel technologies to perform RBC quality assessment. We imagine a future where lab-on-a-chip technologies utilize novel predictive markers of RBC quality identified by -omics and machine learning to usher in a new era of safer and precise transfusion medicine.

Emerging Biomarkers for Monitoring Chimeric Antigen Receptor T-Cell Therapy.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy has revolutionized treatment of hematologic malignancies and holds promise for solid tumors. While responses to CAR T-cell therapy have surpassed other available options for patients with refractory malignancies, not all patients respond the same way. The reason for this variability is not currently understood. Therefore, there is a strong need to identify characteristics of patients as well as cellular products that lead to an effective response to CAR T-cell therapy. CONTENT: In this review, we discuss potential biomarkers that may predict clinical outcomes of CAR T-cell therapy. Based on correlative findings from clinical trials of both commercially available and early-phase products, we classify biomarkers into categories of pre- and post-infusion as well as patient and product-related markers. Among the biomarkers that have been explored, measures of disease burden both pre- and post-infusion, as well as CAR T-cell persistence post-infusion, are repeatedly identified as predictors of disease response. Higher proportions of early memory T cells at infusion appear to be favorable, and tracking T-cell subsets throughout treatment will likely be critical. SUMMARY: There are a growing number of promising biomarkers of CAR T-cell efficacy described in the research setting, however, none of these have been validated for clinical use. Some potentially important predictors of response may be difficult to obtain routinely under the current CAR T-cell therapy workflow. A collaborative approach is needed to select biomarkers that can be validated in large cohorts and incorporated into clinical practice.

Local manufacturing processes contribute to variability in human mesenchymal stromal cell expansion while growth media supplements contribute to variability in gene expression and cell function: a Biomedical Excellence for Safer Transfusion (BEST) collaborative study.

BACKGROUND AIMS: Culture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods, source material and culture media. The purpose of this multicenter study was to assess the impact on MSC expansion, gene expression and other characteristics when different laboratories expanded MSCs from cultures initiated with bone marrow-MSC aliquots derived from the same donor source material yet with different growth media. METHODS: Eight centers expanded MSCs using four human platelet lysate (HPL) and one fetal bovine serum (FBS) products as media supplements. The expanded cells were taken through two passages then assessed for cell count, viability, doubling time, immunophenotype, cell function, immunosuppression and gene expression. Results were analyzed by growth media and by center. RESULTS: Center methodologies varied by their local seeding density, feeding regimen, inoculation density, base media and other growth media features (antibiotics, glutamine, serum). Doubling times were more dependent on center than on media supplements. Two centers had appropriate immunophenotyping showing all MSC cultures were positive for CD105, CD73, CD90 and negative for CD34, CD45, CD14, HLA-DR. MSCs cultured in media supplemented with FBS compared with HPL featured greater T-cell inhibition potential. Gene expression analysis showed greater impact of the type of media supplement (HPL versus FBS) than the manufacturing center. Specifically, nine genes were decreased in expression and six increased when combining the four HPL-grown MSCs versus FBS (false discovery rate [FDR] <0.01), however, without significant difference between different sources of HPL (FDR <0.01). CONCLUSIONS: Local manufacturing process plays a critical role in MSC expansion while growth media may influence function and gene expression. All HPL and FBS products supported cell growth.

Assessment of the LOVO device for final harvest of novel cell therapies: a Production Assistance for Cellular Therapies multi-center study.

BACKGROUND AIMS: The final harvest or wash of a cell therapy product is an important step in manufacturing, as viable cell recovery is critical to the overall success of a cell therapy. Most harvest/wash approaches in the clinical lab involve centrifugation, which can lead to loss of cells and decreased viability of the final product. Here the authors report on a multi-center assessment of the LOVO Cell Processing System (Fresenius Kabi, Bad Homburg, Germany), a cell processing device that uses a spinning filtration membrane instead of centrifugation. METHODS: Four National Institutes of Health Production Assistance for Cellular Therapies cell processing facilities (CPFs) assessed the LOVO Cell Processing System for final harvest and/or wash of the following three different cell products: activated T cells (ATCs), tumor-infiltrating lymphocytes (TILs) and bone marrow-derived mesenchymal stromal cells (MSCs). Each site compared their current in-house, routinely used method of final cell harvest and/or wash with that of the LOVO device. RESULTS: Final harvest and/or wash of ATCs, TILs and MSCs using the LOVO system resulted in satisfactory cell viability and recovery with some substantial improvement over the in-house methods of CPFs. Processing time was variable among cell types/facilities. CONCLUSIONS: The LOVO Cell Processing System provides an alternative to centrifuge-based technologies. The system employs a spinning membrane filter, exposing cells to minimal g-forces compared with centrifugation, and is automated and closed. This small multi-center study demonstrated the ability of the LOVO device to yield satisfactory cell viability and recovery of T cells and MSCs.

Healthy versus inflamed lung environments differentially affect mesenchymal stromal cells.

BACKGROUND: Despite increased interest in mesenchymal stromal cell (MSC)-based cell therapies for acute respiratory distress syndrome (ARDS), clinical investigations have not yet been successful and our understanding of the potential in vivo mechanisms of MSC actions in ARDS remains limited. ARDS is driven by an acute severe innate immune dysregulation, often characterised by inflammation, coagulation and cell injury. How this inflammatory microenvironment influences MSC functions remains to be determined. AIM: The aim of this study was to comparatively assess how the inflammatory environment present in ARDS lungs versus the lung environment present in healthy volunteers alters MSC behaviour. METHODS: Clinical-grade human bone marrow-derived MSCs (hMSCs) were exposed to bronchoalveolar lavage fluid (BALF) samples obtained from ARDS patients or from healthy volunteers. Following exposure, hMSCs and their conditioned media were evaluated for a broad panel of relevant properties, including viability, levels of expression of inflammatory cytokines, gene expression, cell surface human leukocyte antigen expression, and activation of coagulation and complement pathways. RESULTS: Pro-inflammatory, pro-coagulant and major histocompatibility complex (self-recognition) related gene expression was markedly upregulated in hMSCs exposed ex vivo to BALF obtained from healthy volunteers. These changes were less apparent and often opposite in hMSCs exposed to ARDS BALF samples. CONCLUSION: These data provide new insights into how hMSCs behave in healthy versus inflamed lung environments, and strongly suggest that the inflamed environment in ARDS induces hMSC responses that are potentially beneficial for cell survival and actions. This further highlights the need to understand how different disease environments affect hMSC functions.

Comparative analysis of cell therapy infusion workflows at clinical sites.

BACKGROUND AIMS: Cell therapies are an emerging treatment option for a variety of diseases, especially with the success of chimeric antigen receptor T-cell therapies. With 18 FDA-approved cell therapy products as of December 2020 and a growing number in clinical trials, standards for most aspects of the cell therapy lifecycle are well-established by professional organizations like AABB and FACT; however, there are limited standardized protocols regarding the day-of infusion. METHODS: Infusions were observed at three academic medical centers in the United States, and the workflows were analyzed and compared based on factors including facility layout, product verification processes, cryobag design, timing restrictions, and use of electronic medical records. RESULTS: Variations between the facilities were identified with product thawing location and cell therapy lab location being the most important factors in time from thaw to infusion. CONCLUSIONS: Based on this analysis, opportunities were identified for standardization and streamlining the infusion workflow which may help facilitate adoption of new and existing cell therapies at a wider range of hospitals.

Infusion reactions in natural killer cell immunotherapy: a retrospective review.

BACKGROUND AIMS: The use of natural killer (NK) cells as a cellular immunotherapy has increased over the past decade, specifically in patients with hematologic malignancies. NK cells have been used at the authors' institution for over 15 years. Most patients have a reaction to NK cell infusion. The authors retrospectively analyzed the reactions associated with NK cell infusions to characterize the types of reactions and investigate why some patients have higher-grade reactions than others. METHODS: A retrospective chart review of NK cell infusions was performed at the authors' institution under nine clinical protocols from 2008 to 2016. An infusion reaction was defined as any symptom from the time of NK cell infusion up to 4 h after infusion completion. The severity of infusion reactions was graded based on Common Terminology Criteria for Adverse Events, version 4. Two major endpoints of interest were (i) infusion reaction with any symptom and (ii) grade ≥3 infusion reaction. Multivariable logistic regression models were used to investigate the association between variables of interest and outcomes. Odds ratios (ORs) and 95% confidence intervals (CIs) were obtained for each variable. RESULTS: A total of 130 patients were receiving NK cell infusions at the authors' institution. The most common reported symptom was chills (n = 110, 85%), which were mostly grade 1 and 2, with only half of patients requiring intervention. There were 118 (91%) patients with infusion reactions, and only 36 (28%) were grade 3. There was one life-threatening grade 4 reaction, and no death was reported due to infusion reaction. Among grade ≥3 reactions, cardiovascular reactions (mainly hypertension) were the most common, and less than half of those with hypertension required intervention. NK cell dose was not associated with any of the grade 3 infusion reactions, whereas monocyte dose was associated with headache (grade ≤3, OR, 2.17, 95% CI, 1.19-3.97) and cardiovascular reaction (grade ≥3, OR, 2.13, 95% CI, 1.13-3.99). Cardiovascular reaction (grade ≥3) was also associated with in vitro IL-2 incubation and storage time. Additionally, there was no association between grade ≥3 infusion reactions and overall response rate (OR, 0.75, 95% CI, 0.29-1.95). CONCLUSIONS: The majority of patients who receive NK cell therapy experience grade 1 or 2 infusion reactions. Some patients experience grade 3 reactions, which are mainly cardiovascular, suggesting that close monitoring within the first 4 h is beneficial. The association of monocytes with NK cell infusion reaction relates to toxicities seen in adoptive T-cell therapy and needs further exploration.

Differential effects of the cystic fibrosis lung inflammatory environment on mesenchymal stromal cells.

Growing evidence demonstrates that human mesenchymal stromal cells (MSCs) modify their in vivo anti-inflammatory actions depending on the specific inflammatory environment encountered. Understanding this better is crucial to refine MSC-based cell therapies for lung and other diseases. Using acute exacerbations of cystic fibrosis (CF) lung disease as a model, the effects of ex vivo MSC exposure to clinical bronchoalveolar lavage fluid (BALF) samples, as a surrogate for the in vivo clinical lung environment, on MSC viability, gene expression, secreted cytokines, and mitochondrial function were compared with effects of BALF collected from healthy volunteers. CF BALF samples that cultured positive for Aspergillus sp. (Asp) induced rapid MSC death, usually within several hours of exposure. Further analyses suggested the fungal toxin gliotoxin as a potential mediator contributing to CF BALF-induced MSC death. RNA sequencing analyses of MSCs exposed to either Asp+ or Asp- CF BALF samples identified a number of differentially expressed transcripts, including those involved in interferon signaling, antimicrobial gene expression, and cell death. Toxicity did not correlate with bacterial lung infections. These results suggest that the potential use of MSC-based cell therapies for CF or other lung diseases may not be warranted in the presence of Aspergillus.

Evaluation of post-thaw CFU-GM: clinical utility and role in quality assessment of umbilical cord blood in patients receiving single unit transplant.

BACKGROUND: The CFU assay is considered the only in vitro assay that assesses the biologic function of hematopoietic stem and progenitor cells (HSPC). STUDY DESIGN AND METHODS: To investigate the impact of post-thaw CFU-GM counts on the quality of umbilical cord blood (UCB), we studied transplant outcomes in 269 patients receiving single UCB transplant. We also correlated the post-thaw CFU-GM counts of 1912 units with the pre-freeze and post-thaw graft characteristics, hoping to optimize selection criteria of UCB. Data analysis included: total nucleated cells, viability, CD34+, nucleated red blood cells (NRBC), hematocrit, frozen storage time, and cord blood bank (CBB). RESULTS: We demonstrated an association between post-thaw CFU-GM dose and the speed of neutrophil and platelet engraftment (p < 0.01). Higher post-thaw CFU-GM dose showed an increased benefit for neutrophil and platelet engraftment (p < 0.01). Post-thaw CD34+ cell dose and CFU-GM dose were strongly correlated (r = 0.78). However, CFU-GM dose showed additional benefit for patients receiving the lowest quartile of CD34+ dose. HLA disparity did not adversely impact either neutrophil or platelet engraftment. Post-thaw CFU-GM/million nucleated cells plated showed moderate correlation with pre-freeze and post-thaw CD34+ and weak correlation with other parameters. Post-thaw CFU-GM was not influenced by storage time, but was impacted by the CBB from which the unit is obtained (p < 0.01). CONCLUSION: Post-thaw CFU-GM is an effective measure of the quality and efficacy of the UCB graft, particularly adding valuable clinical information when the CD34+ cell dose is low. Consideration of pre-freeze CD34+ cell content and CBB as additional selection criteria is warranted.

A multicenter evaluation of heterogeneity in cellular therapy processing laboratory procedure times to assess workload capacity.

BACKGROUND: Growth in size and complexity of clinical hematopoietic progenitor cell (HPC) transplant programs necessitates parallel increases in cellular therapy laboratory (CTL) workload. Typically individually developed, HPC product processing is labor and time intensive. Variation in procedure type and numbers across CTLs complicates direct comparisons, and benchmark data are not readily available. STUDY DESIGN AND METHODS: Studies were undertaken at seven CTLs. Transplant volume and staff numbers were determined. Staff recorded time performing tasks broken down into steps: paperwork, product acceptance, transport/infusion, processing, and cryopreservation. Times were added to obtain total times for 15 common CTL procedures. RESULTS: Annual transplant volume ranged from 53.4 to 463.2, with products processed by a range of 2 to 10 dedicated CTL staff. Paperwork time constituted 23.7% to 62.3% total time; product processing time accounted for 1.8 (for National Marrow Donor Program product receipt) to 62.6% (for red blood cell reduction of allogeneic HPC products from bone marrow) of total processing time. Mean time for 15 procedures ranged from 1.27 to 8.28 hours (standard deviation range, 0.35-2.71 hr). Mean time for products from bone marrow versus peripheral blood was 6.6 ± 2.0 versus 5.5 ± 1.1 hours (p = 0.02). Cryopreservation (6.5 ± 1.6 vs. 4.4 ± 0.85 hr; p < 0.01) and manipulation (6.4 ± 1.5 vs. 4.4 ± 0.85 hr; p < 0.01) added time. CONCLUSION: CTL procedures are time intensive, with wide intra- and inter-CTL variation. Paperwork accounted for substantial portion of total time across procedures. Bone marrow source, cryopreservation, and manipulation contributed to longer times. These findings provide concrete data on which to build regarding CTL workload capacity.

Lung inflammatory environments differentially alter mesenchymal stromal cell behavior.

Mesenchymal stromal (stem) cells (MSCs) are increasingly demonstrated to ameliorate experimentally induced lung injuries through disease-specific anti-inflammatory actions, thus suggesting that different in vivo inflammatory environments can influence MSC actions. To determine the effects of different representative inflammatory lung conditions, human bone marrow-derived MSCs (hMSCs) were exposed to in vitro culture conditions from bronchoalveolar lavage fluid (BALF) samples obtained from patients with either the acute respiratory distress syndrome (ARDS) or with other lung diseases including acute respiratory exacerbations of cystic fibrosis (CF) (non-ARDS). hMSCs were subsequently assessed for time- and BALF concentration-dependent effects on mRNA expression of selected pro- and anti-inflammatory mediators, and for overall patterns of gene and mRNA expression. Both common and disease-specific patterns were observed in gene expression of different hMSC mediators, notably interleukin (IL)-6. Conditioned media obtained from non-ARDS BALF-exposed hMSCs was more effective in promoting an anti-inflammatory phenotype in monocytes than was conditioned media from ARDS BALF-exposed hMSCs. Neutralizing IL-6 in the conditioned media promoted generation of anti-inflammatory monocyte phenotype. This proof of concept study suggest that different lung inflammatory environments potentially can alter hMSC behaviors. Further identification of these interactions and the driving mechanisms may influence clinical use of MSCs for treating lung diseases.

Preservation of cell-based immunotherapies for clinical trials.

In the unique supply chain of cellular therapies, preservation is important to keep the cell product viable. Many factors in cryopreservation affect the outcome of a cell therapy: (i) formulation and introduction of a freezing medium, (ii) cooling rate, (iii) storage conditions, (iv) thawing conditions and (v) post-thaw processing. This article surveys clinical trials of cellular immunotherapy that used cryopreserved regulatory, chimeric antigen receptor or gamma delta T cells, dendritic cells or natural killer (NK) cells. Several observations are summarized from the given information. The aforementioned cell types have been similarly frozen in media containing 5-10% dimethyl sulfoxide (DMSO) with plasma, serum or human serum albumin. Two common freezing methods are an insulated freezing container such as Nalgene Mr. Frosty and a controlled-rate freezer at a cooling rate of -1°C/min. Water baths at approximately 37°C have been commonly used for thawing. Post-thaw processing of cryopreserved cells varied greatly: some studies infused the cells immediately upon thawing; some diluted the cells in a carrier solution of varying formulation before infusion; some washed cells to remove cryoprotective agents; and others re-cultured cells to recover cell viability or functionality lost due to cryopreservation. Emerging approaches to preserving cellular immunotherapies are also described. DMSO-free formulations of the freezing media have demonstrated improved preservation of cell viability in T lymphocytes and of cytotoxic function in natural killer cells. Saccharides are a common type of molecule used as an alternative cryoprotective agent to DMSO. Improving methods of preservation will be critical to growth in the clinical use of cellular immunotherapies.

Umbilical cord blood-derived T regulatory cells to prevent GVHD: kinetics, toxicity profile, and clinical effect.

We studied the safety and clinical outcomes of patients treated with umbilical cord blood (UCB)-derived regulatory T cells (Tregs) that expanded in cultures stimulated with K562 cells modified to express the high-affinity Fc receptor (CD64) and CD86, the natural ligand of CD28 (KT64/86). Eleven patients were treated with Treg doses from 3-100 × 10(6) Treg/kg. The median proportion of CD4(+)FoxP3(+)CD127(-) in the infused product was 87% (range, 78%-95%), and we observed no dose-limiting infusional adverse events. Clinical outcomes were compared with contemporary controls (n = 22) who received the same conditioning regimen with sirolimus and mycophenolate mofetil immune suppression. The incidence of grade II-IV acute graft-versus-host disease (GVHD) at 100 days was 9% (95% confidence interval [CI], 0-25) vs 45% (95% CI, 24-67) in controls (P = .05). Chronic GVHD at 1 year was zero in Tregs and 14% in controls. Hematopoietic recovery and chimerism, cumulative density of infections, nonrelapse mortality, relapse, and disease-free survival were similar in the Treg recipients and controls. KT64/86-expanded UCB Tregs were safe and resulted in low risk of acute GVHD.

Optimal Storage Conditions for Apheresis Research (OSCAR): a Biomedical Excellence for Safer Transfusion (BEST) Collaborative study.

BACKGROUND: Cell therapy products are often stored and transported between sites. The aim of this study was to determine the effect of storage temperature, solution, and cell concentration on nonmobilized, peripheral blood-derived mononuclear cells (MNCs). STUDY DESIGN AND METHODS: This was a multicenter prospective study involving healthy volunteers who underwent nonmobilized MNC collection by apheresis. Products were processed at local laboratories and concentrated to either 100 × 106 or 300 × 106 nucleated cells/mL in 5% human serum albumin (HSA) or HypoThermosol FRS (HT; BioLife Solutions). Products were stored at room temperature (RT; 20-25°C) or refrigerated temperatures (2-8°C) with assessment at 0, 24, 48, and 72 hours. NC and MNC concentration, viability, and flow cytometric analysis for CD3, CD4, CD8, CD14, CD19, CD25, and CD56 were measured. RESULTS: Viability decreased over time for all conditions tested. Refrigerated storage preserved viability greater than RT storage, especially for products with a higher cell concentration. RT maintenance with a high cell concentration was associated with a relative loss of CD14- and CD4-positive cells, whereas the concentration of cells positive for other markers tested did not vary. Finally, there was delayed decrease in pH when using HT compared with HSA; however, there was no difference in viability between the two solutions. CONCLUSION: Low cell concentrations (approx. 100 × 106 cells/mL), refrigerated temperatures, and HT storage solution appear to be the optimal conditions for storing nonmobilized, peripheral blood-derived MNC products.

Clinical-scale production of cGMP compliant CD3/CD19 cell-depleted NK cells in the evolution of NK cell immunotherapy at a single institution.

BACKGROUND: Allogeneic natural killer (NK) cell adoptive immunotherapy is a growing therapeutic option for patients. Clinical-scale production of NK cells using immunomagnetic selection complies with current good manufacturing practices (cGMPs) and allows for closed-system, automated purification. We report our experience with CD3/CD19 cell-depleted (CD3/CD19dep ) NK cell production and compare to previous methods of CD3 cell depletion and CD3 cell depletion/CD56 cell enrichment. STUDY DESIGN AND METHODS: Nonmobilized mononuclear cells collected by apheresis were incubated with anti-CD3/anti-CD19 microbeads and depleted in an automated cell selection system (CliniMACS, Miltenyi). The NK cell-enriched products were incubated overnight in interleukin (IL)-2 or IL-15, washed, and resuspended prior to lot release testing and infusion. RESULTS: Since 2010, 94 freshly infusible CD3/CD19dep NK cell products were manufactured in support of eight clinical trials. Sixty-six products were incubated in IL-2 and 28 products in IL-15. Processing resulted in a mean NK cell recovery of 74% and viability of 95.8%; NK cells, T cells, B cells, and monocytes accounted for 47%, 0.2%, 0.08%, and 49% of the final products, respectively. Seven products required dose adjustments to meet lot release. The specification for purity changed throughout the evolution of manufacturing. IL-2 or IL-15 activation enhanced in vitro cytotoxicity compared to preactivated cells. There was no difference in final product composition or cytotoxicity between cytokine cohorts. CONCLUSION: Clinical-scale/cGMP production of NK cells using CD3/CD19 cell-depletion effectively minimized T-cell and B-cell contamination in a single manipulation without compromise to NK-cell recovery. Cytokine activation increased in vitro cytotoxicity compared to column-depleted, preactivated NK cells.

Current practices for viability testing of cryopreserved cord blood products: an international survey by the cellular therapy team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.

BACKGROUND: Viability testing is a common practice in laboratories. The goal of this study was to ascertain current laboratory practices internationally for performing viability testing for cryopreserved cord blood (CB) products and glean information about how to standardize the method to improve interlaboratory reproducibility. STUDY DESIGN AND METHODS: A survey to evaluate current laboratory practices for viability testing was designed and distributed internationally. The question topics included sampling and testing methods, responses to unexpected results, and the rating of the reliability of the CB quality tests, together with expectations for standardization. RESULTS: There were 32 respondents to the survey, of whom 28 responded to the more detailed questionnaire about viability methods. Overall, responses indicated that various stains were used among the laboratories, and when multiple sites used the same viability stain the methods differed. The majority of the respondents were in favor of standardizing the viability testing methods. A wide variety of preferences were communicated about how to standardize the method, but a majority did advocate the use of 7-aminoactinomycin D (7-AAD) with flow cytometry. CONCLUSIONS: The survey results revealed a variety of tests and inconsistent interlaboratory practices for performing the viability assay. Flow cytometry with a 7-AAD dye was suggested as a first step toward standardization.

Impact of Delayed Infusion Time in Umbilical Cord Blood Transplantation.

In umbilical cord blood (UCB) transplantation, UCB units are typically thawed, washed, and infused into the patient as rapidly as possible. In some instances there is a delay in the time from the unit thaw and wash procedure to infusion into the patient. Therefore, we examined the effect of thaw duration time on engraftment outcomes in 567 patients undergoing UCB transplantation. With a range of 32 to 523 minutes, a prolonged thaw duration had no obvious effect on the incidence of neutrophil engraftment or time to recovery. This was true for recipients of single UCB transplantation (incidence: 97% versus 93%, P = .13; time to neutrophil recovery: 21 days versus 21 days, P = .32; and platelet recovery: 79% versus 78%, P = .48), and similar results were observed in double UCB transplantation (time to neutrophil engraftment: 20 days versus 19 days, P = .71). However, there was a trend toward better platelet recovery in recipients of double UCB transplants with prolonged thaw duration (HR, 1.28; P = .06). In conclusion, this study demonstrates prolonged thaw duration has no detrimental effect on engraftment after single or double UCB transplantation.

Optimization of cGMP purification and expansion of umbilical cord blood-derived T-regulatory cells in support of first-in-human clinical trials.

BACKGROUND AIMS: Thymic-derived regulatory T cells (tTreg) are critical regulators of the immune system. Adoptive tTreg transfer is a curative therapy for murine models of autoimmunity, graft rejection, and graft-versus-host disease (GVHD). We previously completed a "first-in-human" clinical trial using in vitro expanded umbilical cord blood (UCB)-derived tTreg to prevent GVHD in patients undergoing UCB hematopoietic stem cell transplantation (HSCT). tTreg were safe and demonstrated clinical efficacy, but low yield prevented further dose escalation. METHODS: To optimize yield, we investigated the use of KT64/86 artificial antigen presenting cells (aAPCs) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture. RESULTS: aAPCs increased UCB tTreg expansion greater than eightfold over CD3/28 stimulation. Re-stimulation with aAPCs increased UCB tTreg expansion an additional 20- to 30-fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following current Good Manufacturing Practice (cGMP) validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared with that obtained with anti-CD3/28 mAb coated-bead expansion and Foxp3 expression was stable during in vitro expansion and following transfer to patients. Increased expansion did not result in a senescent phenotype and GVHD was significantly reduced. DISCUSSION: Expansion culture with cGMP aAPCs and re-stimulation reproducibly generates sufficient numbers of UCB tTreg that exceeds the numbers of T effector cells in an UCB graft. The methodology supports future tTreg banking and is adaptable to tTreg expansion from HSC sources. Furthermore, because human leukocyte antigen matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease.