Total Nucleated Cell Viability Research Articles

Effect of temporary storage of cryopreserved cellular therapy products at −80⁰ celsius on cell recovery and viability

Cellular therapy (CT) involving the transplantation of hematopoietic progenitor cells (HPC) is a treatment modality for both benign and malignant disorders. All autologous products require cryopreservation while allogeneic product cryopreservation became more common during the Coronavirus disease 2019 pandemic. Cells are stored in liquid nitrogen (LN2) freezers which can malfunction and products may have to be temporarily stored in a mechanical −80 °C freezer if additional LN2 freezer space is not available. The practice of temporary short-term −80 °C storage is present but there is no study to show that the product is unaffected by the temporary storage at a significantly warmer temperature. In this study, we identified previously collected CT products that were cryopreserved for now-deceased recipients that had remaining cryovials with aliquots of products for quality control purposes. Vials from 20 collections were split into 4 groups of 5 in with one vial placed in temporary storage at −80 °C for 2–5 weeks before returning to LN2 storage while another vial remained in LN2 storage for the entire duration of the study. The vials were then simultaneously thawed, processed, and evaluated for total nucleated cell (TNC) and CD34 + cell count and TNC and CD34 + cell viability to determine if there were any differences induced by temporary −80 °C storage. No statistically significant differences were seen after 4 weeks of −80 °C storage; however, after 5 weeks, a statistically significant decrease in TNC viability and viable TNC count, but not CD34 + cell viability and viable CD34 + cell count was observed. These results provide some reassurance to CT processing labs that if there is a failure in their LN2 storage for cryopreserved products, these products may be safely stored at −80 °C for up to 4 weeks and returned to LN2 storage without compromising CD34 + cell viability.

A critical assessment of dose effects of post-thaw CD34 on autologous stem cell transplantation treatment of haematological malignancies.

A consensus threshold of pre-cryopreservation CD34-positive cells (CD34s) has been used as the minimum dose to initiate autologous stem cell transplantation (ASCT). Advances in cryopreservation posed a debate whether post-thaw CD34s might be a superior surrogate instead. We addressed the debate in this retrospective study of 217 adult ASCTs in five different haematological malignancies treated at a single centre. We showed that post-thaw CD34s was highly correlated with pre-cryopreservation CD34s (r=0.97) and explained ∼2.2% (p=0.003) of the variation of the post-thaw total nucleated cell viability that however had no power to predict engraftment outcomes. After stratifying the ASCT cases into four dose groups based on post-thaw CD34s reinfused, stepwise multivariate regression analyses detected significant effects in dose group and interactions with diseases for neutrophil and platelet recovery respectively. The significant dose effects and interactions were triggered by two technical outliers in the low dose group, and disappeared in the repeated regressions after exclusion of the outliers where disease and age were the significant predictors remained. Our data clearly support the validity of the consensus threshold in ASCT applications but also highlight neglected conditions where monitoring post-thaw CD34s and clinical attributes are valuable.

The Viability of Hematopoietic Progenitor Cell Grafts after Cryopreservation Does Not Predict Delayed Engraftment in Allogeneic Hematopoietic Stem Cell Transplantation.

Due to the COVID-19 pandemic, more peripheral blood stem cell (PBSC) allogeneic grafts are being frozen and infused thawed. Our objective was to study the influence of graft viability on engraftment outcome in patients treated with PBSCs. Using trypan blue stain, we compared total nucleated cell (TNC) viability of both fresh and thawed grafts in allogeneic PBSCs. The viability of thawed PBSC grafts median was 74%, and fresh was 99.0%. The median number of CD34 + cells/kg infused thawed was 6.3 × 106/kg and median time to neutrophil and platelet engraftment was 17.5 and 20 days. Median number of CD34 + cells/kg infused fresh was 5.1 × 106/kg and median time to neutrophil and platelet engraftment was 18 and 19 days. There were no statistically significant differences in the time to engraftment between the 2 groups. A low TNC viability of thawed PBSC grafts does not have an effect on time to neutrophil and platelet engraftment when more than 2.85 × 106 CD34 + cells/kg are infused.

Validation of automated fluorescent-based technology for measuring total nucleated cell viability of hematopoietic progenitor cell products.

A reliable rapid method for measuring total nucleated cell (TNC) viability is essential for cell-based products manufacturing. The trypan blue (TB) exclusion method, commonly used to measure TNC viability of hematopoietic progenitor cell (HPC) products, is a subjective assay, typically uses a microscope, and includes a limited number of cells. The NucleoCounter NC-200 is an automated fluorescent-based cell counter that uses pre-calibrated cartridges with acridine orange and DAPI dyes to measure cell count and viability. This study describes the validation of the NC-200 for testing HPC's viability. Samples from 189 fresh and 60 cryopreserved HPC products were included. Fresh products were tested for viability after collection by both TB and NC-200. 7-aminoactinomycin D (7AAD) CD45+ cell viability results were obtained from a flow cytometry test. Cryopreserved products thawed specimens were tested for viability by both TB and NC-200. The NC-200 viability results were compared with the other methods. Acceptability criteria were defined as ≤10% difference between the NC-200 method and the other methods for at least 95% of the samples. Fresh products' mean viability difference between NC-200 and TB or 7AAD CD45+ method was 4.9% (95%CI 4.6-5.4) and 2.8% (95%CI 2.2-3.4), respectively. Thawed products' mean viability difference between NC-200 and TB was 3.0% (95%CI 0.4-5.6). The NC-200 automated fluorescent-based method can be used effectively to determine HPC's viability for both fresh and cryopreserved products. It can help eliminate human bias and provide consistent data and operational ease.

Effects of gestational diabetes mellitus on the quality and quantity of blood hematopoietic stem cells: a case-control study

AimTo evaluate the effects of gestational diabetes mellitus (GDM) on the quantity and quality of hematopoietic stem cells (HSC).MethodsIn this case-control study, HSC were isolated from umbilical cord blood (UCB) procured at delivery from 63 mothers with GDM and 67 healthy mothers. Total nucleated cells (TNC) and CD34+ cells were quantified using BD FACSCalibur flow cytometer. The quantity and quality of stem cells were determined.ResultsThe GDM group had lower total cord blood volume and lower number of nucleated HSC compared with healthy mothers. Regarding stem cell quantity parameters, they had significantly lower UCB volume (P = 0.041), TNC count (P = 0.022), total viable NC count (P = 0.014), and CD34+ percentage (P = 0.014). Regarding the quality of stem cells, they had significantly lower viable TNC percentage (P = 0.015). The predictors for total TNC count were longer labor duration (adjusted B coefficient [p]: 0.031 [0.046]), greater estimated blood loss (0.089 [0.005]), female neonates (12.322 [0.049]), and higher placenta weight (0.080 [0.033]). The predictors of total viable NC count were greater estimated blood loss (0.092 [0.003]), female neonates (13.16 [0.035]), and greater placenta weight (0.083 [0.026]).ConclusionThe GDM group had much lower quantity and quality of UCB stem cells. Our results should be taken into consideration when drawing cord blood for unrelated stem cell banking in an obstetric unit to ensure the obtaining of optimal cord blood samples and to avoid unnecessary expenses.

Monitoring of Stored Hematopoietic Stem/Progenitor Graft Stability Program in a Single Institute

Cryopreservation of hematopoietic stem/progenitor cell (HPC) grafts and other cell based products to be infused in the future is a common practice. However, cryopreservation and conditions of storage can influence post thaw recovery of cellular products. HPC graft is composed of heterogeneous blood cells. Various assays detect viability based on different principles and their sensitivity differs. Less than 1 in 100,000 CD34+ cells possess in vivo hematopoietic reconstitution potential, therefore determination of viability and recovery need to be interpreted accordingly. We analyzed total 38 graft aliquots stored in liquid nitrogen (auto 10 yrs = 7, Auto 1 yr = 12, allo 10 yrs = 5, Allo 5 yrs = 5, allo 1 yr = 9). The viable CD34+ cell frequency in allo HPC grafts was higher than auto grafts cryopreserved 10 years ago (Auto 10 yrs = 0.39 ± 0.24%, Allo 10 yrs = 0.96 ±0.31%, Allo 5 yrs = 0.82 ± 0.3%, p = 0.018). When we compared both single and dual ISHAGE platform methods it displayed comparable post thaw viable CD34+ cell numbers (Figure 1A). The recovery of viable total nucleated cells (TNC) based on trypan blue dye exclusion was 46.83± 4.98% auto 10 years, 43.11 ± 7.76% allo 10 years, 48.74 ± 4.94% allo 5 years, 87.56 ± 6.93% allo 1 year. The viable CD34+ cell recovery between the groups were comparable (Figure 1B). Auto HPC grafts stored for more than 10 years displayed 64.05±16% while auto grafts stored less than a year yielded 85.4±15.15% viable CD34 recovery. The effects of storage duration on grafts is presented below by monitoring hematopoietic engraftment after transplantation. Interestingly, auto grafts in comparison to allo grafts stored for 5 to10 years displayed higher red fraction (JC-1); indicative of higher mitochondrial membrane potential (MMP) and viability (Auto 10 yrs = 72.24 ± 12.45%, Allo 10 yrs = 40.6 ± 13.92%, Allo 5 ysr = 47.2 ± 15.36%, p = 0.011). The higher fraction of cells with intact MMP (red fraction) within CD34+ cell compartment of post thaw suggesting likely higher resilience of auto grafts. Samples stored up to10 years displayed positive colony forming unit (CFU) growth but had poor CFU recovery. CFU recovery for HPC grafts stored for less than 1 year displayed 82.93 ± 25.68% for auto grafts while allo graft displayed 72.46 ± 34.76%. HPC grafts stored for 5 to 10 years which were utilized here to determine graft stability were transplanted within two months of storage and displayed timely neutrophil and platelet engraftment. When single ISHAGE platform method was used to enumerate viable CD34+ cells and HPC grafts are thawed within one year, viable CD34 recovery for auto HPC graft was 85.4 ± 15.15% and for allo graft recovery was 86.85 ±18.0%. Four patients which were transplanted (CD34+ cells infused mean ± SD, 4.35 ± 1.43 x 10e6/KG body weight) with autologous grafts stored for 5 to 8 years in liquid nitrogen (vapor phase) displayed timely neutrophil (9.5 ± 1 day) and platelet (16.5 ± 5 day) engraftment. Taken together, viability assays detect various aspects of cell integrity. The post thaw viable CD34 recovery corresponds well with in vivo hematopoietic reconstitution but in some instances functional potency assays particularly for samples stored longer than 5 years may be crucial. The relatively higher resilience of auto HPC grafts stored for longer than10 years is of note which will need to be further validated. Disclosures Patel: Celgene: Speakers Bureau; Janssen: Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

Abbreviated T-Cell Activation on the Automated Clinimacs Prodigy Device Enhances Bispecific CD19/22 Chimeric Antigen Receptor T-Cell Viability and Fold Expansion, Reducing Total Culture Duration

Introduction: As clinical applications for Chimeric Antigen Receptor (CAR) T-cell therapy expand, cell manufacturing incorporating closed-system, automated instruments are supplanting traditional open-system, labor-intensive culture methods. At our institution and others, the CliniMACS Prodigy (Miltenyi Biotec), a closed-system automated device, has demonstrated success in the production of CAR T-cells from T-cell enrichment, activation, viral transduction, and expansion to downstream harvest for cryopreservation/fresh infusion. However, the duration of T-cell activation/viral transduction, and total T-cell culture duration are variable across centers (2-5 days and 7-13 days) and merit evaluation prior to routine use.Methods: Following the opening of our clinical protocol (Clinicaltrials.gov NCT03448393), CD19/CD22 Bispecific CAR T-cell products were manufactured on the Prodigy for 4 patients (Original Method, OM) but CAR T cell manufacturing was felt to be suboptimal. Consequently, we investigated a modified processing method (Modified Method, MM), for the manufacture of clinical grade products using the Prodigy. Specifically, TransAct CD3/CD28 reagent mediated T-cell activation/stimulation and lentiviral transduction (MSCV-CAR1922-WPRE; Lentigen Inc.) was terminated with a wash step at Day 3 (instead of the wash step at Day 5, as in the OM). Overstimulation of the relatively more sensitive patient cells was proposed as a likely cause of suboptimal cell viability and expansion in the OM runs. Final cell harvest was planned between culture days 7-12. A total of 4 apheresis products were evaluated using this MM and compared with the 4 prior runs using the OM. All products were obtained from live or deceased patients with disease profiles similar to patients on the clinical trial. Other process parameters (enrichment for CD4/CD8 subsets, in-process media changes with GMP-TexMACS Medium supplemented with human IL-2 (200IU/mL) and 3% human AB serum) were kept unchanged across the 2 methods. Transduction by Protein L expression, viability and cell phenotype (CD3, CD4/CD8) were measured by flow cytometry.Results: From ~0.1x109 CD4/8 enriched T-cells placed into the Prodigy culture chamber on day 0, the mean viable Total Nucleated Cells (TNC) obtained in the final product was 1.93x109 ± 0.27x109 in the 4 MM runs. This cell dose was accomplished by culture Day 7. In contrast, in the OM runs, the mean viable TNC obtained in the final products between Days 9 and 12 was 0.8x109 ± 0.7x109 (Figure 1a). Viable CAR transduced CD3+ fold increase was calculated for days 0-7 of the MM cultures and 0-9 and 0-12 of the OM cultures depending on day of harvest and for the 4 MM products the average fold increase was 15.3 ± 4.2 by Day 7 (Figure 1b) which was ~3 fold greater than OM products harvested on day 9 or 12. Viability of transduced cells was >80% throughout MM culture. In contrast, viability was about 31% during manufacturing of one of the OM products (Figure 1c). On the day of harvest, >99% of the cells were CD3+ T-cells for all 4 MM products (Figure 1d) with no remaining CD19+CD22+ cells. The CD4/CD8 ratio was as expected and favored CD4 T-cells over CD8 T-cells (Figure 1e). Transduction efficiency based on Protein L binding was >70% for the MM products and passed clinical release criteria (Figure 1f). A head-to-head comparison of the 2 methods from the same starting fraction in one patient product (Figure 1, 3A, 3B) also confirmed all findings above.Conclusions: Our data demonstrate that the modified CD19/CD22 Bispecific CAR T-cell manufacturing method (MM) which terminated T-cell activation/transduction by culture Day 3, resulted in reproducible and robust CAR T cell production, even in the relatively more sensitive patient cells. Viability, Viable TNC recovery, CD3% and Protein L expression were consistently higher with the MM compared to the OM. All final products in the MM met product release criteria. In addition, final product dose requirements were consistently met by culture Day 7 when using the MM, augmenting process efficiency. Consequently, we have adopted the MM for the manufacture of clinical CD19/CD22 Bispecific CAR T cells. However, to determine if this change effects CAR T cell potency, studies have been initiated to compare differences in T-cell subsets, activation/exhaustion/senescence and differentiation markers, and the metabolic activity of cells manufactured by the 2 methods. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Infusion of a Cryopreservable Human Megakaryocyte-Biased Cell Product Results in Sustained Platelet Reconstitution In Vivo

The demand for platelet (PTL) transfusions has steadily increased, straining a supply that is limited by its dependency on donors, short lifespan, risk of infections and alloimmunization. This stimulated the search for alternative PTL sources including PTLs generated ex vivo from primary CD34+ cells and immortalized pluripotent stem cells. These approaches, however, are associated with obstacles such as scalability and encounter identical limitations as donor PTLs: short shelf life, storage at ambient temperature and limited lifespan after infusion. These obstacles lead us to focus our efforts on not producing PLTs but rather a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTLs after transfusion into patients.Umbilical cord blood units (CBU) are readily available sources for stem cell for transplantation. We created an efficient and cost effective culture system in which CB-derived CD34+ cells are first expanded then allowed to mature into MKs. Initially, we determined the optimal culture period (10-11 days) resulting in the greatest number of CD41+/CD42b- and CD41+/CD42b+ MKs which are capable of PTL production. Next, we used research and clinical grade CBU to generate clinically relevant doses of MK. The median number of CD34+ cells selected from one CBU was 2.5x106 with a purity of 90% (n=4). Following expansion and MK maturation, these cells generated 5.8x107 viable total nucleated cells (TNC)/CBU. Out of these, 3.3 x 107 were CD41+ MKs which corresponds to a median cell dose of 4.1x105 CD41+ cells/Kg of body weight. 92% of CD41+ MKs were mature CD42b+ cells which we previously showed that are capable of ex vivo platelet production. Finally, we performed clonogenic assays and found that one CBU can generate ~1.5x106 CFU-MK.One half of these MK-biased cultures was characterized and assessed immediately after culture and the other half was cryopreserved. The fresh product was infused into sublethally irradiated NSG mice and the presence of human PTLs in the mouse peripheral blood (mPB) was evaluated weekly for 8 weeks at which time the animals were also analyzed for hMK chimerism in the bone marrow (BM), spleen (SP) and lung. The results demonstrate that 87% of animals (13 out of 15) had a robust hPTLs population in their PB. hPTL were detected as early as week 1 post infusion and their number peaked on week 4 (median, 6x103 hPTL/μl) after which it plateaued. The release of hPTL in the mPB was accompanied by the presence of hCD41+ MKs in the mBM, SP and the lung indicating that the infused cells provided both early hPTL release and a reservoir of MK precursors for continuous hPTL production. We also found that in addition to MKs, these same organs contained hCD34+, CD45+ and myeloid CD15+ cells. These findings underscore the capabilities of this product which might have broader clinical applicability such as utilization during myeloablative or suppressive chemo/radiotherapy to improve the time and duration for both neutrophil and platelet engraftment. Equally important, we provide novel evidence that the lung is a site for hMK engraftment after transplantation, in line with recent reports recognizing the pulmonary bed as site for platelet production in the mouse.The major advantage of developing a MK-based product over ex vivo generated PTLs is the amenability of the former to cryopreservation thus becoming a readily available cellular therapy which would be amenable to stock-piling. Therefore, portions of the same MK products described above were cryopreserved then subjected to ex vivo and in vivo studies identical to these performed on their fresh counterparts. Following thawing, the average recovery rate was 71% for TNC and 74.3% for CD41+ cells. MK phenotype and morphology as well as the number CFU-MK generated ex vivo were identical to that found in the fresh product. Although the number of TNC in the thawed product was lower than that of its fresh counterpart, the number of hPTL detected after its infusion ranged from 0.4 to 20.5x103 hPTL/μl which is comparable to that detected after infusion of the fresh equivalent, 0.7-16x103 hPTL/μl.In summary, we created a potent transfusable MK cell product that provides robust and sustained PTL and hematopoietic engraftment in vivo and maintains this capability after cryopreservation. Clinical development of such product is now being pursued for the treatment of thrombocytopenia in acute leukemia patients undergoing chemotherapy. DisclosuresHoffman:Summer Road: Research Funding; Janssen: Research Funding; Formation Biologics: Research Funding; Merus: Research Funding; Incyte: Research Funding. Iancu-Rubin:Incyte: Research Funding; Merck: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding.

In vitro and in vivo evaluation of cord blood hematopoietic stem and progenitor cells amplified with glycosaminoglycan mimetic.

BackgroundExpansion protocols aim at both increasing the number of umbilical cord blood (UCB) hematopoietic stem cells and progenitor cells (HSPCs) and reducing the period of neutropenia in UCB HSPC graft. Because glycosaminoglycans (GAGs) are known to be important components of the hematopoietic niche and to modulate growth factor effects, we explored the use of GAG mimetic OTR4131 to potentiate HSPC’s in vitro expansion and in vivo engraftment.MethodsUCB CD34+ cells were expanded with serum-free medium, SCF, TPO, FLT3-lig and G-CSF during 12 days in the absence or the presence of increasing OTR4131 concentrations (0-100 μg/mL). Proliferation ratio, cell viability and phenotype, functional assays, migration capacity and NOD-scid/γc-/- mice engraftment were assessed after expansion.ResultsAt Day 12, ratios of cell expansion were not significantly increased by OTR4131 treatment. Better total nucleated cell viability was observed with the use of 1 μg/mL GAG mimetic compared to control (89.6 % ± 3.7 % and 79.9 % ± 3.3 %, respectively). Phenotype analysis showed a decrease of monocyte lineage in the presence of OTR4131 and HSPC migration capacity was diminished when GAG mimetic was used at 10 μg/mL (10.9 % ± 4.1 % vs. 52.9 % ± 17.9 % for control). HSPC clonogenic capacities were similar whatever the culture conditions. Finally, in vivo experiments revealed that mice successfully engrafted in all conditions, even if some differences were observed during the first month. Three months after graft, bone marrow chimerism and blood subpopulations were similar in both groups.ConclusionsUCB HSPCs ex-vivo expansion in the presence of OTR4131 is a safe approach that did not modify cell function and engraftment capacities. In our experimental conditions, the use of a GAG mimetic did not, however, allow increasing cell expansion or optimizing their in vivo engraftment.

Long-term effects of cryopreservation on clinically prepared hematopoietic progenitor cell products

Background aimsThe question of how long hematopoietic progenitor cells (HPCs) destined for clinical applications withstand long-term cryopreservation remains unanswered. To increase our basic understanding about the stability of HPC products over time, this study focused on characterizing long-term effects of cryopreservation on clinically prepared HPC products. MethodsCryovials (n = 233) frozen for an average of 6.3 ± 14.2 years (range, 0.003–14.6 years) from HPC products (n = 170) representing 75 individual patients were thawed and evaluated for total nucleated cells (TNCs), cell viability, viable CD34+ (vCD34+) cells and colony-forming cells (CFCs). TNCs were determined by use of an automated cell counter, and cell viability was measured with the use of trypan blue exclusion. Viable CD34 analysis was performed by means of flow cytometry and function by a CFC assay. ResultsSignificant losses in TNCs, cell viability, vCD34+ cells and CFC occurred on cryopreservation. However, once frozen, viable TNCs, vCD34+ cells and CFC recoveries did not significantly change over time. The only parameter demonstrating a change over time was cell viability, which decreased as the length of time that an HPC product was stored frozen increased. A significant negative correlation (correlation coefficient = −0.165) was determined between pre-freeze percent granulocyte content and post-thaw percent viability (n = 170; P = 0.032). However, a significant positive correlation was observed between percent viability at thaw and pre-freeze lymphocyte concentration. ConclusionsOnce frozen, HPC products were stable for up to 14.6 years at <−150°C. Post-thaw viability was found to correlate negatively with pre-freeze granulocyte content and positively with pre-freeze lymphocyte content.

Expansion Culture Of Hematopoietic Stem & Progenitor Cells From Frozen-Thawed, Non-Enriched Human Umbilical Cord Blood In Animal Component– & Serum–Free Medium Enhances Engraftment & Reduces Graft-Versus-Host-Disease

Hematopoietic stem cell transplantation in adults using umbilical cord blood (UCB) is limited by low cell dosage &amp; post-thaw viability. In several clinical trials cytokine supplementation &amp; stromal cell support have been shown to enhance total nucleated cells (TNC). However, clinical safety is compromised due to source inconsistency &amp; population heterogeneity of stromal cells along with animal components of the conventional growth media. In this study, we demonstrate effective use of an animal component– &amp; serum–free growth medium to enhance the viability &amp; ex vivo expansion of SCID repopulating cells (SRC) from frozen-thawed, non-enriched UCB–mononucleated cells (UCB-MNC). UCB-MNC were cultured in a commercially available animal component– &amp; serum–free medium, StemSpanTM–ACF (ACF), while StemSpanTM–SFEM (SFEM), a conventional serum–free medium with human and bovine components served as control. Both media (from STEMCELL Technologies INC. Vancouver, Canada) were supplemented with clinical grade SCF, Flt-3 ligand, TPO, &amp; IGFBP2. The expansion effects were characterized based on cell viability, phenotypic stem &amp; progenitor cells &amp; functional in vitro &amp; in vivo assays. After 3-days of culturing, viability of CD45+ UCB-MNC was maintained at a significantly higher level in ACF (90.7±0.2%) compared to SFEM (75.4±0.1%) (p&lt;0.0001; n=3). Culturing for 11-days significantly (p&lt;0.0001; n=6) increased CD45+CD34+CD38– hematopoietic progenitors in ACF (90.6±13.5 fold) compared to control (4.8±0.4 fold). Further phenotypic study of ACF expanded cells showed significant increases of 4.1-fold for CD45+CD34+C38–CD90+ stem cells (p&lt;0.0001), 2.1-fold for CD45+CD34+CD13+CD33+ myeloid progenitors (p&lt;0.01) and 2.3-fold for CD45+CD34+C38–CD7+(p&lt;0.01) lymphoid progenitors compared to SFEM (n=6). Viable TNC expansions were 4.3±0.2 fold and 5.9±0.7 fold in ACF and SFEM respectively (n=6; p&lt;0.05). Colony forming unit (CFU) assay showed that ACF supported significantly higher expansion of GM progenitors than SFEM (60.1±7.9 vs. 14.6±2.1 fold; p&lt;0.00001; n=16). The numbers of multi-potent progenitors, CFU-GEMM, were maintained in ACF but decreased in SFEM (0.83±0.21 vs. 0.09±0.04 fold relative to non-expanded UCB; p&lt;0.01; n=16). UCB-MNC cultured for 11 days reconstituted the bone marrow (BM) of sub-lethally irradiated NOD/SCID gamma (NSG) mice with human CD45+/71+ cells as measured 16 weeks after transplantation at a dosage of 1x108 cells/kg. The frequency of human cells was higher for UCB expanded in ACF (38.1±15.4%; n=5) than for UCB expanded in SFEM (3.4±2.1; n=14; p&lt;0.01). Human CD34+ progenitors were also detected in BM of the engrafted mice at frequencies of 2.4±1.4% and 0.2±0.1% for ACF and SFEM expanded cells respectively (p&lt;0.05). Human hematopoiesis was multi-lineage with significantly higher numbers of CD45+/71+ &amp; CD15+/66b+ granulopoietic cells (71.4-fold; p&lt;0.001) and CD19+/20+ B-lineage cells (23.1-fold; p&lt;0.001) in mice transplanted with cells expanded in ACF (n=5) as compared to SFEM (n=14). At a transplantation dosage of 2.5x107 cells/kg, non-expanded grafts (n=10) had similar engraftment of CD45+/71+cells compared to ACF expanded grafts (n=5; p=0.14), while engraftment was lower for SFEM expanded grafts (n=12; p&lt;0.01). Limiting dilution analysis revealed that SRC frequencies were increased, on average, 7.9– and 1.2–fold in ACF relative to SFEM expanded &amp; non-expanded grafts respectively. NSG mice transplanted with non-expanded grafts had a significantly lower (p&lt;0.001) survival rate (40.4%, n=47) compared to those transplanted with grafts expanded in ACF (90.9%, n=11) or SFEM (92.3% n=26), or injected with saline only (100%, n=7). The high mortality rate in recipients of non-expanded grafts was due to higher incidence of graft-versus-host-disease (GVHD) associated with significantly (p&lt;0.01; n=6) higher CD45+CD7+T cells in comparison to expanded grafts. In conclusion, expansion of freeze-thawed, non-enriched UCB-MNC in animal component– &amp; serum–free medium improves in vivo repopulation and reduces mortality due to GVHD in a xenotransplantation model. These findings could set the platform for developing safer, cheaper &amp; time efficient clinical transplantation, since no animal components, in the form of serum albumin or stromal cells, are required to achieve desired ex vivo expansion of hematopoietic stem &amp; progenitor cells &amp; pre-clinical outcomes. Disclosures: No relevant conflicts of interest to declare.

A new system for quality control in hematopoietic progenitor cell units before reinfusion in autologous transplant

In our Center, the cell viability, the integrity of the bag, and the clonogenic assay were evaluated before the reinfusion of hematopoietic progenitor cells-apheresis (HPC-A). This quality control (QC) should be made 14 days before the reinfusion to the patient to have the result of the functional test on the proliferative capacity of hematopoietic progenitors. This study was designed to assess the potential of an automatic cell counting system (NucleoCounter NC-3000, ChemoMetec) in our clinical routine as a support of the clonogenic assay and the cytofluorimetric analysis for the QC of the cryopreserved HPC-A. The cell viability was evaluated by flow cytometry using the modified International Society of Hematotherapy and Graft Engineering protocol. The proliferative potential was assessed by specific clonogenic tests using a commercial medium. Furthermore, we evaluated the cellular functionality with NucleoCounter NC-3000, by using two protocols: "vitality assay" and "mitochondrial potential assay." The evaluation of the total nucleated cells in preapoptosis measured by 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine iodide (JC-1) assay showed a negative correlation (r=-0.43) with the total number of colonies (colony-forming unit [CFU]-granulocyte-macrophage progenitors plus burst-forming unit-erythroid progenitors plus CFU-granulocyte, erythroid, macrophage, megakaryocyte progenitors) obtained after seeding of 50 × 10(6) /L viable total nucleated cells. We observed a significant difference (p<0.0001) comparing the median number of colonies (166.70; SD, ± 136.36) obtained with a value of JC-1 less than 30% to the number of colonies (61.75; SD, ± 59.76) obtained with a value of JC-1 more than 30%. The evaluation of cell functionality by the use of the NucleoCounter NC-3000 is in agreement with results from clonogenic assay and can be considered an effective alternative in the routine laboratory.

Characteristics of thawed autologous umbilical cord blood

Autologous umbilical cord blood (AutoUCB) has historically been cryopreserved for potential use in hematopoietic transplantation. Increasingly, private AutoUCB banking is performed for therapies unavailable today. A Phase I trial using AutoUCB treatment for early pediatric Type 1 diabetes afforded us an opportunity to analyze characteristics of AutoUCBs. Twenty AutoUCBs from AABB-accredited private cord blood banks (CBBs) were evaluated for collection, processing, cryopreservation, and thaw characteristics. Using a standardized thaw-wash method, AutoUCBs were assessed for viable total nucleated cells (vTNCs), viable CD34+ (vCD34+), and colony-forming unit-granulocyte-macrophage counts. Postthaw %vTNC recoveries were compared against processing characteristics and analyzed according to processing method, cryopreservation volume, concentration, container, and length of storage. AutoUCB collection volumes (19.9-170 mL), cryopreserved TNC counts (7.6 × 10(7) -3.34 × 10(9)), %TNC processing recoveries (39%-100%), postthaw %vTNC recoveries (58%-100%), and %vCD34+ recoveries (26%-96%) varied widely. Regarding cell dose requirements, only 11% of evaluable AutoUCBs achieved the minimum TNC count of at least 9.0 × 10(8) to meet the National Cord Blood Inventory banking threshold, and only 50% met the minimum of 5.0 × 10(8) TNC count for Food and Drug Administration cord blood licensure eligibility. %vTNC recoveries correlated with %vCD34+ recoveries (R = 0.7; p = 0.03). Length of storage, cryopreservation volume, concentration, and container type did not affect postthaw %vTNC recoveries. CBB processing method, however, was associated with %vTNC postprocessing recoveries, with unmanipulated and plasma-depleted AutoUCBs having the highest postthaw %vTNC recovery, followed by RBC-depleted and density gradient-separated AutoUCBs. The high variability and low counts found in AutoUCB banking suggest that further standardization of characterization, collection, and processing procedures is needed.

The Effects of Anticoagulants on Cord Blood Viability From Collection to Processing

Abstract 1923 Background:Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells for transplantation and regenerative medicine applications. UCB collection bags must contain an anticoagulant to prevent coagulation and to maintain viability during transport to the processing facility. Although Citrate Phosphate Dextrose (CPD) has been widely used based on the commercial availability of pre-filled collection bags, heparin is also accepted as an anticoagulant for processing hematopoietic stem cells. Data is lacking on the optimal anticoagulant for UCB collections particularly in the volume range of cord blood collected for family banking purposes. CPD was originally formulated for use in whole blood collections at a dilution ratio of 1:7 CPD to blood. Commercially available UCB bags contain 35 mL of CPD and are intended to allow a maximum collection volume of 210 mL in a 250 mL bag, creating up to a 1:6 dilution. However, typical cord blood collections rarely reach this volume. Given the wide variability in UCB collection volumes and the standard volume of 35 mL CPD pre-loaded in each bag regardless of collection volume, dilution ratios for UCB collections could drop as low as 1:1 CPD to blood. Concentrated exposure to CPD could adversely impact cell viability and blood chemistry. Methods:UCB collections from 15 individual donors were each approximately equally divided (p>0.05) between a 300 mL collection bag containing 500 units of lyophilized heparin (LH) and a 250 mL bag with 35 mL liquid CPD. Samples were placed on an orbital mixer at room temperature for a simulated shipping time of 48 hours. Aliquots of the samples were assessed for total nucleated cell (TNC) count and cell viability at time of collection, and at 24 and 48 hour time points. Viable TNC were obtained utilizing acridine orange/propidium iodide staining. Total cell counts were obtained using a Sysmex hematology analyzer. The values at each time point were compared to the previous measurement to detect changes over time. Results:Shown in Table 1. Samples collected in CPD showed a statistically significant decrease in TNC yield at 24 hours (p=0.004) and again at 48 hours (p=0.002). There was a significant but lesser decrease in TNC yield in samples collected in LH between hours 0 and 24 (p=0.04) and no significant difference thereafter. There was a significant decrease in TNC viability in CPD samples at both time points. The viability of samples collected in LH vs. CPD was 96.6±3.0% vs. 85.9±9.0% (p<0.001) at hour 24 and 95.9±3.0% vs. 66.9±17.8% (p<0.001) at hour 48.Table 1:Cell Yield and Viability of LH and CPD at hours 0, 24 and 48 Post CollectionTNC (10^6)/mL% Viable TNCTime from collection0 Hrs24 Hrs48 Hrs0 Hrs24 Hrs48 HrsLH Mean (std dev)14.01 (4.06)13.2 (3.92)12.96 (4.72)95.6 (5.6)96.6 (3.0)95.9 (3.0)p value for change between time points0.04NSNSNSCPD Mean (std dev)14.54 (4.45)13.0 (4.71)9.87 (3.93)96.7 (3.9)85.9 (9.0)66.9 (17.8)p value for change between time points0.0040.002<0.001<0.001p value LH to CPDNSNS0.005NS<0.001<0.001 Conclusion:The TNC yield and viability of samples collected in LH compared to CPD suggest that UCB quality was impacted by anticoagulant selection. The results of this study indicate LH was more biocompatible than CPD as measured by cell viability endpoints. During the study, it was also noted that CPD exposure typical of family cord blood banking conditions caused significant acidosis of the blood. In a family banking setting, where the time from collection to processing could be as long as 48 hours and beyond, prolonged exposure to an acidic environment could be detrimental. LH was found not to cause acidosis or other dilution related issues. Because family cord blood banking preserves genetically unique stem cells for each individual newborn and his or her family, it is important to ensure that UCB unit quality will be maintained during transport regardless of the size of the collection volume. Disclosures:No relevant conflicts of interest to declare.

Does Stored Hpc, Apheresis Product Need an Expiration Date? Graft Cell Viability After Long Term Storage

Regulatory & accrediting organizations require that stored HPC products be labeled with an expiration date. Stem Cell Transplant Program at Children's Memorial Hospital has been collecting HPC, Apheresis products since 1992 with signed donor consent for storage and discard. Though products were labeled with a 5 year expiration date, cells were not discarded until 2006, for want of storage space. We identified 54 products which could be discarded as their intended recipients had died. Products were in liquid nitrogen storage for a period of upto 15.2 yrs. Cells were thawed & viability testing done, as per written procedure. We undertook this opportunity to discard products to document effect of long term storage on graft cell viability. Donor products were collected by HPC, Apheresis between 1992 & 2003. Pre-freezing viability done using Trypan Blue Dye Exclusion test was between 96-100% (median 97.3%). Products were mixed with equal quantity of cryoprotectant [3 parts TC-199 without phenol red and 2 parts each of 10% Dimethylsulfoxide (DMSO) and Autologous serum]. Heparin was used as anti-coagulant. Cells were frozen using a controlled rate freezing program and stored in liquid nitrogen. Product bags were thawed in a water bath between 37 - 40oC, gently kneading the contents. Post thaw samples were re-tested for viability, using Trypan Blue. Statistical evaluations were done on SigmaStat 3.5. Table 1Years in Storage# of ProductsViability RangeViability MedianP-vlauePearson's Correlation (R)Total (0.1- 15.2)5468- 9990.29%0.484-0.09720.1- 5.02777- 9791.03%0.478-0.1425.1- 10.01268- 9888.58%0.526-0.20410.1- 15.21579- 9990.33%0.723-0.0999 Open table in a new tab Post thaw median viability was 90.3% (range68- 99%). Of 54 products, 37 products (69%) had viability > 90%; 51 (94%) had viability > 80%; only 3 (6%) had viability below 79%. 15 products (28%) were in storage for < 3.0 yrs; 12 products (22%) for 3.1 to 5.0 yrs; 12 products (22%) for 5.1 to 10.0 years; & 15 products (28%) were stored for > 10.1 yrs. Statistical evaluations revealed no significant relationship between storage time & cell viability. Total nucleated cell viability was measured as an indicator of graft composition. 94% of products retained a viability of > 80% on long term storage. Difference in pre freezing & post thaw median vibility (7%) can be attributed to the freezing process itself. Since HPC, A grafts are capable of engraftment regardless of storage time or percentage of viable cells, stored HPC, A products may actually have a long expiration period. Long term storage of HPC, A products in liquid nitrogen does not result in any significant cell loss and may not need an expiration date.

Long-Term Cryopreservation of Autologous Stem Cell Grafts: Impact of Patient Age, Initial Collection, and Stem Cell Mobilization Regimen.

AbstractAbstract 1178 Background:Effectively cryopreserving hematopietic peripheral blood progenitor cells (HPC(A)) until time of transplant is critical for successful autologous stem cell transplant. It is important to demonstrate long-term HPC(A) viability and recovery and also to identify patient characteristics that may influence this. We evaluated HPC(A) products cryopreserved over 16 year period. We analyzed potential correlations between total nucleated cell (TNC) and CD34+ recovery and viability and patient age, initial collection, and impact of stem cell mobilization regimen. Method:Samples were obtained from expired patients, chosen based on storage duration but randomly selected in regard to patient and product characteristics; 23 samples were obtained from 18 patients. Five patients had 2 samples each; for duplicate samples, data were averaged (collection dates and thaw dates were similar). All HPC(A) products were frozen in autologous plasma and 10% DMSO, then stored in liquid phase or vapor phase nitrogen. Products were uniformly thawed and washed to remove DMSO. Analysis included 7-Amino-actinomycin D viability, TNC and CD34+ cell counts and recovery. Some samples had post-thaw CD34+ cell counts that exceeded pre-thaw count; for these, the recovery was set to 100%. Spearman rank correlations were used to analyze association between the different parameters. Wilcoxon rank sum test and analysis of covariance were used to compare patient groups. Result:Median patient age at time of stem cell collection was 54 (range 2–75) years. A majority of patients had lymphoma (61%) or multiple myeloma (22%). Most (56%) were mobilized using G-CSF+VP16, 33% with G-CSF alone, and 11% other. Samples were cryopreserved for a median of 8 years (range 1–16). Median (range) for total CD34+ × 106 collected was 547 (43-5845). Median (range) for CD34+/kg × 106 was 8 (1-59). Median (range) count/bag for TNC × 108 was 144 (37-285) and for CD34+ × 106 was 105 (8-805). Median (range) recovery and viability were 85% (32-213%), and 70% (46-85%) respectively for TNC; and 97% (16-359%) and 80% (61-98%), respectively for CD34+ cells.Pre- and post- thaw TNC and CD34+ counts were both highly correlated (r=0.95, p<.0001 in both cases – figures 1a and b). There was no significant association between TNC recovery and viability (r=-0.07, p=.79) or between CD34+ cell recovery and viability (r=-0.21, p=.41). Storage duration did not impact CD34+ recovery or viability (r=-0.01, p=.98 and -0.18, p=.47, respectively); or TNC viability (r=-0.13, p=.62). There was, however a significant positive correlation between the storage duration and TNC recovery (r=0.81, p<.0001).Age did not significantly impact TNC or CD34+ recovery (r=-0.05, p=0.86, and r=-0.26, p=0.29, respectively); however there was suggestion of negative impact on viability (r=-0.45, p=0.06 and r=-0.42, p=0.08, respectively, figures 1a and 1b). Overall, there did not appear to be a correlation between the initial TNC count/bag or total CD34+ cell dose on recovery or viability (r=0.29, r=-0.22, r=0.18, r=-0.38, respectively; all p>0.12). However, when patients were stratified according to total collection >25×106 CD34+ cells/kg or ≤25 × 106 CD34+ cells/kg, there was better %CD34+ cell count recovery between groups (p = 0.07, median recovery 81% for 13 patients with ≤25 × 106 CD34+ cells/kg and 100% for 5 patients with >25×106 CD34+ cells/kg). For mobilization regimen, there was no significant difference in TNC recovery or viability (p = 0.19 and p=0.76, respectively) or CD34+ viability (p = 0.53), however there was suggestion that CD34+ recovery was greater with G-CSF+VP16 (p=0.06, see figure 2), independent of storage time. Conclusion:Products cryopreserved for 16 years retain acceptable recovery and viability. Unexpectedly, storage duration positively correlated with TNC recovery, and CD34+ cell recovery of >100% was noted in several samples. Reasons for this are unclear, but are likely related to changes in enumeration method or use of methods validated for counting fresh cells. Patient age was suggested to negatively impact post-thaw HPC(A) viability. Our data also suggested that the mobilization regimen or the CD34+ cell collection yield may affect the CD34+ cell recovery, possibly reflecting differences in graft characteristics. The number of patients included is relatively small and these findings warrant further studies for validation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Improved Cell Viability, Viable Cell Recovery and Faster Engraftment of Products Thawed Using a Premixed Solution of Human Serum Albumin and Dextran 40 Compared to Sequential Addition During the Thawing Procedure

AbstractAbstract 2248HPC, Apheresis products collected for autologous use must be cryopreserved prior to hematopoietic progenitor cell transplantation. Improved recovery of cryopreserved cord blood cells was demonstrated by Rubinstein et al. (PNAS:92, 10119, 1995) when the thawed cells were diluted with an equal volume of a solution containing 2.5% (wt/vol) of human serum albumin (HSA) and 5% (wt/vol) Dextran 40, the cells centrifuged, the supernatant removed, and the cells restored to their original volume with the same Dextran/Albumin solution as compared to thawing the product without dilution or washing. This method has the additional advantage of allowing cells to be thawed under controlled conditions within the laboratory and greatly reduces reactions to DMSO. Several variations in this procedure have been published including one employed at our center that has been used to thaw all cryopreserved products since 1996 in which 5% HSA and 10% Dextran 40 was added sequentially to the thawed cells each at 1/2 the frozen volume. The cells and solution were mixed and after 10 minutes of incubation the bag was filled to capacity (300 mL total) with Dextran 40 prior to centrifugation. Reconstitution to the original volume for infusion was also by sequential addition of HSA and Dextran 40 (Method 1). A variation of this method is to use a premixed solution of 2.5% HSA and 5% Dextran 40 both before and after centrifugation (Method 2). A third method, which is currently recommended by the Blood and Marrow Transplantation Clinical Trials Network (BMT-CTN) for thawing HPC, Cord Blood is similar to method 2 but uses a higher concentration of HSA resulting in 4.2% HSA in the premixed Dextran/Albumin solution (Method 3). We directly compared the three methods using autologous products (n=3) that were no longer needed for infusion and that were each frozen in multiple bags to assess viability (7-AAD method), along with recovery of viable total nucleated cells (TNC), CD3+ T-cells, and CD34+ cells. No significant differences were seen for any of the outcomes between method 2 and method 3. However, both method 2 and method 3 resulted in better overall viability, viable TNC recovery and viable recovery of CD34+ cells and CD3+ T-cells than method 1. Starting in January 2010 we modified our standard thawing procedure to method 3 (to conform to CTN requirements) and have compared the results of 96 bags thawed using this method with 173 bags thawed using method 1. Thaw viability (Trypan blue method) using method 1 was 72.7%±11.8% compared to 77.5%±9.1% for method 3, p=0.0005 and viable cell recovery was 62.1%±12.6% versus 68.5%±10.2%, p=<0.0001, respectively. When compared to product quality control vials thawed without a Dextran/Albumin wash, viability averaged 11.1% higher for products thawed using method 3 compared to 9.5% higher for products thawed using method 1. Engraftment data was available for 46 patients receiving products thawed using method 3 and was compared with 159 patients transplanted with products thawed using method 1. Patients receiving products thawed using method 1 achieved an ANC of 500 at a median of day 12 compared to day 11 for method 3, p=0.0003 (Log Rank test). The median time to platelets of 20K of 20 days for method 1 compared to 21 days for method 3 was not significantly different (p=0.07). In summary, thawing HPC, Apheresis products using a Dextran/Albumin wash method resulted in superior cell recovery and viability compared to a direct thaw method and using a premixed solution of Dextran/Albumin was superior to adding the two reagents sequentially during the thawing procedure. This resulted in a small but significant decrease in the time to granulocyte recovery post transplant. No advantage was seen when the thawing solution contained a final concentration of 4.2% versus 2.5% HSA. Disclosures:No relevant conflicts of interest to declare.

Evaluation of an automated cell processing device to reduce the dimethyl sulfoxide from hematopoietic grafts after thawing

The direct transfusion of thawed hematopoietic progenitor cells (HPCs) is associated to transfusion-related side effects that are thought to be dose-dependent on the infused dimethyl sulfoxide (DMSO). Both the effectiveness of a fully automated cell processing device to washing out DMSO and the effects of DMSO elimination over the recovered cells were evaluated. Twenty cryopre-served peripheral blood HPC bags (HPC apheresis [HPC-A]) were thawed and processed for washing with an automated cell-processing device. Viability, colony-forming units (CFUs), and absolute count of recovered cells were evaluated by flow cytometry immediately after washing as well as at different times after washing and compared with a sample taken just after thawing (control) but maintained at 4 degrees C. DMSO content was measured by high-performance liquid chromatography and the osmolarity with an osmometer. The median recovery of viable total nucleated cells, viable CD34+ cells, and CFU colonies was 89 (range, 74-115), 103 (range, 62-126), and 91 percent (range, 46%-196%), respectively, in the washing group. Recovery of viable CD3+ cells was 97 percent (range, 42%-131%) and CD14+ cells was 82 percent (54%-119%). The percentages of DMSO elimination and osmolarity reduction were 98 (range, 96-99) and 90 percent (range 86%-95%), respectively. Moreover, elimination of the cryoprotectant improved CFU count, viability, and cell recoveries along the time when compared with the control group. Washing out DMSO in thawed HPC-A by use of this approach is safe and efficient in terms of recovery and viability of nucleated and progenitor cells. Additionally, the removal degree of DMSO is very high and therefore might ameliorate the transfusion-related side effects.