Storage characteristics of cord blood progenitor cells: report of a multicenter study by the cellular therapies team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative
ABSTRACT Most hematopoietic progenitor cell (HPC) products are infused or processed shortly after collection, but in some cases this may be delayed for up to 48 hours. A number of variables such as temperature and cell concentration are of critical importance for the integrity of HPCs during this time.
We evaluated critical variables using cord blood HPC units that were divided equally and stored at 4 °C versus room temperature (RT) for up to 96 hours. Total nucleated cell (TNC) and mononuclear cell (MNC) counts, viable CD34+ cell counts, and CD45+ cell viability as well as colony-forming unit-granulocyte-macrophage (CFU-GM) present over time at each temperature were determined.
Overall, the data indicate that with the exception of viable CD34+ cells, there was a significant decrease in each variable measured for 72 to 96 hours and, with the exception of viable CD34+ cells and CFU-GM, the reductions were significantly greater in RT units than 4 °C units. There was an increase in viable CD34+ count for units where TNC count was greater than 8.5 × 10(9) /L, compared with units where TNC count was less than 8.5 × 10(9) /L, that was different for each storage temperature.
Cord blood HPC collections maintained at 4 °C retained higher TNC counts, MNC counts, and CD45+ cell viability over a 72- to 96-hour storage period.
SourceAvailable from: Zoran Ivanovic[Show abstract] [Hide abstract]
ABSTRACT: During storage and transportation of collected cord blood units (CBU) to the bank prior to their processing and cryopreservation it is imperative to preserve the functional capacities of a relatively small amount of cells of interest (stem and progenitor cells) which are critical for graft potency. In order to improve CBU storage efficiency, we conceived an approach based on two principles: 1) to provide a better nutritive and biochemical environment to stem and progenitor cells in CB and 2) to prevent the hyperoxygenation of these cells transferred from a low- (1.1 to 4% O2 in the cord blood) to a high-oxygen (20 to 21% O2 in atmosphere) concentration. Our hypothesis is confirmed by the functional assessment of stem cell (hematopoietic reconstitution capacity in immunodeficient mice - SRC assay) and committed progenitor activities (capacity of in vitro colony formation and of ex vivo expansion) after the storage period with our medium (HP02) in gas-impermeable bags.This storage procedure maintains the full functional capacity of a CBU graft for 3 days with respect to Day 0. Furthermore, using this procedure a graft stored three days at +4°C exhibits better functional capacities than one currently used in routine storage (CBUs stored at +4°C for one day in gas-permeable bags and without medium). We provided the proof of principle of our approach, developed a clinical-scale kit and performed a preclinical assay demonstrating the feasibility and efficiency of our CBU preservation protocol through all steps of preparation (volume-reduction, freezing and thawing).Stem cells and development 04/2014; DOI:10.1089/scd.2014.0046 · 4.20 Impact Factor
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ABSTRACT: BACKGROUND: Umbilical cord blood (UCB) is a good source of hematopoietic stem cells for transplantation and cell therapy. In 2006, the Brazilian Public Network of Cord Blood Banks was founded; however, because our country is large, logistic problems could hamper the collection of numerous samples. Our aim was to evaluate the viability of several UCB cell subsets until 96 hours after collection, to examine whether this delay would be acceptable for processing and freezing the samples. STUDY DESIGN AND METHODS: Two experiments were performed: in the first one, volume reduction of the UCB units was carried out before analysis. In the second one, analysis was carried out with no previous manipulation. Samples were stored at room temperature and one aliquot was taken daily for analysis. We examined CD34+ cell, B-cell precursor, mature B and T lymphocyte, monocyte, granulocyte, and mesenchymal stem cell (MSCs) concentrations. RESULTS: Thirty-six UCB units were analyzed. CD34+ cells and mature T lymphocytes increased (viability 99%). Mature B lymphocytes and MSCs decreased, maintaining viability. Granulocytes decreased with loss of viability. Monocytes and immature B lymphocytes remained stable. Clonogenic assays showed a decrease in colony-forming unit (CFU) number in UCB units stored for 96 hours. CONCLUSION: UCB manipulation did not influence cell viability. All cell subsets remained viable until 96 hours after collection. CD34+ cells and T lymphocytes increased, probably due to the loss of other subsets. CFU growth during the period analyzed and confirmed stem cell functionality, despite the decrease at 96 hours. Results demonstrated that UCB units could probably be processed up to 96 hours after collection.Transfusion 01/2013; 53(9). DOI:10.1111/trf.12078 · 3.57 Impact Factor
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ABSTRACT: Cryopreserved umbilical cord blood (CB) is increasingly used as a cell source to reconstitute marrow in hematopoietic stem cell transplant patients. Delays in cryopreservation may adversely affect cell viability, thereby reducing their potential for engraftment after transplantation. The impact of delayed cryopreservation for up to 3 days on the viability of both CD45+ and CD34+ cell populations in 28 CB donations with volumes of 58.40 ± 15.4 mL (range, 39.4-107.4 mL) was investigated to establish whether precryopreservation storage time could be extended from our current time of 24 to 48 hours in line with other CB banks. Viability was assessed on 3 consecutive days, both before and after cryopreservation, by flow cytometry using 7-aminoactinomycin D (7-AAD) and annexin V methods. The results using 7-AAD and annexin V indicated the viability of CD34+ cells before cryopreservation remained high (>92.33 ± 4.11%) over 3 days, whereas the viability of CD45+ cells decreased from 86.36 ± 4.97% to 66.24 ± 7.78% (p < 0.0001) by Day 3. Storage time significantly affected the viability of CD34+ cells after cryopreservation. Using 7-AAD, the mean CD34+ cell viability decreased by approximately 5% per extra day in storage from 84.30 ± 6.27% on Day 1 to 79.01 ± 7.44% (p < 0.0057) on Day 2 and to 73.95 ± 7.54% (p < 0.0001) on Day 3. With annexin V staining CD34+ cell viability fell by approximately 7% per extra day in storage from 77.17 ± 8.47% on Day 1 to 69.56 ± 13.30% (p < 0.0194) on Day 2 and to 62.89 ± 15.22% (p < 0.0002) on Day 3. This study demonstrates that extended precryopreservation storage adversely affects viability and should be avoided.Transfusion 11/2013; 54(5). DOI:10.1111/trf.12481 · 3.57 Impact Factor