Difference in Viability of CD34+ Cells in Cryopreserved Cord Blood According to Evaluation Methods
01 Jun 2009-The Korean Journal of Hematology (The Korean Society of Hematology)-Vol. 44, Iss: 2, pp 92-99
TL;DR: After thawing the cryopreserved UCB, 89% of the total MNCs and 84% of CD34+ cells were viable as identified by trypan blue exclusion assay, and the cell death rate was found to be 47% by Annexin-V/PI staining and less than 5% by DNA contents analysis.
Abstract: Background: On performing umbilical cord blood (UCB) transplantation, faster engraftment may lead better clinical outcome. Because transplanted viable cell count in UCB is related to the engraftment, accu- rate evaluation of viability of CD34+ cells in cryopreserved UCB has clinical implication. We examined the difference in viability of cells in cryopreserved UCB according to the duration of cryopreservation and different methods. Methods: A total of 60 UCB samples which were cryopreserved for 1 to 4 years were used in this study. Viability of cryopreserved cells were examined with trypan blue exclusion assay, DNA contents analysis, caspase-3 activation test, intracellular esterase activity and Annexin-V/PI staining. Results: After thawing the cryopreserved UCB, 89% of the total MNCs and 84% of CD34+ cells were viable as identified by trypan blue exclusion assay. In the CD34+ cell population, the cell death rate was found to be 47% by Annexin-V/PI staining and less than 5% by DNA contents analysis. However, cspase-3 activity failed to document apoptosis. The intracellular esterase activity test also showed a cell death rate of about 10 ∼20% at 2, 4, and 6 hours after thawing.
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TL;DR: It is concluded that cryopreservation-thawing of human sperm from patients and donors was associated with membrane change, as revealed by membrane translocation of phosphatidylserine, while having no major impact on DNA fragmentation.
Abstract: The objective of these studies was to evaluate the effect of cryopreservation-thawing of human spermatozoa on DNA fragmentation and membrane integrity. This was a prospective, controlled cohort study, performed at a university-based infertility center. Ejaculates were examined from 5 donors and 16 men undergoing infertility evaluation. Purified sperm populations were prepared by gradient centrifugation, cryopreserved using a manual method and TEST-yolk buffer and glycerol (TYB-G), followed by quick-thaw. Annexin V binding was used for assessing membrane translocation of phosphatidylserine, and terminal deoxynucleotidyl transferase-me-diated dUTP nick end labeling (TUNEL) was utilized for the evaluation of DNA fragmentation. The results were as follows: the percentage of live cells with intact membranes (annexin V−, live) was significantly reduced after cryopreservation-thawing. On the other hand, the percentages of live cells with phosphatidylserine translocation (annexin V+, live) and of necrotic (dead) cells increased significantly after thawing. TUNEL revealed percentages of cells with DNA fragmentation in the prefreeze and postthaw samples that were not significantly different. In a further attempt to examine differences in response to various cryoprotection protocols, experiments were carried out using no cryoprotection, glycerol alone, or TYB-G. Samples frozen with TYB-G demonstrated significantly higher percentages of live cells without phosphatidylserine translocation than the other conditions. We concluded that cryopreservation-thawing of human sperm from patients and donors was associated with membrane change, as revealed by membrane translocation of phosphatidylserine, while having no major impact on DNA fragmentation.
118 citations
TL;DR: Cryopreservation-thawing was associated with induction of membrane PS translocation; postthaw ROS levels were lower than before freezing; and neither annexin V binding results nor the generation of ROS were able to accurately predict sperm cryosurvival rates.
90 citations
TL;DR: The vital stain SytoR16 in combination with the permeability marker 7-amino actinomycin D, (7-AAD) has been used to identify an early stage of apoptosis, not detected with trypan blue or 7-A AD alone or with conventional apoptosis tests and not consistently and only partly detected by the early apoptosis marker annexin V.
Abstract: Apoptosis is the common cell death pathway which is initiated by a variety of different stimuli. The recognition of early apoptotic events would markedly improve reliability and convenience of apoptosis assays. In the present study the vital stain SytoR 16 in combination with the permeability marker 7-amino actinomycin D, (7-AAD) has been used to identify an early stage of apoptosis, not detected with trypan blue or 7-AAD alone or with conventional apoptosis tests and not consistently and only partly detected by the early apoptosis marker annexin V. The method was established using solid tumour cell lines treated with TNF. Subsequently we applied it to determine apoptotic populations in CD34(+) peripheral blood progenitor cells obtained from growth factor and/or chemotherapy mobilised patients and frozen/thawed according to standard stem cell transplantation protocols. In a cell line model as well as CD34(+) progenitor cells, different subpopulations with decreased SytoR 16 fluorescence (SytoR 16int or SytoR 16low, compared with the normal SytoR 16high) appeared which are not, or only partly, apoptotic using conventional techniques including morphology or 7-AAD staining: eg percentages of SytoR 16(int)/7-AAD(-) and SytoR 16(low)/7-AAD(-) may amount to the majority of cells present in a particular CD34(+) sample. Second, upon further incubation these subpopulations become late apoptotic/secondary necrotic much faster than the unmodified SytoR 16high population, as determined with 7-AAD staining and morphology. Third, these cells have strongly or completely reduced clonogenic capacity for committed (CFU-GM) and early (LTC-IC, determined only for CD34(+) cells) progenitors. This technique needs the inclusion of a blocker of P-glycoprotein, which is highly active in CD34(+) progenitor cells. This prevents the interference of the detection of SytoR16(low) apoptotic cells by SytoR 16low cells resulting from P-glycoprotein activity. By comparison with other apoptosis markers we found that early apoptotic subpopulations were detected in the order SytoR 16 > annexin V > 7-AAD. In conclusion, the combination of SytoR 16 and 7-AAD detects apoptotic events earlier than conventional apoptosis techniques or annexin V. Compared to the presently available viability tests, it allows a much better estimation of the number of viable clonogenic CD34(+) cells after freeze/thawing.
83 citations
TL;DR: This research presents a novel and scalable approach called “Smart Marrow Donor Selecting” that allows for rapid and efficient decision-making in the selection of a single donor for a single organ.
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TL;DR: The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for a more correct determination of the number of viable nonapoptotic cells in stem cell grafts, and should allow its incorporation into the routine CD 34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.
Abstract: Quality assessment of stem cell grafts is usually performed by flow cytometric CD34+ enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of ea...
65 citations