Viability of CD34+ Cells in Cryopreserved Cord Blood

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4138-4138
Author(s):  
Young-woo Eom ◽  
Seong Hyun Jeong ◽  
Jin-Hyuk Choi ◽  
Seok Yun Kang ◽  
Hyun Woo Lee ◽  
...  
Keyword(s):  
Cord Blood ◽  
Esterase Activity ◽  
Death Rate ◽  
Trypan Blue ◽  
Caspase 3 ◽  
Annexin V ◽  
Trypan Blue Exclusion ◽  
Cell Death Rate ◽  
Cd34 Cells ◽  
Dna Contents

Abstract Purpose: 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, we examined cryopreserved UCB cells with several methods after thawing. Methods: Viability of cryopreserved cells were examined with trypan blue, DNA contents analysis, caspase-3 activation test, intracellular esterase activity and Annexin-V/PI staining. Results: A total of 60 samples were used in this study. After thawing, 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. Caspase-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. Conclusion: Viable cells in UCB should be measured by several compensatory techniques rather than a single method. Discordance among Annexin-V/PI staining versus trypan blue exclusion, DNA contents analysis, and the caspase-3 activation test or intracellular esterase activity should be clarified in order to apply these techniques for actual cord blood transplantation.

Inhibition of FcγRIIA Mediated Platelet Phagocytosis with Syk-Specific siRNA Affects PECAM-1 Mediated Signal Transduction and Cell-Cell Detachment in Human Macrophages.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 517-517
Author(s):  
Diana S. Beardsley ◽  
Bing-guan Chen ◽  
Oleg Kruglov
Keyword(s):  
Signal Transduction ◽  
Monoclonal Antibody ◽  
Tyrosine Kinase ◽  
Trypan Blue ◽  
Annexin V ◽  
Cell Spreading ◽  
Cell Detachment ◽  
Intercellular Interaction ◽  
Trypan Blue Exclusion ◽  
Cell Cell

Abstract We have previously shown that Syk-specific siRNA inhibits FcγRIIA mediated platelet phagocytosis in the human macrophage cell line, THP-1. Surprisingly, we observed that THP-1 cell-cell interactions were affected by Syk-specific siRNA suggesting that Syk may be important in the signaling pathway for physiological cell spreading. As reported by Brown et al. (Nature418:200, 2002), homophilic ligation of PECAM-1 on macrophages triggers cell-cell detachment, prompting us to speculate that Syk may be involved in PECAM-1 mediated signaling. PECAM-1 is a member of the immunoglobulin superfamily that contains an immunoreceptor tyrosine inhibitory motif (ITIM) required for inhibitory signaling and cell spreading via binding protein tyrosine phosphatase. The structure of Syk reveals considerable flexibility in the relative orientation of its tandem SH2 domains providing a molecular basis for the potential involvement of Syk in a variety of signal transduction pathways. Thus, the five tyrosine residues present in the cytoplasmic domain of human PECAM-1 could serve as docking sites for recruitment of other intracellular signaling molecules such as the protein tyrosine kinase, Syk. The present studies test the hypothesis that Syk may mediate an activation influence on intercellular interaction upon PECAM-1 ligation. METHODS: Association of Syk with PECAM-1 was studied in THP-1 cells by immunoprecipitation and immunoblotting with anti-PECAM-1 monoclonal antibody with or without Syk-specific siRNA. The effect of Syk-specific siRNA on THP-1 cells was evaluated by morphology, cell growth kinetics, Trypan Blue exclusion, annexin V staining, and DNA fragmentation analysis. A series of synthetic peptides including sequences within the ITIM region of PECAM-1 were evaluated for their ability to bind Syk directly in THP-1 lysates and to phosphorylate Syk intracellularly. RESULTS: First, Syk protein was co-immunoprecipitated from THP-1 cell lysates by anti-PECAM-1 monoclonal antibody, and Syk-specific siRNA knocked down PECAM-1 associated Syk in THP-1 cells. Second, knock down of Syk by siRNA resulted in decreased cell spreading and cell proliferation. Trypan Blue exclusion, annexin V staining, and DNA fragmentation were identical in THP-1 cells transfected with control dsRNA and Syk-specific siRNA. Third, the synthetic peptide of residues 658-691 within the ITIM region of PECAM-1 that contains two phosphotyrosines (N-S-D-V-Q-pY-T-E-V-Q-V-S-S-A-E-S-H-K-D-L-G-K-K- D-T-E-T-V-pY-S-E-V-R-K) bound directly to Syk. Fourth, ligation of PECAM-1 on THP-1 cells by anti-PECAM-1 monoclonal antibody resulted in Syk phosphorylation. Fifth, the dual phosphotyrosine 658-691 PECAM-1 peptide triggered direct phosphorylation of Syk in permeabilized THP-1 cells. CONCLUSIONS: Our novel observations suggest that signal transduction after PECAM-1 ligation may trigger an activation pathway through the tyrosine kinase, Syk, and facilitate intercellular adhesion. The decreased proliferation observed in THP-1 cells transfected with Syk-specific siRNA is not a result of cell death or apoptosis. Based on our findings, we propose that PECAM-1 may exhibit both ITIM (inhibitory) and ITAM (activation) properties and that the balance of phosphatase and Syk activity triggered by PECAM-1 homophilic binding may affect the fate of intercellular interaction. These processes are expected to influence the net degree of phagocytosis of opsonized platelets by the reticuloendothelial system in patients with immune thrombocytopenia.

The Effect of Physical Conditions on the Transportation of Peripheral Blood Progenitor (PBPC) Products.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2315-2315
Author(s):  
Jan Jansen ◽  
Pamela L Nolan ◽  
Margaret I Reeves ◽  
Luke Paul Akard ◽  
James M. Thompson ◽  
...  
Keyword(s):  
Trypan Blue ◽  
Room Temperature ◽  
Trypan Blue Exclusion ◽  
Physical Conditions ◽  
Cd34 Cells ◽  
Wide Range ◽  
Duration Of Storage ◽  
Higher Temperature ◽  
Storage Times ◽  
And Storage

Abstract The viability of transported PBPC products has not been studied extensively. Commonly, PBPC products are transported at a concentration of >200 x109/l in containers with −20oC ice packs. Continuous temperature monitoring has shown that the temperatures of these products stays at <10oC for less than 24 hours and reaches room temperature by 48 hours. Samples of freshly collected PBPC from 12 allogeneic donors were studied for various viability parameters during storage for up to 96 hours. The effects of storage time, concentration of cells, temperature, and storage in gas-permeable bags were studied. Trypan-blue exclusion and double fluorescence for 7-AAD and CD34 were used for viability assessment. Over a wide range of temperatures and storage times, the viable CD34+ assay was more sensitive to damage than trypan-blue exclusion (mean Δ 10.7%, p<0.0001 in paired t-test). The viable CD34+ assay was routinely used in parallel with CFU-GM cultures. No difference in survival of viable CD34+ cells or CFU-GM was found whether cells were incubated for 48hr in test-tubes or in gas-permeable bags. When cells at 200 x 109/l were incubated for 48hr at room temperature, the mean viability decreased to 19% and 6% of starting values of viable CD34+ cells and CFU-GM, respectively. Serial dilution to 25 x 109/l improved the survival to 81% and 51% respectively. Similarly, incubation at lower temperatures led to better survival of CD34+ cells and CFU-GM: 67% and 18% at 17oC, 80% and 50% at 13oC, and 95% and 86% at 4oC. At 200 x109/l and 22oC the survivals of CD34+ cells and CFU-GM were 74% and 21% at 24hr, 19% and 7% at 48hr, 7% and 6% at 72hr, and 3% and 13% at 96hr. The effects of concentration, temperature and duration of storage were all significant (p<0.05). Transportation at 4oC leads to the best survival of CD34+ cells and CFU-GM, in particular at a low concentration. If transportation at a slightly higher temperature is necessary, dilution of the PBPC product will enhance the survival of CD34+ cells and CFU-GM. Proliferative assays such as CFU-GM appear the most sensitive parameters of PBPC survival, and should be included in the validation process of PBPC transportation.

The p55 TNF receptor mediates TNF inhibition of osteoblast differentiation independently of apoptosis

2005 ◽  
Vol 288 (5) ◽  
pp. E1011-E1018 ◽  
Author(s):  
Linda C. Gilbert ◽  
Janet Rubin ◽  
Mark S. Nanes
Keyword(s):  
Cell Viability ◽  
Caspase 3 ◽  
Annexin V ◽  
Inhibitory Effect ◽  
Tnf Receptor ◽  
Trypan Blue Exclusion ◽  
Total Dna ◽  
Factor Α ◽  
Tnf Inhibition

After menopause, increased tumor necrosis factor-α (TNF-α) stimulates bone resorption while inhibiting differentiation of new bone-forming osteoblasts (OB). TNF receptors, p55 and p75, signal similar intracellular pathways, but only p55 activates apoptosis. To evaluate the relationship between the TNF receptor mediating inhibition of OB differentiation and the role of apoptosis, marrow stromal cells (MSC) were cultured from mice deficient in either or both receptors. Cells grown in ascorbate and β-glycerophosphate produce alkaline phosphatase and osteocalcin and mineralize matrix. Treatment of wild-type or p55+/+/p75−/− MSC with murine TNF (binds p55 and p75) or human TNF (binds only p55) inhibited OB differentiation. TNF did not inhibit OB differentiation in p55−/− MSC. Expression of p75 modestly attenuated sensitivity to TNF. To determine the role of apoptosis, changes in total DNA, cell viability, caspase 3, and percentage of annexin V-positive cells were measured in MSC and preosteoblastic MC3T3 cells. TNF treatment that reduced differentiation by 50% did not decrease cell viability or increase apoptosis, as determined by alamar blue reduction, trypan blue exclusion, and percentage of annexin V-positive cells. TNF increased caspase 3 activity 1.5-fold in MC3T3 and insignificantly in MSC cells compared with >4-fold after 4 h actinomycin D. Treatment of MSC or MC3T3 cells with three caspase inhibitors failed to reverse the inhibitory effect of TNF on OB differentiation despite inhibition of caspase activity. These results suggest that the p55 receptor is essential, and p75 dispensable, for TNF inhibition of OB differentiation through a mechanism that does not require apoptosis.

Expression of caspase-3 in cord blood CD34+ cells during culture in vitro

2003 ◽  
Vol 2 (3) ◽  
pp. 166-168
Author(s):  
Ma Yanping ◽  
Li Rongping ◽  
Zou Ping ◽  
Xiao Juan ◽  
Huang Shiang ◽  
...  
Keyword(s):  
Cord Blood ◽  
Caspase 3 ◽  
Culture In Vitro ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Flow cytometry assessment of apoptotic CD34+ cells by annexin V labeling may improve prediction of cord blood potency for engraftment

Transfusion ◽  
2011 ◽  
Vol 52 (3) ◽  
pp. 549-559 ◽  
Author(s):  
Richard C. Duggleby ◽  
Sergio Querol ◽  
Robert C. Davy ◽  
Laura J. Fry ◽  
Daniel A. Gibson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cord Blood ◽  
Annexin V ◽  
Cd34 Cells

scholarly journals Induction of Necrosis and DNA Fragmentation during Hypothermic Preservation of Hepatocytes in UW, HTK, and Celsior Solutions

2003 ◽  
Vol 12 (1) ◽  
pp. 59-68 ◽  
Author(s):  
Salomon L. Abrahamse ◽  
Pieter Van Runnard Heimel ◽  
Robin J. Hartman ◽  
Rob A. F. M. Chamuleau ◽  
Thomas M. Van Gulik
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Dna Fragmentation ◽  
Trypan Blue ◽  
Caspase 3 ◽  
Trypan Blue Exclusion ◽  
Hypothermic Preservation ◽  
Mtt Reduction ◽  
Ldh Release ◽  
Porcine Hepatocytes

Donor cells can be preserved in University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), or Celsior solution. However, differences in efficacy and mode of action in preventing hypothermia-induced cell injury have not been unequivocally clarified. Therefore, we investigated and compared necrotic and apoptotic cell death of freshly isolated primary porcine hepatocytes after hypothermic preservation in UW, HTK, and Celsior solutions and subsequent normothermic culturing. Hepatocytes were isolated from porcine livers, divided in fractions, and hypothermically (4°C) stored in phosphate-buffered saline (PBS), UW, HTK, or Celsior solution. Cell necrosis and apoptosis were assessed after 24- and 48-h hypothermic storage and after 24-h normothermic culturing following the hypothermic preservation periods. Necrosis was assessed by trypan blue exclusion, lactate dehydrogenase (LDH) release, and mitochondrial 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. Apoptosis was assessed by the induction of histone-associated DNA fragments and cellular caspase-3 activity. Trypan blue exclusion, LDH release, and MTT reduction of hypothermically preserved hepatocytes showed a decrease in cell viability of more than 50% during the first 24 h of hypothermic preservation. Cell viability was further decreased after 48-h preservation. DNA fragmentation was slightly enhanced in hepatocytes after preservation in all solutions, but caspase-3 activity was not significantly increased in these cells. Normothermic culturing of hypothermically preserved cells further decreased cell viability as assessed by LDH release and MTT reduction. Normothermic culturing of hypothermically preserved hepatocytes induced DNA fragmentation, but caspase-3 activity was not enhanced in these cells. Trypan blue exclusion, LDH leakage, and MTT reduction demonstrated the highest cell viability after storage in Celsior, and DNA fragmentation was the lowest in cells that had been stored in PBS and UW solutions. None of the preservation solutions tested in this study was capable of adequately preventing cell death of isolated porcine hepatocytes after 24-h hypothermic preservation and subsequent 24-h normothermic culturing. Culturing of isolated and hypothermically preserved hepatocytes induces DNA fragmentation, but does not lead to caspase-3 activation. With respect to necrosis and DNA fragmentation of hypothermically preserved cells, UW and Celsior were superior to PBS and HTK solutions in this model of isolated porcine hepatocyte preservation.

Quality and Safety of Hematopoietic Progenitor Cell (HPC) Products Cryopreserved by Simple Passive Gradual Freezing Method

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4410-4410
Author(s):  
Salem Akel ◽  
Christianna Henderson ◽  
Donna Regan ◽  
Deepika Bhatla ◽  
William S. Ferguson
Keyword(s):  
Cord Blood ◽  
Cooling Rate ◽  
Trypan Blue ◽  
Outcome Data ◽  
Nitrogen Storage ◽  
Quality And Safety ◽  
Transplant Outcome ◽  
Cd34 Cells ◽  
Crf System ◽  
Liquid Vapor

Abstract Abstract 4410 To provide quality HPC products for transplantation, harvests of bone marrow (BM), Peripheral Blood Stem Cells (PBSCs) and Cord Blood (CB) are routinely cryopreserved by gradual cooling from ambient temperature to −90°C using Controlled Rate Freezing (CRF) systems before placement in liquid/vapor nitrogen storage. CRF systems generally utilize a freezing chamber and computer-controlled freezing cycle with an established sequence of steps where timing, target temperatures and cooling rates are defined. System failures that endanger product quality may occur due to impurities in liquid nitrogen, mechanical/software problems and probe malfunction. A more convenient and economical freezing may be performed by simple “passive freezing” (PF) where products are placed directly into an ultra-low freezer (−80°C). Continuous temperature monitoring during the freeze process is achieved using individual data loggers. In the PF process, products remain undisturbed during the freeze process, and are removed for placement in liquid/vapor nitrogen storage when the target product temperature is reached (−80°C). Probe specific freezing curves are generated from downloaded data logger information. In an internal validation study, the St. Louis Cord Blood Bank (SLCBB) investigated quality and safety of HPC products cryopreserved using this PF method. Evaluation was based on the analysis of freezing kinetics, post-thaw cell recoveries, and available transplant outcome data in reference to those obtained by the conventional CRF method. A total of twelve (12) red blood cell and plasma reduced CB products cryopreserved in 10% DMSO/Dextran by PF were thawed according to a validated albumin/dextran reconstitution thaw method. Compared to the programmed CRF system, PF product cooling rate to a temperature of −60°C was faster (1.2– 2.0°C/min compared to 1°C/min), ultimately followed by slower cooling rate to a product target temperature of −80°C (approximately 0.4°C/min compared to 10°C/min). The kinetics of PF curves resulted in longer freezing cycles with an average freeze time of 180 minutes compared to 80 minutes by CRF method. Post-thaw cell recoveries of the PF data set were comparable to those obtained from an established thaw control group of RBC and plasma reduced CB cryopreserved by CRF system (n = 25). Average percent recoveries for CB products cryopreserved by PF methodology compared to CRF system were reported as follows, respectively: viable nucleated cells (NC) by trypan blue = 84% +/− 5% compared to 71% +/− 6%, viable (7AAD) CD34+ cells = 67% +/− 7% compared to 63% +/− 15%, and colony forming unit (CFU) = 73% +/− 7% compared to 72% +/− 14%. Subsequently, the SLCBB reviewed post-thaw cell recoveries of ten (10) PBSC harvests cryopreserved by PF and thawed at the SLCBB for clinical transplantation. Similar to the CB evaluation, results indicated a high quality HPC product post-thaw with average percent recoveries reported as follows: viable nucleated cells (NC) by trypan blue = 79% +/− 10%, viable (7AAD) CD34+ cells = 81.2% +/− 20.2, and CFU = 73.6% +/− 21.9%. All PBSC products were thawed and infused with no reports of infusion-related adverse events. These PBSC products engrafted shortly within predicted time; absolute neutrophil count (ANC) > 500/uL was reported within 12 days, while platelets > 20,000/uL was achieved within 22 days. Retrospective review of more than 1,800 CB products distributed by the SLCBB for transplant showed that five (5) PF products were transplanted as singlet products to treat patients with myeloid malignancies. Transplant outcome data received by CIBMTR for those cases indicated 100% success of engraftment and 100% survival at 100 days post-infusion. Engraftment data reports indicated ANC > 500 was achieved within 6–30 days, and platelet count > 20,000 was achieved within 25–163 days. The transplant outcome of PF product is comparable to that reported historically for CRF CB products. Conclusions: In vitro results and clinical findings support the quality and safety of HPC products cryopreserved using PF methodology and would recommend PF as valid alternative to the use of CRF systems. Disclosures: No relevant conflicts of interest to declare.

Design, Synthesis and In Vitro Anti-Cancer Evaluation of Novel Derivatives of 2-(2-Methyl-1,5-diaryl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-3- yl)acetamide

2020 ◽  
Vol 16 (3) ◽  
pp. 340-349
Author(s):  
Ebrahim S. Moghadam ◽  
Farhad Saravani ◽  
Ernest Hamel ◽  
Zahra Shahsavari ◽  
Mohsen Alipour ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Peripheral Neuropathy ◽  
Cell Line ◽  
Normal Cell ◽  
Caspase 3 ◽  
Annexin V ◽  
Normal Cell Line ◽  
Anti Cancer ◽  
Mcf 7

Objective: Several anti-tubulin agents were introduced for the cancer treatment so far. Despite successes in the treatment of cancer, these agents cause toxic side effects, including peripheral neuropathy. Comparing anti-tubulin agents, indibulin seemed to cause minimal peripheral neuropathy, but its poor aqueous solubility and other potential clinical problems have led to its remaining in a preclinical stage. Methods: Herein, indibulin analogues were synthesized and evaluated for their in vitro anti-cancer activity using MTT assay (on the MCF-7, T47-D, MDA-MB231 and NIH-3T3 cell lines), annexin V/PI staining assay, cell cycle analysis, anti-tubulin assay and caspase 3/7 activation assay. Results: One of the compounds, 4a, showed good anti-proliferative activity against MCF-7 cells (IC50: 7.5 μM) and low toxicity on a normal cell line (IC50 > 100 μM). All of the tested compounds showed lower cytotoxicity on normal cell line in comparison to reference compound, indibulin. In the annexin V/PI staining assay, induction of apoptosis in the MCF-7 cell line was observed. Cell cycle analysis illustrated an increasing proportion of cells in the sub-G-1 phase, consistent with an increasing proportion of apoptotic cells. No increase in G2/M cells was observed, consistent with the absence of anti-tubulin activity. A caspase 3/7 assay protocol showed that apoptosis induction by more potent compounds was due to activation of caspase 3. Conclusion: Newly synthesized compounds exerted acceptable anticancer activity and further investigation of current scaffold would be beneficial.

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