Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation

Yang, Hang; Acker, Jason P.; Cabuhat, M; McGann, L.E.

doi:10.1038/sj.bmt.1704247

Cited by 29 publications

(31 citation statements)

References 16 publications

(31 reference statements)

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“…The present study demonstrates that deep-freezing (-70°C) may allow banking of murine neurospheres, with preserved functionality yielding a minimum of 50% NPC recovery. In comparison, cryopreservation of HSCs, which have been widely used clinically, retrieved 50%-60% HSCs [24,25]. We used a rate-controlled freezing device, which was affordable and easy to handle.…”

Section: Discussionmentioning

confidence: 99%

Cryopreservation Does Not Affect Proliferation and Multipotency of Murine Neural Precursor Cells

2005

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Stem cell research offers unique opportunities for developing new medical therapies for devastating diseases and a new way to explore fundamental questions of biology. Establishing an efficient freezing protocol for neural precursor cells (NPCs) is of great importance for advances in cell-based therapies. We used fluorescence-activated cell sorter-based cell death/survival analysis and Western blot analysis of proliferation markers (proliferating cell nuclear antigen) and prosurvival proteins (Bcl-2) to study the effect of a variety of cryoprotective agents on fetal mouse forebrain NPCs. Neurospheres frozen at -70ºC or in liquid nitrogen in a ratecontrolled manner and thawed after 5 days retained viability of 60%-70% measured 24 hours after thawing. However, 1 week after thawing, viability dropped to 50%-60%. Using a clonogenic sphere formation assay, we showed that recovery rate of frozen NPCs was approximately 26% and did not significantly differ between dimethyl sulfoxide (DMSO)-and glycerol-supplemented samples. Application of the caspase inhibitor zVAD-fmk during freezing or in the first week after thawing resulted in protection of cryopreserved neurospheres after thawing but not during the freezing process, indicating that apoptosis limits recovery of NPCs. Cell survival was not reduced in cells that were enzymatically separated before cryopreservation. Optimal protection of NPCs was achieved when 10% DMSO alone or in a combination with 10% fetal calf serum (FCS) was used. However, 10% glycerol alone was equally effective. Using these protocols, NPCs retained their multipotency and differentiated into both glial (GFAP-positive) and neuronal (Tuj1-positive) cells. Percentage of Tuj1-positive cells in 5% and 10% DMSO, in 10% DMSO + 10% FCS, and in 10% glycerol remained at the same level as before freezing and varied from 5%-7%. We conclude that cryopreservation (up to 1 month at -70°C and up to 1 year in liquid nitrogen) does not markedly alter the rate of proliferation and multipotency of murine neural precursor cells. Stem Cells

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Section: Discussionmentioning

confidence: 99%

Cryopreservation Does Not Affect Proliferation and Multipotency of Murine Neural Precursor Cells

2005

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“…However, these studies showed considerable variability (0-304%) after cryopreservation 4 and an increased mean of 1.22% viable CD34 þ cells before cryopreservation to a mean of 2.16% after cryopreservation. 5 Other studies 4,[6][7][8][9][10][11][12][13][14][15][16][17][18][19] have shown more convincing results, but the washing of the postcryopreservation component for the enumeration of CD34 þ cells does not represent the actual number of CD34 þ cells in the DMSO-containing HPC products that are infused in the patients. The above procedures used for CD34 þ cell enumeration and CFU-GM assay (if performed) had not been validated and standardized, and the increased viability after cryopreservation (4100%) made the proposal even more dubious.…”

Section: Cd34mentioning

confidence: 99%

“…4,8 This wide variation of the post-cryopreservation viability resulted from the post-thaw assessment assays being based on the number of nucleated cells remaining after cryopreservation. 9,10 However, the number of nucleated cells, particularly neutrophils, is decreased during cryopreservation and during post-thaw incubation due to cell loss and clumping of the damaged cells. 11,12 In dual-platform CD34 þ enumeration assays, this decrease in the number of nucleated cells results in an apparent increase in the percentage of CD34 þ cells when nucleated cell count is used as the denominator to calculate the total post-cryopreservation CD34 þ cells.…”

Section: Cd34mentioning

confidence: 99%

“…Even when using a validated single-platform CD34 þ enumeration technique to obtain absolute CD34 þ cells, assays did not account for cells lost during cryopreservation or post-thaw washing, 13 nor were comparisons made with proliferation of progenitor cells (granulocyte-macrophage colony-forming units (CFU-GM)). 7 In previous studies, 9,10 we validated the use of post-cryopreservations assays for assessing postcryopreservation viability of CD34 þ cells and CFU-GM with high accuracy. 7 It is our hypothesis that post-thaw assessment of CD34 þ cell viability and CFU-GM may be a more accurate method to predict hematopoietic engraftment.…”

Section: Cd34mentioning

confidence: 99%

“…4,5,20 Our previous studies have validated the assays for the post-cryopreservation assessment of CD34 þ cells and CFU-GM in HPC by eliminating variability inducing factors and defining assay conditions. 9,10,13 In this study, we used validated assays to assess HPC components (52 patients) stored in quality control vials, and correlated the percent of recovery and infused dose of viable CD34…”

Section: Cd34mentioning

confidence: 99%

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Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment

Yang

Acker

Cabuhat

et al. 2005

Bone Marrow Transplant

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Summary:In all, 78 peripheral hematopoietic progenitor cell collections from 52 patients were evaluated using our previously published validated post-thaw assays at the time of collection and following transplantation by assessment of viable CD34 þ cells, and granulocytemacrophage colony-forming units (CFU-GM) cryopreserved in quality control vials. The median (range) post-thaw recovery of viable CD34 þ cells and CFU-GM was 66.4% (36.1-93.6%) and 63.0% (28.6-85.7%), respectively, which did not show significant correlation with the engraftment of either neutrophils (P ¼ 0.136 and 0.417, respectively) or platelets (P ¼ 0.88 and 0.126, respectively). However, the reinfused viable CD34 þ cells/ kg of patient weight pre-or post-cryopreservation showed significant correlation to engraftment of neutrophils (P ¼ 0.0001 and 0.001, respectively) and platelets (P ¼ 0.023 and 0.010, respectively), whereas CFU-GM pre-or post-cryopreservation was significantly correlated to neutrophils (P ¼ 0.011 and 0.007, respectively) but not to platelets (P ¼ 0.112 and 0.100, respectively). The results show that post-cryopreservation assessment of viable CD34 þ cells or CFU-GM is as reliable a predictor of rapid engraftment as that of pre-cryopreservation measures. Therefore, the post-cryopreservation number of viable CD34 þ cells or CFU-GM should be used to eliminate the risks of unforeseen cell loss that could occur during cryopreservation or long-term storage.

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Cryopreservation of Hematopoietic Stem/Progenitor Cells for Therapeutic Use

Watt

Austin

Armitage

2007

Cryopreservation and Freeze-Drying Protocols

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To date, more than 25,000 hematopoietic transplants have been carried out across Europe for hematological disorders, the majority being for hematological malignancies. At least 70% of these are autologous transplants, the remaining 30% being allogeneic, which are sourced from related (70% of the allogeneic) or unrelated donors. Peripheral blood mobilized with granulocyte colony stimulating factor is the major source of stem cells for transplantation, being used in approx 95% of autologous transplants and in approx 65% of allogeneic transplants. Other cell sources used for transplantation are bone marrow and umbilical cord blood. One crucial advance in the treatment of these disorders has been the development of the ability to cryopreserve hematopoietic stem cells for future transplantation. For bone marrow and mobilized peripheral blood, the majority of cryopreserved harvests come from autologous collections that are stored prior to a planned infusion following further treatment of the patient or at the time of a subsequent relapse. Other autologous harvests are stored as backup or "rainy day" harvests, the former specifically being intended to rescue patients who develop graft failure following an allogeneic transplant or who may require this transplant at a later date. Allogeneic bone marrow and mobilized peripheral blood are less often cryopreserved than autologous harvests. This is in contrast to umbilical cord blood that may be banked for directed or sibling (related) hematopoietic stem cell transplants, for allogeneic unrelated donations, and for autologous donations. Allogeneic unrelated donations are of particular use for providing a source of hematopoietic stem cells for ethnic minorities, patients with rare human leukocyte antigen types, or where the patient urgently requires a transplant and cannot wait for the weeks to months required to prepare a bone marrow donor. There are currently more than 200,000 banked umbilical cord blood units registered with the Bone Marrow Donors Worldwide registry. In this chapter, we describe several protocols that we have used to cryopreserve these different sources of hematopoietic stem/progenitor cells, keeping in mind that the protocols may vary among transplant processing centers.

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Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation

Cited by 29 publications

References 16 publications

Cryopreservation Does Not Affect Proliferation and Multipotency of Murine Neural Precursor Cells

Cryopreservation Does Not Affect Proliferation and Multipotency of Murine Neural Precursor Cells

Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment

Cryopreservation of Hematopoietic Stem/Progenitor Cells for Therapeutic Use

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