Successful cryopreservation of purified autologous CD34+ cells: influence of freezing parameters on cell recovery and engraftment

Beaujean, F.; Bourhis, J.; Bayle, C; Jouault, Hélène; Diviné, Marine; Rieux, Claire; Janvier, Maud; Forestier, C. Le; Jl, Pico

doi:10.1038/sj.bmt.1701494

Cited by 40 publications

(37 citation statements)

References 23 publications

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“…Recently, there have been several reports suggesting that cells can be successfully cryopreserved with minimal concentrations of DMSO [39][40][41][42][43][44][45][46][47][48][49][50][51]. For example, Zhao et al [46] showed that the use of 5% or 10% DMSO in combination with either 20% or 70% serum produced similar results during cryopreservation of fetal human liver CD34 + cells.…”

Section: Discussionmentioning

confidence: 74%

Evaluation of Methylcellulose and Dimethyl Sulfoxide as the Cryoprotectants in a Serum-Free Freezing Media for Cryopreservation of Adipose-Derived Adult Stem Cells

Thirumala

Gimble

Devireddy

2010

Stem Cells and Development

101

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Developing effective techniques for the cryopreservation of human adipose-derived adult stem cells (ASCs) could increase the usefulness of these cells in tissue engineering and regenerative medicine. To this end, we investigated the post-freeze/thaw viability and apoptotic behavior of Passage 1 (P1) adult stem cells (ASCs) in 11 different media: (i) the traditional media containing Dulbecco's modifi ed Eagle's medium (DMEM) with 80% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO), (ii) DMEM with 80% human serum (HS) and 10% DMSO, (iii) DMEM with 1% methyl cellulose (MC) and 10% of either HS or FCS or DMSO, and (iv) DMEM with 0%, 2%, 4%, 6%, 8%, or 10% DMSO. Approximately 1 mL (10 6 cells/mL) of P1 ASCs were frozen overnight in a −80°C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37°C water bath (1-2 min of agitation), resuspended in culture media, and seeded in separate wells of a 6-well plate for a 24-h incubation period at 37°C. After 24 h, the thawed samples were analyzed by bright-fi eld microscopy and fl ow cytometry. The results suggest that the absence of DMSO (and the presence of MC) signifi cantly increases the fraction of apoptotic and/or necrotic ASCs. However, the percentage of viable cells obtained with 2% DMSO and DMEM was comparable with that obtained in freezing media with 10% DMSO and 80% serum (HS or FCS), that is, ~84% ± 5% and ~84% ± 8%, respectively. Adipogenic and osteogenic differentiation behavior of the frozen thawed cells was also assessed using histochemical staining. Our results suggest that post-thaw ASC viability, adipogenic and osteogenic differentiability can be maintained even when they are frozen in the absence of serum but with a minimal concentration of 2% DMSO in DMEM.

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Section: Discussionmentioning

confidence: 74%

Evaluation of Methylcellulose and Dimethyl Sulfoxide as the Cryoprotectants in a Serum-Free Freezing Media for Cryopreservation of Adipose-Derived Adult Stem Cells

Thirumala

Gimble

Devireddy

2010

Stem Cells and Development

101

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“…These results are comparable with recovery data reported after cryopreservation of PBSC (70-90% for nucleated cells, 33-80% for committed progenitors. 12,[30][31][32][33][34][35] In the experiments described in Table 4, three washes were performed before the addition of medium. As a consequence, recovery data were variable.…”

Section: Discussionmentioning

confidence: 99%

Storage of unprocessed G-CSF-mobilized whole blood in a modified Leibovitz's L15 medium preserves clonogenic capacity for at least 7 days

Kreuk

Jonkhoff

Zevenbergen

et al. 2001

Bone Marrow Transplant

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Summary:Autologous stem cell transplantation using unprocessed, G-CSF-mobilized whole blood (WB) is a simple, cost-reducing procedure and supports high-dose chemotherapy regimens not exceeding 72 h. Thereafter, clonogenic capacity rapidly decreases if routine anticoagulants are used for storage. In order to increase clinical applicability, we investigated the requirements for optimal preservation of unprocessed WB for 7 days. During storage at 22؇C in CPDA-1, a decrease in pH was noted, which was at least partially responsible for the low recovery of clonogenic cells. Subsequently, WB cells were stored in various cell culture media (RPMI 1640, ␣-MEM, X-VIVO15, CellGro SCGM and Leibovitz's L15 medium) containing either serum, serum-free substitutes or no additives. Leibovitz's L15 showed significantly better CFU-GM recoveries than the other media. Using a calcium-free modification of L15 medium (added 3:10 to WB), 94 ؎ 24% of CD34 ؉ cells, 41 ؎ 14% of BFU-E, 56 ؎ 17% CFU-GM and 90 ؎ 14% of LTC-IC were preserved during storage for 7 days at 22؇C. Storage at 4؇C was also feasible, but showed less optimal recoveries of 52 ؎ 29% (CD34), 32 ؎ 10% (BFU-E), 13 ؎ 7% (CFU-GM) and 58 ؎ 9% (LTC-IC). The expression of CD38, Thy-1, c-kit, AC133, L-selectin and CXCR4 on CD34-positive cells remained unchanged. In conclusion, a modified Leibovitz's L15 medium better meets the metabolic requirements of a high-density cell culture and allows safe storage of G-CSF mobilized WB for at least 7 days. The results encourage further exploration of WB transplants stored for 7 days for clinical use. Bone Marrow Transplantation (2001) 28, 145-155.

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“…However, the inevitable loss of CD34 þ cells between collection and transplant, especially during cryopreservation, is difficult to predict without assessment of the sample after cryopreservation. 4,5,7,8,18 This leads to the current debate as to whether the quantification of HPC should be performed before or after cryopreservation.…”

Section: Cd34mentioning

confidence: 99%

“…5 In addition, the reported viability of CD34 þ cells varied from 0 to 304%. 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.…”

Section: Cd34mentioning

confidence: 99%

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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Successful cryopreservation of purified autologous CD34+ cells: influence of freezing parameters on cell recovery and engraftment

Cited by 40 publications

References 23 publications

Evaluation of Methylcellulose and Dimethyl Sulfoxide as the Cryoprotectants in a Serum-Free Freezing Media for Cryopreservation of Adipose-Derived Adult Stem Cells

Evaluation of Methylcellulose and Dimethyl Sulfoxide as the Cryoprotectants in a Serum-Free Freezing Media for Cryopreservation of Adipose-Derived Adult Stem Cells

Storage of unprocessed G-CSF-mobilized whole blood in a modified Leibovitz's L15 medium preserves clonogenic capacity for at least 7 days

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

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