Svenja Schwandt1, Stefanie Liedtke2, Gesine Kogler2. 1. Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University Medical Center, Duesseldorf, Germany. Electronic address: svenja.schwandt@uni-duesseldorf.de. 2. Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University Medical Center, Duesseldorf, Germany.
Abstract
BACKGROUND: Hematopoietic stem cell (HSC) viability and potency is crucial for qualified cord blood (CB) transplants. This study analyzes time and temperature condition before cryopreservation for the viability of CD34+/CD45+ cells after cryopreservation. METHODS: Cell viabilities were determined by antibody co-staining with 7-aminoactinomycin D detecting necrotic cells, and subsequent flow cytometric analysis. Additionally, Annexin V staining for determination of apoptotic cells and colony-forming unit (CFU) assays for testing functional potency of HSCs were performed. RESULTS: For all cell types assessed (CD45+/CD34+ cells, lymphocytes and granulocytes), the highest viabilities were obtained for CB maintained at 4°C or room temperature (RT; 22 ± 4°C) and cryopreserved directly after collection. Starting material were CB units with an age of 24.7 ± 3.5 h after birth. Post-thaw CD34+ cell results were > 90% after temperature treatment of t = 24 h (48 h total age) and > 70% after t = 48 h (72 h total age) at 4°C (48 h, 91.4 ± 5.5%; 72 h, 75.0 ± 12.0%) and RT (48 h, 84.2 ± 9.7%; 72 h, 72.6 ± 0.6%). Viabilities for 30°C samples were < 80% after t = 24 h (48 h total age, 79.8 ± 3.1%) and < 50% after t = 48 h of treatment (72 h total age, 46.8 ± 14.3%). Regarding CFU recovery of pre-freeze (without volume reduction) and thawed CB, a trend toward the highest recoveries was observed at 4°C/RT. The difference between 4°C (77.5 ± 12.0%) and 30°C samples (53.9 ± 4.8%) was shown to be significant in post-thaw samples after t = 24 h treatment (48 h total age; P = 0.0341). DISCUSSION: Delays between collection and cryopreservation should be minimized because increasing time reduces numbers of viable cells and CFUs before/after cryopreservation. CB units should be maintained at 4°C/RT to retain the highest possible potency of the cells after thawing.
BACKGROUND: Hematopoietic stem cell (HSC) viability and potency is crucial for qualified cord blood (CB) transplants. This study analyzes time and temperature condition before cryopreservation for the viability of CD34+/CD45+ cells after cryopreservation. METHODS: Cell viabilities were determined by antibody co-staining with 7-aminoactinomycin D detecting necrotic cells, and subsequent flow cytometric analysis. Additionally, Annexin V staining for determination of apoptotic cells and colony-forming unit (CFU) assays for testing functional potency of HSCs were performed. RESULTS: For all cell types assessed (CD45+/CD34+ cells, lymphocytes and granulocytes), the highest viabilities were obtained for CB maintained at 4°C or room temperature (RT; 22 ± 4°C) and cryopreserved directly after collection. Starting material were CB units with an age of 24.7 ± 3.5 h after birth. Post-thaw CD34+ cell results were > 90% after temperature treatment of t = 24 h (48 h total age) and > 70% after t = 48 h (72 h total age) at 4°C (48 h, 91.4 ± 5.5%; 72 h, 75.0 ± 12.0%) and RT (48 h, 84.2 ± 9.7%; 72 h, 72.6 ± 0.6%). Viabilities for 30°C samples were < 80% after t = 24 h (48 h total age, 79.8 ± 3.1%) and < 50% after t = 48 h of treatment (72 h total age, 46.8 ± 14.3%). Regarding CFU recovery of pre-freeze (without volume reduction) and thawed CB, a trend toward the highest recoveries was observed at 4°C/RT. The difference between 4°C (77.5 ± 12.0%) and 30°C samples (53.9 ± 4.8%) was shown to be significant in post-thaw samples after t = 24 h treatment (48 h total age; P = 0.0341). DISCUSSION: Delays between collection and cryopreservation should be minimized because increasing time reduces numbers of viable cells and CFUs before/after cryopreservation. CB units should be maintained at 4°C/RT to retain the highest possible potency of the cells after thawing.
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