The influence of temperature treatment before cryopreservation on the viability and potency of cryopreserved and thawed CD34+ and CD45+ cord blood cells.

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.

Cord blood collection and processing with hydroxyethyl starch or non-hydroxyethyl starch.

BACKGROUND: Collection and processing characteristics influencing quality of cord blood (CB) units play an essential role to cord blood banks (CBBs). At many CBBs, volume reduction is performed using hydroxyethyl starch (HES) and the Sepax (Biosafe) automated cell processing system. Due to the withdrawal of HES from the European market, a validation of the nonHES protocol was performed. METHODS: This partially retrospective study identified CB characteristics such as gestational age and CB volume/cell count correlated with higher quality. For the nonHES validation, CB was analyzed for total nucleated cell (TNC), mononuclear cell (MNC) recovery, hematocrit (HCT) and colony-forming units (CFUs). Viabilities of CD34(+) and CD45(+) cells were determined by 7-aminoactinomycin D (7-AAD) and AnnexinV (AnnV) staining and compared for 21 mL and 42 mL buffy coat (BC) samples applying the HES/nonHES protocol. RESULTS: Factors affecting the potency of CB transplants were the gestational age and the volume reduction to a defined BC volume. High initial cell counts and CB volumes correlated negatively with post-processing TNC recovery for lower BC volumes. Post-processing HES and nonHES results were comparable, but nonHES revealed a significantly lower post-thaw recovery of viable CD34(+) cells measured by 7-AAD/AnnV (21 mL: 45.4 ± 16.4%; 42 mL: 67.3 ± 14.5%) as compared with HES (21 mL: 72.7 ± 14.4%, P = 0.0164; 42 mL: 83.4 ± 14.7%, P = 0.0203). DISCUSSION: Due to the lower post-thaw CD34(+) cell viability (AnnV(-)/7-AAD(-)) for nonHES samples, the use of HES is recommended, ideally combined with a high BC volume. The post-processing HCT has no statistically significant impact on the post-thaw CD34(+) cell viability (AnnV(-)/7-AAD(-)).