Abstract

Studies on the optimal conditions to store fresh cord blood (CB) in order to optimise haematopoietic stem cell (HSC) recovery prior to processing and cryopreservation have produced conflicting data. In this study we investigate the effects of time and temperature on CB HSC after collection. We also investigate whether the process of cryopreservation and thawing aggravates stress created by storage prior to cryopreservation. 30 CB units were collected and transported to the laboratory within 5 hours of collection. An aliquot of each CB unit was tested for nucleated cell count (NCC), CD34 count, CD34 viability and CFU-GEMM, and then frozen with 10% DMSO. Each CB unit was then separated into three 10 ml portions and incubated at 3 different temperatures (4–8°C, 21–24°C and 29–31°C). After 24, 36 or 48 hours, samples were taken and tested. An aliquot of each of the portions was then cryopreserved. After a minimum of 1 week, the cryopreserved samples were thawed and tested. For fresh samples incubated for 24 hrs or 36 hrs there was no difference over time when CB was incubated at 4–8°C, whereas, at temperatures above 20°C, there was either a trend or significant decrease in CD34 and CFU colonies with time. For thawed samples, viable CD34 and CFU colonies were decreased below baseline at either one or both 24 hr and 36 hr time points. This was particularly apparent for CFU colony measurements for samples kept at 29–31°C. At 48 hrs, at each of the temperature ranges (4–8°C, 21–24°C and 29–31°C), CFU were significantly lower for fresh (21.1, 16.8 and 14.0; baseline 31.0) and thawed (9.3, 8.5 and 3.0; baseline 2.7) samples and viable CD34 were significantly lower for thawed (2.0, 2.46 and 1.8; baseline 2.7) samples respectively (p<0.001). In conclusion, the best survival /recovery was seen with CB stored at 4–8° C and for ≥ 36 hrs. Temperatures above room temperature (21–24°C) appeared detrimental. The data also indicate that this damage becomes more evident when tested after the CB has gone through the cryopreservation and thawing process, and is even more evident the higher the temperature and the greater the time of storage prior to cryopreservation. These results have implications for quality and safety of CB stored for clinical use.

Viable CD34 and CFU related to incubation time and temperature, tested pre- and post-cryopreservation.

Viable CD34 cells x 10e6CFU-GEMM /12,500 cells plated
Storage temperature4–8C°21–24°C29–31°C4–8°C21–24°C29–31°C
 Baseline 3.4 3.4 3.4 20.9 20.9 20.9 
Fresh 24 hrs 3.3 3.67 2.45 20.4 18.8 21.6 
 36 hrs 3.7 2.45 2.74 20.5 19.6 17.4 
Frozen / Baseline 3.0 3.0 3.0 11.0 11.0 11.0 
Thawed 24 hrs 2.7 2.8 2.5 11.8 11.8 6.1 
 36 hrs 2.5 2.84 2.1 10.6 10.1 4.7 
Viable CD34 cells x 10e6CFU-GEMM /12,500 cells plated
Storage temperature4–8C°21–24°C29–31°C4–8°C21–24°C29–31°C
 Baseline 3.4 3.4 3.4 20.9 20.9 20.9 
Fresh 24 hrs 3.3 3.67 2.45 20.4 18.8 21.6 
 36 hrs 3.7 2.45 2.74 20.5 19.6 17.4 
Frozen / Baseline 3.0 3.0 3.0 11.0 11.0 11.0 
Thawed 24 hrs 2.7 2.8 2.5 11.8 11.8 6.1 
 36 hrs 2.5 2.84 2.1 10.6 10.1 4.7 

Author notes

Corresponding author