Controlled rate freezing is recognized as a method of choice for the cryopreservation of blood cells. DMSO, the cryoprotectant most commonly used for cryopreservation of cells, is well known to affect cell viability. It is therefore imperative to insure that cells are frozen without any appreciable delay after the addition of DMSO. Addition of cryoprotectant may take between 5–10 minutes per processed cord blood unit. In situations involving large scale processing, such as 5–10 cord blood units simultaneously, delay will occur, which could cause adverse affects on cell viability when controlled rate freezing is utilized

This study was designed to compare the effect of controlled with two non-controlled rate freezing methods on cord blood stem cell viability.

After adding DMSO to final concentration of 10%, each cord blood unit was split into two 25 ml volumes and enclosed in metal freezing canisters. The UCB units were covered by styrofoam sleeves and then placed directly into either a −80°C or a −140°C mechanical freezer. A corresponding portion of each unit was frozen utilizing a Forma Scientific, model 1010 controlled rate freezer.

Approximately 30 days after, matching units were thawed in a 37°C water bath and tested without washing.

Total Nucleated Cell count, Total CD34 count, 7AAD viability and colony forming units assay were performed on fresh and cryopreserved/thawed samples. Statistical pair analysis using student-t test was performed to indicate any statistically significant differences between each procedures.

(table1)

(table2)

Results indicate that all three methods produces similar cell viability as measured by total nucleated cell recovery, total CD34 counts, 7AAD viability and colony forming assays.

Analysis of fresh and frozen samples did not indicate that controlled rate freezing produces a better quality product after thawing compared to two uncontrolled freezing methods.

Controlled rate freezing vs −80oC dump method°

N=5Fresh SampleControlled Rate FreezingNon controlled Freezing at −80oC
TNC (x10^6) 412.4 ±37.06 397.6±15.65 406.7±24.78 
Total CD34+ (10^6) 0.23±0.08 0.19±0.08 p<0.05 0.19±0.07 p<0.05 
Viability (%) 97.5±1.67 94.8±2.28 p<0.05 93.4±3.51 p<0.05 
Total # of Colonies (x10^6) 0.27±0.09 0.21±0.10 p<0.05 0.20±0.08 p<0.05 
N=5Fresh SampleControlled Rate FreezingNon controlled Freezing at −80oC
TNC (x10^6) 412.4 ±37.06 397.6±15.65 406.7±24.78 
Total CD34+ (10^6) 0.23±0.08 0.19±0.08 p<0.05 0.19±0.07 p<0.05 
Viability (%) 97.5±1.67 94.8±2.28 p<0.05 93.4±3.51 p<0.05 
Total # of Colonies (x10^6) 0.27±0.09 0.21±0.10 p<0.05 0.20±0.08 p<0.05 
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Controlled rate freezing vs −140oC dump method

N=5Fresh SampleControlled Rate FreezingNon Controlled −140oC
TNC (10^6) 365.4±100.87 372.1±102.18 365.9±104.9 
Total CD34+ (x10^6) 0.28±0.08 0.023±0.05 p<0.05 0.23±0.05 p<0.05 
Viability (5) 96.0±3.94 96.2±2.28 p<0.05 96.2±1.79 
Total # of Colonies (10^6) 0.27±0.13 0.27±0.11 0.24±0.15 
N=5Fresh SampleControlled Rate FreezingNon Controlled −140oC
TNC (10^6) 365.4±100.87 372.1±102.18 365.9±104.9 
Total CD34+ (x10^6) 0.28±0.08 0.023±0.05 p<0.05 0.23±0.05 p<0.05 
Viability (5) 96.0±3.94 96.2±2.28 p<0.05 96.2±1.79 
Total # of Colonies (10^6) 0.27±0.13 0.27±0.11 0.24±0.15 
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Topics:
freezing, umbilical cord blood, cryopreservation, dimethyl sulfoxide, cd34 antigens, cryoprotective agents, colony-forming units assay, metals, water bath, erythroblasts

Author notes

Corresponding author

2005, The American Society of Hematology
2004
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