We recently incorporated the use of the automated Sepax Cell Processing System (Biosafe SA, Switzerland) for red cell and volume reduction of cord blood units (CBU) before cryopreservation. Now that we have been routinely using this new technique in the laboratory for about six months, we decided to compare the results of this method with our standard manual processing method (

Rubinstein, et al, PNAS 1995,; 92: 10119–22
).

For both methods, hespan is added to the cells at final concentration of 20% (v/v). With the Sepax system, after addition of hespan, the cell bag is connected to the Sepax tubing set with the final freeze bag pre-attached to the set. After completion of the automated procedure, buffy coat is collected in the final freeze bag. Cryoprotectant solution is then added directly to the freeze bag. In the manual method, buffy coat and white cell rich plasma layer is collected after first centrifugation step and the white cells are separated from the plasma after the second centrifugation step. Cryoprotectant solution is then added to the cells before transfer to the final freeze bag for cryopreservation.

The following are summary of results for each method:

Table 1
Manual (n=1160)Sepax (n=311)
Pre-Processing volume (ml) 107±30 114±28 
Pre-Processing TNC (×10e6) 1196±577 1315±519 
TNC Recovery (%) 80±8 83±8 
TNC Viability (%) (7-AAD) 97±3 98±3 
Total CD34 (×10e6) 4.3±4 4.9±3.6 
Total DFU (×10e6) 70±0.9 61.5±20 
Post Processing RBC Volume (ml) 9±2 7.3±2 
Total Processing Time (including Setup) 60 minutes 30 minutes 
Manual (n=1160)Sepax (n=311)
Pre-Processing volume (ml) 107±30 114±28 
Pre-Processing TNC (×10e6) 1196±577 1315±519 
TNC Recovery (%) 80±8 83±8 
TNC Viability (%) (7-AAD) 97±3 98±3 
Total CD34 (×10e6) 4.3±4 4.9±3.6 
Total DFU (×10e6) 70±0.9 61.5±20 
Post Processing RBC Volume (ml) 9±2 7.3±2 
Total Processing Time (including Setup) 60 minutes 30 minutes 

It is important to note that there was not a significant difference in TNC Recovery over a wide range of Pre-Processing Volume (66–206ml) or Pre-Processing TNC (440 – 3559×106).

Since the Sepax device is an automated procedure, issues could arise (i.e. short term loss of electrical power) that would require us to reprocess the CBU before cryopreservation. The Sepax system allows for recovery and reprocessing of the cell using the ‘Purge mode’. We used 5 CBU units to evaluate TNC recovery and viability after purging the cells once and reprocessing.

Table 2
TNC Recovery (%)TNC Viability (%)
First Buffy Coat 85±5 98±0.9 
Post Purge and reprocessing 76±5 98±0.9 
TNC Recovery (%)TNC Viability (%)
First Buffy Coat 85±5 98±0.9 
Post Purge and reprocessing 76±5 98±0.9 

Although the TNC recovery was lower after the second procedure, it was still within acceptable limits and the viability of the cells had not changed. These data demonstrates that both methods are equivalent with respect to cell recovery. However, the Sepax System substantially reduces processing time and hands-on operator intervention. Additionally system provides, closed-system processing, bar code reading capability and run data print-out suitable for GMP manufacturing settings.

Disclosures: JDM on Biosafe Board of Directors.

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