Enrichment of Mononuclear Cells From Cryopreserved Cord Blood Units Using the Purecell” Select System.

Many procedures for manufacturing clinical products for cellular therapy involve enrichment of mononuclear cells (MNC). The most common procedure is density gradient separation. Disadvantages of this procedure are low yield of cells especially with cryopreserved products and the open events during processing. We evaluated use of the Purecell” Select System (PALL Medical, NY) for enriching MNC from cryopreserved Cord Blood Units (CBU).

Initial experiments were performed to optimize the system for recoveries of total nucleated cells (TNC), MNC, neutrophils, lymphocytes, monocytes and CD34+/CD3+ cells. We evaluated thaw/predilute/filter vs thaw/filter, starting volumes (30 — 95mls) and three different methods for harvesting cells from the filter (standard method, input bag rinse and harvest port rinse). Once conditions were optimized, cryopreserved CBU were thawed and split into two fractions. One half of the product was diluted and processed on the Purecell” Select System. The other half was washed and ficolled. The MNC fraction was CD3+ enriched using CD3/28 beads and then cultured with rIL-2 (200 units/ml) for 14 days. The absolute number of CD3+ cells post culture, fold expansion and viabilities of these cells were determined.

The Purecell” procedure was 15 times faster than the ficoll method. Optimal volume to load onto the filter was 50ml. When MNC were harvested by the three recommended procedures, there was no difference in recoveries of TNC, MNC, CD34+ and CD3+ cells and neutrophils. However, the lymphocyte and monocyte recoveries were higher (p<0.05 and p=0.001) when harvested with Input bag rinse compared to the standard procedure. Monocyte recoveries were also higher with the harvest port rinse (p=0.004) when compared to the standard procedure. The direct comparison studies of the two MNC enrichment systems demonstrated that the Purecell” Select System gave significantly higher recovery of TNC (p= 0.003), MNC (p=0.029), CD34+ cells (p<0.001) and granulocytes (p<0.001). There was no statistical difference in T cell recoveries, however, there was a significant difference in the recovery of T cells after CD3/28 enrichment.. Interestingly, T cells began to proliferate earlier from the PALL system compared to ficoll isolated T cells (day 4 vs day 7). Although the fold expansion was greater for the ficolled prepared cells, the absolute numbers of T cells obtained after 14 days of culture with rIL-2 was greater for the Purecell Select System in all experiments. The viabilities of the cells from both cultures were comparable.

PALL's Purecell” Select System can be used for clinical processing since it is a functionally closed system. The advantage of this system compared to the ficoll method are the reduced time for processing, increase yield in T cells (post processing), the earlier expansion time These benefits result in an increase in absolute number of T cells in post culture. A clinical trial using this system is about to be initiated.

Karandish:Pall Medical: Employment. McMannis:Pall Medical: Research Funding.