Abstract 21
Introduction
Success in transplantation is dependent upon cell dose (a minimum of 25 million cells/kg body weight). Thus, most cord blood units will contain insufficient cells to treat larger patients. Viable white cell recoveries vary between isolation methods. To date, processes for isolating the white cell fraction (WCF or buffy coat) result in substantial cell loss, with up to 45% of cells removed pre‐freeze. Furthermore, often greater losses take place during thawing. Additionally, current processing methods usually result in the almost total loss of potentially important, low abundance cellular subsets.
Objectives
The aim of this study was to provide a new cord blood‐derived stem cell isolation method that delivers enhanced pre‐ and post‐thaw viable cell recovery.
Methods
We have developed a novel solution, TotiCyte (TC), that, upon combination with blood, results in the selective sedimentation of erythrocytes by gravity within 30 minutes. The WCF remains in solution and can be easily separated from the erythrocyte sediment. The WCF can then be concentrated by gentle centrifugation into a small volume containing <1% of the original erythrocyte content. The addition of DMSO for cryogenic storage and controlled freezing using standard procedures then completes this simple process.
Results
We have clearly demonstrated that this method allows almost the entire WCF to be isolated and/or concentrated with only modest loss of any of the cellular subsets. In addition to improving pre‐freeze yields, post‐thaw recoveries of cells are increased, with a yield >90%. Possibly more important, the CFU assay results reproducibly yield higher counts of CFU‐GM, CFU‐GEMM, and BFU colonies (Table 1) which is a strong indicator that this method will improve patient outcomes. In addition, megakaryocyte‐like cells (CD45+, fCD61+) and early progenitor cells expressing Oct4 and Nanog (VSELs), cells usually lost using current separation techniques, are recovered.
Table 1.
Cell isolation and colony‐forming unit analysis comparison with other frequently used method
| Analysis | TotiCyte | System 1 | System 2 | System 3 |
|---|---|---|---|---|
| Cell isolation analysis | ||||
| Number of CD45+ cells pre‐processing | 300,000,000 | 300,000,000 | 300,000,000 | 300,000,000 |
| CD45+ recovery post‐thaw | 47.20% | 43.81% | 44.48% | 23.91% |
| Number of viable CD45+ | 141,586,163 | 131,440,024 | 133,444,567 | 71,730,135 |
| Number of CD34+ cells pre‐processing | 1,000,000 | 1,000,000 | 1,000,000 | 1,000,000 |
| CD34+ initial viability | 96.83% | 96.85% | 94.32% | 98.06% |
| Post‐thaw CD34+ viable recover | 63.22% | 40.87% | 29.83% | 25.99% |
| Comparison with TC (recovery) | 1.5 | 2.1 | 2.4 | |
| Post‐thaw number of viable CD34+ cells | 632,180 | 408,690 | 298,256 | 259,936 |
| Post‐thaw CD34+ as percentage of post‐thaw CD45+ | 0.45% | 0.31% | 0.22% | 0.36% |
| Colony‐forming unit analysis | ||||
| Plate number of CD45+ cells | 15,000 | 15,000 | 15,000 | 15,000 |
| Number of CD34+ cells plate | 66.97 | 46.64 | 33.53 | 54.36 |
| CFUs per 1.5 × 104 CD45+ cells | 49.85 | 16.50 | 20.60 | 29.95 |
| Number of CFUs per CD34+ cells plated | 0.74 | 0.35 | 0.61 | 0.55 |
| Comparison with TC (CFUs) | 2.1 | 1.2 | 1.4 | |
| Number of CFUs for post‐thaw viable CD34+ | 470,538 | 144,584 | 183,264 | 143,198 |
| Comparison with TC (CFUs for viable CD34+) | 3.3 | 2.6 | 3.3 | |
| Normalized TC performance relative to initial CD34+ viability | 3.3 | 2.5 | 3.3 |
Abbreviations: CFU, colony‐forming unit; TC, TotiCyte.
Discussion
Our method achieves larger WCF than current methods, independent of volume, as well as higher percentage recoveries of all cell types and higher post‐thaw recovery of viable nucleated cells. Therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults. Until now, it has been suppressed due to the limited cell yields delivered by existing methods.