Table 1.
CD34+ Cell selection workflow and troubleshooting
| Issue | Solution |
|---|---|
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| Pre-processing HPC(A) Product Storage | Products are diluted with 2.5% human serum albumen buffer to a cell concentration of <200 × 10E6 mL whenever possible. |
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| Platelet Washes | Perform two (2) platelet washes using the COBE 2991 cell washing device (Terumo BCT, Lakewood, CO) to remove the majority of platelets, prior to the antibody incubation phase. Excess platelet contamination causes product clumping at subsequent stages of processing which can interfere with successful cell selection and cell recovery. |
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| Human IVIG | Add to the washed product at a concentration of 1.5mg/mL and incubate product five (5) minutes at room temperature on an orbital rotator. This step is intended to reduce non-specific binding of the target cell antibody during antibody incubation step. Follow manufacturer’s instructions for antibody incubation. |
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| Post-incubation Wash Step | Wash product once to remove any excess unbound antibody, by centrifugation using a refrigerated floor model centrifuge. (e.g. Sorvall RC3BP, 655 × g, 10 minutes, Thermo Fisher, Waltham, MA) |
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| Product Resuspension & Examination | Resuspend product to the appropriate volume for loading on the machine. Examine product for signs of clumping. If clumping is observed, pass product through a platelet filter (Blood Component Recipient Set with Standard Blood Filter and Luer Adapter, 170 to 260 micron filter). We use as many filters as necessary. Load filtered product on the device following standard procedures. |
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| Product at upper limit of total nucleated cells (TNC) for device tubing set (120×10E8 cells) and/or product is still clumpy after several rounds of filtering | Split the product and run over two large scale columns. Allows us to successfully avoid fluidic problems during the selection run and retain high levels of pure cell recovery. |
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| Concentration of final CD34+ selected cell fraction | At completion of CD34+ selection procedure, transfer CD34+ cell fraction to four 50 mL conical centrifuge tubes. Spin tubes at 840 × g for 10 minutes at room temperature. Resuspend cell pellets and pool into one tube at a predetermined volume. Perform nucleated cell count manually using a hemacytometer. Samples are removed for other testing, following standard procedures. |
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| Product Testing Performed: | |
| ➢ Pre Selection Tests |
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| ➢ Post Selection Tests |
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