Cryopreserved Cd34 Cells: Measuring Viability Pre-Thaw and Viability over Time Post-Thaw

Abstract

Common practices associated with the use of cryopreserved hematopoietic progenitor cells (HPC) include measuring the viability of the thawed cells from a sample derived from the thawed product, and thawing cells at the patient's bedside out of concern for possible toxicity of DMSO in the post-thaw liquid cell suspension. The former practice carries the risk of discovering excess cell death when it is too late to correct, and the concomitant risk of non-engraftment. The latter practice carries all of the risks associated with manipulating cells in a non-controlled environment including risk of contamination and lack of suitable containment in the event of failure of a cryostorage container. In an effort to reduce risks associated with cell cryostorage, we are currently examining the validity of measuring CD34 viability from cells frozen in retention vials concomitantly with product freezing. We are also examining the stability of HPC being held in cryostorage medium from 1 to 24 hours post thaw. We present here our results from a pilot study performed on six bags of cells, previously identified for discard due to death of the intended recipients. Six bags of HPC and associated retention vials, frozen for 5 to 12 years, were selected at random from the pool of discarded products and de-identified per guidelines for discard of medical waste. CD34+ cells were identified using a modification of the ISHAGE method, and dead cells were identified by infiltration with the fluorochrome dye 7-AAD. CD34+ cells stored in retention vials were 94±11 percent viable, and CD34+ cells stored in bags were 96±7 percent viable. A Wilcoxan matched pairs test yielded p=0.6, indicating that these results were not different. Percent viable CD34+ cell recovery, relative to the known number of CD34+ cells per mL originally frozen, was determined immediately following thawing, and following 1, 2, 4 and 24 hours remining in thawed cryostorage medium, with recoveries (mean ± std. dev.) of 88 ± 31, 76 ± 33, 62 ± 28, 88 ± 41, and 49 ± 26 percent respectively. These preliminary results suggest that viable CD34 percent as measured from a retention vial may serve as a predictor of viability at thawing of it's associated product, and also that HPC held in cryostorage medium post thaw retain viability for at least four hours post thawing. Based on these results we are continuing these studies.