Winter Jacob M, Jacobson Pam, Bullough Brandon, Christensen Austin P, Boyer Michael, Reems Jo-Anna
University of Utah Cell Therapy and Regenerative Medicine Facility, Salt Lake City, Utah, USA.
University of Utah Cell Therapy and Regenerative Medicine Facility, Salt Lake City, Utah, USA; University of Utah Division of Hematology & Hematologic Malignancies, Salt Lake City, Utah, USA.
Cytotherapy. 2014 Jul;16(7):965-75. doi: 10.1016/j.jcyt.2014.02.005.
The question of how long hematopoietic progenitor cells (HPCs) destined for clinical applications withstand long-term cryopreservation remains unanswered. To increase our basic understanding about the stability of HPC products over time, this study focused on characterizing long-term effects of cryopreservation on clinically prepared HPC products.
Cryovials (n = 233) frozen for an average of 6.3 ± 14.2 years (range, 0.003-14.6 years) from HPC products (n = 170) representing 75 individual patients were thawed and evaluated for total nucleated cells (TNCs), cell viability, viable CD34+ (vCD34+) cells and colony-forming cells (CFCs). TNCs were determined by use of an automated cell counter, and cell viability was measured with the use of trypan blue exclusion. Viable CD34 analysis was performed by means of flow cytometry and function by a CFC assay.
Significant losses in TNCs, cell viability, vCD34+ cells and CFC occurred on cryopreservation. However, once frozen, viable TNCs, vCD34+ cells and CFC recoveries did not significantly change over time. The only parameter demonstrating a change over time was cell viability, which decreased as the length of time that an HPC product was stored frozen increased. A significant negative correlation (correlation coefficient = -0.165) was determined between pre-freeze percent granulocyte content and post-thaw percent viability (n = 170; P = 0.032). However, a significant positive correlation was observed between percent viability at thaw and pre-freeze lymphocyte concentration.
Once frozen, HPC products were stable for up to 14.6 years at <-150°C. Post-thaw viability was found to correlate negatively with pre-freeze granulocyte content and positively with pre-freeze lymphocyte content.
用于临床的造血祖细胞（HPCs）能够耐受长期冷冻保存的时长问题仍未得到解答。为了增进我们对HPC产品随时间稳定性的基本认识，本研究着重于描述冷冻保存对临床制备的HPC产品的长期影响。
从代表75例个体患者的HPC产品（n = 170）中取出平均冷冻6.3±14.2年（范围为0.003 - 14.6年）的冻存管（n = 233）进行解冻，并对其总核细胞（TNCs）、细胞活力、存活的CD34 +（vCD34 +）细胞和集落形成细胞（CFCs）进行评估。使用自动细胞计数器测定TNCs，通过台盼蓝拒染法测量细胞活力。通过流式细胞术进行存活CD34分析，并通过CFC测定法评估其功能。
冷冻保存后，TNCs、细胞活力、vCD34 +细胞和CFC均出现显著损失。然而，一旦冷冻，存活的TNCs、vCD34 +细胞和CFC的回收率并不会随时间显著变化。唯一显示随时间变化的参数是细胞活力，它随着HPC产品冷冻保存时间的延长而降低。在冷冻前粒细胞含量百分比与解冻后活力百分比之间确定了显著的负相关（相关系数 = -0.165）（n = 170；P = 0.032）。然而，在解冻时的活力百分比与冷冻前淋巴细胞浓度之间观察到显著的正相关。
一旦冷冻，HPC产品在<-150°C下可稳定保存长达14.6年。发现解冻后的活力与冷冻前的粒细胞含量呈负相关，与冷冻前的淋巴细胞含量呈正相关。
