Analysis of cord blood CD34+ cells purified after cryopreservation.
Many practical issues regarding processing blood samples for cord blood banking remain. After cryopreservation, a reduction in clonogenicity has been reported, although it is unknown whether this is associated with lower potential for long-term engraftment. CD34+ cell purification of cryopreserved cord blood (CB) may be important for the clinical application of in vitro expansion. We compared purity, yield, clonogenicity, and growth in long-term stromal-based culture of fresh and cryopreserved CD34+ purified cells (n = 12) using the miniMACS separation system. Mean purity of CD34+ cells was 93% when processed before and 73% when processed after cryopreservation. Fresh CD34+ cells had higher clonogenic potential than cryopreserved cells (45 vs 20%, p < 0.05) in CFU-Mix assays, indicating that progenitor cell loss during cryopreservation is due in part to reduced cloning efficiency of viable CD34+ cells. In long-term culture (LTC) on irradiated normal human bone marrow stroma (n = 7), CFU-GM production in the two groups was the same over 12 weeks, suggesting identical long-term culture-initiating cell (LTC-IC) numbers. We conclude that apparent clonogenic cell loss during cryopreservation is associated with relative sparing of the more primitive LTC-ICs. CFU-Mix assays may therefore underestimate the transplant potential of cryopreserved CB. Purification of CD34+ cells following cryopreservation gives sufficient purity for detailed evaluation of CD34+ cells and for stem cell expansion.
Stem Cell Research Laboratory, University of Bristol Department of Transplantation Science, UK.
A
A
Nicol
M
M
Nieda
C
C
Donaldson
P
P
Denning-Kendall
B
B
Bradley
J
J
Hows
D018952:Antigens, CD34 / Q000032:analysis*; D002455:Cell Division; D002469:Cell Separation; D002478:Cells, Cultured; D002999:Clone Cells / Q000502:physiology; D003114:Colony-Forming Units Assay; D015925:Cryopreservation; D005312:Fetal Blood / Q000166:cytology*; D006085:Graft Survival; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology / Q000502:physiology; D006801:Humans; D013997:Time Factors
D018952:Antigens, CD34