Evaluation of the effect of cryopreservation on ex vivo expansion of hematopoietic progenitors from cord blood

Abstract

Summary:In previous studies, we identified a cytokine cocktail including thrombopoietin, Flt-3 ligand, interleukin (IL)-6 and IL-11 in serum-free medium, suitable to induce significant and sustained ex vivo expansion of primitive hematopoietic stem cells (HSCs) from cord blood (CB) for up to 10 weeks. The aim of the present study was to evaluate the effects of cryopreservation on ex vivo expansion of HSCs and their committed progenitors. CD34 ؉ cells were purified from CB units, each of which was processed in part as such and in part as cryopreserved and thawed, then expanded for 5 weeks in serum-free medium with the cytokine cocktail described above. We determined the number of nucleated cells in the last few years several protocols for ex vivo expansion of HSCs from CB have been developed 5-8 and tested successfully in an animal model. 9-11 Despite the positive findings so far collected, the clinical use of such protocols requires additional work. First, the expansion procedures must be scaled-up to fit the body size requirements of human recipients, with due consideration and strict adherence to regulatory issues. Second, the positive results obtained with fresh CB must be confirmed with CB stored in the cryopreserved state, which is the standard storage condition of CB units banked for transplantation purposes. In this regard, it was shown that the proliferative capacity of immature HSCs was not impaired by cryopreservation and storage in liquid nitrogen for up to 10 years. 12 In the present work, we compared the expansion obtained with our protocol 8 using in parallel fresh and cryopreserved aliquots of CB units. Moreover, as the prolonged time to platelet reconstitution remains a significant problem in CB transplantation, we evaluated the number of megakaryocyte (Mk) progenitors in cultures expanded from both fresh and cryopreserved CB units. Materials and methods Collection of CBCB was collected after informed consent of the mother from full-term newborns. After delivery of the baby, the umbilical cord was clamped and disinfected and CB was recovered with the placenta in utero into sterile CB collection bags containing 29 ml of citrate-phosphate dextrose (CPD) as anticoagulant (Macopharma, Tourcoing, France). Freezing and thawing of CBCB samples were processed within 24 h of collection. Twothirds of a CB unit were resuspended vol/vol in ice-cold freezing medium containing 70% RPMI-1640 w/o phenol red (Sigma, St Louis, MO, USA), 20% dimethylsulfoxide (DMSO; Cryoserv, Research Industries Corporation, Salt Lake City, UT, USA) and 10% human serum albumin (Farma Biagini, Lucca, Italy) and cryopreserved with a controlled-rate freezing procedure. CB was thawed as described by Rubinstein et al. 13 Briefly, the CB bags were placed in the gas phase of liquid

Clinical scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells

“…Frozen cells achieved similar levels of expansion as observed for freshly isolated cells (data not shown), confirming previous reports (De Bruyn et al, 2003;Lazzari et al, 2001). As described by Sandstrom et al (1995), we also observed a significant (Wilcoxon signed rank test, P ¼ 0.0313) increase in expansion for cultures initiated with MNC (11,200 AE 4,320; mean AE SD, n ¼ 6) compared to those initiated with selected CD34 þ cells (6,250 AE 3,050; (Fig.…”

Ex vivo expansion of cord blood

“…48–50 IL-11 has also been incorporated into ex vivo expansion cocktails. 38,51–55 The optimal combination has yet to be defined.…”

Stem Cells Expressing Homing Receptors Could be Expanded From Cryopreserved and Unselected Cord Blood

“…There may be some concerns regarding the detrimental effects of cryopreservation on the engraftment potential of expanded CB, however, DiGiusto et al (8) and Rice et al (19) have demonstrated that cryopreservation does not affect the engraftment potential of the frozen cells. Lazzari et al (20) have also recently observed similar clonogenic efficiencies after ex vivo expansion of both fresh and cryopreserved CD34+ cells.…”