Prior Cryopreservation of Ex Vivo-Expanded Cord Blood Cells Is Not Detrimental to Engraftment as Measured in the NOD-SCID Mouse Model

Abstract

Cytokine-mediated expansion has been proposed and successfully used to facilitate engraftment post transplantation. This study examined whether cryopreservation following expansion has a detrimental effect on the ability of cells to engraft, using the NOD-SCID mouse model. Cord blood (CB) CD34(+) cells were incubated for 7 days with stem cell factor (SCF), flt-3 ligand (FL), and megakaryocyte growth and development factor (MGDF). Expanded CD34(+) cells were transplanted into NOD-SCID mice either fresh or following cryopreservation and thawing. After thawing, recovery of nucleated cells was 94%, of CD34 cells was 63%, and of day-14 progenitors was 17%. The loss of day-14 progenitor cells among the thawed expanded cells did not influence the kinetics of human engraftment in the mouse. Bone marrow (BM) of mice transplanted with thawed expanded CD34(+) cells (14 +/- 3.9%) showed significantly higher levels of human engraftment than mice transplanted with fresh expanded CD34(+) cells (1.5 +/- 0.5%, p = 0.0064). Thawed expanded CD34(+) cells had significantly higher SCID Engrafting Potential (SEP) than freshly expanded CD34(+) cells (p < 0.001). Results suggest that prior cryopreservation does not prevent expanded cells engrafting in NOD-SCID mice.

Cryopreservation of cord blood CD34+ cells before or after thrombopoietin expansion differentially affects early platelet recovery in NOD SCID mice

“…[13][14][15][16] The effect of cryopreservation on the outcome of ex vivo expansion is, however, indistinct and mainly compared by in vitro yields and viability variables and less often in clinical studies. [17][18][19][20] Although adequate proliferation and differentiation ability of CD341 cells in culture with TPO has been observed after thawing of CB CD341 cells, 9,11 several studies have reported a decrease in various colony formation assays after cryopreservation and thawing of expanded megakaryocyte progenitor cells. 14,21 Moreover, this resulted in a high cell death and a dramatic loss of postthaw granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units (CFU-GEMM) and megakaryocyte colony-forming unit (CFU-Mk) progenitors.…”

“…The time and the storage temperature before cryopreservation of CB units are well recognized as important determinants affecting in vitro characteristics such as postthaw viability, recovery and hematologic engraftment after transplant in human recipients . The effect of cryopreservation on the outcome of ex vivo expansion is, however, indistinct and mainly compared by in vitro yields and viability variables and less often in clinical studies . Although adequate proliferation and differentiation ability of CD34+ cells in culture with TPO has been observed after thawing of CB CD34+ cells, several studies have reported a decrease in various colony formation assays after cryopreservation and thawing of expanded megakaryocyte progenitor cells .…”

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

“…Although Briddell et al (10) demonstrated CD34+ cell selection is necessary for the optimal expansion of clonogenic cells, other studies have revealed that CD34+ cells and clonogenic cells could be expanded in unselected samples, as in contrast to the selected samples (13, 14). 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.…”