Selection of CD34+ cells from cryopreserved PBPC can be significantly improved by the addition of recombinant human DNase (Pulmozyme).
BACKGROUND
It has been reported previously that PBPC can be recovered from cryopreservation and can be efficiently CD34-selected, to provide a product of high purity (> 80% CD34) with good yield (> 50% recovery).


METHODS
In this study, we have investigated the effects of thawing and CD34-selecting cryopreserved PBPC in the presence of recombinant human deoxyribonuclease (rhDNase; Pulmozyme) and magnesium chloride (MgCl2 injection).


RESULTS
The addition of Pulmozyme and MgCl2 significantly improves the yield of CD34+ cells, compared with the standard procedure (65.2% and 39.7%, respectively). Following CD34 selection, significantly greater recovery of CFC in the selected fraction can be obtained from Pulmozyme-treated cells, compared with standard cells. The use of recombinant human Pulmozyme and i.v. grade MgCl2 should facilitate the application of this procedure to the clinical setting. CD34+ cells selected from cryopreserved PBPC, can in turn be cryopreserved for a second time. When thawed, these cells still retained good viability (> 80%).


CONCLUSIONS
Cells originally processed in the presence of Pulmozyme gave significantly superior yields of CD34+ cells and CFC compared with standard cells. The functional ability of these CD34+ cells was demonstrated further in an ex vivo expansion culture system with extensive proliferation of cells and CFC. In addition, the presence of significant numbers of primitive hemopoietic cells could be readily demonstrated in a cobblestone-area forming assay.
Alcorn
M J
MJ
Department of Haematology and BMT Unit, Glasgow Royal Infirmary, University NHS Trust, Glasgow, UK.
Richmond
L J
LJ
Farrell
E
E
Barr
J
J
Pearson
C
C
Schupp
R
R
Franklin
I M
IM
D018952:Antigens, CD34 / Q000378:metabolism*; D001773:Blood Cells / Q000166:cytology* / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods; D003850:Deoxyribonuclease I / Q000494:pharmacology*; D004526:Efficiency; D006801:Humans; D007163:Immunosorbent Techniques; D011994:Recombinant Proteins / Q000494:pharmacology
D018952:Antigens, CD34; D011994:Recombinant Proteins; C085693:DNASE1 protein, human; D003850:Deoxyribonuclease I