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KMID : 0391319960060020269
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Korean Journal of Biological Response Modifiers
1996 Volume.6 No. 2 p.269 ~ p.276
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Effective Isolation of Human Hematopoietic Stem Cells Using Counterflow Centrifugal Elutriation
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Han Chi-Hwa
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Abstract
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Background: Counterflow centrifugal elutriation(CCE) has been proposed as a effective method for separating heterogeneous cell populations on the basis of different sedimentation characteristics. We evaluate the morphological characteristics and the effective isolation of CD34(+) hematopoietic stem cells using CCE in G-CSF mobilized peripheral blood stem cells(PBSC).
Materials and Methods: The CD34(+) cells in bone marrow(BM) were isolated by AIS MicroCELLector™ T-25 cell culture flasks and identified by flow cytometry with FITC-antiHPCA. The morphological characteristics of isolated CD34(+) cells Background: Counterflow centrifugal elutriation(CCE) has been proposed as a effective method for separating
was identified with cytocentrifuge and Wright-Giemsa staining. The G-CSF mobilized PBSC were separated by CCE according to changes of flow rate. The morphology and immunological markers of each fractions was idenfied by cytocentrifuge and flow cytometry.
Results: In the morphological characteristics, the majority of CD34(+) cells in BM through panning method was monocytes and large lymphoblastoid cells and the minority was shown small lymphocyte like morphology. The flow cytometric characteristics of isolated CD34(+)cell in BM was both high and low-to-intermediate forward light scattering area within the low-to-intermediate right angle scattering. The yields of separated CD34(+) cells in PBSC using CCE were 86.2%, 90.2% of loading CD34(+) cells. In hematopoietic stem cell separating by CCE, the majority of separated CD34(+) cells was identified in large cell fraction and the minority in small lymphocyte fraction.
Conclusion: The CD34(+) cells separating by CCE in PBSC was identified in both large myelomonocytic cells and small lymphocyte-like cells. The CD34(+) cells of small lymphocyte-like cells may be the primitive hematopoietic stem cells. Therefore, We conclude that CCE may be a effective method for separating the hematopoietic stem cells in heterogeneous cell populations of bone marrow or peripheral blood stem cell collection.
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KEYWORD
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CD34(+) cells, Counterflow centrifugal elutriation
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