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KMID : 0366219840190010015
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Korean Journal of Hematology
1984 Volume.19 No. 1 p.15 ~ p.20
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A Report on Technical Problerns of in Vitro Semisolid Agar Culture of Hemopoietic Cells
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조현찬(趙顯燦)/Hyoun Chan Cho
김상인(金相仁)/안우성(安祐成)/조농익(趙濃翊)/Sang In Kim/Woo Sung Ahn/Han Ik Cho
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Abstract
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In vitro culture of hemopoietic cells using semisolid agar culture techniques has been
established after long and labrous trials. Followings are the details of optimun culture
conditions by which we have solved the technical difficulties.
1. Human tone marrow samples of 2-3 ㎖ and peripheral blood samples of 10 ㎖
collected in the tubes containing 200 units of preserve-free heparin gave the adequate
number of nucleated cells in most cases except a few cases of markedly hypocellular
marrow.
The nucleated cells could be separated from red cells(1) by taking out red cell-free
supernatant with a Pasteur pipette after standing for 1-2 hours or (2) by layering the
sample over the Ficoll-Hypaque solution(S.G. = 1.098) and centrifugation at 1,500 g for
15 minutes.
2. Compared to peripheral blood leukocyte underlayer as a source of colony stimulating
factor, the human placental conditioned medium (HPCM) was easier to prepare and more
accurate to standardize.
3. Proper gelling of agar media was one of the most troublesome problems at the
initial period of bone marrow cell culture studies. However the optimum gelling was
obtained simply by placing the culture dishes in the refrigerator briefly.
Heat-inactivated fetal calf serum yield more colony formation comparing to
noninactivated serum.
The fungus was the most common contaminant, but it could be successfully
elintinated by frequent disinfection of the clean bench with phenol solution.
4. Acetoorcein in situ stain of colonies made it easy to score and identify the colony
cells, although it was difficult to differentiate neutrophils from eosinophils.
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