High-throughput evaluation of quiescent hematopoietic progenitor cells using a micro-multiwell plate.
Conventional assays for hematopoietic progenitor cells (HPCs) require long-term culturing, a labor-intensive procedure, and technique proficiency. We aimed to develop a high-throughput method to determine frequency of quiescent primitive HPCs by a combination of the micro-multiwell plate and 5-fluorouracil (5-FU) treatment. The micro-multiwell plate was made of a silicone sheet with a 6 x 6 array of 1-mm diameter holes and a glass substrate. To enrich primitive HPCs in a CD34 population, CD34 cells and stromal cells were applied to micro-multiwells and cultured in the presence of 5-FU for 2 days. The quiescent primitive HPCs that survived after 5-FU treatment were then expanded with cytokines in the absence of 5-FU for a further 10 days. After culturing, cells were immunostained and the number of primitive HPCs in inoculated CD34 cells was estimated from fluorescent intensity for each well under a stereoscopic fluorescent microscope. The frequencies of primitive HPCs correlated well with frequencies of cobblestone area-forming cells for two CD34 cell lots. Our method allows high-throughput screening for primitive HPCs in CD34 cells.
Fujita
Satoshi
S
Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Morita
Yutaka
Y
Iwata
Hiroo
H
D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D002452:Cell Count; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods*; D002460:Cell Line; D018920:Coculture Techniques; D005472:Fluorouracil / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008832:Microchemistry; D017154:Stromal Cells / Q000166:cytology
D018952:Antigens, CD34; D005472:Fluorouracil