Analytical and Bioanalytical Chemistry

, Volume 391, Issue 8, pp 2753–2758

High-throughput evaluation of quiescent hematopoietic progenitor cells using a micro-multiwell plate

Technical Note

DOI: 10.1007/s00216-008-1921-3

Cite this article as:
Fujita, S., Morita, Y. & Iwata, H. Anal Bioanal Chem (2008) 391: 2753. doi:10.1007/s00216-008-1921-3
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Abstract

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 × 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.

Figure

Representative image of a micro-multiwell plate fordetecting primitive hematopoietic stem cells

Keywords

High-throughput screening Hematopoietic progenitor cell 5-Fluorouracil Microwell 

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Department of Reparative Materials, Institute for Frontier Medical SciencesKyoto UniversityKyotoJapan
  2. 2.Regenerative Medicine Research CenterItabashi Chuo Medical CenterKobeJapan
  3. 3.Department of Obstetrics and GynecologyItabashi Chuo Medical CenterTokyoJapan

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