A novel three-dimensional culture system for isolation and clonal propagation of neural stem cells using a thermo-reversible gelation polymer.
In the present study, we examined the possible utility of a three-dimensional culture system using a thermo-reversible gelation polymer to isolate and expand neural stem cells (NSCs). The polymer is a synthetic biologically inert polymer and gelates at temperatures higher than the gel-sol transition point ( approximately 20 degrees C). When fetal mouse brain cells were inoculated into the gel, spherical colonies were formed ( approximately 1% in primary culture and approximately 9% in passage cultures). The spheroid-forming cells were positive for expression of the NSC markers nestin and Musashi. Under conditions facilitating spontaneous neural differentiation, the spheroid-forming cells expressed genes characteristic to astrocytes, oligodendrocytes, and neurons. The cells could be successively propagated at least to 80 poly-D-lysines over a period of 20 weeks in the gel culture with a growth rate higher than that observed in suspension culture. The spheroids formed by fetal mouse brain cells in the gel were shown to be of clonal origin. These results indicate that the spheroid culture system is a convenient and powerful tool for isolation and clonal expansion of NSCs in vitro.
Yang
Xin-Zhi
XZ
Department of Cell Biology, Okayama University Graduate School of Medicine , Dentistry and Pharmaceutical Sciences, Okayama, Japan .
Kataoka
Ken
K
Medina
Reinhold
R
Yamamoto
Ken-Ichi
K
Than
Swe Swe
SS
Miyazaki
Masahiro
M
Huh
Nam-Ho
NH
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001921:Brain / Q000166:cytology; D001973:Bromodeoxyuridine / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D002999:Clone Cells; D005260:Female; D005333:Fetus / Q000166:cytology; D005782:Gels; D007381:Intermediate Filament Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D011108:Polymers / Q000494:pharmacology*; D018874:Spheroids, Cellular / Q000166:cytology / Q000187:drug effects; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects; D013696:Temperature
D015415:Biomarkers; D005782:Gels; D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D011108:Polymers; D001973:Bromodeoxyuridine