Characterization of long-term mouse brain aggregating cultures: evidence for maintenance of neural precursor cells.
An extensive characterization of fetal mouse brain cell aggregates has been performed using immunohistochemical and stereological methods. Single cell suspensions from mechanically dissociated cortex and hippocampus were cultured in serum-free, B27-supplemented medium under constant gyratory agitation for up to 56 days. Three-dimensional aggregates started to form immediately after seeding and reached a final average size of 500 microm in diameter. Among the cell types identified, neurons were the most abundant cells in the aggregates, followed by astrocytes, microglia, and oligodendrocytes. Western blotting for synaptophysin and immunostaining for neurotransmitter-related molecules indicated the presence of well-defined phenotypic characteristics of the neurons in this culture system, suggesting functionality. Proliferating cells, many with neural precursor cell properties, were seen throughout the culture period and could be isolated from the aggregates even after 2 months in culture. Neural precursor cells were isolated from the aggregates after more than 1 month in culture; these cells were successfully differentiated into neurons, astrocytes, and oligodendrocytes. The aggregate culture system may provide a versatile tool for molecular dissection of processes identified in mouse models, including transgenic animals and manipulation of neural precursor cells.
Berglund
C Mikaela D
CM
Karolinska Institutet, Department of Medicine and Center for Molecular Medicine, Karolinska Hospital, SE-171 76 Stockholm, Sweden.
Aarum
Johan
J
Haeberlein
Samantha L Budd
SL
Nyengaard
Jens R
JR
Hökfelt
Tomas
T
Sandberg
Kristian
K
Näslund
Jan
J
Persson
Mats A A
MA
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D015153:Blotting, Western; D001921:Brain / Q000166:cytology* / Q000378:metabolism; D002449:Cell Aggregation; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D005333:Fetus; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D051379:Mice; D017628:Microglia / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors