Establishment of three-dimensional culture of neural stem/progenitor cells in collagen Type-1 Gel.
OBJECTIVE
Recent studies demonstrated that transplanting neural stem/precursor cells (NSPCs) into an injured spinal cord of adult rat promotes functional recovery. The functional recovery from spinal cord injury might be enhanced by transplanting NSPCs with a scaffold that fills the cavity, entraps the NSPCs in the cavity, and acts as an attachment for neurite extension. We recently focused on collagen type-1 as a scaffold for NSPC transplantation into the injured spinal cord. In the present study, we determined the optimal conditions for culturing NSPCs in 3D collagen type-1 gel with respect to cell survival and cell migration. We, then, evaluated the ability of NSPCs to differentiate under the optimal condition.


METHODS
NSPCs were derived from the striatum of rat embryos. To determine the optimal cell density and collagen concentration for 3D collagen gel culture for NSPC, we performed viability assay and migration assay. Then, we examined the proportion of phenotypes differentiated from NSPC in that optimal condition.


RESULTS
In viability assay, the viability rate increased as the NSPC density increased, and peaked at 1 x 10(7) to 5 x 10(7) cell/ml. For collagen concentration, the viability rate increased as the collagen concentration decreased. In migration assay, cell migration was most extensive at collagen concentrations between 0.3 and 0.75 mg/ml. Migration distances gradually declined as collagen concentrations increased. In the optimal condition, NSPCs differentiated into neurons (40.1%), astrocytes (53.1%), and oligodendrocytes (5.3%) in 3D collagen gel culture.


CONCLUSIONS
The optimal conditions for NSPC culture in 3D collagen gel was a cell density between 1 x 10(7) and 5 x 10(7) cells/ml and a collagen concentration between 0.5 and 0.75 mg/ml. Under the condition, NSPCs could differentiate into neurons, astrocytes, and oligodendrocytes.
Watanabe
Kota
K
Department of Orthopaedic Surgery, Keio University 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan.
Nakamura
Masaya
M
Okano
Hideyuki
H
Toyama
Yoshiaki
Y
D000818:Animals; D030801:Animals, Genetically Modified; D001253:Astrocytes / Q000166:cytology; D002452:Cell Count; D002454:Cell Differentiation; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D024042:Collagen Type I / Q000008:administration & dosage / Q000494:pharmacology; D003584:Cytological Techniques; D004305:Dose-Response Relationship, Drug; D004628:Embryonic Structures; D005782:Gels; D049452:Green Fluorescent Proteins / Q000235:genetics; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D009836:Oligodendroglia / Q000166:cytology; D009994:Osmolar Concentration; D051381:Rats; D017207:Rats, Sprague-Dawley; D018874:Spheroids, Cellular; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology
D024042:Collagen Type I; D005782:Gels; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins