Neural stem cell differentiation in a cell-collagen-bioreactor culture system.
Neural stem cells and neural progenitors (NSCs/NPs) are capable of self-renewal and can give rise to both neurons and glia. Such cells have been isolated from the embryonic brain and immobilized in three dimensional collagen gels. The collagen-entrapped NSCs/NPs recapitulate CNS stem cell development and form functional synapses and neuronal circuits. However, the cell-collagen constructs from static conditions contain hypoxic, necrotic cores and the cells are short-lived. In the present study, NSCs/NPs isolated from embryonic day 13 rat cortical neuroepithelium are immobilized in type I collagen gels and cultured in NASA-designed rotating wall vessel (RWV) bioreactors for up to 9 weeks. Initially, during the first 2 weeks of culture, a lag phase of cellular growth and differentiation is observed in the RWV bioreactors. Accelerated growth and differentiation, with the cells beginning to form large aggregates (approximately 1 mm in diameter) without death cores, begins during the third week. The collagen-entrapped NSCs/NPs cultured in RWV show active neuronal generation followed by astrocyte production. After 6 weeks in rotary culture, the cell-collagen constructs contain over 10 fold greater nestin+ and GFAP+ cells and two-fold more TuJ1 gene expression than those found in static cultures. In addition, TuJ1+ neurons in RWV culture give rise to extensive neurite outgrowth and considerably more synapsin I+ pre-synaptic puncta surrounding MAP2+ cell bodies and dendrites. These results strongly suggest that the cell-collagen-bioreactor culture system supports long-term NSC/NP growth and differentiation, and RWV bioreactors can be useful in generating neural tissue like constructs, which may have the potential for cell replacement therapy.
Lin
Hsingchi J
HJ
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Code 6920. 4555 Overlook Ave. SW, Washington, DC 20375, USA. jlin@cbmse.nrl.navy.mil
O'Shaughnessy
Thomas J
TJ
Kelly
Jeremy
J
Ma
Wu
W
D000818:Animals; D001253:Astrocytes / Q000502:physiology; D019149:Bioreactors; D002454:Cell Differentiation / Q000502:physiology*; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003094:Collagen / Q000502:physiology*; D017364:Collagenases / Q000737:chemistry; D003584:Cytological Techniques / Q000295:instrumentation; D003720:Densitometry; D004996:Ethidium; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009457:Neuroglia / Q000502:physiology; D009474:Neurons / Q000502:physiology*; D010206:Papain / Q000494:pharmacology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000502:physiology*; D013820:Thermolysin / Q000737:chemistry
D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM; D003094:Collagen; D010206:Papain; D017364:Collagenases; C106635:Liberase; D013820:Thermolysin; D004996:Ethidium