The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.
Note: For routine culturing of our hNSPCs, we change 50-100% of the media every other day and usually passage them 1:2 once a week. The culture media contains 20% BIT-9500, 1X antibiotic/antimycotic, and growth factors (EGF, FGF, and PDGF each at 40 ng/ml) in DMEM:F12 base media.
Preparing the Coated Flask and Dissociating the Cells
Centrifuging, Resuspending, and Plating Cells
We have found that this protocol provides reliable cultures of hNSPCs. One critical factor for our cells is that they need to be kept as fairly dense cultures and cannot be passaged to the point that the cells are sparse. In our hands, sparse cultures grow very slowly or completely cease dividing. For this reason, we usually split our cultures 1:2 or 1:3 when a culture is extremely dense. Coating the surface of a culture dish with fibronectin is important since it promotes good cell attachment and migration, but, unlike laminin, it also permits the use of Cell Dissociation Buffer (CDB) to detach cells. Cell attachment to laminin is too strong and requires the use of a proteolytic enzyme (e.g. trypsin) for cell detachment. Non-enzymatic CDB is preferred to trypsin since proteolytic cleavage of cell surface proteins may lead to morphological changes in hNSPCs over time. Also, culturing hNSCPs in conditioned media helps maintain these cells in their undifferentiated state.
The authors gratefully acknowledge Dr. Philip H. Schwartz of the National Human Neural Stem Cell Resource at the Children’s Hospital, Orange County Research Institute for providing hNSPCs and initial instruction in their culturing.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
BIT-9500 (BSA, Insulin, Transferrin) | Reagent | Stem Cell Technologies | 09500 | |
Antibiotic-Antimycotic | Reagent | Invitrogen | 15240-062 | |
DMEM:F12 (1X) | Reagent | Invitrogen | 11330-032 | |
EMEM (1X) | Reagent | Mediatech | MT-10-010-CV | Distributed by Fisher under the indicated catalog # |
Fetal Bovine Serum | Reagent | Invitrogen | 10437-028 | |
FGF, human basic recombinant | Reagent | Peprotech | 100-18B | |
PDGF-AB | Reagent | Peprotech | 100-00AB | |
EGF, human recombinant | Reagent | BD Biosciences | CB40052 | Distributed by Fisher under the indicated catalog # |
Cell Culture Flask, 25 cm2 | Tool | Corning | 10-126-28 | Distributed by Fisher under the indicated catalog # |
Fibronectin, human, natural | Reagent | BD Biosciences | CB40008A | Distributed by Fisher under the indicated catalog # |
Human Neural Stem/Precursor Cells | Cells | National Human Neural Stem Cell Resource http://www.nhnscr.org/default.htm | ||
Cell Dissociation Buffer | Reagent | Invitrogen | 13150-016 |