Figure 1. Expansion of rat fetal NSCs in adherent and neurosphere cell culture.(Left) Rat Fetal NSCs at passage 3 (P3) in adherent cell culture using StemPro NSC SFM media. (Right) Rat fetal NSCs at P3 in neurosphere suspension culture using StemPro NSC SFM.
Ready-to-use neural stem cells, with superior proliferation and multipotent differentiation potential. Each vial of cell contains 2 x 106 cells, isolated from the cortex of Sprague-Dawley E14 rats.
Components of the Gibco Rat Fetal NSC Kit
The kit contains:
- Rat fetal neural stem cells (2 x 106 cells)
- StemPro NSC SFM Media (KnockOut DMEM/F-12, StemPro NSC SFM supplement, basic FGF recombinant protein, and EGF recombinant protein)
StemPro NSC SFM enables superior expansion of rat fetal NSCs in both adherent cell culture as well neurosphere suspension cell culture (Figure 1).
We offer detailed protocols for both conditions:
Gibco Rat Fetal Neural Stem Cells can be expanded in StemPro NSC SFM media up to three passages without differentiation, with more than 75% of rat fetal NSCs retaining their undifferentiated phenotype. (Figure 2)
Figure 2. Retains undifferentiated phenotype. Fluorescence images (20x) of rat fetal NSCs at P3 cultured in StemPro NSC SFM for 10 days and stained for the appropriate phenotypic marker using fluorescence-conjugated antibodies. Nuclei were stained with DAPI (blue) in all images. While approximately (A) 90% of the cells stain positive for the undifferentiated NSC marker Nestin, less than 10% of the cells are positive for differentiated cell type markers (B) Dcx, (C) GalC, and (D) GFAP.
After proliferating the rat fetal NSCs, the cells were expanded from 2 x 106 cells to 300 x 106 cells and then differentiated into multipotent lineages. Rat fetal NSCs spontaneously differentiate into neurons, oligodendrocytes, or astrocytes upon withdrawal of bFGF from culture media. The images below show the differentiation potential of Gibco Rat Fetal NSCs cultured up to P3 in StemPro NSC SFM (Figure 3).
Figure 3. Differentiation potential of Gibco Rat Fetal NSCs. Fluorescence images (20X) of rat fetal NSCs cultured in StemPro NSC SFM for up to P3 and then allowed to differentiate into into neurons, oligodendrocytes, or astrocytes. Upon differentiation, (A) cells start to lose undifferentiated NSC marker Nestin, but stain positive for differentiated cell type markers (B) Dcx, (C) GalC, and (D) GFAP. Nuclei were stained with DAPI (blue) in all images.
1. From which strain of the rat and which tissue are these cells isolated?
NSC cells were isolated from the cortex of Sprague Dawley
2. From what age rats are these cells derived?
The cells are derived from day E14 of gestation.
3. At what passage were the cells cryopreserved?
Cells were isolated from the fetus without passage and frozen down at passage 0.
4. How many passages can I expand these cells?
Rat NSC have been tested for proliferation and differentiation potential up to passage 3 after thawing and we have seen around 300 million cells from 1 vial after the 3rd passage. We have not expanded the cells beyond the third passage, but we encourage you to try expanding further.
5. How do we know that these cells are indeed NSC?
We evaluated cells with two criteria. One is to express phenotype marker of Nestin and do not express differentiated phenotype markers such as GFAP, Dcx or GalC.
The second criteria was to demonstrate their differentiation potential toward all three downstream lineages such as neurons, astrocytes and Oligodendrocytes.
6. What is the % purity of these cells?
Cell purity was measured by the expression of NSC marker of Nestin and devoid of expression of differentiated marker. More than 75% of the cells were positive to Nestin and expressions of the differentiated markers were less than 10%.
7. What viability should I get from the cells once thawed?
Upon thawing, the vial has more than 50% viability which will give more than 2 millions live cells.
8. What is the doubling time of the cells?
The doubling time for the cells is around 20-30 hours. This tends to increase with passage number.
9. Cells do not attach to the CELLstart coated plate but float to form a sphere. Is this normal?
Please ensure that the buffer used for CELLstart has Calcium and Magnesium. Please review the product insert for our recommended buffer. If the right buffer is used, increase the concentration of CELLstart up to 1:50 dilution rate. If CELLstart with our recommended buffer does not work, some users had success with Poly-L-Ornithine–coated plates with StemPro NSC-SFM. If you use Poly-L-Ornithine, please perform overnight incubation.
10. Can we use Neurobasal/B27 or DMEM instead of NSC-SFM?
To maintain the cells in NSC status, we recommend using NSC-SFM. We have validated and optimized the culture conditions in NSC-SFM to ensure superior growth and performance of these cells.
11. How do fetal derived NSCs compare with adult derived NSCs?
Adult derived NSC tends to be regionally restricted and have limited plasticity compared to fetal derived NSC.
12. What are some of the key applications for these cells?
NSC is a valuable source not only for neuroscience but also for clinical use to treat neurodegenerative disease or neurological disorder. These cells can be used for a wide range of differentiation studies.
13. Where can I find more detailed protocol for directed differentiation?
Please contact our technical support team for the details. Our product manual will however guide you in the protocol for spontaneous differentiation. All three lineages can be obtained from spontaneous differentiation. However the % of the various cell types (neurons, astrocytes and oligodendrocytes) will be few and will depend on the passage number.
14. What size container do you recommend that we use for culture?
Any format of culture vessel (e.g. flask, 6-well plates, etc) can be used. Seed cells at a density of 5 x 104 cells/cm2. Depending on the surface area of your culture vessel, calculate how many cells you will need to have on hand. Example: For one 60 mm dish, you will need 5 x 104 cells/cm2 x 20 cm2 = 1 x 106 cells total.
15. Are the cells derived from rats of a particular gender?
Gibco Rat Fetal NSC’s are derived from a mixed pool of embryos; therefore, cells will be derived from rats of both sexes.
16. Is there any difference between NSCs isolated from cortex vs hippocampus?
Cortical NSC’s contain a greater mixture of cells and have different neurotransmitters. They have a wider set of applications. Fewer NSCs can be obtained from hippocampal tissue, but the cells are more pure. They are used by a smaller hippocampal focused audience.
17. What are the estimated % neurons, astrocytes, and oligodendrocytes that we might get from using a spontaneous differentiation protocol vs. a directed differentiation protocol?
| Marker | Spontaneous differentiation (% positive) | Directed differentiation (% positive) |
|---|---|---|
| Dcx (neuronal) | 10 | 40 |
| GFAP (astrocyte) | 30 | 40 |
| O4 (oligodendrocyte) | 2 | 32 |
18. Can we grow cells on cover slips?
If you are growing cells on cover slips to do immunocytochemistry, we recommend using Permanox chamber slides:
- Permanox chamber slides, 4-well, pk/16 (VWR, Cat. No. 62407-330)
- Permanox chamber slides, 8-well, pk/16 (VWR, Cat. No. 62407-335)
If you are growing cells on cover slips for other purposes and require coating, use this procedure to double coat the cover slips:
- Incubate cover slips overnight at room temperature in poly-ornithine solution (final concentration = 100 mg/ml in cell culture water)
- Rinse twice with sterile water
- Incubate cover slips overnight at 4℃ in laminin solution (final concentration = 15 mg/ml in cell culture water)
19. What related products can be used with these cells?
- Temple, S. The development of neural stem cells. Nature 414, 112–117 (2001).
- Gage, FH et al. Mechanisms and Functional Implications of Adult Neurogenesis. Cell 132, 645–660 (2008).
- Shin S and Vemuri M. Protocols for Neural Cell Culture, 4rd edn. (eds. Laurie Doering, in press 2009).
- Wu YY, Mujtaba T, Rao MS. Isolation of stem and precursor cells from fetal tissue. Methods Mol Biol 198:29-40 (2002).
- Bjorklund A, Lindvall O. Cell replacement therapies for central nervous system disorders. Nat Neurosci 3:537-44 (2000).
- Gage FH. Mammalian neural stem cells. Science 287:1433-8 (2000).
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