实验概要
Glial precursor cells (GPCs), also known as glial restricted progenitors (GRP) or oligodendrocyte progenitor cells (OPCs), are cells that have the potential to differentiate into oligodendrocytes or astrocytes. The GPC population is derived from tissue or is generated from pluripotent cells by differentiation, which is induced by exogenously applied factors. Here we described a culture system that can be adjusted to favor differentiation into either astrocytes or oligodendrocytes.
主要试剂
Neurobasal® Medium
D-MEM
GlutaMAX™-I
B-27® Serum-Free Supplement
N-2 Supplement
T3 (Liothyronine)
FBS
Geltrex™ Reduced Growth Factor Basement Membrane Matrix
Laminin
Poly-L-Ornithine
Water, distilled
KnockOut™ D-MEM/F-12
Dulbecco’s Phosphate-Buffered Saline (D-PBS) without Ca2 and Mg2
Dulbecco’s Phosphate-Buffered Saline (D-PBS)
StemPro® NSC SFM
实验材料
GIBCO® Rat Glial Precursor Cells
实验步骤
1. Preparing Matrix
1) Matrix for Oligodendrocyte Differentiation
i. Prepare a 1:500 dilution of poly-L-ornithine in distilled water for a final concentration of 20 μg/mL.
ii. Add 2 mL of 20 μg/mL poly-L-ornithine solution to a 35-mm dish (0.5 mL for 4-well plate or slide, 0.25 mL for 8-well slide).
iii. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for at least 1 hour.
iv. Rinse the culture vessel once with distilled water.
v. Prepare a 1:1,000 dilution of laminin in distilled water for a final concentration of 1 μg/mL.
vi. Add 2 mL of 1 μg/mL laminin solution to a 35-mm dish (0.5 mL for 4-well plate or slide, 0.25 mL for 8-well slide).
vii. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for at least 1 hour. Store it at 4°C until use.
Note: You may coat the plates in advance and store them at 2-8°C, wrapped tightly with Parafilm®, for up to 4 weeks.
2) Matrix for Astrocyte Differentiation
i. Thaw the Geltrex™ bottle at 4°C overnight to prevent polymerization. The next day, prepare a 50X stock solution of Geltrex™ by diluting to a final concentration of 10 mg/mL with D-MEM/F-12. Use an ice bucket to keep the bottle at 4°C. Quickly prepare 1 mL aliquots in 50-mL conical tubes (pre-chilled on ice), and store the tubes at –20°C.
ii. Thaw one tube of Geltrex™ (1 mL aliquot described above) slowly at 4°C, and add 49 mL of cold D-MEM/F-12. Mix gently.
iii. Cover the whole surface of each culture plate with the Geltrex™ solution (1.5 mL for a 35-mm dish, 3 mL for 60-mm dish, 5 mL for a T-25 culture flask).
iv. Seal each dish with Parafilm® to prevent drying, and incubate 1 hour at room temperature in a laminar flow hood. Store at 4°C until use.
v. Immediately before use, remove all Geltrex™ solution, wash once with D-PBS with calcium and magnesium, and replace it with pre-warmed complete medium.
2. Differentiation of GRPs
1) Differentiation to Oligodendrocytes
i. Plate glial precursor cells on poly-L-ornithine and laminin coated plate in StemPro® NSC SFM at a density of 2.5 × 104-5 × 104 cells/cm2.
ii. Culture the cells for 2 days, then change the medium to Oligodendrocyte Differentiation Medium.
iii. Change the medium every 3-4 days.
2) Differentiation to Astrocytes
i. Plate glial precursor cells on Geltrex™ coated plate in StemPro® NSC SFM at a density of 2.5 × 104 cells/cm2.
ii. Culture the cells for 2 days, then change the medium to Astrocyte Differentiation Medium.
iii. Change the medium every 3-4 days.