Thawing and Establishing Rat Glial Precursor Cells (GPCs)
- Rat Glial Precursor Cells (Cat. no. N7746-100), stored in liquid nitrogen
- CELLStart™ or poly-L-ornithine coated, tissue-culture treated flasks, plates, or Petri dishes
- Complete GPC growth medium*, pre-warmed to 37°C *Complete StemPro® NSC SFM (Cat. no. A1050901) supplemented with 2 mM GlutaMAX™-I (Cat. no. 35050-061) and 10 ng/mL PDGF-AA (Cat. no. PHG0035)
- Disposable, sterile 15-mL conical tubes, pre-rinsed with growth medium
- 37°C water bath
- 37°C incubator with humidified atmosphere of 5% CO2
- Flame-polished and autoclaved glass Pasteur pipettes, or plastic Pasteur pipettes pre-rinsed with growth medium
- Hemacytometer, cell counter and Trypan Blue (Cat. no. 15250-061), LIVE/DEAD® Cell Vitality Assay Kit (Cat. no. L34951), or the Countess™ Automated Cell Counter (Cat. no. C10227)
- Pre-rinse your culture flasks, plates, or Petri dishes with growth medium to coat the plastic surface. Make sure to pre-rinse any other material that will come in contact with the cells to prevent cells from sticking to the plastic.
- Remove the cells from liquid nitrogen storage, and immediately transfer the cells to a 37°C water bath to prevent crystal formation.
- Quickly thaw the vial of cells by gently swirling it in the 37°C water bath and removing it when the last bit of ice has melted, typically < 2 minutes. Do not submerge the vial completely. Do not thaw the cells for longer than 2 minutes. Do not introduce bubbles into the cell suspension as it decreases cell viability.
- When thawed, transfer the tube containing the cells into the laminar flow hood, and wash the outside of the tube with 70% isopropanol.
- Rinse the pipette tip with media, and very gently transfer the cells into a pre-rinsed 15-mL centrifuge tube.
- Rinse the vial with 1 mL of growth medium, and dropwise add to the cells in the 15-mL centrifuge (one drop per second). Mix by gentle swirling after each drop.
- Slowly add 2 mL of growth medium to the cell solution, and mix gently.
- Determine the viable cell count using your method of choice. The viability of thawed cells should be >50%, and the total live cell number should be >1 x 106.
- Plate the cells at a seeding density of 3 x 104–5 x 104 cells per cm2 on a CELLStart™ or poly-L-ornithine coated, tissue-culture treated culture dish. If necessary, gently add growth medium to the cells to achieve the desired cell concentration and recount the cells.
- Incubate at 37°C, 5% CO2, and 90% humidity and allow cells to adhere for at least 24 hours.
- The next day, replace the medium with an equal volume of fresh, pre-warmed complete growth medium. Change the medium every other day, and passage cells when the culture is 75–90% confluent.