Subculturing Rat Alk Phos MSC Cells
Follow the protocol below to culture StemPro Alk Phosexpressing Rat MSCs. Subculture cells when needed (before colonies start contacting each other), typically every 7–10 days.
The following materials are required:
- Culture vessels containing StemPro Alk Phosexpressing Rat MSCs
- Tissue-culture treated flasks, plates or dishes
- Alpha-MEM medium with GlutaMAX™-I supplemented with 10% MSC-Qualified FBS and containing antibiotic/antimycotic or gentamycin, pre-warmed to 37°C
- Disposable, sterile 50-ml tubes
- 37°C incubator with humidified atmosphere of 5% CO2
- Dulbecco’s Phosphate Buffered Saline (DPBS), containing no calcium, magnesium, or phenol red
- TrypLE™ Express, pre-warmed to 37°C
- Hemacytometer, cell counter and Trypan Blue,
- LIVE/DEAD Cell Vitality Assay Kit, or the Countess™ Automated Cell Counter
- Aspirate the complete alpha-MEM medium from the cells.
- Rinse the surface of the cell layer with DPBS without Ca2+ and Mg2+ (approximately 2 ml DPBS per 10 cm² culture surface area) by adding the DPBS to the side of the vessel opposite the attached cell layer, and rocking back and forth several times.
- Aspirate the DPBS and discard.
- To detach the cells, add a sufficient volume of pre-warmed TrypLE™ Express to cover the cell layer (approximately 0.5 ml/10 cm²).
- Incubate at 37˚C for approximately 5–8 minutes.
- Observe the cells under a microscope. If the cells are less than 90% detached, continue incubating and observe within 2 minutes for complete detachment of the cells. Tap the vessel gently to expedite cell detachment.
- When ≥ 90% of the cells have detached, tilt the vessel for a minimal length of time to allow the cells to drain. Add the equivalent of 2 volumes (twice the volume used for TrypLE™ Express) of pre-warmed complete alpha-MEM medium. Disperse the medium by pipetting over the cell layer surface several times.
- Transfer the cells to a 50-ml conical tube and centrifuge at 300 x g for 5 minutes at room temperature. Aspirate and discard the medium.
- Resuspend the cell pellet in a minimal volume of pre-warmed complete alpha-MEM medium and remove a sample for counting.
- Determine the total number of cells and percent viability using your method of choice. If necessary, add complete alpha-MEM medium to the cells to achieve the desired cell concentration and recount the cells.
- Determine the total number of vessels to inoculate by using the following equation: Number of vessels = Number of viable cells ÷ (growth area of vessel in cm² × 5,000 cells per cm² recommended seeding density)
- Add complete alpha-MEM medium to each vessel so that the final culture volume is 0.2–0.5 ml per cm².
- Add the appropriate volume of cells to each vessel and incubate at 37°C, 5% CO2 and 90% humidity.
- 3–4 days after seeding, completely remove the medium. Replace with an equal volume of complete alpha-MEM medium.