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.
- 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.