To begin, retrieve two cryopreservation tubes containing frozen human adipose-derived mesenchymal stem cells at passage three from liquid nitrogen. Agitate them in a 37 degree Celsius water bath until fully thawed. Then disinfect the tubes using 75%alcohol.
Place the disinfected tubes on an ultra-clean table. Using a pipette, aspirate the cell suspension and transfer it to a 15 milliliter centrifuge tube. Centrifuge the tube in a low speed centrifuge at 250 G and four degrees Celsius for five minutes.
Meanwhile, prepare four 10-centimeter culture dishes and add nine milliliters of medium to each dish. Mark the name, cell type, generation, and the experiment date on each dish. Then preheat them in a 37 degree Celsius thermostatic cell incubator.
Once the five minute centrifugation is over, disinfect the centrifuge tube before moving it to an ultra-clean table. Using a pipette, remove the supernatant and add four milliliters of medium to achieve the desired cell concentration. Gently agitate the solution until it is evenly dispersed.
Next, add one milliliter of cell suspension to each preheated dish and shake it by agitating the same on a level surface in a cross pattern. Observe the cells at 40x magnification under an optical microscope before placing the dishes into the 37 degree Celsius incubator. On day two, observe the cell state under the optical microscope at 40x magnification before proceeding for exchange medium preparation.
After disinfection, transfer the culture dishes to an ultra-clean table with Biosafety Level 2. Remove the medium from each culture dish. Rinse the dish gently with two milliliters of PBS solution twice and add 10 milliliters of medium to each dish.
Transfer the cells to the incubator until they're ready for supernatant collection.
