How do I perform an explant culture?

Explant culture is performed in 4 main steps:

1. Obtaining the Explant: To obtain an explant, sterile equipment is used to surgically collect tissue from mammals, avian organs, and rodents. For instance, adipose tissue can be used to establish mesenchymal stem cells.
2. Cutting and Cleaning the Explant: Place the tissue in a petri dish with 1-2 mL of incomplete medium. Using a sharp surgical blade, cut the tissue into pieces (typically around 1×1 mm). Collect the explant pieces with sterile forceps and wash gently. To wash the explant, transfer the tissue pieces into a centrifuge tube with approximately 0.5 mL of incomplete medium. Gently mix the contents by pipetting the medium 4 to 5 times. Allow the tissue pieces to settle, then carefully take off the upper medium. Repeat washing 2-3 times.
3. Culturing the Explants: Aseptically place the explants on a coated surface and allow them to attach in the presence of a rich culture medium. Use basal minimal media with 10-15% serum. Culture the explants under standard tissue culture conditions (pH 7.2-7.4, 5% CO2 and humidity, temperature 37°C) to facilitate cell migration and growth. Change the media every 3 days without interfering with the explants. Cells should grow from the explant within 15-30 days. Once this begins, add 5 mL of medium to the flask on the succeeding days.
4. Subculturing and Establishing Cell Line: Once cells completely encompass the explants, trypsinize the cells and subculture. Use a lower concentration of trypsin (e.g., <0.25% trypsin for 5 min). Use a proper sized flask for seeding based on the total number of cells obtained.