Hello and welcome to the video. Today we are going to focus on cell culture, normally with regards to culture of human dermal mesenchymal stromal cells. What we already know about mesenchymal stromal cells?

Mesenchymal stromal cells, MSC, are also known as mesenchymal stem cells and multipotent stromal cells. It can differentiate into a variety of cell types. MSC can be isolated from different tissue sources, bone marrow, adipose tissue, skin, tendons, menstrual blood, placenta, umbilical corn blood, marten jelly, etc.

A high therapeutic potential of MSC is also provided by paracrine activity of a wide spectrum of release-grow factors, cytokines and other biological active molecules. MSC are the most attractive cell type for experimental and clinical medicine.

Let's start with towing. Remove the vial of human mesenchymal stem cells from liquid nitrogen and incubate in a 37 degrees water bath. Stir the test tube in a water bath, but do not vortex the cells. Do not warm the cells completely if a small piece of ice in the test tube.

As soon as the cells are ready, disinfect the outside of the vial with 70% ethanol. Proceed immediately to the next step. In a laminar flow hood, use a 1 or 2 ml pipette to transfer the cells to a sterile 15 ml conical tube. Be careful, do not introduce any bubbles during the transfer process.

Using a 10 ml pipette, slowly add twice 9 ml of mesenchymal stem cells, called to medium, performed to 37 degrees Celsius, to the 15 ml conical tube. Do not add the entire volume of media all at once to the cells. This may result in decreased cell viability due osmotic shock.

Gently mix the cell suspension by slowly pipetting up and down. Be careful, do not introduce any bubbles. Then refuge the tube at 300 G for 5 minutes to epalate the cells.

Decunt as much of the supplementat as possible. It is necessary to remove CPA. Resuspend the cells in a total volume of 10 m of medium with 10% of phytoboyl serum, performed to 37 degrees Celsius.

Plate the cell suspension out on a tissue culture plate or a tissue culture flask. Maintain the cells at 37 degrees Celsius in an incubator, equilibrated with 5% CO2. The next day, exchange the medium with fresh, warm, full media, replaced with fresh medium every 2 to 3 days.

When cells are approximately 80% confluency, they can be desolated with strips in the day and passage future for frozen for later years. Depending of seeding density and passage number, cells may take longer to reach 80% confluency.

Expansion Subculture cells once they have reached approximately 80% confluency and are achievely proliferating. Carefully remove the medium from the tissue culture plate containing the confluenced layer of human mesenchymal stem cells. Apply 5 milliliters of trypsin-a-DTA solution and incubate in a 37-degree Celsius incubator for 5 minutes.

Inspect the plate and ensure the completely de-attach of cells by gently typing the side of the plate with the palm of your hand. Add 5 milliliters medium to the plate.

Gently rotate the plate to mix the cell suspension. Transfer the desolated cells to a 15-milliliters conical tube. Centrifuge the tube at 300 G for 5 minutes to balance the cells. Discard the supplementat. Apply 2 milliliters warm medium containing 10% VBS to the conical tube and resuspend the cells.

Count the number of cells using GOREV's camera and microscope or other alternative modern methods to subcounty. Plate the cells into appropriate flask plates or veils in medium containing 10% VBS.

Cells can be frozen in mesenchymal stem cell growth media plus 10% VBS at a density of 1 million cells per vial. Regarding freezing, you can refer to Diyat Hayat's video on electroporation. Thank you for watching. I hope you find this video helpful.

See you in the next videos. Bye.