Mesenchymal Stem Cells (MSCs) are the most commonly used cells in stem cell therapy and regenerative medicine, due to their high and multi-potency. Mesenchymal Stem Cells (MSCs) can be isolated from different tissues in the body.
In this article, you’ll be learning about culture-expanded MSCs, how MSCs can be expanded, The potency of MSCs and the type of cells they can differentiate into.
What are culture expanded Mesenchymal Stem cells?
Mesenchymal stem cells are high potent cells used for cellular therapy and isolated from different parts of the body. Mesenchymal stem cells can be used to improve the patient outcome in diseases and conditions such as autoimmune diseases, degenerative diseases, nerve damage, diabetes mellitus, bone problems etc.
For every patient, millions of mesenchymal stem cells are needed and the exact amount varies according to disease, route of administration, administration frequency, weight, and age of patient.
Mesenchymal stem cells are expanded in a culture media, on a large scale in order to obtain the required quantity of cells needed for cellular therapy.
Culture expanded MSCs: How does it work?
Expanding Mesenchymal stem cells in a media involves step by step process of isolation and expansion.
Mesenchymal Stem Cells Isolation
Mesenchymal stem cells can be isolated from different tissues in the human body such as adipose tissues, dental pulp, human bone marrow, umbilical cord tissue, umbilical cord blood, peripheral blood and synovium.
Mesenchymal stem cells are expanded in culture to increase their yield and amplify their desired functions and potency.
Although the population of Mesenchymal Stem Cells obtained will vary from donor to donor, here are some steps to follow:
· Acquire fresh tissue extracts in strictly aseptic conditions, to maintain purity.
· To remove any cell clusters, you have to filter the cell suspension with a 70-mm filter mesh
· Use a centrifuge to roll the cells for about 5 minutes at 500g
· Suspend the cells again the cells to measure the cell viability and yield using Trypan blue exclusion
· Use in T75 culture dishes to culture the cells in 10 mL of complete MSC medium at a density of 25 × 106 cells/mL. You can then go on to Incubate the plates at 37 °C with 5% CO2 in a humidified chamber without any interruption.
· When it’s past 3 h, remove the non-adherent cells that accumulate on the surface of the dish by changing the medium and replacing it with 10 mL fresh complete medium.
· After an additional 8 h of culture, add 10ml fresh complete medium as a replacement for the existing medium. You’ll have to repeat this step every 8 h for up to 72 h of initial culture.
· Cells can be frozen in MSC growth media plus 10% DMSO (D2650) at a density of 2X106 cells/vial.
Expansion of Mesenchymal Stem Cells in a culture media
Culture expanded mesenchymal cells undergo various stages from the preparation of the culture plate, thawing of Mesenchymal stem cells, and the actual expansion of Mesenchymal stem cells.
The reason behind the cultural expansion of Mesenchymal stem cells is to get them to differentiate into other cell types such as osteoblast, adipocyte, and mesenchymal stromal cells.
In preparation, to expand MSCs in a culture media, you need a culture ware. You can get one plastic or glassware plate and coat it with a sufficient amount of 0.1% gelatin. Don’t forget to aspirate the gelatin solution from the coated plate or flask before you use it.
The next step involves the thawing of the Mesenchymal stem cells, and here are a few steps for you to follow:
After the recommended culture medium and coated culture ware is ready and on standby, remove the vial of Mesenchymal Stem Cells from liquid nitrogen and incubate in a 37C water bath and pay close attention to it, until all the cells are completely thawed. The extent of completely thawed frozen cells and how fast, are what determines the cell viability.
Once the cells have thawed completely, take steps to avoid contamination by disinfecting the walls with 70% ethanol, before you proceed to the next step.
Place the cells in a hood, and carefully transfer the cells to a sterile tube with a pipette (1 or 2ml pipette), Do this in such a way to prevent bubbles.
Then, add drops of Mesenchymal Stem cell expansion medium that have been pre-warmed to 37C to the tube containing the Mesenchymal stem cells.
Be careful to take your time when adding the medium to avoid osmotic shock which could lead to decreased viability.
Proceed to mix the suspension slowly by pipetting up and down two times while avoiding any bubbles.
Place the tube in a centrifuge and centrifuge the tube at 300 x g for 2-3 minutes to roll the cells, and you should not vortex the cells.
After this, then decant as much of the supernatant as possible. These steps are necessary to remove residual cryopreservative (DMSO).
Suspend the cells in a total volume of 10 mL of Mesenchymal Stem Cell Expansion Medium again or any alternative of choice, pre-warmed to 37 °C, containing freshly added 8 ng/mL FGF-2 (F0291).
The next step involves placing the cell suspension onto a 10-cm tissue culture plate or a T75 tissue culture flask.
Maintain the cells in a humidified incubator at 37 °C with 5% CO2.
The next day, exchange the medium with fresh Mesenchymal Stem Cell Expansion Medium (pre-warmed to 37 °C) containing 8 ng/mL FGF-2*. Replace with fresh medium containing FGF-2 every two to three days thereafter.
Isolate the cells when they are approximately 80% confluent, using Trypsin-EDTA and passaged further or frozen for later use.
Expansion of Mesenchymal Stem Cells
Once the cells are actively proliferating and have reached a confluence of approximately 80% (before 100%), you should subculture the cells.
Then remove the medium from the 10-cm tissue culture plate containing the confluent layer of human mesenchymal stem cells, carefully and apply 3-5 mL of Trypsin-EDTA Solution, before proceeding to incubate in a 37 °C incubator for 3-5 minutes.
Crosscheck the culture to see if all the cells are completely detached. Then, add 5 mL Mesenchymal Stem Cell Expansion Medium to the plate.
Swirl the plate mildly to mix the cell suspension. Transfer the separated/isolated cells to a 15 mL conical tube.
Centrifuge the tube at 300 x g for 3-5 minutes to pellet the cells.
Throw the supernatant away and apply 2 mL Mesenchymal Stem Cell Expansion Medium (pre-warmed to 37 °C) containing 8 ng/mL FGF-2 to the conical tube and completely suspend the cells again. Remember not to vortex the cells.
Then, use a hemocytometer to count the number of cells.
Plate the cells at a density of 5,000-6,000 cells per cm2 into the appropriate flasks, plates, or wells in a Mesenchymal Stem Cell Expansion Medium containing 8 ng/mL FGF-2.
Cells can be frozen in MSC growth media plus 10% DMSO (D2650) at a density of 2X106 cells/vial.
Functions of Culture Expanded MSCs
Mesenchymal stem cells are required to be expanded in order for them to be used clinically for therapeutic purposes.
The culture expanded MSCs can be induced to differentiate into adipocytes, osteocytes, hepatocytes, chondrocytes, tenocytes and cardiomyocytes.
Because of its potential to differentiate into different kinds of cells in the body, it can be used to manage liver problems, heart problems, joint and bone problems etc.
Mesenchymal stem cells are also used in tissue regeneration and modulation of the immune system. They possess anti apoptotic, angiogenic, anti fibrotic, and anti-oxidative properties.
However, the quantity of MSCs isolated from body tissues is not enough for clinical and therapeutic applications.
This is why MSCs are expanded in culture to increase their yield for desired therapeutic effect.