Mesenchymal stem cells abbreviated as MSCs are isolated from a six to eight week old rag dolly, female rat, and cultured in a 10 centimeter Petri dish until they reach about 80%co fluency. To enrich the MSCs, the cells are stained with biotin conjugated primary antibodies, followed by streptavidin conjugated magnetic beads. Labeled cells are then separated into two fractions, the negative fraction and the positive fraction by magnetic activated cell sorting.
Subsequently, flow cytometry analysis is used to confirm surface marker expression. Finally, cloning cylinders are used to obtain single colony derived MSCs. Hi, I'm Lee from the laboratory of Christina Chang in the Department of Chemical Engineering and the material science at Michigan State University.
Today we'll show you a procedure for isolation and enrichment of chy stem cells as well as obtaining single colony derived cells. We use this procedure in our laboratory for studying the proliferation and the differentiation behaviors of red stem cells. So let's get started.
Mesenchymal stem cells or MSCs will be isolated from a six to eight week old spra dolly female rat. To begin this procedure, cut off the femurs and tibia from the back limbs of the euthanized animal. Remove the skin and muscles, put the dissected femurs and tibia in 70%isopropanol for a few seconds, and then transfer them to one X-D-P-B-S.
The following steps should be performed in a biosafety cabinet. First, transfer the femur and tibia to a 10 centimeter dish containing DMEM. Then while holding each bone with tweezers, cut open the two ends with scissors.
Attach a 22 gauge needle to a three milliliter syringe and fill it with DMEM. Insert the needle into one opened end of the bone and flush the marrow into a 50 milliliter tube. Repeat two to three times for each bone when all the marrow has been obtained.
Resus suspend the cells, then pass the cell suspension through a 70 micrometer cell strainer to remove bone debris and blood aggregates. Spin down the cells at 200 GS at four degrees Celsius for five minutes. After removing the supernatant by aspiration, resuspend the cells in 25 milliliters of MSC medium seed.
10 milliliters of cell suspension into each of two 10 centimeter culture dishes. Incubate the culture dishes in a 37 degrees Celsius 5%carbon dioxide incubator for one to two weeks. Once the previously isolated rat MSCs, reach a co fluency of about 80%Aspirate the medium and at four to five milliliters of trypsin EDTA to each dish.
Return the dishes to the incubator and incubate for about five minutes. To allow cell detachment, add an equal amount of culture medium to inactivate the trypsin. Collect cell suspensions in a 15 milliliter tube and spin cells down at 200 GS four degrees Celsius for five minutes.
Now we will demonstrate the enrichment of MSCs by two surface markers, CD 54 and CD 90. Using the BD I magnet a cell separation magnet from BD Biosciences. Begin by Resus suspending the cell pellet in cell staining buffer at 20 million cells per milliliter.
Next, add biotinylated CD 54 antibody and a biotinylated CD 90 antibody to the cell suspension and to mix gently add after incubation on ice for 15 minutes, wash the labeled cells with an excess volume of one X BD IMAG buffer. Spin down the labeled cells at 200 GS four degrees Celsius for five minutes. Prepare the BD imag streptavidin particles by vortexing thoroughly, and then add 40 microliters of particles for every 10 million cells.
Mix the particles with the labeled cells thoroughly and incubate the cells at six to 12 degrees Celsius for 30 minutes. During this incubation time, label a round bottom test tube for collecting the positive fraction. When the incubation is done, bring the labeling volume to 20 million cells per milliliter.
With one Expedia I mag buffer. Transfer the labeled cells to the positive fraction collection tube. Place the positive fraction tube onto the BDI magnet and let it stay for six minutes.
Then with the positive fraction tube still on the BDI magnet, remove the supernatant with a glass pastier pipette. Next, remove the positive fraction tube from the BDI magnet and place it on ice. Add one milliliter of ice called one XBDI mag buffer and resuspend the cells by gentle mixing.
Place the tube back onto the BDI magnet and let it stay for two to four minutes. Remove the supernatant with a new glass pastier pipette. Repeat the washing as described previously.
One more time. Finally, remove the supernatant with a new glass pastier pipette. And then remove the tube from the BDI magnet reus.
Suspend the cells in one milliliter of culture medium and transfer the cell suspension to a 50 milliliter tube containing 25 milliliters of medium seed. 1 75 centimeter square flask for maintaining the cells. And a 10 centimeter dish for flow cytometry.
To begin the procedure for separating single colony derived MSCs plate cells at about 50 to 100 cells per 10 centimeter dish and incubate in a 37 degree Celsius and 5%carbon dioxide incubator for one to two weeks. During this one to two week period, examine well isolated colonies with an inverted microscope. Once the colonies have reached a sufficient size of more than 100 cells in each colony, mark the colonies with a permanent marker at the bottom of the dish.
When picking a colony to be marked, make sure there are no surrounding colonies nearby. Aspirate medium and wash the dish once with one X-D-P-B-S using forceps. Pick up a sterile cloning cylinder and gently place it around the marked colony.
Repeat this until every marked colony has a cloning cylinder placed over it. Add 100 microliters of tripsin EDTA to each cloning cylinder and return the dish to the incubator. After five minutes, check the cells under the microscope to see whether they are rounding up.
When the cells have lifted up, add an equal amount of culture medium to inactivate the trypsin. Mix the cell suspension with the 200 microliter micro pipetter and transfer the cell suspension to a 60 millimeter dish containing three milliliters of prewarm culture. Medium labeled the dishes and put them back into the incubator Bader, after their isolation plastic adherent.
MSCs should be visible the next day after plating. As the cells continue to proliferate. Confluence cells should look like the cells shown in this phase contrast image where the majority are spin alike or starlike culturing is appropriate when cells reach about 80%co fluency.
After trypsin ization detached cells should look round and floating in the medium. Once MSCs are enriched by magnetic cell sorting flow cytometry is performed to verify the surface marker expressions. If the enrichment is good, the cells should show positive staining against the MSC markers, CD 54 and CD 90, but negative against the HSC marker CD 45 isotype controls IgG two A and IgG one are used as negative controls.
When cells are seated at a proper clonal density, colonies should rise from single cells as shown in this example. Cloning cylinders can then be used to separate the colonies and cells derived from the colonies can be cultured separately. In these two examples, the cells derived from one colony are spin alike, whereas the cells derived from another colony are round.
So we've shown you how to isolate and enrich random and common stem cells and also how to obtain single colony derived cells. When during this procedure, it is important to remember to get rid of the blood clots and bone debris during isolation. Use the optimal amount of antibody during enrichment and mark well isolated colonies during colony separation.
So that's it. Thanks for watching and good luck with your experiments.
