An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

The overall goal of this procedure is to make use of clotted bone marrow samples by digesting the clots with urokinase. This is accomplished by first filtering the bone marrow sample over a cell strainer. Next, the bone marrow clot is cut into small pieces.

Then the clot is transferred to a Falcon tube and digested using urokinase. Finally, the digested clot material is seeded for expansion of mesenchymal stem cells or MSCs. Ultimately, the plates are compared to a control plate using em sustain to assess the efficacy of the digest.

The implication of this technique extends toward autologous stem cells therapy or diagnosis. Because graft need expansion and differentiation of a large number of stem cells. We first had the idea for this method when you realize that a lot of bone marrow samples from DOC were clotted when they arrived in our lab.

We simply did not want to trash such precious samples To reconstitute urokinase. Inject 10 milliliters of sterile PBS into the septum inlet flask and vortex to dissolve to a concentration of 50, 000 units per milliliter. Aliquot 500 microliters of 25, 000 units each into sterile tubes and store at negative 20 degrees Celsius for up to six months to carry out a digestion reaction Thaw one aliquot of urokinase per clouded bone marrow aspirate of up to 25 milliliters.

Preheat a water bath or shaking incubator to 37 degrees Celsius under a laminar flow biosafety cabinet. And while wearing gloves, place a 100 micrometer cell strainer on top of a 50 milliliter tube. Carefully pour the bone marrow aspirate through the cell strainer, tilting the strainer, or moving around the clot material.

Carefully pour the bone marrow aspirate through the cell strainer without tilting the strainer or moving around the clot material. Use a sterile pipette for better flow through the filter mesh with sterile forceps. Transfer the bone marrow clot material into an empty cell culture dish.

Then use a sterile scalpel to cut the debris into two cubic millimeter pieces. Next, with a five milliliter microtiter pipette, tritrate the clot by pipetting up and down five times. Then add one aliquot of urokinase to the sample and incubate at 37 degrees Celsius for 30 minutes in a water bath or shaking incubator.

After the incubation in the biosafety cabinet, tritrate again is just demonstrated and incubate for an additional 30 minutes. Then tri rate five more times and pass the sample over a fresh 100 micrometer cell strainer and pool with the initial filtrate centrifuge the cell suspension at 500 times G and room temperature for 10 minutes. After discarding the supernatant, use 50 milliliters of basal medium to resuspend the pellet.

Using Trian blue solution, dilute an ALI quad of resuspended cells one to 10, and add the suspension to a neubauer chamber to count them. After counting the cells, plate them in. Cell culture flasks at a density of five times 10 to the seven cells per cubic centimeter for the CFU assay control plate.

10 to the ninth cells In a 10 centimeter diameter round cell culture dish, incubate in a humidified chamber at 37 degrees Celsius. Following three days of culturing MSCs will be attached to the cell culture dish while other cells remain in suspension. Use basal medium containing five nanograms per milliliter of basic fibroblast growth factor to replace the medium in both the experimental and control plates.

Continue to culture for a total of two weeks. Changing the medium three times per week. If the cells exceed 80%co fluency, trypsin eyes the culture and split to carry out the CFU assay.

Prepare 10 milliliters of GSA solution per dish by using sterile water to dilute the stock solution. One to 10. Remove the medium from the cell culture dish and use PBS to carefully wash the cells with pure methanol.

Fix the cells at room temperature for five minutes. Then discard the methanol and add the gemsa solution. Incubate in a humidified chamber at 37 degrees Celsius for 60 minutes after the incubation.

Use PBS to wash the cells twice. Then air dry the plate. Head first on a paper towel with a marker on the back of the plate.

Manually count the colonies for adipogenic differentiation. Culture MSCs in a monolayer at four times 10 to the fifth cells per square centimeter. Alternating between two different culture conditions for the first condition.

Culture cells, an adipogenesis maintenance medium, and for the second condition culture cells. An adipogenesis inducing medium with maintenance medium according to the recipes outlined in the text protocol. To visualize the lipid droplets, use 10%formaldehyde to briefly fix the cells.

Then with PBS, wash the cells before using 0.35%oil red O in isopropanol to stain the cells for osteogenic differentiation culture MSCs in a monolayer at seven times 10 of the third cells per square centimeter. After stimulating the cells using the medium outlined in the text protocol, use 10%formaldehyde to briefly fix the cells. Once the cells have been washed with PBS use Von cost sustain to identify mineral deposits to carry out congenic differentiation from a sponge shaped medical device constituted from lyophilized collagen type one cut cubes three millimeters per side seed MSCs on top of the cubes at a concentration of four times 10 to the eighth cells per milliliter after allowing the cells to adhere to the cubes for 30 minutes at room temperature.

Maintain MSC collagen constructs in congenic medium as listed in the text protocol. Add 0.4%Ian Blue solution to the sections and incubate overnight at four degrees Celsius. The next day use 40%DMSO and 0.05 molar magnesium chloride to wash the cells for 30 minutes before using nuclear fast red to counterstain the cells as demonstrated here.

A simple DAPI stain section of clot material confirms the presence of nucleated cells at high density, indicating that a considerable number of MSCs are trapped within the clots. Using the protocol described in this video. Treatment with urokinase allowed the digestion of all but 15%by weight of dog and 9%by weight of human blood clots to determine the efficacy of the urokinase digestion cells from clotted sample filtrates and digested clots were seated separately and incubated for two weeks.

Em sustain of the cells showed 3.8 times more CFUs from urokinase digested canine samples than from corresponding initial filtrates. Human digested samples showed 1.6 more CFUs than the undigested filtrates. In this experiment, MSCs from bone marrow clots were compared to MSCs derived from UNC clotted bone marrow samples for their ability to differentiate under the appropriate culture conditions.

After four weeks in culture, it was possible to differentiate MSCs from digested samples into adipogenic, osteogenic and congenic lineages. Furthermore, von Casa oil, red O and Sian blue staining indicated no differences in the grade of differentiation between the groups. Once mastered, Mr.Nick can be done in about one and a half hours if it is performed properly.

After watching this video, you should have a good understanding of how to isolate me hemo stem cells for expansion culture, even if your samples are clotted instead of discarding the sample. Our protocol uses the enzyme kinase to digest the clot and ES stem cells for expansion culture.