The overall goal of this method is to isolate and culture bone marrow mesenchymal stromal cells in autologous conditions, such as a patient serum to supplement the medium and fibrin for cell delivery in orthopedics. This method can help with the clinical translation of mesenchymal stromal cell therapies in the fields of regenerative medicine and tissue engineering. The main advantage of this techinique is the completely autologous context that avoids negative effects like infective and inflammatory risks.
Furthermore, fibrin clot is a reabsorbable scaffold for cell delivery. Demonstrating the procedure will be myself and Sara Savelli, a biologist from my laboratory. After collecting bone marrow and isolating plasma from the patient according to the text protocol, use a spike to transfer the unit of plasma into a transfer bag.
Disconnect the filled bag by welding, and weigh the bag to calculate the volume of plasma. Coagulate the autologous plasma by injecting 100 milligrams per milliliter of calcium gluconate at 10%weight per volume through the port connector. After mixing the bag, place it at four degrees Celsius without shaking overnight to facilitate clot formation.
The following day, centrifuge the bag at 4, 900 times G at room temperature for 15 minutes. Next, isolate the clot just above the clamp to facilitate filtration. Then, use a spike placed into the outlet port of the filtration kit to connect the filtration kit to the transfer bag.
Hang the bag, and open the blue clamp to let the serum flow by gravity. To prevent transfer of fibrin into the bag, avoid applying pressure to the filter. After filtration, seal the tubing and remove the bag.
Under a laminar flow bench to avoid microbial contamination, connect a dedicated line to the final bag, and transfer serum into 50-milliliter tubes. Transfer the bone marrow sample from the syringe into a 50-milliliter conical tube, and use sterile saline to dilute it at a one to four ratio. Vortex the 50-milliliter tube for 30 seconds to disaggregate the cell clusters.
Warm a 15-milliliter density gradient to room temperature, and gently layer 20 milliliters of the diluted bone marrow sample onto the gradient. Centrifuge the gradient at 400 times G without a break and 25 degrees Celsius for 30 minutes. Then, remove fat from the upper layer, and collect the mononuclear fraction at the liquid-liquid interface using a sterile five-milliliter pipette.
Transfer the fraction to a new 50-milliliter tube. Using complete proliferation medium, wash the collected mononuclear fraction twice, and centrifuge the tube at 400 times G and 25 degrees Celsius for 10 minutes to obtain cell pellets. After the second wash, discard the supernatant, and use five milliliters of complete proliferation medium to resuspend the pellet.
Then, dilute an aliquot of the cell suspension, one to 100, and after a one to one dilution with Trypan Blue, use a hemocytometer to check viability and to count the cells. Fill two or more 75-centimeter squared tissue culture flasks with 15 milliliters of fresh complete proliferation medium, and incubate them at 37 degrees Celsius and 5%carbon dioxide. Then, seed 0.3 to 0.5 times 10 to the six cells per centimeter squared in the flasks, and incubate them at 37 degrees Celsius and 5%carbon dioxide for 48 hours.
At the same time, seed one times 10 to the six cells in a 25-centimeter squared flask with five milliliters of fresh complete proliferation medium for the Colony Forming Unit Fibroblast assay, and culture the cells for two weeks. After the 48 hour incubation, discard the medium from the flasks, and wash the cells using complete proliferation medium to remove nonadherent cells and debris. Then, add fresh medium to the 75-centimeter squared flasks.
Replace half of the medium twice a week until the cells reach 70 to 80%confluency. Recover the cells by adding a specific recombinant animal-free protease to the flasks, and incubate at 37 degrees Celsius and 5%carbon dioxide for 10 minutes. Then, add six milliliters of complete proliferation medium per flask, and collect all the cells in one 50-milliliter tube.
After diluting and counting the cells with Trypan Blue as before, further expand the cells by replating the cell suspension at 2, 000 to 3, 000 cells per centimeter squared in new 75-centimeter squared flasks. Prepare fresh osteogenic medium by combing DMEM-LG with 10%autologous serum, 50 micrograms per milliliter of ascorbic acid, and 0.4 micrograms per milliliter of hydrocortisone. Filter sterilize the medium.
96 hours before harvesting the cells, completely remove the proliferation medium, and add osteogenic medium. Then, change the medium again with osteogenic medium 24 hours before harvesting. To harvest the cells, use the recombinant animal-free protease to detach the cells as before.
After pelleting the cells in a 50-milliliter tube, use two milliliters of complete proliferation medium to resuspend the pellet. Once the cells are counted and washed, gently resuspend the pellet at one to two times 10 to the six viable cells per two milliliters of autologous plasma per 50-milliliter tube. To prepare the hMSC fibrin clot construct, add 150 microliters of 100 milligrams per milliliter calcium gluconate to the cells, and gently resuspend them.
Then, incubate the cells to obtain hMSC fibrin clot constructs. To carry out a cell viability assay, remove the liquid from the tubes, and add one milliliter of complete proliferation medium containing 10%volume to volume cell viability reagent according to the manufacturer's guidelines. Measure the absorbance at 570 nanometers with a reference wavelength of 600 nanometers.
Discard the supernatant, and add two milliliters of fresh complete proliferation medium. The following day, remove the medium from the tubes, and add one milliliter of complete proliferation medium with 10%cell viability reagent. Incubate the tubes for three hours, and read the absorbance at 570 nanometers again.
Carry out histologic evaluation according to the text protocol. By plating bone marrow mononuclear cells isolated using a density gradient, it is possible to isolate hMSCs and their progenitors. Under autologous culture conditions, approximately 10%of the cells, called MPCs, are detected at passage zero depending on patient variability.
As seen here, MPCs are still present after replating of the proteases used for cell passaging. In this figure, von Kossa, osmium tetroxide and Alcian Blue staining were carried out to identify osteogenic, adipogenic, and chondrogenic differentiation respectively to compare the hMSC population identity with cells obtained using non-autologous culture conditions. Inside plasma clots, at 24 hours the hMSCs are viable, as demonstrated by the color change of the cell viability dye from blue to pink.
The cell viability inside the clot is confirmed by H&E staining, showing a well-preserved cell morphology. Furthermore, as seen by von Kossa staining, a minimal osteoinduction just before the second cell harvest gives rise to preliminary calcium deposition by stimulated hMSCs. As demonstrated in this example, eight compassionate cases of severe upper limb non-union were treated with hMSC fibrin clot constructs.
Long-term assessment confirmed successful clinical and functional outcomes for all patients without evidence of tissue overgrowth or tumor formation. Once mastered, this protocol can be performed in about three weeks. When attempting this procedure, remember to manipulate the osteoinduced cell gently.
Following this technique, other method like the colony forming unit fibroblast assay can be performed in order to evaluate the stemness potential of the sample. After its development, this technique paved the way for researchers in the field of regenerative medicine to explore cartilage and tendon cell therapies in orthopedic patients. After watching this video, you should have a good understanding of how to prepare a fully autologous MC product for bone regeneration.
Don't forget that working with bone marrow and peripheral blood samples can be hazardous and precautions like individual protection and sample screening for precautions should be taken while performing this procedure.
