This technique enables development of a standardized system that is easy to set up from human cell lines or primary cell populations and provides features of a well-organized, bone marrow-like structure. This model allows cell harvest post fixation to characterize in situ cell localization within the system via imaging, or 3D structure dissociation to collect each cellular component for molecular or functional studies. Demonstrating the procedure will be Kevin Geistlich, an assistant engineer from my laboratory.
Weigh 35 to 40 milligrams of BCP beads in a 1.5-milliliter tube for each insert. That is, a small cylindrical plastic dish with a polycarbonate membrane at the bottom. Bore a small hole with a clean needle through the top of the 1.5-milliliter tube to avoid the lid popping open during the autoclave cycle, and place the tubes in a vertical position in a closed, sterile box.
Put sterile polycarbonate cell culture inserts within the wells of a six-well plate. Pour sterile BCP beads from a 1.5-milliliter tube into the insert. Wash the beads carefully by dripping 350 microliters of 1X PBS inside the insert and then add 4.5 milliliters of 1X PBS to the well.
Discard the PBS using a vacuum or a five-milliliter pipette by placing the tip in a corner of the well. Repeat the washing step twice. Then, use 1X PBS supplemented with 10%FBS to block the system.
Carefully add 350 microliters of the blocking solution to the insert and 4.5 milliliters to the well, and place the whole plate in an incubator overnight. Remove the blocking solution from the system. Mix 1 times 10 to the 6 HMEC-1 cells and 2 times 10 to the 6 HS27A cells, representing the vascular and mesenchymal stromal cell compartments respectively, in a 15-milliliter tube and centrifuge for five minutes at 1, 575 G at room temperature.
Discard the supernatant and add 175 microliters of HMEC-1 medium, supplemented with 175 microliters of ODM that will promote mesenchymal stromal cell differentiation into osteoblasts. Pour 350 microliters of the cell suspension containing 3 times 10 to the 6 total cells inside each insert. Then, pour 4.5 milliliters of the combined media without cells into the wells of the plate.
Gently aspirate and dispense the mixture several times with a one-milliliter micropipette to homogenize the cells and BCP beads within the insert. Allow the whole mix to settle at the bottom of the insert. Once the endothelial and mesenchymal cells are homogenized inside the insert, keep the six-well plate inside the incubator for three weeks.
Change the medium inside the insert in the well twice a week by carefully removing the medium using a one-milliliter micropipette and placing the tip at the inner insert wall to avoid disturbing the corner of the insert. Resuspend the hematological cells of interest in the HMEC-1 and RPMI combination medium and add the cell suspension to the insert. Other cell types of interest with dedicated media can be added at this step.
If a drug treatment protocol is required, wait 24 hours after adding the cells of interest before treating them, giving them time to adhere to the bone-like structure. Refresh the medium with 50%HMEC-1 complete medium and 50%RPMI complete medium twice a week. After changing the culture medium twice a week, store the retrieved supernatant from the inside of the insert at minus 80 degrees Celsius to evaluate protein production within the system.
To dissociate the cells from the 3D structure, mix 4.5 milliliters of RPMI with 0.5 milliliters of FBS, supplemented with 0.4 milligrams per milliliter of collagenase in a 15-milliliter tube. Then, warm the solution at 37 degrees Celsius for 10 minutes. Place the insert upside down on a sterile, six-well plate cover to cut the membrane out properly.
Use a sterile scalpel to cut the membrane from the sides, thus retrieving a white solid disc. Place the disc inside the warm 15-milliliter tube containing the solution mixture prepared earlier. Place the tube in an active wheel in a 37 degrees Celsius incubator and incubate for 10 minutes to allow digestion.
Apply the same incubation time for all membranes to avoid a skewed analysis. After the incubation, centrifuge the 15-milliliter tube for five minutes at 1, 575 G at room temperature. Discard the supernatant and resuspend the pellet in five milliliters of warm 1X PBS.
Resuspend the pellet in 300 microliters of warm trypsin and mix well by pipetting for 10 seconds, and place it in a water bath set at 37 degrees Celsius for one minute. Stop the enzymatic digestion by adding one milliliter of warm, pure FBS. With a one-milliliter micropipette, mechanically aspirate and dispense the medium to completely dissociate the disc.
Centrifuge the tube for five minutes at 1, 575 G and resuspend the pellet in three milliliters of 1X PBS supplemented with 10%FBS. Leave the beads to sediment for five seconds before collecting the supernatant and filtering it through a 35 micron cell strainer placed on a collection tube. Centrifuge the retrieved filtrate for five minutes at 1, 575 G and count the cells with trypan blue to evaluate the viability.
Resuspend the pellets in the antibody mix and incubate for 30 to 40 minutes on ice away from light. Add one milliliter of PBS supplemented with 2%FBS to wash the cells and centrifuge for five minutes at 1, 575 G.To initiate the flow cytometry analysis, resuspend the retrieved pellet in 200 microliters of 1X PBS with 10%FBS, supplemented with DAPI in a 1 to 2000 solution. Analyze the tubes in a cytometer using the parameters described in the text manuscript.
Use an FSC-H versus FSC-W graph plot to gate the singlet population. In the singlet fraction, use a DAPI-A versus FSC-A graph plot to determine the viable cell population. With the DAPI-negative population previously gated, use an APCA-A versus FSC-A graph plot to determine the CD45-positive population.
To fix the BM-like structure, transfer the insert from the old plate to a new six-well plate filled with 1X PBS. Then, gently replace the medium inside the insert with 1X PBS. Once the insert is washed, place it within a new six-well plate and fill both the well and the insert with freshly-opened paraformaldehyde.
Allow the fixation process to proceed for four hours at room temperature, away from light. Next, wash the insert once with 1X PBS and store it at four degrees Celsius away from light. After the desired exposure, the results suggested that it was possible to maintain hematological cells for at least six weeks within the 3D model before retrieving viable cells.
It was possible to substitute the HS27A cell line with primary normal bone marrow MSC or acute myeloid leukemia MSC in this 3D BM-like model, or replace the hematopoietic cell line with primary HSCs. Paraffin-embedding and immunochemistry successfully allowed the analysis of CD45 and CD73 contents using the 3D models. This 3D systems requires three weeks of bone marrow-like structures and additional time with cells of interest.
And sterile conditions must be maintained as sometimes, we can see medium contaminations with the twice-weekly medium changes.
