The overall goal of this procedure is to set up a culture system that closely recapitulates the cellular and intracellular microenvironment of human bone marrow. First, the surface of a tissue culture well is coated with the industrial matrix. Then bone marrow mononuclear cells, or other cells of interest are mixed with the bone marrow matrix.
Later, the industrial matrix is removed and the cell matrix mixture is added to the well. Once the matrix has jelled, it is overlaid with growth medium. Ultimately, cells can be visualized directly in the matrix or can be isolated for downstream analyses Compared to existing methods for culturing cells.
For example, standard tissue culture methods or the co-culture models that where cells are grown on plastic. The main benefit of using a 3D culture method is that cells are grown within the physiological micro environment and hence it allows you to study cells within the native conditions of the tissue. Prepare a 48 well plate in which the cells will be plated.
By first adding 65 microliters of industrial coating solution into each, well spread the solution evenly to cover the entire surface of the well. This method is scalable to other surfaces as well. Allow the plates to incubate at room temperature for at least 30 minutes.
In the meantime, prepare the cell suspension of 50, 000 cells per microliter of PBS. Prepare enough to add 10 microliters to each well then in an einor tube, mix the cell suspension with 100 microliters of reconstructed bone matrix per. Well mix gently and avoid introducing bubbles.
Keep the mixture on ice so the matrix does not gel when the plate has finished. Incubating, remove all the liquid and dote coating solution via aspiration. Then add 100 microliters of well mixed cell suspension with matrix to each.
Well quickly tilt the plate using a pipette to cover the entire surface of the well evenly. Keep mixing the suspension between well fills as cells will clump up once fully loaded. Incubate the plate for 30 to 60 minutes without agitation until the matrix solidifies.
Next, prewarm the bone marrow growth medium in a water bath set to 37 degrees Celsius. After 30 minutes of incubation, check to see that the matrix has set properly. A good matrix forms a soft gel-like layer that does not move when the plate is tilted.
When the matrix is ready, gently overlay it with a milliliter of medium dropwise using a serological pipette. Then return the assembled reconstructed bone matrix culture to the incubator for up to 30 days with no medium changes. If a medium change is required, only change half of the medium at once.
Remove the medium gently without disturbing the matrix layer. Tip the plate and drain the medium gently Using a pipette. Do not aspirate the media over the matrix later.
When staining the cells. Use this technique for solution changes. For removing media from the wells of a 3D culture, it is very crucial that it is removed from the side of the well.
Otherwise, the matrix will be disturbed and the cells might be lost Sticking to the same technique. Wash the cells twice with sterile one XPBS. Then add a half milliliter of ice cold cell recovery solution to each.
Well remove the entire matrix layer along with the cells by vigorously pipetting up and down. For each, well transfer the collection to a micro centrifuge tube and place it on ice. Then add an additional half milliliter of recovery solution to the same well and collect all the remaining material.
Vortex all the collections, and incubate them for an hour on ice. During the hour, vortex the tubes every 15 minutes to facilitate cell release from the matrix. After an hour, check the mixture for visible clumps of matrix.
None should be present, but if there are, transfer the mixture to a larger tube and add an additional two to five milliliters of cell recovery solution. Then incubate the cells for another 30 to 60 minutes. Vortexing periodically, and check again for an absence of matrix clumps.
When the matrix is no longer clumped. Centrifuge the mix at 1000 RPM for five to 10 minutes at four degrees celsius. Then remove the supernatant and resuspend the cell pellet in a milliliter of cold one XPBS.
Repeat the centrifugation. Replace the supernatant with fresh PBS and repeat the wash. A third time.
After the third PBS wash, recover the cells in the appropriate solution for the next procedure. Bone marrow cells were grown in reconstructed bone matrix for 30 days and imaged periodically by light microscopy at 200 times magnification. Stromal elements were first detected on day five, but they were clearly evident by day 14.
Over the course of the culture period, cells migrated through the matrix and aggregated into distinct clusters. Masses of malignant cells expanded over time to evaluate the composition of the stromal components that outgrow in RBM. The morphology of cells at the transition between RBM and the industrial coating was evaluated.
The cells were stained for fibronectin, actin, and nuclei. Visualization cells with stromal morphology were shown to be pres osteoblasts with high levels of alkaline phosphatase activity. They were capable of mineralizing calcium as determined by zarin red staining cells with a visible presence of lipid droplets were identified as adipocytes based on positive staining with oil.
Red cells with occasional multiple nuclei were recognized as pres osteoclasts and were positive for tartrate resistant acid phosphatase. Following isolation of cells from A 3D culture, you can do a variety of things. For example, you can study the cellular phenotypes of various cells grown in the microenvironment.
You can study the gene and protein expression and you can even determine what kind of molecules are secreted by these cells under 3D conditions.
