The overall goal of this procedure is to obtain mature ramified osteocytes from primary osteoblasts. This is achieved first by isolating primary osteoblasts from bones of newborn mice and culturing them to allow osteoblast maturation. Next confluent osteoblasts are tryps.
Inized then counted and replated at a defined density to ensure success. Then all trans retinoic acid or ATRA is added to the cell medium to reduce cell proliferation and induce complete differentiation towards osteocyte morphology Results are obtained that show that in the presence of ATRA cells rapidly acquire a ramified morphology, stop producing extracellular matrix and differentiate towards a mature osteocyte phenotype as shown by expression of osteocyte markers. The main advantage of this technique over existing methods, such as the isolation of primary osteocytes or the differentiation of osteoblasts in a three dimensional matrix is that it produces sufficient numbers of osteocytes in a few days.
The absence of surrounding metrics facilitates the use of these cells in N cell biology and molecular biology assays. Furthermore, the method can be applied to transgenic mice, allowing functional studies on molecules of interest. Demonstrating the procedure will be Deborah, a PhD from my lab.
To begin prepare digesting medium by adding 0.1%collagenase P and 0.05%tryin to hank's balanced salt solution or HBSS. After sacrificing and decapitating three to four day old mice, use 70%ethanol to spray the head and place it on ice. Then with tweezers and dissecting scissors, remove the skin and muscle layers.
Gently remove the parietal bones and separate them two halves by following the sagittal suture. After removing any sutures and adherent tissue, place the individual bone pieces in HBSS medium. Next, discard the HBSS and add the digesting medium to each.
Well then incubate at 37 degrees Celsius for 15 minutes. After discarding the supernatant, add fresh digesting medium and repeat the incubation. After the second incubation, collect the supernatant and combine it with alpha mam supplemented with FBS and pen strep.
Repeat the digestion two more times. Combining the supernatant with the medium, then centrifuge the pooled fractions at 425 Gs for five minutes, discard the supernatant and resuspend the pellet in supplemented alpha mam, before transferring the cell suspension to a 25 square centimeter flask. After incubating at 37 degrees Celsius for 24 hours, remove the debris and dead cells.
Then add alpha mem, followed by 50 micrograms per milliliter as ORIC acid or aa, and three millimolar glycerol, two phosphate di, sodium salt, hydrate, or GP to the culture before returning to the incubator. After removing the medium from sub confluence cells and using HBSS to wash them, add freshly prepared trypsin and incubate for three minutes. At 37 degrees Celsius, when the cells have detached, add three milliliters of Alpha M medium to inactivate the trypsin.
Recover the cells and centrifuge at 425 Gs for 15 minutes before resus, suspending the cell pellet in fresh medium, place a drop of the suspension in a hemo cytometer. Allow it to settle for a few minutes and count the cells using alpha men plus A A GP.Transfer the cells to 25 square centimeter flasks at a density of 10, 000 cells per square centimeter. A higher cell density will impair proper development of cell processes, and lower cell numbers can result in cell death due to the absence of intercellular contacts, while incubating add ATRA to the medium at a concentration of five micromolar every 24 hours.
Morphological changes are generally observed after 48 hours. A homogeneous population of mature osteocytes should be present after four to five days. The cells can be used for up to 12 to 15 days as shown here.
A A GP treated primary cells have mostly cobblestone like features. Characteristic of mature osteoblasts interspersed. Ramified cells indicated by the red arrows likely represent some osteocytes.
After two days of ATRA treatment, cells rapidly start displaying ramifications as seen in this figure. Ramified cells require space to extend their dendrites as the treatment with ARA proceeds ramifications progressively increase in number and complexity. Staining a filamentous actin by rho domine labeled foid in highlights cell processes.
Primary osteoblasts produce large amounts of calcified matrix, which accumulates over and among cells and can be visualized by alizarin red staining. In contrast, osteocytes do not produce calcified extracellular matrix and alazar and red staining is completely negative in ATRA incubated cells. Accordingly, none or minimal alazar and red can be measured after the extraction procedure.
As shown here. Immunos staining for sclerostin reveals intense cytoplasmic expression and the presence of positive dots along cell processes. In addition, FGF 23 is highly detectable in ATRA treated cells in both the cell body and along cell processes.
While lati this, this procedure, it's important to remember that you have dealing with primary cells, therefore adjustment of aro concentration and replacing of cell. If proliferation remains elevated after a tradition have to be taken in consideration, especially when dealing with transgenic animals. Remember that cell morphology is critical in guiding all step of this method.
Check the cell every day under inverted microscope.
