The overall goal of this novel cell culture method is to generate dedifferentiated fat cells with greater efficiency. Our method may help researchers working in the field of regenerative medicine answer key questions about the several processes involved in producing adipogenic, osteogenic, and chondrogenic cells. Since the primary advantage of our cell culture protocol is that it enhances the quality of DEFAT cells, it may be applied in the near future to improve current applications in cell therapy and tissue engineering.

After bringing the patient's subcutaneous fat sample to the lab from the operating room, weigh the tissue and place a one to two gram sample of adipose tissue into a 15 milliliter tube. Then wash the sample with five milliliters of PBS and then transfer the sample to a 10 centimeter plastic dish. Remove the PBS.

Then add 10 milliliters of sterile 0.1%weight per volume collagenase to the dish containing the sample. Next, use surgical scissors to mince the tissue until it reaches a pureed consistency. Transfer the minced tissue and collagenase solution to a 15 milliliter centrifuge tube and digest at 37 degrees Celsius for 30 minutes, with shaking at 60 RPM.

Following the digestion, filter the digested sample through a 100 micron filter into a fresh 15 milliliter tube. Then, pass 10 milliliters of DEFAT culture medium supplemented with 2%FBS through the filter. To isolate the fat cells from the cell suspension, first centrifuge the filtered cells at 100 times g for one minute at room temperature.

During the centrifugation, prepare five milliliters of DCM supplemented with 2%FBS in a 15 milliliter tube. Next, use a 1000 microliter pipette to draw up the floating fat cells from the centrifuged tube and add the cells to a 15 milliliter tube containing medium. Shake the tube gently to mix the DCM medium with the cells.

Then, centrifuge the tube at 100 times g for one minute at room temperature. After centrifuging, use an 18 gauge needle and a 20 milliliter syringe to remove the medium at the bottom of the tube. Then add 10 milliliters of DCM supplemented with 2%FBS to the cells and mix by shaking the tube gently.

Centrifuge the cells again at 100 times g for one minute at room temperature. To begin plating, add 41 milliliters of DCM or DMEM supplemented with 20%FBS to a 12.5 centimeter flask. Then use a 200 microliter pipette to draw up 40 microliters of fat cells and add them to the flask.

Then fill the flask with DCM or DMEM supplemented with 20%FBS. It is critical that the cells are spread throughout the medium. Place the flask on the lid of a round Petri dish to keep it flat.

Finally, place the flask inside an incubator set to 5%carbon dioxide and 37 degrees Celsius and leave it undisturbed for seven days. After a week of incubation, invert the flask and check for dedifferentiated fat cells. Next, aspirate the media and then add five milliliters of DCM or DMEM supplemented with 20%FBS to the flask.

Allow the DEFAT cells to grow for another week after changing the medium. After a week, use a cell counter to count the number of DEFAT cells generated under various cell culture conditions. This image shows DEFAT cell colonies following 14 days of culture in DMEM.

Here the DEFAT cells are shown after 14 days of culture in DCM. In each case, the scale bar represents 200 microns. DEFAT cell proliferation was quantified using a cell counter, and the efficiency of DCM and DMEM in generating DEFAT cells, was compared.

As seen here, enhanced cell proliferation is observed with the use of DCM. Here, human DEFAT cells, generated using DCM, were cultured for three weeks in the appropriate induction media to evaluate the redifferentiation and differentiation abilities of DEFAT cells. The cells were positive for osteogenic capacity by alkaline phosphatase stain and by staining with Alizarin Red S.Finally, adipogenic capacity was shown with an Oil Red O stain.

It's quite very important to ensure that collagenase with rudiment and fat cell isolation are both performed with the utmost of care in order to successfully generate high quality DEFAT cells. Through these efforts, our validated cell culture method aims to facilitate both current and future research efforts in the fields of developmental biology and regenerative medicine.