Efficient Isolation of Adipose-derived Stem Cells and Adipocytes from Porcine Adipose Tissue

The purpose of this research is to isolate cells from adipose tissue efficiently and rapidly. Traditional digestion is time cost and may over digest. Our enzyme and mechanical wave measure offers gentle dispersion and reduces isolation time.

To begin, put a piece of back subcutaneous adipose tissue of a euthanized pig in a culture dish. Dissect along the natural plane between the adipose tissue and dermis to obtain a complete two to three millimeters thick ULB sheet. Rinse the ULB once in ice cold sterile saline.

After tearing a sterile culture dish, weigh the tissue in the sterile dish on a pre sterilized balance. For every 1.8 grams of tissue, add 10 milliliters of ice cold D-Hank's buffer, and keep the tissue fully submerged. Use sterile surgical scissors to identify and excise all visible blood vessels from the tissue.

Remove any connective tissue and fibrotic components. Discard the excised materials into designated biohazard containers. Rinse the tissue within D-Hank's buffer to remove residual blood and debris.

Weigh the tissue in the sterile culture dish on a pre sterilized balance after tearing the dish. Transfer 1.8 grams of the rinse tissue fragments to a sterile 1.5 milliliter micro centrifuge tube. Add 800 microliters of A type tissue dissociation buffer to the tube.

Use sterile ophthalmic scissors to mince the tissue in the tube into pieces approximately one to two cubic millimeters in size. Transfer the mince tissue along with the surrounding buffer to a new micro centrifuge tube. Add 200 microliters of A type tissue dissociation agent to the tube.

Gently rotate and shake the tube by hand three times to mix the contents. Ensure that the adipose fragments are fully immersed in the dissociation solution. Check if the tube cap is tightly closed.

Place the tube in a 37 degrees Celsius metal bath and incubate for five to 10 minutes. Transfer the partially digested adipose tissue in buffer into a sterile 1.5 milliliter micro centrifuge tube designated for the mechanical dissociation system. Run the preset program for adipose tissue to perform mechanical dissociation using a 200 hertz sinusoidal wave.

Collect the cell suspension and pass it through a 200 micrometer cell strainer into a new 50 milliliter centrifuge tube. Add five milliliters of pre-cool D-Hank solution at four degrees Celsius to the strainer. Rinse the strainer twice with the D-Hank solution.

Centrifuge the filtered cell suspension at 500 G for five minutes at four degrees Celsius. Use a sterile pasteur pipette to aspirate and discard the supernatant lipid layer while leaving one milliliter of buffer to protect the adipocyte layer. Collect the second layer containing the adipocytes without disturbing the third and fourth layers.

Aspirate and discard the third layer. Add one milliliter of D.Hank's buffer to the bottom pellet containing the stromal vascular fraction or SVF. Resuspend the cell pellet gently in the buffer to achieve a cell concentration of two to five times 10 to the power of six cells per milliliter.

Prepare the SVF cell suspension in a 1.5 milliliter micro centrifuge tube. Keep the suspension on ice until use. Add 10 microliters of the SVF cell suspension and 10 microliters of 0.4%trypan blue solution in a sterile 0.5 milliliter micro centrifuge tube.

Gently pipette up and down three times. Transfer 10 microliters of the stained cell mixture to a counting slide, appropriate for the automated cell counter. Verify that the sample fills the chamber completely without overflowing.

Insert the counting slide into the automated cell counter. Select the trypan blue assay type and adjust the cell concentration range if necessary. Press count to initiate automated cell counting.

Monitor the counting chamber images and observe the automatic distinction of live and dead cells. Record the cell viability percentage and the total number of viable cells displayed on the instrument screen. Perform three independent measurements for each sample.

Calculate the mean viability and the cell yield per gram of adipose tissue. Prepare a staining solution with five micrograms per milliliter huksed and one micromolar BODIPY in D.Hank's. Incubate the isolated adipocytes in the staining solution for 15 minutes at 37 degrees Celsius.

Use a self-made adipocyte friendly slide cover glass system for slide preparations suitable for fluorescence microscopy. The homemade adipocyte friendly mounting system enabled uniform distribution of intact adipocytes for clear imaging. Unlike traditional methods that produced crowded, multi-layered arrangements, the extracted adipocytes showed intact structure and normal morphology.

The survival rate of adipocytes obtained by the mechanical dissociation method was significantly higher compared to the traditional enzymatic hydrolysis method. The purified and cultured adipose-derived stem cells exhibited a uniform fibroblast like morphology with elongated spindle shaped cells arranged in an orderly manner. Most cells remained unstained, indicating viability, whereas only a small proportion stained blue, representing dead cells.

This protocol allows researchers to compare the isolated cell characters across adipose depot. The isolated SVFs from the protocol can be used for organoid construction and adipose stem cell purification. Future study could further optimize the isolation method, considering the adipose tissue variations in extracellular matrix across depot.