Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

The overall goal of this procedure is to isolate adipose-derived stem cells from porcine subcutaneous white adipose tissues for evaluation of their ability to differentiate into mesenchymal adipocytes, osteocytes, and chondrocytes. This method can help answer the key questions of obesity research. It is a good tool to uncover the molecular mechanism involved in regulating adipose tissue development.

The main advantage of this technique is that an abundant number of stromovascular cells can be harvested allowing in depth analysis of adipocyte differentiation and biology. The implication of this technique stands toward stem cell therapy research as pig adipose-derived stem cells exhibit multipotency for transdifferentiation into multiple cell lineages. Begin by laying the piglet in the prone position on a clean surgical table.

Shave all of the hair down the midline from the neck to the tail, and scrub the dorsal skin with 7.5%Povidone iodine three times. After the third scrub, allow the iodine to sit on the surface of the skin for about 10 minutes. Then, spray the skin with 70%ethanol.

And use ethanol-soaked gauze pads to wipe the skin in one direction to remove the disinfectant until no obvious color is observed. Next, make an incision from the neck region down to the leg. Then, holding up the fat and skin with forceps, use a scalpel to separate the porcine dorsal fat layer of subcutaneous adipose tissues and the adjoining skin layer from the muscles.

Immediately immerse the skin and fat tissue in a sterilized 200-milliliter beaker, containing serum-free, 37-degrees Celsius DMEM and spray the outside of the beaker with 70%ethanol. Then, place the beaker in a laminar flow cell culture hood and place a 40 by 30-centimeter sterilized, triple-layer foil cover next to the beaker. Transfer the tissue, skin facing down, onto the foil.

And use forceps and scissors to trim the remaining muscle tissue off of the adipose tissue. Cut the fat into square 7 by 7-centimeter pieces and then transfer the fat pieces into a new sterilized 200-milliliter beaker containing serum-free, 37-degrees Celsius DMEM. Now, place a customized slice holder, equipped with a carbon-steel slicer blade onto the cover foil and place one of the fat pieces onto the slicer with the fat layer facing down.

Slice the fat into approximately 1-millimeter thick pieces as close as possible to, but without cutting the skin. Then, use scissors to mince the adipose tissue pieces as finely as possible, and add the pieces to a 250-milliliter Erlenmeyer flask containing filtered digestion medium supplemented with collagenase. Inclubate the flask with swirling for 90 minutes at 45 RPM, in an orbital shaker at 37 degrees Celsius.

When the digestion medium becomes a slurry, without significant tissue clumps, stop the digestion with an equal volume of culture medium containing DMEM F-12 with 10%FBS. To collect the pig adipose-derived stem cells, filter the digested adipose tissue suspension through a single layer of chiffon into a sterilized 250-milliliter serological bottle, using forceps to depress the middle of the chiffon, to guide and assist the passage of the digest. Drain and twist the chiffon with the forceps to complete the passage and distribute the medium into four 50-milliliter conical centrifuge tubes.

Collect the stromal-vascular cells by centrifugation. Then, decamp the supernatant without disturbing the pellets to remove most of the top fat layer containing the mature adipocytes. Next, re-suspend the pellets in 10 milliliters of DMEM with pipetting and gentle shaking.

Spin down the cells again and re-suspend the pellets in 10 milliliters of ACK lysis buffer. After seven minutes at room temperature, stop the red blood cell lyisis with the addition of 10 milliliters of DMEM to each tube, gently shaking the tubes to mix. And then, spin down the cells again.

Wash the cells two more times with 10 milliliters of DMEM and then pool the supernatants through a 100-micron strainer into a single 50-milliliter conical tube. Gently pipette the cell suspension several times to evenly distribute the cells and count the cells by Trypan Blue exclusion. Finally, seed the pig adipose-derived stem cells at a 6 times 10 to the 4th per centimeter-squared density into the appropriately-sized culture container in cell culture medium.

And incubate the cells at 37 degrees Celsius and 5%CO2, to facilitate the formation of a monolayer. The morphology of the stromal-vascular fraction-derived pig adipose-derived stem cells is similar to mouse or human adipose-derived stem cells with an expanded fiberglass-like morphology observed 24 hours after seeding. The pig adipose-derived stem cells become confluent within 72 hours, at which point they are ready for adipocyte or other mesenchymal-type differentiation.

These cells also exhibit a strong adipogenic potential after chemical induction and mature adipocytes can be observed after nine days of differentiation, with over 90%of the pig adipose-derived stem cells demonstrating adipogenic differentiation. Mesenchymal stem cells surface markers including CD 29, CD 44, CD 90, and MHC 1, are highly expressed on the pig adipose-derived stem cells, while CD 4a, CD 31, CD 45, and MHC II are barely detectable, demonstrating that these pig adipose-derived stem cells exhibit mesenchymal-type characteristics without significant endothelial or hematopoietic stem cell contamination. Further, the differentiated pig adipose-derived stem cells can be identified as adipocytes, osteocytes, or chondrocytes, through the use of specific tissue-staining dyes, demonstrating that these stromal-vascular fraction-derived cells retain a multipotency typical of mesenchymal-type progenitors.

While performing this procedure, it is important to remember to have a good aseptic technique for cell culture and to perform all the steps as quickly as possible. To maximize the viability of the cells. Once mastered, the cells can be harvested from two to three pigs in six hours, if the technique is performed properly.

This standard technique paved the way for obesity researchers in the field of diabetes to explore the molecular switches, such as transcription factors that control adipocyte differentiation. After watching this video, you should have a good understanding of how to isolate pig adipose-derived stem cells and to evaluate the multi-ability of these cells to differentiate into adipocytes, osteocytes, and chondrocytes.