Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

Infrapatellar Fat Pad-derived Mesenchymal Stem Cells, or in short, IFP-MSCs, is a relatively less studied stem cell type. This protocol is a simple, reliable, and reproducible method demonstrating the isolation of IFP-MSCs from goat stifle joint. The main advantage of the technique is that a large number of high-quality IFP-MSCs can be obtained.

Isolated IFP-MSCs can differentiate into multiple lineages and possess extensive regenerative potential. Due to their anatomical location, IFP-MSCs have gained interest in repairing cartilage defects and alleviating cartilage degradation in osteoarthritis. The method has implications for cell-based therapy in the field of tissue engineering and regenerative medicine.

The procedure for isolation, expansion, and differentiation of IFP-MSCs will be demonstrated by Dr.Sugata Hazra and Mr.Aman Mahajan. To begin the isolation of Infrapatellar Fat Pad Mesenchymal Stem Cells, or IFP-MSCs, collect goat femorotibial joints encompassing around 15 centimeters of the femoral and tibial regions of the hind limbs in a sterilized collection sample box. Store the sample at four degrees Celsius for transportation to the laboratory for further processing.

Place the sample on a 150-millimeter Petri dish and ensure to process the sample aseptically and a bio-safety cabinet throughout the procedure. Rinse it thoroughly with autoclaved PBS supplemented with antibiotics. Keep the sample hydrated using PBS.

Carefully examine the anatomical regions of the sample. For easy access, remove the extra tissues from both the long bones completely by holding the end surface of the femur bone with one hand and cutting it longitudinally towards the joint using sharp scissors. Then, without disturbing the tissue surrounding the joint capsule, remove the muscles and adipose tissue from the bone.

Similarly, make another incision at the tibial end of the sample and remove the muscles and adipose tissue from the tibial bone. Ensure that both the long bones are completely exposed and that the joint capsule is visible. Next, make an incision from either side of the synovial membrane to cut open the articulating joint.

Cut free the patella from the synovial membrane and patellar ligaments using fresh sterilized scissors and forceps. Immediately place the separated patella in a Petri dish containing PBS. From the separated patella, remove the entire fat pad present on the inner surface using scissors and forceps.

Collect it in another fresh Petri dish, and mince it using a scalpel. Include other surgical tools to obtain the small fat segments of around two to three millimeters. Use a spatula to transfer the minced tissue to a 50-milliliter centrifuge tube and wash it thrice with PBS supplemented with antibiotics for 15 minutes at room temperature using a test tube mixer.

For enzymatic digestion, incubate around five grams of the minced tissue in Dulbecco's Modified Eagle Media, or DMEM, low-glucose media containing 1.5 milligrams per milliliter of Type II collagenase and 2%FBS at 37 degrees Celsius for 12 to 16 hours. Carefully aspirate the digested tissue and filter it through a 70-micron cell strainer to remove any undigested part. Centrifuge the filtrate in a 15-milliliter centrifuge tube at 150 times g for five minutes.

Remove the supernatant before washing the cell pellet with DMEM low-glucose twice. Re-suspend the pellet in complete DMEM, also known as expansion media. Seed the cells on a 150-millimeter Petri dish and culture them in the expansion media supplemented with five nanograms per milliliter of Basic Fibroblast Growth Factor, or BFGF, and 50 micrograms per milliliter 2-Phospho-L-ascorbic acid trisodium salt.

Incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator to allow the cells to adhere and proliferate. Monitor the attachment and growth of the cells daily. For proper maintenance and expansion of the IFP-MSCs, change the media every three days.

While changing the media, add fresh five nanograms per milliliter BFGF and 50 micrograms per millimeter of 2-Phospho-L-ascorbic acid trisodium salt directly to the Petri dish. Once the cells become 80%to 90%confluent, subculture them by removing the expansion media from the Petri dish and washing the cells with 1X PBS. Then add four milliliters of 0.25%Trypsin-EDTA to the dish and incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator.

After four minutes, tap the plate on its side to dislodge the cells. Confirm the complete detachment of all the cells under a microscope. Once confirmed, add an equal volume of expansion media to neutralize the trypsin.

Use a pipette to collect these dissociated cells in a fresh 15-milliliter polypropylene tube and centrifuge them at 150 times g for five minutes at room temperature. Discard the supernatant and resuspend the pellet in the desired volume of expansion media. Count the cells using the standard cell counting method and subculture them for further expansion at a seeding density of 10, 000 cells per square centimeter in a 150-milliliter Petri dish.

For all the assays, use a confluent monolayer of cells of passage number P2 to P5.When the expanded cells reach 80%to 90%confluency, dissociate the cells using Trypsin-EDTA solution before pelleting them down in a 15-milliliter centrifuge tube at 150 times g for five minutes. Then resuspend the pellet in goat plasma at a density of 2 million cells per 50 microliters. Add 50 microliters of plasma containing the cells onto a sterilized 90-millimeter Petri dish, followed by the addition of calcium chloride at a final concentration of 0.3%Mix well to fabricate cross-linked plasma hydrogel.

After incubating the fabricated hydrogel at 37 degrees Celsius for 40 minutes, transfer the hydrogels to 24-well tissue culture plates containing chondrogenic media. Change the media every three days for 14 days. The hydrogels cultured in complete DMEM media without TGF-beta 1 are considered un-induced controls.

After 14 days of chondrogenic differentiation of cells in plasma hydrogel, wash the hydrogels thrice with PBS for five minutes each, and fix the samples in neutral buffered formalin for three hours. Isolated IFP-MSCs were homogenically adherent and attained elongated morphology within 24 hours of in vitro culture. The cells efficiently proliferated 80%to 90%confluency within six days of expansion and displayed clonogenic capacity, indicating efficient proliferative and self-renewal capabilities.

When treated to differentiate into adipogenic lineage, the isolated cells produced lipid droplets, which was confirmed by Oil Red O staining on days 14 and 21. Such oil droplets were not visible in the cells without adipogenic inducing factors. The isolated cells encapsulated in plasma hydrogel showed the formation of white and glossy neo-tissue after 14 days in the induced group displayed the chondrogenic potential.

The un-induced group had pale white tissue with reduced glossiness. Additionally, positive histological staining for Alcian Blue and Safranin O in the induced group indicated that the cells could secrete sulfated glycosaminoglycans, one of the major components of the cartilage matrix. In contrast, the hydrogel sections from the un-induced groups were negative for Safranin O and Alcian Blue staining, confirming the chondrogenic differentiation potential of the mesenchymal stem cells only in the presence of chondrogenic stimuli.

The isolated cells induced with osteogenic media showed positive staining for alkaline phosphatase, confirming mineralization at the end of 14 days. After 28 days of osteogenic induction, the calcified depositions were observed with Alizarin Red S staining. The results underlined the differentiation potential of induced cells into adipogenic, chondrogenic, and osteogenic lineages.

It is essential to process the sample under aseptic conditions to avoid contamination. It is also important to identify the correct location of the patella. This procedure opens the avenue to isolate various cell types, such as articular chondrocytes, ligament fibroblasts, and stem cells from synovium and synovial fluid, having wide applications in regenerative medicine.

After isolation of IFP-MSCs using this technique, its effectiveness was particularly investigated for regeneration of musculoskeletal tissues, such as cartilage, bone, and ligament using scaffold and scaffold-free approaches.