Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

In the field of cartilage tissue engineering and regenerative medicine, there is still a requirement to enhance biological and functional outcomes in terms of improving ongoing treatment and developing new therapeutic strategies. Current preclinical research points towards the potential use of cartilage-derived chondroprogenitors as a viable option for cartilage healing. Current research includes strategies to further enhance chondrogenic potential while maintaining lower hypertrophic potency through the use of additional growth factors and gene recombinant techniques.

Chondroprogenitors face experimental challenges, including a lack of consensus on nomenclature, diverse isolation techniques, characterization difficulties, variability in culture conditions, and limited understanding of underlying mechanisms. Clinically demonstrating that chondroprogenitors yield positive outcomes compared to commonly used cells could significantly reduce the global burden associated with cartilage-related pathologies. Evolution of improved therapeutics using cell-free alternatives, such as extracellular vesicles derived from progenitors, and using recombinant strategies for the regeneration of genuine hyaline-like cartilage.

To begin, obtain tibiofemoral or knee joints from human donors in 1X PBS under sterile conditions, then transfer the harvested joints onto the sterile underpad in the hood. To stabilize the joints, hold the subchondral bone with the cartilage facing upward. Using a number 22 scalpel blade, harvest rectangular-shaped cartilage shavings from the non-weight-bearing areas.

Cut the cartilage into sections measuring eight millimeters by 10 millimeters from the superficial layer to the deeper layer. After washing the slices with PBS, place them in a Petri dish containing one to two milliliters of plain Dulbecco modified Eagle's medium, or DMEM. Next, using a number 22 scalpel blade, mince the cartilage slices to a size smaller than one millimeter cubed.

To begin, obtain minced cartilage from extracted human knee joints. Place the minced cartilage into an upright T25 flask containing 10 milliliters of serum-free DMEM/F-12 with 0.15%collagenase type II for enzymatic digestion. Leave the flask undisturbed in a carbon dioxide incubator for 12 to 14 hours.

Following overnight digestion, transfer the medium containing the released chondrocytes into a fresh sterile centrifuge tube. Use a cell strainer to separate the cells from the debris. Add an equal volume of DMEM/F-12 with 10%fetal bovine serum, or FBS.

Centrifuge the filtered cells at 1, 200 G for five minutes at 37 degrees Celsius. After discarding the supernatant, reconstitute the pellet in one milliliter of medium. Count the viable cells using a trypan blue exclusion assay.

Next, load the chondrocytes into a T25 flask at a concentration of 10, 000 cells per square centimeter. Expand the cells to the required passage number using DMEM/F-12 containing 10%FBS. Refresh the medium every three days before harvesting the cells at subconfluence using 0.125%trypsin containing EDTA.

Chondrocytes adhered promptly after loading and transitioned from a rounded cobblestone shape to a fibroblastic appearance as they expand. To begin, obtain cartilage shavings from extracted human tibiofemoral or knee joints. Subject the cartilage shavings to sequential overnight enzymatic digestion, first using 0.2%pronase for three hours, followed by 0.04%collagenase type II for 12 hours in a shaking water bath maintained at 37 degrees Celsius.

Then, coat the required number of wells of a six-well plate with 1.5 milliliters of the PBS fibronectin solution. Seal the plate tightly and refrigerate overnight at four degrees Celsius. The following day, remove the fibronectin-coated six-well plate from the refrigerator.

After removing excess fibronectin, add two to three milliliters of plain DMEM/F-12 medium into the coated wells. Seed the released chondrocytes onto the coated wells at a density of 4, 000 cells per well and leave the plate undisturbed for 20 minutes. After incubation, aspirate the excess media and non-adherent cells.

Add two to three milliliters of DMEM/F-12 with 10%fetal bovine serum to the wells. Maintain the adherent cells under standard culture conditions for 10 to 12 days to obtain chondrogenic progenitor cell, or CPC, clones, then isolate the CPC clones using 0.125%trypsin EDTA for 180 seconds, then replay at a ratio of one clone per five square centimeters. Expand the enriched polyclonal CPCs to the required confluence.

Chondrocytes subjected to a fibronectin adhesion assay display clonal growth, reaching a population doubling of five by day 10. To begin, obtain the shaved cartilage explants from the harvested articular joint in supplemented DMEM/F-12 medium. Place the plate with explants inside a carbon dioxide incubator set at 37 degrees Celsius for 48 hours.

After two days, transfer the explants to a centrifuge tube containing 10 milliliters of 0.1%collagenase solution for enzymatic digestion. Incubate the tube at 37 degrees Celsius for two hours. Post-digestion, rinse the explants with 1X PBS and return them to the same wells in the plate containing fresh supplemented DMEM/F-12 medium.

Maintain the plate in the incubator under standard culture conditions and monitor for the migration of chondroprogenitors over the subsequent days. Once the explants reach subconfluence, harvest the mesenchymal chondroprogenitors cells, or MCPs, using 0.125%trypsin EDTA solution. Expand the MCPs using the supplemented DMEM/F-12 medium.

Chondroprogenitors start migrating from the edge of the explant by day 10 of culture, exhibiting a spindle-shaped growth pattern with further expansion.