Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint disease demand advanced cell-based therapies to regenerate damaged tissues efficiently. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. Cartilage repair in chronic joint disease demands advanced cell-based therapies to regenerate damaged tissue sufficiently.

This protocol provides a step-by-step method for differentiating induced pluripotent stem cells into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The main challenges are minimizing type one collagen expression in iPSC-derived cells to prevent fibrous cartilage, creating conditions for mature highline cartilage formation, improving 3D culture methods for spheroid stability, and developing scalable approaches for producing clinical volumes of cellular material. The protocol provides an advantage by using iPSCs as an alternative source of chondrocytes, allowing for scalable production without invasive procedures.

It ensures better maintenance of the chondrocyte phenotype through 3D culturing, and mimics natural cartilage development, resulting in cells closely resembling native chondrocytes with high expression of cartilage-specific markers. Develop effective methods to enhance iPSC-derived chondrocyte maturity and functionality, ensure long-term stability in vivo, create fibrosed three-dimensional culture materials, and develop scalable production of cellular material for clinical application. To begin, sterilize the scissors in an autoclave set to 121 degrees Celsius at 15 pounds per square inch for 30 minutes.

After the scissors dry, slice centrifuge tubes into rings that are seven to eight millimeters in height. Now crush low adhesion, untreated, or microbiological Petri dishes into small pieces using an appropriate crushing tool. Dissolve approximately one gram of plastic particles in 10 milliliters of chloroform overnight inside a fume hood.

Verify the viscosity of the liquid plastic solution to ensure it is suitable for pipetting. If the solution is too thin, add one to three grams of additional plastic particles. If it becomes too thick, dilute it with around five milliliters of chloroform.

Place an autoclave plastic ring in the center of a 60-millimeter Petri dish. Then apply liquid plastic inside the ring to form a plastic knob. Let the dishes dry uncovered in a laminar flow hood for two to three hours.

After drying, expose the dishes to ultraviolet radiation for 20 to 30 minutes. To begin, obtain the modified Petri dishes containing plastic knobs. Two days after cartilage harvest, wash cartilage pieces in Hank's solution using 60-millimeter Petri dishes.

Then cut the cartilage into fragments approximately one to two millimeters in diameter using autoclave scissors. Transfer cartilage pieces into a 15-milliliter tube and digest them in a 0.01%collagenase type two solution in DMEM for eight to 12 hours in an orbital shaker incubator. After digestion, add the cartilage fragments with DMEM and centrifuge the tube at 300 G for 10 minutes.

After discarding the wash solution, resuspend the fragments in a chondrocyte culture medium and seed them onto an adhesive T25 flask pre-coated with 0.1%gelatin solution and incubate. Next, cultivate induced pluripotent stem cells in six-well plates pre-coated with a matrix containing extracellular proteins. To initiate differentiation towards the chondrocytic lineage, culture iPSCs in medium A for the first two days.

On day three, replace medium A with a solution that excludes CHIR-99021 and rho-kinase inhibitor while keeping all other components unchanged. On day 10, transfer chondrocyte-like cells to medium B formulated to facilitate chondrogenesis, then melt 1.5%argarose in a microwave for about 60 to 90 seconds at 700 watts. Once liquified, add 75 microliters of the agarose to each well of a 96-well cell suspension culture plate and allow it to harden at room temperature.

Next, add 150 microliters of DMEM to each well and incubate the plate in a carbon dioxide incubator for at least 12 hours. On day 12, observe the phenotypical changes associated with androgenesis to proceed for spheroid initiation. Detach cells at 80%confluency from the plate using a 0.05%trypsin solution.

After adding equal volumes of DMEM with 10%FBS, transfer the cells to a 15-milliliter tube and centrifuge at 200 G for five minutes. Resuspend the cells in one milliliter of medium B and transfer them to a 75 square centimeter cell culture flask pre-coated with 0.1%gelatin solution. Once the cells reach 80%confluency as a monolayer, detach them again with trypsin and resususpend them in medium B.Now dispense the cells into a 96-well plate coated with 1.5%agarose at a density of 100, 000 cells per well, along with 150 microliters of medium B.Incubate the cells for a minimum of one day and a maximum of three days until spheroids are formed.

Then transfer the spheroids from the wells using a one-milliliter pipette tip with a trimmed end to a 15 milliliter tube. Let the spheroids settle for two to three minutes and remove the supernatant. Now immerse the spheroids in freshly thawed, undiluted basement membrane matrix solution tempered at four degrees Celsius.

After 30 minutes, centrifuge the tube at 100 G for one minute to collect the spheroids. Finally, transfer the spheroids into the prepared mini bioreactors and add six milliliters of medium B.Place the mini bioreactor inside a carbon dioxide incubator. After 19 days of differentiation, 99%of cells expressed chondrogenic markers aggrecan, collagen type one, collagen type two, and SOX9.

High quality chondrospheres were identified as those with at least 90 to 95%expression of chondrocytic gene markers, assessed through gene expression analysis on day 30 of differentiation. Mature spheroids exhibited consistent morphogenesis, growing to a typical diameter of one to two millimeters after two months, with larger spheroids showing central zones with cavities or necrosis.