Mesenchymal Stem Cell Differentiation into Three Lineages: Optimised Explant Isolation, Morphology Assessment and Osteogenic, Adipogenic and Chondrogenic Induction Protocols

Mesenchymal Stem Cell Three-Lineage Differentiation: Practical Protocols

1. Explant Method for MSC Isolation from Umbilical Cord

Mesenchymal stem cells are obtained from several sources, with umbilical cord being the most widely used. The tissue explant method is the most common isolation approach for umbilical cord-derived MSC.

Step 1. Under aseptic conditions, take fresh umbilical cord and wash away blood with DPBS. Note that umbilical cords are generally obtained in a non-sterile environment, so a brief rinse with 75% alcohol is acceptable.

Step 2. Place the cord in a 10 cm culture dish, cut it into 1-2 cm segments, remove the vessels, wash away residual blood with DPBS, then cut into 1 mm3 tissue fragments. Fig. 1 illustrates removal of the three arteries and vein from the cord.

Step 3. Using a sterile pipette, distribute small tissue fragments evenly over two wells of a 6-well plate.

Step 4. Incubate at 37 degrees C with 5% CO2 for 30 minutes to allow the fragments to attach to the well surface.

Step 5. Add 0.8 ml complete medium to each well, dispensing carefully along the well wall without disturbing the tissue fragments.

Step 6. Incubate overnight at 37 degrees C with 5% CO2 and add a further 1 ml complete medium to each well on day 1.

Step 7. Continue culture at 37 degrees C with 5% CO2 and perform a half-medium change on day 6. Cells are usually not yet visible at this point, although outgrowth from the fragment edge may be seen as early as day 3. Fig. 2 shows cells crawling out from the fragment edge.

Step 8. Change half the medium again after a further 5 days. Cells are normally visible at this stage, though outgrowth may take up to 14 days. Fig. 3 shows rapid proliferation of the outgrowing cells and Fig. 4 the appropriate passage timing.

Step 9. Change medium every 2 days and continue for 2-4 days, then passage once confluence reaches approximately 80%.

Note: the key to explant culture is to keep the fragments permanently attached; medium changes should be as gentle as possible to avoid dislodging them, and excessive medium volume should be avoided so that fragments do not float.

2. Distinguishing Healthy from Unhealthy Mesenchymal Stem Cells

2.1 Characteristics of healthy MSC

Fibroblast-like morphology; polarised arrangement with a swirling pattern; even distribution without clumping (see accompanying micrographs).

2.2 Characteristics of unhealthy MSC

Uneven distribution; clumping; grid-like patterning; poor attachment (see accompanying micrographs).

Poor MSC morphology most often reflects an unsuitable culture system, such as batch-to-batch serum variation, or inappropriate digestion conditions, since these cells require recombinant trypsin. If a grid-like clumping pattern appears, these two aspects should be examined first.

3. MSC Three-Lineage Differentiation: Experimental Protocols

3.1 Osteogenic differentiation

Preparation. Add an appropriate volume of coating solution C to the culture vessel and gently shake to cover the entire bottom surface. In a laminar flow hood, leave the vessel undisturbed for 30-60 minutes, discard the solution and air-dry at room temperature. Six-well plates are recommended with 1 ml of coating solution C per well; results obtained with other vessels should be validated for stability. The whole procedure is carried out in a laminar flow hood to avoid contamination.

Procedure.

1. Prepare MSC for induction. When confluence reaches approximately 85%, digest the cells and resuspend in MSC complete medium.

2. Seed at 2 x 10^4 cells/cm2 in coated 6-well plates with 2 ml MSC complete medium per well and incubate at 37 degrees C with 5% CO2.

3. At 60% confluence, discard the spent medium and add 2 ml MSC osteogenic induction complete medium per well. Note that cells readily detach after osteogenic induction, so the medium must be equilibrated to room temperature for 30 minutes before use, and addition should be gentle along the vessel wall without blowing the cells off.

4. Replace with fresh osteogenic induction medium every 3 days and maintain for 2-4 weeks. With good cell quality the medium typically turns cloudy around day 5, which should not be confused with contamination, and calcium deposits appear at approximately 7-10 days. Larger, denser deposits generally appear within 2 weeks and become increasingly opaque. Deposits may appear later in some cultures, usually not beyond 3 weeks; if no obvious deposit is evident after 3 weeks, the procedure and other factors should be reviewed. The endpoint can be set according to experimental requirements but is generally not extended beyond 4 weeks.

5. At the end of induction, discard the spent medium, wash twice with DPBS and fix the cells with 2 ml of 4% paraformaldehyde for 20 minutes at room temperature.

6. Discard the fixative, wash twice with DPBS and add 1 ml Alizarin Red staining solution per well for 15 minutes.

7. Discard the staining solution, wash 3 times with DPBS and add fresh DPBS. The wells should appear visibly red after staining. Throughout the staining procedure, avoid allowing the cells to dry, since drying impairs microscopic observation.

8. Observe and photograph under the microscope. A high-power objective is generally used for photography, selected according to the requirement.

Result interpretation. Red-stained nodules visible at low power indicate calcium deposits and demonstrate osteogenic capacity of the MSC; absence of such deposits indicates no osteogenic capacity. Staining results are shown for umbilical cord-, adipose- and placenta-derived osteogenic differentiation.

3.2 Adipogenic differentiation

Preparation. Coat the culture vessel with coating solution C as described above, then leave to dry in the laminar flow hood. Six-well plates with 1 ml of coating solution C per well are recommended.

Procedure.

1. Prepare MSC for induction. At approximately 85% confluence, digest the cells and resuspend in MSC complete medium.

2. Seed at 3 x 10^4 cells/cm2 in coated 6-well plates with 2 ml MSC complete medium per well and incubate at 37 degrees C with 5% CO2.

3. At 90% confluence, discard the spent medium and add 2 ml induction medium B per well. Cells detach readily after adipogenic induction, so medium must be pre-warmed to room temperature and added gently along the vessel wall without disturbing the cells. The confluence at the start of induction is critical to the outcome, so cells must be grown to sufficient density before induction begins.

4. After 72 hours, discard the spent medium and add 2 ml induction medium C per well, continuing incubation at 37 degrees C with 5% CO2.

5. After a further 24 hours, discard the medium and add 2 ml induction medium B per well.

6. Repeat steps 4 and 5 approximately 3-5 times, then switch to induction medium C once obvious lipid droplets are visible.

7. At high power, numerous small round bright vacuoles are usually visible in the cytoplasm at around day 4, coalescing into larger vacuoles by day 5-9; these structures surround the nucleus and the larger ones approximate nuclear size. Cultures with good cell quality may be concluded within 10 days. If no lipid droplets appear by day 10, the procedure and other factors should be reviewed.

8. Change induction medium C every 2 days and continue until the lipid droplets are sufficiently large. Induction medium must be pre-warmed, since insufficient warming impairs the induction outcome and may cause cell detachment. If the adipogenic area or droplet size becomes excessive and merges into a sheet, the result is less well presented, so induction should be terminated earlier. The timing of lipid droplet appearance varies between laboratories, and if large areas of adipogenesis appear early, induction medium C can be introduced sooner.

9. At the end of induction, discard the spent medium, wash twice with DPBS and fix with 2 ml of 4% paraformaldehyde per well for 20 minutes at room temperature.

10. Discard the fixative, wash twice with DPBS and add 1 ml Oil Red O working solution per well for 15 minutes. This step identifies adipogenic capacity; lipid droplets appear red. To obtain well-presented images, the staining duration should be adjusted accordingly.

11. Discard the Oil Red O working solution, wash 3 times with DPBS and add fresh DPBS. The wells should appear visibly red. The staining solution must be prepared as a working solution and used fresh, since it will not perform correctly otherwise. Avoid drying of the cells during staining.

12. Observe and photograph under the microscope.

Result interpretation. Extensive red-stained areas at low power, with red spherical structures inside cells at 20x and 40x magnification, represent lipid droplets and demonstrate adipogenic capacity of the MSC. This assessment is qualitative only; a dedicated protocol is required for quantitative analysis. Staining results are shown for adipose- and umbilical cord-derived adipogenic differentiation.

3.3 Chondrogenic differentiation

Procedure.

1. Prepare MSC for induction. At approximately 85% confluence, digest the cells and resuspend the pellet in chondrogenic induction premix.

2. Centrifuge at 220 x g for 5 minutes, resuspend the pellet in premix at a cell density of approximately 2 x 10^6 cells/ml and centrifuge again, discarding the supernatant.

3. Resuspend the pellet in chondrogenic induction complete medium at a density of 2 x 10^6 cells/ml.

4. Dispense 500 ul of cell suspension into 15 ml centrifuge tubes and centrifuge at 220 x g for 5 minutes. The total cell number per tube should be between 5 x 10^5 and 1 x 10^6; as a rule, one T25 culture flask yields one chondral pellet, too few cells producing a small pellet and too many causing fragmentation. Centrifugation should be judged by cell clumping and the force can be adjusted if necessary; excessive force impairs pellet suspension while insufficient force compromises pellet formation. A pellet of 1-2 mm3 is normally formed.

5. Loosen the tube cap and place the tube upright in an incubator at 37 degrees C with 5% CO2 for static culture. Avoid shaking the pellet during the first 24 hours to ensure aggregate integrity, and use sterile polypropylene tubes.

6. After 24 hours of induction, gently tap the bottom of the tube to resuspend the pellet and return it to the incubator. The timing of resuspension depends on the cells, generally between 24 and 48 hours, using the appearance of clustering around the pellet as the criterion. If the pellet adheres firmly, it can be resuspended by gentle pipetting without dispersing the aggregate.

7. Replace with fresh complete induction medium every 2 days, aspirating the old medium gently and adding 500 ul of freshly prepared chondrogenic induction complete medium per tube. Medium changes should be gentle to avoid aspirating the pellet, and induction medium must be pre-warmed since inadequate warming impairs differentiation. Extensive extracellular matrix rich in chondroitin sulphate and type II collagen appears after 2-3 weeks of induction.

8. After 14-20 days of induction, discard the spent medium, wash twice with DPBS and fix with 2 ml of 4% paraformaldehyde per tube for 1 hour at room temperature. Once the chondral pellet has condensed it can be stained; prolonged induction beyond 20 days may produce internal vacuoles or even cracking within the pellet, so the endpoint should be defined according to the characteristics of the cells used.

9. Discard the fixative and wash the pellet twice with DPBS.

10. Alcian blue staining: add 500 ul Alcian blue staining solution to the pellet and stain for 30 minutes. Discard the staining solution and wash 5 minutes at a time with 5 ml of purified water, repeating the wash 5 times. Because the pellet is difficult to see in the staining solution, the supernatant must be aspirated carefully to avoid losing the sample.

Result interpretation. Deep blue staining at low power corresponds to acidic mucopolysaccharide and demonstrates chondrogenic capacity of the MSC; absence of such staining indicates no chondrogenic capacity. Staining results are shown for umbilical cord-derived chondrogenic differentiation.

Contact Absin

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. worldwide@absin.cn

Follow us on Facebook: Absin Bio