Alcian Blue Staining to Visualize Proteoglycans: A Technique to Detect Proteoglycans in Mesenchymal Stem Cell Differentiated Chondrocytes Culture In Vitro

NOTE: Canine bone marrow aspirates from the iliac crest were collected with dog owner's consent. Canine (approx. 10 ml) bone marrow aspirates were anti-coagulated by the addition of 15 ml of 3.8% sodium citrate immediately after withdrawal in the operation theatre. The samples were transferred to the laboratory environment for processing the same day as withdrawn.

1. Preparation of Urokinase (Prior to 1^st Use)

1. Reconstitute urokinase using sterile phosphate-buffered saline (PBS) according to the manufacturer's instructions: Add 10 ml PBS to the septum-inlet flask using a 10 ml syringe. Dissolve the dry substance by swirling to obtain 50,000 injection units (U) per ml.
2. Prepare 500 μl aliquots (25,000 U each) into sterile tubes. Store aliquots -20 °C and uses it when required for at least 6 months.

2. Preparative Steps (Prior to Every Bone Marrow Treatment)

1. Thaw one aliquot of urokinase (25,000 U) per clotted bone marrow aspirate (aspirate volumes up to 25 ml have been successfully treated using a single aliquot of 25,000 U).
2. Preheat a water bath or shaking incubator to 37 °C.

3. Enzymatic Digest of the Clot

1. Perform all work in a laminar flow biosafety cabinet to avoid microbial contamination of the bone marrow samples during processing. When working with potentially infectious human material, the use of protective gloves, as well as careful handling, is advised.
2. Place a 100 μm cell strainer on top of a sterile 50 ml tube. Carefully pour the bone marrow aspirate through the cell strainer, tilting the strainer or moving around the clot material. Use a sterile pipet tip for better flow through the filter mesh.
   NOTE: Avoid any dilution of the clot, e.g., by washing the clot with PBS. Urokinase acts indirectly and requires components of the serum (i.e., plasminogen) from the biopsy for effective digest. While continuing the procedure with the clot material, the filtrate can be kept at RT until further use in step 3.6.
3. Transfer the bone marrow clot material into an empty cell culture dish using sterile forceps. Cut the debris into small pieces of approximately 2 mm³ using a sterile scalpel.
4. Transfer the small pieces of the clot into a 50 ml reaction tube using the sterile forceps. Ensure that the minced clot has a wet appearance. If it appears to be dry, use some of the filtrate from step 3.1 to moisten.
5. Triturate the clot by pipetting up and down for 5 times using a 5 ml microtiter pipet. Add 1 aliquot of urokinase to the sample. Incubate for 30 min at 37 °C either in a water bath or in a shaking (gently) incubator.
6. Triturate by pipetting up and down for 5 times using a 5 ml pipet. Perform this step in a biosafety cabinet. Incubate for another 30 min at 37°C. After the incubation, triturate again 5 times using a 5 ml pipet. Pass over a fresh 100 μm cell strainer to pool with the filtrate from step 3.2.
7. Centrifuge the cell suspension at ambient temperature for 10 min at 500 x g. Discard the supernatant. Re-suspend the cell pellet in media as described below or according to the standard procedure of your laboratory for MSC expansion culture.

4. Seeding for Expansion Cell Culture and CFU Plates

1. Resuspend the cells (consisting of erythrocytes and mononuclear cells) in 50 ml of basal medium: Alpha-MEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 2.5 mg/ml amphotericin B. Count the cells diluted 1:10 in Trypan Blue solution using a Neubauer chamber.
2. Plate the cells in cell culture flasks at the density of 5 x 10⁷ cells/cm² and incubate in a humidified incubator at 37 °C. If available, use hypoxic (5% oxygen) conditions. For the CFU assay control, plate 10⁹ cells in a cell culture dish of 10 cm diameter.
3. After 3 days of culture, mesenchymal stem cells are attached to the cell culture dish while other cells remain in suspension. Change the media with basal medium supplemented with 5 ng/ml basic fibroblast growth factor (bFGF). For the CFU assay control, treat the cell culture dish likewise.
4. Continue cell culture for a total of 2 weeks, exchanging media three times a week. If cells exceed 80% confluency, split by trypsinization. For the CFU assay control, exchange the media likewise, but do not split the cells for the course of the two weeks.

5. MSC Differentiation

1. Canine MSCs differentiation
   NOTE: Canine MSCs were differentiated into chondrogenic, osteogenic and adipogenic lineages by stimulation with the appropriate media for four weeks.
2. Chondrogenic differentiation
   1. Cut cubes (3 mm per side) from a sponge-shaped medical device, constituted from lyophilized collagen type I, and used as scaffold material to support cells.
   2. Seed MSCs on the top of the cubes at the concentration of 4 x 10⁶ cells/ml (~70,000 cells/cube).
   3. Prior to the addition of media, allow the cells to adhere to the cubes for 30 min.
   4. Maintain MSC-collagen constructs in chondrogenic media consisting of DMEM/F12 + GlutaMAX, 2.5 % FBS, 100 units/ml penicillin,100 mg/ml streptomycin, 2.5 μg/ml amphotericin B, 40 ng/ml dexamethasone, 50 μg/ml ascorbic acid 2-phosphate, 50 μg/ml L-proline,1x Insulin-Transferrin-Selenium X, and 10 ng/ml transforming growth factor-β1.
   5. Use alcian blue staining to visualize accumulation of proteoglycans in constructs sections. Briefly, stain the sections O/N with 0.4% alcian blue dissolved in 0.01% H₂SO₄ and 0.5M guanidine hydrochloride. Next, wash the sections were washed for 30 min in 40% DMSO and 0.05M MgCl₂. Cells were counterstained with nuclear fast red.