Culture of chondrocytes with alginate microbeads
Culture of chondrocytes with alginate microbeads
Alginate microbead cultures based on the gelling action of calcium chloride in chondrocyte alginate suspensions.
Operation method
Scheme 23.16 Culturing chondrocytes with alginate microbeads
Principle
Alginate microbead cultures based on the gelling action of calcium chloride in chondrocyte alginate suspensions.
Materials and Instruments
Chondroectomy culture solution Growth culture solution Enzyme solution for chondrocyte isolation Trypsin and EDTA mixture Sodium alginate solution Gelatinizing solution Dissolving solution Move osteotomy (medicine) Caveat 1. The effects of batches of serum on chondrocyte growth and differentiated cellular phenotypes should be compared, e.g., the serum should be evaluated by immunolabeling collagen type II. When selecting a batch of serum, several months' supply should be purchased to avoid frequent serum changes. 2. The effectiveness of the new collagenase in isolating chondrocytes should be tested by comparing it with the previously used one. For more product details, please visit Aladdin Scientific website.
Sterile magnet
1. Cartilage is removed from the knee, shoulder and hip joints. Cartilage from embryonic or juvenile donors is preferable to that from adult donors because the cartilage from embryonic or juvenile donors acquires more cells than adult donors and senesces over a longer period of time. Fauve de Bourgogne or New Zealand rabbits one month after birth are preferable. However, surgically removed hip joints from adults are also a suitable source of chondrocytes.
2. Prior to cartilage dissection, the hip joints were treated with CDM (cartilage dissection medium (CDM): Ham's F12 culture (In-vitrogen) with 20 μg/ml zetimibine (Sigma), 10 μg/ml vancomycin (Sigma) and 30 μg/ml cefotaxime (In-vitrogen)). 30 μg/ml ceftazidime (Sigma). CDM is used to cut the joint and excise cartilage, and is also used for enzymatic digestion) to thoroughly clean the joint tissue. The earlier the cartilage is removed, the better. Remove the skin, muscles and tendons from the joint. Carefully cut off the cartilage without taking the connective tissue with it.
Separation of cells
To prevent cartilage from drying out, work in 100 mm dishes (not treated with TC) containing CDM.
The amount of enzyme solution used in the following procedure applies to cartilage taken from the knee and shoulder joints of one rabbit, but the amount of enzyme solution must be determined according to the amount of cartilage removed.
Enzyme solution for isolation of chondrocytes:
(a) 0.05% porcine trypsin (Sigma), prepared with Ham's F12;
(b) 0.3% collagenase type I (Worthington, CLSI), prepared with Ham's F12;
(c) 0.06% collagenase, prepared with Ham's F12, added to 10 % FCS.
The three enzyme solutions were filtered through a 0.22 μm filter to remove bacteria.
3. The cartilage slices were cut into 1 mm3 pieces with a cross blade.
4. Transfer the cartilage pieces into a 30 ml flat-bottomed vial.
5. Add 10 ml of 0.05% trypsin, seal the vial and digest for 25 min at room temperature with moderate magnetic stirring.
6. After the tissue mass has settled, the trypsin solution is removed.
7. Add 10 ml of 0.3 % collagenase, seal the vial and digest for 30 min at room temperature with moderate magnetic stirring.
8. After the tissue mass has settled, remove the collagenase solution.
9. Add 10 ml of 0.06 % collagenase.
10. Transfer the tissue mass suspension to a 100 mm bacterial culture-grade Petri dish. After rinsing the vial with 10 ml of 0.06 % Collagenase, transfer the rinse solution to a Petri dish. Then, place in an incubator and incubate overnight at 37°C and 5 % CO2.
11. Transfer the cell suspension into a 50 ml centrifuge tube and mix for a few moments with a vortex mixer.
12. Remaining tissue after digestion was filtered through a 70 μm nylon filter (BD Biosciences).
13. Centrifuge the filtrate (400 g for 10 min).
14. Suspend the cells with 20 ml of CDM and count them with a blood cell counter plate.
15. Centrifuge (400 g, 10 min).
Embedded cells
16. Suspend the cells with 1 ml of sodium alginate solution and gradually dilute the cell suspension with sodium alginate solution until the cell density is 2 x 106 cells/ml. gradual dilution is necessary to obtain a homogeneous alginate cell suspension.
Sodium alginate solution: 1.25% (W/V) sodium alginate (Fluka), prepared with 20 mmol/L HEPES and 0.15 mol/L NaCl.
Method of Preparation:
(a) Using a heated stirrer, dissolve HEPES in 0.15 mol/L NaCl and heat to approximately 60°C. The solution should be prepared in the same manner as the sodium alginate solution;
(b) Add sodium alginate and continue stirring until completely dissolved. Dissolution time should be more than 2 h;
(c) If irreversible precipitation occurs due to the addition of an acid (hydrochloric or sulfuric), carefully adjust the pH to 7.4 with 1 mol/L NaOH, but do not exceed 7.4. Dispense the solution and autoclave at 121 °C for 10 min.
Alternatively, sodium alginate is quickly dissolved in a mixture of HEPES and NaCl in a microwave oven for about 10 min. However, care must be taken to avoid scorching.
17. The cell suspension is added dropwise to a gelling solution (102 mmol/L calcium chloride, 5 mmol/L HEPES, pH 7.4) with moderate magnetic stirring through a 21G needle, allowing the alginate to aggregate for 10 min to form beads.
18. Wash the beads three times with 5 times the volume of 0.15 mol/L NaCl.
19. 5 ml of beads (1 × 107 cells) were placed into 75 cm2 culture flasks containing 20 ml of growth medium (DMEM (containing 1 μg/L glucose) and Ham F12 mixture (1 : 1, V/V) with 4 μg/ml gentamicin and 10% FCS).
20. Incubate at 37°C with humidified 5 % CO2 and 95 % air.
Recovery of cells
21. Aspirate the culture solution.
22. Add twice the volume of lysis solution (50 mmol/L EDTA, 10 mmol/L HEPES, pH 7.4) to the microbeads.
23. Incubate at 37°C for 15 min.
24. Centrifuge (400 g, 10 min).
25. Suspend cells in growth medium containing 0.06% collagenase.
26. Incubate for 30 min at 37°C with humidified 5 % CO2 and 95 % air.
27. Centrifuge (400 g, 10 min).
28. Suspend the cells with a mixture of serum-free DMEM and Ham F12, and count them with a hemocyte counter plate.
29. Centrifuge (400 g, 10 min).
30. Repeat steps 28 and 29 without counting the cells.