Synthetic Developmental Biology: Cartilage Tissue Engineering
For this assignment, we did this at the lab
Day 1
we made the Collagenase Solution and Cell preparation according to the following protocol
Collagenase Solution:
1. Prepare 0.5 mg/mL collagenase in 30 mL media
2. Sterile filter into 50 mL tube.
Cell preparation:
1.Wash intact chicken thigh quarters soapy water.
2.Soak intact chicken thigh quarters in 70% ethanol for 15 minutes.
3.Spray aluminum foil with ethanol and wrap thighs in foil.
4.Spray outside of aluminum foil with ethanol and transfer to biosafety cabinet.
6. Prepare 50 mL tubes with 25 mL PBS.
7. Open up aluminum foil and remove chicken.
8. Carefully cut away the flesh from the thighs.
9. Open the joints
10. With a new scalpel, gently shave away the cartilage from the bone.
11. Place cartilage shavings in a tube with PBS.
11. Aspirate and add 25 mL fresh PBS to wash.
12. Repeat step 11 twice.
13. Aspirate PBS and then add 25 mL collagenase solution.
14. Incubate in collagenase overnight.
Day 2
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5. Spray outside of the bottle with ethanol and transfer to biosafety cabinet.
6. Spray thermometer with ethanol and dry in biosafety cabinet by gently shaking until ethanol evaporates
7 Remove cap from the bottle and insert the thermometer.
8. Let agarose cool to 42°C. Occasionally stirring with a thermometer to ensure even cooling.
9. Aspirate solution without disturbing the cell pellets.
10. Add 2 mL of fresh media and mix by pipetting.
11. Cell counting: preview and decide best approach
12. Combine 2 mL each of cells and 2 mL agarose and mix by pipetting. (Lesson: What’s final concentration of cells in 2% agarose?)
13. Pipette 0.5 mL/well cell-agarose solution into 48-well plate.
14. Let solidify at room temperature for 15-20 minutes.
15. Using the biopsy punch to core out the center of wells and transfer cartilage constructs to a fresh 24-well plate containing 2 mL of media.
Look at the little piece of cartilage!
Prepare agarose solution during spin.
a. Prepare 4% (weight/volume) agarose solution (e.g. 4g agarose in 100 mL PBS) in glass bottle.
b. Loosen lid and microwave at 50% power at 30-second intervals until dissolved. Swirl gently between each interval. Do not let boil, this will evaporate the water and increase the agarose concentration, which will make the solution difficult to work with.
Tissue engineering (sometimes called regenerative medicine, though the latter refers primarily to the use of stem cells) is an emerging interdisciplinary research field initially defined in the early 1990s.It uses principles and methods in engineering, material science, biology, and chemistry to develop biological substitutes that restore, maintain, or improve functionality of damaged tissues and organs
Tissue engineering incorporates many critical factors including cells, scaffolds, bioactive factors, and physical stimuli to assemble biomimetic tissue engineered constructs for replacing damaged tissues in humans.
1. Vortex cell solution.
2. Place cell strainer into 50 mL tube.
3. Pipette cell solution over cell strainer. Try to avoid large cartilage chunks
4. Remove the cell strainer and cap tube.5. Spin at 500 g for 15 minutes.
Next week: Live/Dead Assay!
1. Take pictures of 3-4 engineered cartilage then do LIVE/DEAD analysis.
a. LIVE/DEAD Solution: 5 µg Calcein-AM and 20 µg Ethidium homodimer-1 to 10 mL PBS. Alternate: Add 1 drop of Calcein-AM and 4 drops of Ethidium homodimer-1 to 10 mL PBS
b. Add 1 mL LIVE/DEAD Solution into 3-4 well of a new 24-well plate. Place 1 construct/well and incubate at 37°C or room temperature for 20 minutes.
c. Cut samples in half and image cross-section. Image LIVE cells with 490nm LED and DEAD cells with 515nm LED.
Alternative to Live/Dead Assay: Trypan Blue Staining
1.Take pictures of 3-4 engineered cartilage then do Trypan Blue analysis.
a.Add 1 mL LIVE/DEAD Solution into 3-4 well of a new 24-well plate. Place 1 construct/well and incubate at room temperature for 5 minutes.
b.Wash 3 times in PBS by removing old solution and adding fresh PBS
b.Cut samples in half and image cross-section. Dead cell will be dark blue.