synthetic

development bio

Material List

Cartilage Source (whole intact chicken leg quarters from live poultry and supermarket)
Disposable scalpels #22 blade size
Small Forceps
Beakers
50 mL conical tubes
Pipettor
Individually wrapped sterile serological pipettes (5, 10, 25 mL)
50-250mL glass storage bottles (e.g. Kimble™ KC14395100)
70% ethanol in spray bottle
Kimwipes
Aluminum foil
PBS or DPBS (no magnesium, no calcium)
Sterile deionized distilled water
DMEM, high glucose, pyruvate (ThermoFisher Scientific Catalog # 11995065)
Ascorbic acid solution (Sigma-Aldrich Catalog # V-038)
L-Proline solution (Sigma-Aldrich Catalog # 94321)
Dexamethasone solution (Sigma-Aldrich Catalog # D-085)
Antibiotic-Antimycotic solution (ThermoFisher Scientific Catalog #15240096)
Collagenase/Dispase (Sigma-Aldrich Catalog # 10269638001)
LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells (ThermoFisher Scientific Catalog # L3224)
Agarose (Sigma A9045) – 4% agarose in DPBS (4 mg/100 mL –microwave for 30 sec, swirl, microwave for 30 sec, swirl, etc until dissolved)
24-well plates
48-well plates
Biopsy punches (4 mm- Fisher Sci Catalog # NC9129052)
Syringe filter (0.2 um pore size)
Syringes (3-20 cc)
Trypan Blue Solution, 0.4% (ThermoFisher Scientific Catalog # 15250061)
Hemocytometer
Thermometer
70 um cell strainers

lab task

EXPERIMENTAL HOMEWORK

NINA TANDON

Protocol

Media Making:

DME
10% FBS or 1X ITS
50 μg/mL ascorbic acid
1X essential amino acid

Collagenase Solution:

Prepare 0.5 mg/mL collagenase in 30 mL media
Sterile filter into 50 mL tube.

Cell preparation:

Wash intact chicken thigh quarters soapy water.
Soak intact chicken thigh quarters in 70% ethanol for 15 minutes.
Spray aluminum foil with ethanol and wrap thighs in foil.
Spray outside of aluminum foil with ethanol and transfer to biosafety cabinet.
Prepare 50 mL tubes with 25 mL PBS.
Open up aluminum foil and remove chicken.
Carefully cut away the flesh from the thighs.
Open the joints.
With a new scalpel, gently shave away the cartilage from the bone.
Place cartilage shavings in tube with PBS.
Aspirate and add 25 mL fresh PBS to wash.
Repeat step 11 twice.
Aspirate PBS and then add 25 mL collagenase solution.
Incubate in collagenase overnight.
Vortex cell solution.
Place cell strainer onto 50 mL tube.
Pipette cell solution over cell strainer. Try to avoid large cartilage chunks
Remove the cell strainer and cap tube.
Spin at 500 g for 15 minutes.
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.
c. Spray outside of bottle with ethanol and transfer to biosafety cabinet.
d. Spray thermometer with ethanol and dry in biosafety cabinet by gently shaking until ethanol evaporates
e. Remove cap from bottle and insert thermometer.
f. Let agarose cool to 42°C. Occasionally stirring with thermometer to ensure even cooling.
Aspirate solution without disturbing the cell pellets.
Add 2 mL of fresh media and mix by pipetting.
Cell counting: preview and decide best approach.
Combine 2 mL each of cells and 2 mL agarose and mix by pipetting. (Lesson: What’s final concentration of cells in 2% agarose?)
Pipette 0.5 mL/well cell-agarose solution into 48-well plate.
Let solidify at room temperature for 15-20 minutes.
Using the biopsy punch to core out the center of wells and transfer cartilage constructs to fresh 24-well plate containing 2 mL of media.
Take pictures of 3-4 constructs then do LIVE/DEAD analysis.
a. LIVE/DEAD Solution: 5 μg Calcein-AM and 20 μg Ethidium homodimer-1 to 10 mL PBS.
b. Add 1 mL LIVE/DEAD Solution into 3-4 well of a new 48 well plate. Place 1 construct/well and incubate at 37C for 20 minutes.
c. Image LIVE cells with 490nm LED and DEAD cells with 515nm LED.
Incubate remaining constructs at 37C/5% CO2 to grow cartilage for 1 week.
Change media every 3-4 days by removing old media and adding fresh media. (Lesson: Take picture of media before and after media change and not changes in color).
Run LIVE/DEAD again (see step 25).
Take picture of constructs (Lesson: How did the appearance of constructs change during culturing).

Conclusions

The use of live cartilage cells from chicken leg for cell culture served us to understand the concept of cartilage cell growth. Because cartilage does not naturally repair itself, scientists started studying the properties of tissue culture of the cells in a laboratory setting, this meaning an alternative way to solve the issue and essentially repair a defect in any cartilage layer in the future. There are still some questions about this idea, however, this method and technique open a discussion on the subject, allowing the development of new techniques considering that cells can be grown in almost any scaffold.

Introduction

This class is led by Nina Tandon, CEO of Epibone, a Brooklyn based biomedical tech company that grows bones from living cells for regenerative tissue engineering. Epibone's mission is to transform patients’ lives through personalized skeletal reconstruction, precision design, and stem cell technology.

Epibone's innovative approach of using a patient's own living stem cells for tissue engineering, instead of biomaterials, ceramics, synthetic polymers and natural polymers, opens up

a whole new era of personalized healthcare. The technology allows cells to be grown in the precise anatomical shape and size that is needed, without cutting a bone from a patient's body. Because the tissue is made from stem cells, the implant has a better integration without the possible complications of immune rejection. The implanted bone graft is alive, so the implant has the potential to continue growing and remodeling, just like 'normal' bones do. In this assignment, we simulate a simplified workflow by extracting cartilage from chicken bones to grow tissue ourselves.