2.3 Chemicals
1. CPAs of interest, e.g.:
(a) Glycerol (!99.5%).
(b) Dimethyl sulfoxide (!99.5%).
(c) Ethylene glycol (1,2-ethanediol, !99.5%).
(d) Propylene glycol (1,2-propanediol, !99.5%).
2. 1Â Dulbecco’s phosphate-buffered saline (PBS).
3. Dulbecco’s Modified Eagle Medium F12 (DMEM-F12,
Gibco).
4. Newborn calf serum (heat inactivated, Gibco).
5. 100 mM sodium pyruvate (Gibco).
6. Antibiotics (e.g., Antibiotic–Antimycotic, 100Â, including liquid 10,000 U/mL penicillin, 10,000 μg/mL streptomycin,
25 μg/mL amphotericin B, 100 mL, Gibco).
7. Methanol (99.8%).
3 Methods
Safe laboratory practices should be used at all times (see Note 1).
3.1 Preparation
of Articular Cartilage
The objective of the specimen preparation is to have pure tissue
with well-defined dimensions so that an accurate volume of the
tissue can be calculated. This example will demonstrate important
details for the investigation of porcine articular cartilage (see Note
2).
1. Prepare articular cartilage in a fume hood (or in an area designated for animal work).
2. Label several large lab beakers (250 mL) with the condyle
number.
3. Place absorbent underpads beneath the dissection board,
remove the porcine hind leg from the plastic cooler, and place
it on the board.
4. Use a sterile surgical scalpel to dissect the porcine joint, and
expose the femoral condyles.
5. Use the electric saw to remove the femoral condyles from the
femur bone.
6. Immerse the femoral condyles in 1Â PBS solution to keep the
cartilage moist, avoiding prolonged exposure of cartilage to air.
7. Clean the femoral condyles by immersing in sterile 1Â PBS
solution supplemented with antibiotics (100 U/mL penicillin,
100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B)
for 15 min in a biological safety cabinet under sterile
conditions.
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Kezhou Wu et al.
1. CPAs of interest, e.g.:
(a) Glycerol (!99.5%).
(b) Dimethyl sulfoxide (!99.5%).
(c) Ethylene glycol (1,2-ethanediol, !99.5%).
(d) Propylene glycol (1,2-propanediol, !99.5%).
2. 1Â Dulbecco’s phosphate-buffered saline (PBS).
3. Dulbecco’s Modified Eagle Medium F12 (DMEM-F12,
Gibco).
4. Newborn calf serum (heat inactivated, Gibco).
5. 100 mM sodium pyruvate (Gibco).
6. Antibiotics (e.g., Antibiotic–Antimycotic, 100Â, including liquid 10,000 U/mL penicillin, 10,000 μg/mL streptomycin,
25 μg/mL amphotericin B, 100 mL, Gibco).
7. Methanol (99.8%).
3 Methods
Safe laboratory practices should be used at all times (see Note 1).
3.1 Preparation
of Articular Cartilage
The objective of the specimen preparation is to have pure tissue
with well-defined dimensions so that an accurate volume of the
tissue can be calculated. This example will demonstrate important
details for the investigation of porcine articular cartilage (see Note
2).
1. Prepare articular cartilage in a fume hood (or in an area designated for animal work).
2. Label several large lab beakers (250 mL) with the condyle
number.
3. Place absorbent underpads beneath the dissection board,
remove the porcine hind leg from the plastic cooler, and place
it on the board.
4. Use a sterile surgical scalpel to dissect the porcine joint, and
expose the femoral condyles.
5. Use the electric saw to remove the femoral condyles from the
femur bone.
6. Immerse the femoral condyles in 1Â PBS solution to keep the
cartilage moist, avoiding prolonged exposure of cartilage to air.
7. Clean the femoral condyles by immersing in sterile 1Â PBS
solution supplemented with antibiotics (100 U/mL penicillin,
100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B)
for 15 min in a biological safety cabinet under sterile
conditions.
306
Kezhou Wu et al.