8. Transfer the femoral condyles to a complete medium (DMEM
complete: Dulbecco’s Modified Eagle Medium F12 supplemented with 10% heat-inactivated newborn calf serum,
100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/
mL amphotericin B, and 1 mM sodium pyruvate), and store
at 4
C in a refrigerator prior to CPA permeation. The antibiotics are used to prevent bacterial overgrowth with longer
holding periods, while the DMEM complete is important to
help keep the cells viable if that is important for the process.
9. Use the articular cartilage within 24 h after dissection.
10. Clean the fume hood and biological safety cabinet with 70%
ethanol and paper towels.
3.2 Setup
of Holding Baths
1. Dependent on the desired experimental temperature:
(a) Set the incubator shaker temperature to 37
C.
(b) Set the laboratory room temperature to 22
C.
(c) Use the cold room for the 4
C experimental condition.
2. Set the shaker speed (incubator shaker at 37
C and orbital mini
shaker at 22 or 4
C) to 180 rpm.
3. Fill the multi-well plates (6-well tissue culture plate) with 5 mL
CPA in each well, and leave them shaking at the specific temperature condition for at least 1 h.
4. Use the thermocouple probe to double check the operating
temperature of each CPA solution to make sure the display
temperature on the thermocouple thermometer matches the
set temperature.
3.3 Setup
of Osmometer (e.g.,
Micro-Osmette
Osmometer)
1. Plug in the power adapter to a lab AC outlet.
2. Raise the operating head, and place a clean dry sample test tube
in the refrigerator well.
3. Keep the dry sample test tube in the refrigerator well to prevent
frosting of the well.
4. Turn on the osmometer using the on/off switch on the back of
the machine.
5. Choose an appropriate operation mode and range for the
Micro-Osmette osmometer according to the manufacturer’s
manual.
6. Leave the osmometer on for 15 min before starting the
measurement.
7. Use the standard buffer to check the baseline readout of the
osmometer.
8. Calibrate the osmometer if the standard buffer gives a deviated
reading; refer to the manufacturer’s manual.
Osmometric Measurements of Cryoprotective Agent Permeation
307
complete: Dulbecco’s Modified Eagle Medium F12 supplemented with 10% heat-inactivated newborn calf serum,
100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/
mL amphotericin B, and 1 mM sodium pyruvate), and store
at 4
C in a refrigerator prior to CPA permeation. The antibiotics are used to prevent bacterial overgrowth with longer
holding periods, while the DMEM complete is important to
help keep the cells viable if that is important for the process.
9. Use the articular cartilage within 24 h after dissection.
10. Clean the fume hood and biological safety cabinet with 70%
ethanol and paper towels.
3.2 Setup
of Holding Baths
1. Dependent on the desired experimental temperature:
(a) Set the incubator shaker temperature to 37
C.
(b) Set the laboratory room temperature to 22
C.
(c) Use the cold room for the 4
C experimental condition.
2. Set the shaker speed (incubator shaker at 37
C and orbital mini
shaker at 22 or 4
C) to 180 rpm.
3. Fill the multi-well plates (6-well tissue culture plate) with 5 mL
CPA in each well, and leave them shaking at the specific temperature condition for at least 1 h.
4. Use the thermocouple probe to double check the operating
temperature of each CPA solution to make sure the display
temperature on the thermocouple thermometer matches the
set temperature.
3.3 Setup
of Osmometer (e.g.,
Micro-Osmette
Osmometer)
1. Plug in the power adapter to a lab AC outlet.
2. Raise the operating head, and place a clean dry sample test tube
in the refrigerator well.
3. Keep the dry sample test tube in the refrigerator well to prevent
frosting of the well.
4. Turn on the osmometer using the on/off switch on the back of
the machine.
5. Choose an appropriate operation mode and range for the
Micro-Osmette osmometer according to the manufacturer’s
manual.
6. Leave the osmometer on for 15 min before starting the
measurement.
7. Use the standard buffer to check the baseline readout of the
osmometer.
8. Calibrate the osmometer if the standard buffer gives a deviated
reading; refer to the manufacturer’s manual.
Osmometric Measurements of Cryoprotective Agent Permeation
307