3.4 Cryoprotective
Agent Permeation into
Tissue
This procedure documents the permeation of a single type of CPA
into articular cartilage with a fixed concentration of the CPA in the
surrounding solution for different temperatures and exposure
times. We used this method to collect data for the calculation of
permeation kinetics of a single CPA into porcine articular cartilage.
Measurements at a series of temperatures are used to give an
indication of the temperature dependence of the CPA permeation
process.
1. Prepare the CPA loading solutions with selected concentrations of CPAs (e.g., 6.5 M of glycerol, dimethyl sulfoxide
(DMSO), ethylene glycol (EG), or propylene glycol (PG))
according to the desired initial concentration and desired temperature (see Note 3).
2. Use the 10-mm diameter coring device to core osteochondral
dowels from the weight-bearing area of a condyle (see Fig. 1).
Then, carefully isolate the full-thickness articular cartilage
(10-mm diameter round disk) from its bone base with a surgical scalpel (see Note 4).
3. Each tissue sample to be tested should have its dimensions
measured sufficiently to enable calculation of a tissue volume.
In our circumstance (see Fig. 1), we controlled the diameter by
using a fixed diameter (D) cutting corer and measured height at
three equidistant points around the circumference and averaged those three measurements (H 1 , H 2 , H 3 ) to get tissue
height and calculated the tissue volume (see Note 5).
4. Weigh the tissue (W 1 ) before CPA exposure.
5. Immerse the tissue completely in the CPA solution at the
desired temperature for a specified period of time. The amount
of CPA solution should be sufficient that absorption of CPA
into the tested tissue does not significantly decrease the CPA
concentration in the surrounding solution. In our experiments,
we used 5 mL of CPA solution as the cartilage volume was
typically 0.157 mL. A series of exposure times should be
recorded to give a broad range based on the expected permeation rate. For articular cartilage, we selected the following
times: 1 s, 1 min, 2 min, 5 min, 10 min, 15 min, 30 min,
60 min, 120 min, 180 min, and 24 h.
6. After the desired exposure time has elapsed, remove the tissue,
blot dry the tissue with a Kimwipe, weigh the tissue (W 2 ), and
then place the tissue in a PBS solution after measuring the
baseline osmolality of the PBS solution. There should be sufficient PBS solution such that a small change in osmolality can be
measured. In our case, we used 4 mL of PBS solution. Immerse
the tested tissue in the PBS solution for 24 h to equilibrate the
CPA with the surrounding PBS (give sufficient time for the
CPA to fully efflux out of the tissue).
308
Kezhou Wu et al.
Agent Permeation into
Tissue
This procedure documents the permeation of a single type of CPA
into articular cartilage with a fixed concentration of the CPA in the
surrounding solution for different temperatures and exposure
times. We used this method to collect data for the calculation of
permeation kinetics of a single CPA into porcine articular cartilage.
Measurements at a series of temperatures are used to give an
indication of the temperature dependence of the CPA permeation
process.
1. Prepare the CPA loading solutions with selected concentrations of CPAs (e.g., 6.5 M of glycerol, dimethyl sulfoxide
(DMSO), ethylene glycol (EG), or propylene glycol (PG))
according to the desired initial concentration and desired temperature (see Note 3).
2. Use the 10-mm diameter coring device to core osteochondral
dowels from the weight-bearing area of a condyle (see Fig. 1).
Then, carefully isolate the full-thickness articular cartilage
(10-mm diameter round disk) from its bone base with a surgical scalpel (see Note 4).
3. Each tissue sample to be tested should have its dimensions
measured sufficiently to enable calculation of a tissue volume.
In our circumstance (see Fig. 1), we controlled the diameter by
using a fixed diameter (D) cutting corer and measured height at
three equidistant points around the circumference and averaged those three measurements (H 1 , H 2 , H 3 ) to get tissue
height and calculated the tissue volume (see Note 5).
4. Weigh the tissue (W 1 ) before CPA exposure.
5. Immerse the tissue completely in the CPA solution at the
desired temperature for a specified period of time. The amount
of CPA solution should be sufficient that absorption of CPA
into the tested tissue does not significantly decrease the CPA
concentration in the surrounding solution. In our experiments,
we used 5 mL of CPA solution as the cartilage volume was
typically 0.157 mL. A series of exposure times should be
recorded to give a broad range based on the expected permeation rate. For articular cartilage, we selected the following
times: 1 s, 1 min, 2 min, 5 min, 10 min, 15 min, 30 min,
60 min, 120 min, 180 min, and 24 h.
6. After the desired exposure time has elapsed, remove the tissue,
blot dry the tissue with a Kimwipe, weigh the tissue (W 2 ), and
then place the tissue in a PBS solution after measuring the
baseline osmolality of the PBS solution. There should be sufficient PBS solution such that a small change in osmolality can be
measured. In our case, we used 4 mL of PBS solution. Immerse
the tested tissue in the PBS solution for 24 h to equilibrate the
CPA with the surrounding PBS (give sufficient time for the
CPA to fully efflux out of the tissue).
308
Kezhou Wu et al.