amount (at the achievable cooling and warming rates) of CPA at all
locations throughout the tissue. When CPAs are loaded into cells
and tissues, the freezing point of the specimen will decrease.
Keeping the freezing point throughout the tissue below the loading
temperature at each step of a cryopreservation protocol is essential
in preventing ice formation during CPA loading procedures. CPA
permeation has been investigated by several approaches, such as
magnetic resonance spectroscopy [3–5]. However, these techniques require specialized equipment and personnel and are usually
costly or time-consuming, making them difficult to use in many
laboratories. Our group has published an easier method to quantify
CPA permeation which is described here.
Our method [6–8] to measure the CPA permeation is to permeate a series of tissue samples from a surrounding solution at a
specified concentration of CPA, each sample for a different amount
of time, and then to quantitate the amount of CPA that was taken
up in the tissue during that time period. The quantification is
performed by equilibrating the permeated tissue with a surrounding solution and then measuring the osmolality of the solution to
determine the amounts of CPAs that have come out of each tissue
sample corresponding to each permeation time.
An alternative method to measuring the CPA permeation as a
function of time is to measure the CPA efflux as a function of time
[9, 10]. In the efflux method, a CPA-permeated tissue sample is
placed in a surrounding solution, and solution samples are taken at
different time points throughout the efflux to quantitate how much
CPA has left the tissue by each time point [9, 10]. An advantage of
the efflux method is that it requires fewer tissue samples because
one sample can provide an entire time series of measurements,
whereas for the permeation method, a new sample is required for
each time point. We have shown that CPA diffusion coefficients
obtained from permeation experiments can be used to predict
efflux experiments, meaning that permeation and efflux kinetics
are similar [10].
Our methods for measuring CPA permeation [6–8] and efflux
[9, 10] are given in detail below.
2 Materials
2.1 Tissue
Preparation
This will depend on the type of tissue being investigated. In this
example, porcine articular cartilage is the tissue of interest.
1. Cordless electric saw.
2. Surgical scalpel (e.g., #20 feather stainless steel surgical blade).
3. Tissue forceps.
4. Digital caliper.
304
Kezhou Wu et al.
locations throughout the tissue. When CPAs are loaded into cells
and tissues, the freezing point of the specimen will decrease.
Keeping the freezing point throughout the tissue below the loading
temperature at each step of a cryopreservation protocol is essential
in preventing ice formation during CPA loading procedures. CPA
permeation has been investigated by several approaches, such as
magnetic resonance spectroscopy [3–5]. However, these techniques require specialized equipment and personnel and are usually
costly or time-consuming, making them difficult to use in many
laboratories. Our group has published an easier method to quantify
CPA permeation which is described here.
Our method [6–8] to measure the CPA permeation is to permeate a series of tissue samples from a surrounding solution at a
specified concentration of CPA, each sample for a different amount
of time, and then to quantitate the amount of CPA that was taken
up in the tissue during that time period. The quantification is
performed by equilibrating the permeated tissue with a surrounding solution and then measuring the osmolality of the solution to
determine the amounts of CPAs that have come out of each tissue
sample corresponding to each permeation time.
An alternative method to measuring the CPA permeation as a
function of time is to measure the CPA efflux as a function of time
[9, 10]. In the efflux method, a CPA-permeated tissue sample is
placed in a surrounding solution, and solution samples are taken at
different time points throughout the efflux to quantitate how much
CPA has left the tissue by each time point [9, 10]. An advantage of
the efflux method is that it requires fewer tissue samples because
one sample can provide an entire time series of measurements,
whereas for the permeation method, a new sample is required for
each time point. We have shown that CPA diffusion coefficients
obtained from permeation experiments can be used to predict
efflux experiments, meaning that permeation and efflux kinetics
are similar [10].
Our methods for measuring CPA permeation [6–8] and efflux
[9, 10] are given in detail below.
2 Materials
2.1 Tissue
Preparation
This will depend on the type of tissue being investigated. In this
example, porcine articular cartilage is the tissue of interest.
1. Cordless electric saw.
2. Surgical scalpel (e.g., #20 feather stainless steel surgical blade).
3. Tissue forceps.
4. Digital caliper.
304
Kezhou Wu et al.