Chapter 11
Osmometric Measurements of Cryoprotective Agent
Permeation into Tissues
Kezhou Wu, Leila Laouar, Nadia Shardt, Janet A. W. Elliott,
and Nadr M. Jomha
Abstract
Quantification of the amount of cryoprotective agent (CPA) in a tissue is an essential step in the design of
successful cryopreservation protocols. This chapter details two inexpensive methods to measure cryoprotective agent permeation into tissues as functions of time. One of the methods 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, which requires
fewer tissue samples, is to measure the CPA efflux as a function of time. 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.
Key words Osmometer, Osmometry, Cryoprotective agent, Permeation, Diffusion, Tissue
1 Introduction
Cryoprotectants, such as the permeating cryoprotective agents
(CPAs) dimethyl sulfoxide (DMSO) or ethylene glycol (EG), are
widely used in cryobiological applications to protect targeted cells,
tissues, or organs from freezing injuries. The amount of CPA in the
tissue is a critical factor in developing an appropriate cryopreservation protocol for that specific tissue. For instance, cryopreservation
of tissue by vitrification, a technique which transforms a specimen
into an “ice-free” glassy state for long-term storage [1, 2], requires
a high concentration of CPAs to be permeated into the specimen.
The amount of CPA at a given location within the tissue is important for designing vitrification protocols which require a vitrifiable
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
303
Osmometric Measurements of Cryoprotective Agent
Permeation into Tissues
Kezhou Wu, Leila Laouar, Nadia Shardt, Janet A. W. Elliott,
and Nadr M. Jomha
Abstract
Quantification of the amount of cryoprotective agent (CPA) in a tissue is an essential step in the design of
successful cryopreservation protocols. This chapter details two inexpensive methods to measure cryoprotective agent permeation into tissues as functions of time. One of the methods 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, which requires
fewer tissue samples, is to measure the CPA efflux as a function of time. 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.
Key words Osmometer, Osmometry, Cryoprotective agent, Permeation, Diffusion, Tissue
1 Introduction
Cryoprotectants, such as the permeating cryoprotective agents
(CPAs) dimethyl sulfoxide (DMSO) or ethylene glycol (EG), are
widely used in cryobiological applications to protect targeted cells,
tissues, or organs from freezing injuries. The amount of CPA in the
tissue is a critical factor in developing an appropriate cryopreservation protocol for that specific tissue. For instance, cryopreservation
of tissue by vitrification, a technique which transforms a specimen
into an “ice-free” glassy state for long-term storage [1, 2], requires
a high concentration of CPAs to be permeated into the specimen.
The amount of CPA at a given location within the tissue is important for designing vitrification protocols which require a vitrifiable
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
303