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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Rall WF, Fahy GM (1985) Ice-free cryopreservation of mouse embryos at −196 °C by vitrification. Nature 313:573–575
Jomha NM, Elliott JAW, Law GK, Maghdoori B, Forbes JF, Abazari A, Adesida AB, Laouar L, Zhou X, McGann LE (2012) Vitrification of intact human articular cartilage. Biomaterials 33:6061–6068
Abazari A, Elliott JAW, McGann LE, Thompson RB (2012) MR spectroscopy measurement of the diffusion of dimethyl sulfoxide in articular cartilage and comparison to theoretical predictions. Osteoarthr Cartil 20:1004–1010
Carsi B, Lopez-Lacomba JL, Sanz J, Marco F, Lopez-Duran L (2004) Cryoprotectant permeation through human articular cartilage. Osteoarthr Cartil 12:787–792
Hagedorn M, Hsu EW, Pilatus U, Wildt DE, Rall WR, Blackband SJ (1996) Magnetic resonance microscopy and spectroscopy reveal kinetics of cryoprotectant permeation in a multicompartmental biological system. Proc Natl Acad Sci U S A 93:7454–7459
Sharma R, Law GK, Rekieh K, Abazari A, Elliott JAW, McGann LE, Jomha NM (2007) A novel method to measure cryoprotectant permeation into intact articular cartilage. Cryobiology 54:196–203
Jomha NM, Law GK, Abazari A, Rekieh K, Elliott JAW, McGann LE (2009) Permeation of several cryoprotectant agents into porcine articular cartilage. Cryobiology 58:110–114
Abazari A, Jomha NM, Law GK, Elliott JAW, McGann LE (2009) Erratum to “Permeation of several cryoprotectants in porcine articular cartilage” [Cryobiology 58 (2009) 110–114]. Cryobiology 59:369
Yu H, Al-Abbasi KK, Elliott JAW, McGann LE, Jomha NM (2013) Clinical efflux of cryoprotective agents from vitrified human articular cartilage. Cryobiology 66:121–125
Shardt N, Al-Abbasi KK, Yu H, Jomha NM, McGann LE, Elliott JAW (2016) Cryoprotectant kinetic analysis of a human articular cartilage vitrification protocol. Cryobiology 73:80–92
Lemmon EW, McLinden MO, Friend DG (2008) NIST chemistry webbook, thermophysical properties of fluid systems, NIST standard reference database number 69. https://doi.org/10.18434/T4D303
Jomha NM, Anoop PC, Bagnall K, McGann LE (2002) Effects of increasing concentrations of dimethyl sulfoxide during cryopreservation of porcine articular cartilage. Cell Preserv Technol 1:111–120
Acknowledgments
The articular cartilage research projects referred to in this manuscript were funded by the Edmonton Orthopaedic Research Committee and the Canadian Institutes of Health Research. Janet A. W. Elliott holds a Canada Research Chair in Thermodynamics. Kezhou Wu is funded by the Li Ka Shing Sino-Canadian Exchange Program between the University of Alberta and Shantou University. Nadia Shardt acknowledges funding from the Natural Sciences and Engineering Research Council of Canada (NSERC), Alberta Innovates and Alberta Advanced Education, the Government of Alberta, and the University of Alberta.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Wu, K., Laouar, L., Shardt, N., Elliott, J.A.W., Jomha, N.M. (2021). Osmometric Measurements of Cryoprotective Agent Permeation into Tissues. In: Wolkers, W.F., Oldenhof, H. (eds) Cryopreservation and Freeze-Drying Protocols. Methods in Molecular Biology, vol 2180. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0783-1_11
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0783-1_11
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0782-4
Online ISBN: 978-1-0716-0783-1
eBook Packages: Springer Protocols