Cryobiological Challenges of Banking Reproductive Cells, and Tissues
Interspecies Challenges Mammals Domestic species Lab animal species Endangered Species Humans (Reproductive Med) Birds Domestic species Wild species Fish Lab animal species Commercial species Wild species Mollusks Commercial species Others
Reproductive Cells and Tissue Types Cells: Spermatozoa Oocytes Embryonic Stem Cells One-Cell Embryos Somatic Cells (SCNT) Tissues Multicellular Embryos Ovarian Tissue Testicular Tissue
Challenges: Cell Morphology Spermatozoa Non-spherical cell type Small Size Chilling Sensitivity Oocytes Large size MII spindle sensitivity High Intracellular lipid Embryos Large Size High intracellular lipid Chilling Sensitivity Somatic Cells Varies widely
Genetic Challenges Maintaining genetic diversity in outbred populations Maintaining genetic integrity in inbred lines Limits the choice of germplasm format for banking Haploid cells Diploid cells
Using A Fundamental Cryobiology Approach To Develop New/Better Cryopreservation Methods slow cooling rapid cooling
What s the Advantage? Enables one to understand the mechanisms related to success or failure Allows one to determine the sensitivity or robustness of each step in a given procedure Provides a context for QC/QA troubleshooting procedures Facilitates optimization of different steps in the process (processing vs. end-user)
Interactions Cell Specific Characteristics Cold shock Chilling Sensitivity CPA Selection and Addition Osmotic Characteristics/ CPA/Lp Values Cooling Rate Warming Rate CPA Removal
Freezing survival of different cell types at various cooling rates 70 60 Mouse Embryos Stem Cells Hamster Cells RBC 50 Survival (%) 40 30 20 Yeast 10 0 0.1 1 10 100 1000 10000 Cooling Rate ( o C/min) Redrawn from Mazur, 1977.
Intracellular Ice Formation Mouse Oocytes Cooled at 100 o C/min Critser et al., unpublished data
Freezing survival of different cell types at various cooling rates 70 60 Mouse Embryos Stem Cells Hamster Cells RBC 50 Survival (%) 40 30 20 Yeast 10 0 0.1 1 10 100 1000 10000 Cooling Rate ( o C/min) Redrawn from Mazur, 1977.
Damaging solute concentrations Critser et al., unpublished data
Concentrations of salts produced in the unfrozen portions of aqueous solutions of glycerol in saline as a function of temperature From Rall et al., 1976
Cryoprotectant agents cause osmotically driven volume excursions From Pfaff et al. 2001
CPA Dilution: Osmotic Tolerance
Cell Parameters Necessary for Modeling Cell Responses Cell Volume Occupied by Water and Cell Solids (V b ) Water Permeability (L p ) Solute Permeability (ω) Surface Area
Determination of membrane permeability coefficients
Water and CPA permeability measurements of human spermatozoa From Gilmore et al., 1995
CPA Dilution From Gilmore et al., 1995
H 2 O CPA H 2 O CPA H 2 O H 2 O H 2 O CPA CPA H 2 O H 2 O H 2 O H 2 O CPA CPA CPA H 2 O H 2 O CPA H 2 O
From Gilmore et al., 1995 Modeling cell volume excursions
Modeling optimal CPA addition and removal procedures From Gao et al., 1993
Cooling, Warming and Volume Changes From Gilmore et al., 1995
Cell Volume Response
From: Men et al., 2006 Embryos
Pig Blastocyst Osmotic Tolerance Limits Figure 2. Blastocysts with a. Intact actin filaments; b. Partially disrupted actin filaments c. Completely disrupted actin filaments; d. Cytochalasin treated Men et al., 2005
Pig Blastocyst Osmotic Tolerance Limits 100 Blastocysts with intact morpholo 100 90 80 70 60 50 40 30 20 10 0 0h 18 h 75 210 225 285 600 1200 2400 Osmolality (mosm) Blastocysts with intact actin cytoskeleton (%) 90 80 70 60 50 40 30 20 10 0 0h* 18h* 75 150 210 285 600 1200 2400 Osmolality (mosm) Men et al., 2005
Embryo Development Stage and CPA Removal
Intracellular Lipid (1) Immature oocyte (2) Oocyte after 44 hrs maturation (3) 2 Cell embryo (4) blastocyst. All oocytes/embryos were stained with Sudan IV From: Takano and Niimura, J Mamm. Ova Res., 2004
Lipid Removal From Hara et al., Cryobiology, 2005
In Vitro survival of vitrified porcine embryos after being cultured with or without forskolin (F + or F - ) for 24 h before vitrification Percentage of blastocysts (%) 100 90 80 70 60 50 40 30 20 10 0 F- and vitrified F+ and vitrified Unfrozen control Full cavity Partial Cavity Total Men et al., 2005
Advantages of Our Approach Can easily treat large number of embryos; Does not require expensive equipments; Does not damage the zona pellucida reduce the change of pathogen transmission; Can be applied to various types of cells rich in intracellular lipids, for example, adipocytes.
Limitations of Our Approach Because this approach is based on stimulated lipid metabolism; therefore: It takes time to reduce the lipid contents; It is a reversible process; Only can partially reduce the contents of intracellular lipids in embryonic cells
Intracellular Lipid Courtesy of Heide Schatten
Lipid Droplets and Endoplasmic Reticulum From: Martin and Parton, Nature Reviews 2006
What about Vitrification of Cells and Tissues? Advantages No mass transfer issues during cooling Disadvantages Requires very high CPA concentrations Requires very high cooling and warming rates
Crystallization and Vitrification 1.1 mm From: Arav et al., 2002 Partial Crystallization
Factors to Engineer for Vitrification Size of the sample Ovarian tissue + medium+ container Solution characteristics ([CPA]) Determines vitrification tendencies Heat transfer coefficient of the sample Governs cooling and warming rates
Heat Transfer Modes Vial conduction and natural convection Liquid Nitrogen boiling (convection) Analyze heat transfer process on both sides of vial boundary
Heat Transfer Coefficient: What does it mean? Origin is Newton s law of cooling: The dissipation of heat from a solid to a fluid is proportional to the temperature difference between the solid and fluid T a h Ts Moving Fluid q = ha(t s T a ) Some Typical Values: Natural Convection in Water: ~10 2 W/m 2 K Forced Convection in Water: 10 3-10 4 W/m 2 K Pool Boiling: ~10 3 W/m 2 K (e.g. LN 2 plunging) Forced Flow Boiling: ~10 4-10 5 W/m 2 K
Predictions Use of forced LN 2 flows for cortical strips with ~6M CPA Use of ultra-fast cooling methods for cortical strips with ~5M CPA Required CPA for vitrification (M) 11 10 9 8 7 6 5 The effect of sample size on the required CPA concentration h=10 3 W/m 2 K h=10 4 W/m 2 K h=10 5 W/m 2 K h=10 6 W/m 2 K 4 0.0.2.4.6.8 1.0 Sample Dimension (cm) Pool Boiling Forced Flow Boiling New Technology
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