Interspecies Challenges

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Eugenia Spencer
5 years ago
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1 Cryobiological Challenges of Banking Reproductive Cells, and Tissues

Domestic species Wild species Fish Lab animal species

2 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

3 Reproductive Cells and Tissue Types Cells: Spermatozoa Oocytes Embryonic Stem Cells One-Cell Embryos Somatic Cells (SCNT) Tissues Multicellular Embryos Ovarian Tissue Testicular Tissue

sensitivity High Intracellular lipid Embryos Large Size High

4 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

5 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

6 Using A Fundamental Cryobiology Approach To Develop New/Better Cryopreservation Methods slow cooling rapid cooling

7 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)

8 Interactions Cell Specific Characteristics Cold shock Chilling Sensitivity CPA Selection and Addition Osmotic Characteristics/ CPA/Lp Values Cooling Rate Warming Rate CPA Removal

9 Freezing survival of different cell types at various cooling rates Mouse Embryos Stem Cells Hamster Cells RBC 50 Survival (%) Yeast Cooling Rate ( o C/min) Redrawn from Mazur, 1977.

10 Intracellular Ice Formation Mouse Oocytes Cooled at 100 o C/min Critser et al., unpublished data

11 Freezing survival of different cell types at various cooling rates Mouse Embryos Stem Cells Hamster Cells RBC 50 Survival (%) Yeast Cooling Rate ( o C/min) Redrawn from Mazur, 1977.

12 Damaging solute concentrations Critser et al., unpublished data

13 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

14 Cryoprotectant agents cause osmotically driven volume excursions From Pfaff et al. 2001

15 CPA Dilution: Osmotic Tolerance

16 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

17 Determination of membrane permeability coefficients

18 Water and CPA permeability measurements of human spermatozoa From Gilmore et al., 1995

19 CPA Dilution From Gilmore et al., 1995

20 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

21 From Gilmore et al., 1995 Modeling cell volume excursions

22 Modeling optimal CPA addition and removal procedures From Gao et al., 1993

23 Cooling, Warming and Volume Changes From Gilmore et al., 1995

25 Cell Volume Response

26 From: Men et al., 2006 Embryos

27 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

28 Pig Blastocyst Osmotic Tolerance Limits 100 Blastocysts with intact morpholo h 18 h Osmolality (mosm) Blastocysts with intact actin cytoskeleton (%) h* 18h* Osmolality (mosm) Men et al., 2005

29 Embryo Development Stage and CPA Removal

30 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

31 Lipid Removal From Hara et al., Cryobiology, 2005

32 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 (%) F- and vitrified F+ and vitrified Unfrozen control Full cavity Partial Cavity Total Men et al., 2005

33 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.

34 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

35 Intracellular Lipid Courtesy of Heide Schatten

36 Lipid Droplets and Endoplasmic Reticulum From: Martin and Parton, Nature Reviews 2006

37 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

38 Crystallization and Vitrification 1.1 mm From: Arav et al., 2002 Partial Crystallization

39 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

40 Heat Transfer Modes Vial conduction and natural convection Liquid Nitrogen boiling (convection) Analyze heat transfer process on both sides of vial boundary

41 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: W/m 2 K Pool Boiling: ~10 3 W/m 2 K (e.g. LN 2 plunging) Forced Flow Boiling: ~ W/m 2 K

42 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) 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 Sample Dimension (cm) Pool Boiling Forced Flow Boiling New Technology

43 Thank you

Cryopreservation of human oocytes with slow freezing techniques

ESHRE Campus Symposium Cryobiology and cryopreservation of human gametes and embryos Athens, Greece 25-26 September 2009 Cryopreservation of human oocytes with slow freezing techniques Giovanni Coticchio