Method for cryopreparing biological tissue

• US 4,865,871 A
• Filed: 07/11/1988
• Issued: 09/12/1989
• Est. Priority Date: 08/23/1983
• Status: Expired due to Term

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Livesey, Stephen A., Linner, John G.
• Primary Examiner(s): LUSIGNAN, MICHAEL R
• Application Number:
  US07/217,561
• Time in Patent Office: 428 Days
• Field of Search: 34/5, 62/64, 62/78, 427/4, 427/250, 435/1
• US Class Current: 435/1.3
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
• CPC Class Codes: A01N 1/0226 Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
• CPC Class Codes: A01N 1/0289 Pressure processes, i.e. using a designated change in pressure over time
• CPC Class Codes: F26B 5/06 the process involving freezing
• CPC Class Codes: G01N 1/42 Low-temperature sample treatment, e.g. cryofixation

Abstract

This invention relates to apparatus for the cryopreparation of biological tissue samples. The method comprises treating a biological tissue sample with a cryoprotectant which raises the glass transition temperature range of the sample followed by vitrification under cryogenic temperature conditions and ultra low vacuum conditions. The depressurized, vitrified tissue sample is brought to equilibrium in a sample holder. The tissue sample is then dehydrated while maintained in a state of thermal nonequilibrium.

Claims

1. A method for cryopreparing a biological tissue sample comprising:

treating a biological tissue sample with a cryoprotectant;

vitrifying the tissue sample at a rate and under conditions so as to preclude the formation of significant resolvable ice crystals;

equilibrating said vitrified tissue sample at temperature and pressure conditions below the glass transition range of said sample

adding energy to said equilibrated tissue sample at a rate sufficient to cause vitrified water in said sample to change physical state directly to the vapor phase without substantially disturbing said equilibrium or completing the induced transition; and

removing water from said tissue sample by molecular distillation by the addition of energy until substantially no vaporized water can be detected in the atmosphere surrounding said tissue sample.

2. A method of preparing a biological tissue sample as defined in claim 1 wherein the cryoprotectant increases the glass transition characteristics of the sample.

3. A method of cryopreparing a biological tissue sample as defined in claim 2 wherein the cryoprotectant is selected from the group consisting of DMSO, dextran, sucrose, 1,2-propanediol, glycerol, sorbitol, fructose, trehalose, hydroxyethyl starch, propylene glycol, and polyvinylpyrolidine.

4. A method for cryopreparing a biological tissue sample as defined in claim 3 wherein the cryoprotectant comprises trehalose.

5. A method for cryopreparing a biological tissue sample as defined in claim 1 wherein the vitrification step comprises rapidly supercooling said tissue sample.

6. A method for cryopreparing a biological tissue sample as defined in claim 5 wherein the vitrifcation step comprises contacting the tissue sample with a supercooled metal block.

7. A method for cryopreparing a biological tissue sample for ultrastructural analysis comprising:

treating a biological tissue sample with a cryoprotectant which increases the glass transition temperature range of the sample;

vitrifying the tissue sample under conditions so as to preclude the formation of significant resolvable ice crystals;

bringing the vitrified tissue sample to equilibrium at a temperature below the glass transition temperature range;

adding energy to said equilibrated tissue sample at a rate sufficient to cause vitrified water in said sample to change physical state directly to the vapor phase without substantially disturbing said equilibrium; and

removing water from said tissue sample by molecular distillation by the addition of energy until substantially no vaporized water can be detected in the atmosphere surrounding said biological tissue sample.

8. A method of preparing a biological tissue sample as defined in claim 7 wherein the cryoprotectant increases the glass transition temperature of the sample.

9. A method of cryopreparing a biological tissue sample as defined in claim 8 wherein the cryoprotectant is selected from the group consisting of DMSO, dextran, sucrose, 1,2-propanediol, glycerol, sorbitol, fructose, trehalose, hydroxyethyl starch, propylene glycol, and polyvinylpyrolidine.

10. A method for cryopreparing a biological tissue sample as defined in claim 9 wherein the cryoprotectant comprises trehalose.

11. A method for cryopreparing a biological tissue sample as defined in claim 7 wherein the vitrification step comprises rapidly supercooling the tissue sample.

12. A method for cryopreparing a biological tissue sample as defined in claim 11 wherein the vitrification step comprises contacting the tissue sample with a supercooled metal block.

13. A method of preparing a biological tissue sample as defined in claim 7 further comprising coating said sample with osmium vapors.

14. A method of preparing a biological tissue sample as defined in claim 7 further comprising infiltrating said sample with a degassed resin followed by polymerization of the resin to form an embedded tissue sample.