A method and system for cryopreservation to achieve uniform viability and biological activity

a cryopreservation and biological activity technology, applied in the field of cryopreservation process, can solve the problems of cell damage during cryopreservation, significant loss of cell viability, and 80% or more loss of cell activity and viability

Inactive Publication Date: 2013-05-09
PRAXAIR TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0023]The invention may be characterized as a method of controlling a chilling or freezing process of biological material disposed in a plurality of containers, comprising the steps of: (i) placing said plurality of containers of said biological materials in a cooling area defined as an area between parallel porous surfaces within a cooling chamber; (ii) mixing a liquid cryogen with a warmer gas to produce a cold cryogenic gas at a selected temperature profile, said temperature profile corresponding to a desired cooling rate of said biological materials within said cont

Problems solved by technology

Previous attempts to freeze biological materials, such as living cells often results in a significant loss of cell viability and in some cases as much as 80% or more loss of cell activity and viability.
Cell damage during cryopreservation usually occurs as a result of intracellular ice formation within the living cell during the freezing step or during subsequent recrystallization.
Rapid cooling often leads to formation of more intracellular ice since water molecules are not fully migrated out of the cell during the short timeframe associated with the rapid cool-down rates.
If too much water remains inside the living cell, damage due to initial ice crystal formation during the rapid cooling phase and subsequent recrystallization during warming phases can occur and such damage is usually lethal.
On the other hand, slow cooling profiles during cryopreservation often results in an increase in the solute effects where excess water is migrated out of the cells.
Excess water migrating out of the cells adversely affects the cells due to an increase in osmotic imbalance.
Thus, cell damage occurs as a result of osmotic imbalances which can be detrimental to cell survival and ultimately lead to cell damage and a loss of cell viability.
Such equipment, however, is only suitable for relatively small volume capacities and is not suitable for commercial scale production and preservation of biological materials such as therapeutic cell lines.
Such existing controlled rate freezers, including the Kryo 1060 series, also suffer from the non-uniformity in cooling vial to vial due, in part, to the non-uniform flow of cryogen within the freezers and the requirement for close packing of the vials within the freezer.
The size of individual conventional freezers is limited due to these non-uniform effects.
As conventional controlled rate freezers are scaled up in size, the non-uniformities in cooling increase.
Consequently, the size of conventional controlled rate freezers must be limited to prevent non-uniform sample-to-sample properties due to non-uniform cooling of each sample.
Such convection based cooling or freezing systems cannot achieve temperature uniformity as the vials are often located at various distances from the internal fan or packed in the shadow of other vials or trays.
However, it is impossible to provide a uniform conductive surface area on the bottom of each glass vial since most glass vial bottoms are concave.
Therefore, temperature variations during the freezing process from vial to vial are the biggest drawback for these types of equipment.
Furthermore, the coo

Method used

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  • A method and system for cryopreservation to achieve uniform viability and biological activity
  • A method and system for cryopreservation to achieve uniform viability and biological activity
  • A method and system for cryopreservation to achieve uniform viability and biological activity

Examples

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Effect test

example 1

Controlling the Nucleation Temperature

[0172]Four separate vials were filled with 2.5 mL of 5 wt % mannitol solution. The predicted thermodynamic freezing point of the 5 wt % mannitol solution is approximately −0.5° C. The four vials were placed on a freeze-dryer shelf in close proximity to one another. The temperatures of the four vials were monitored using surface mounted thermocouples. The freeze-dryer was pressurized with argon to 14 psig.

[0173]The freeze-dryer shelf was cooled to obtain vial temperatures of between approximately −1.3° C. and about −2.3° C. (+1° C. measurement accuracy of the thermocouples). The freeze-dryer was then depressurized from about 14 psig to about atmospheric pressure in less than five seconds to induce nucleation of the solution within the vials. All four vials nucleated and began freezing immediately after depressurization. Results are summarized in Table 1 below.

[0174]As seen in Table 1, the controlled nucleation temperatures in this example (i.e., ...

example 2

Controlling the Nucleation Temperature

[0175]In this example, ninety-five vials were filled with 2.5 mL of 5 wt % mannitol solution. The thermodynamic freezing point of the 5 wt % mannitol solution is approximately −0.5° C. The ninety-five vials were placed on a freeze-dryer shelf in close proximity to one another. The temperatures of six vials positioned at different locations in the freeze-dryer shelf were continuously monitored using surface mounted thermocouples. The freeze-dryer was pressurized in an argon atmosphere to about 14 psig. The freeze-dryer shelf was then cooled to obtain vial temperatures of near −5° C. The freeze-dryer was then depressurized from about 14 psig to about atmospheric pressure in less than five seconds to induce nucleation of the solution within the vials. All ninety-five vials were visually observed to nucleate and begin freezing immediately after depressurization. Thermocouple data for the six monitored vials confirmed the visual observation. The resu...

example 3

Controlling the Depressurization Magnitude

[0177]In this example, multiple vials were filled with 2.5 mL of 5 wt % mannitol solution. Again, the predicted thermodynamic freezing point of the 5 wt % mannitol solution is approximately −0.5° C. For each test run, the vials were placed on a freeze-dryer shelf in close proximity to one another. As with the earlier described examples, the temperatures of vials were monitored using surface mounted thermocouples. The argon atmosphere in the freeze-dryer was pressurized to differing pressures and the freeze-dryer shelf was cooled to obtain vial temperatures of about −5° C. In each test run, the freeze-dryer was then rapidly depressurized (i.e., in less than five seconds) from the selected pressure to atmospheric pressure in an effort to induce nucleation of the solution within the vials. Results are summarized in Table 3.

[0178]As seen in Table 3, the controlled nucleation occurred where the pressure drop was about 7 psi or greater and the nuc...

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PUM

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Abstract

A method and system for controlled rate freezing and nucleation of biological materials is provided. The presently disclosed system and method provides the ability to rapidly cool the materials contained in vials or other containers within a cooling unit via forced convective cooling and optionally simultaneous pressure drop using uniform and unidirectional flow of cryogen in proximity to the plurality of vials disposed within a cooling unit. The rapid cooling of the biological materials is achieved by precisely controlling and adjusting the temperature of the cryogen being introduced to the system as well as the chamber pressure as a function of time.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present invention is a continuation-in-part application of U.S. patent application Ser. No. 12 / 266,760 filed Nov. 7, 2008 and also claims priority from U.S. provisional patent application Ser. No. 61 / 480,647 filed Apr. 29, 2011 the disclosure of both applications are also fully incorporated by reference herein.FIELD OF THE INVENTION[0002]The present invention broadly relates to a cryopreservation process, and more particularly, to a method and system for providing controlled rate freezing and nucleation control of biological materials to minimize cell damage resulting from intercellular ice formation and solute effects that arise during the cryopreservation process.BACKGROUND[0003]Cryopreservation is a process used to stabilize biological materials at very low temperatures. Previous attempts to freeze biological materials, such as living cells often results in a significant loss of cell viability and in some cases as much as 80% or mo...

Claims

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Application Information

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IPC IPC(8): F25D13/00F25D3/10
CPCF25D13/00F25D3/10F25D29/001F25D3/102A01N1/0252A01N1/0257F25D2600/06B01L1/025B01L7/50B01L2300/0829B01L2300/14B01L2300/1838
Inventor GRINTER, NIGEL J.CHENG, ALAN T.ZHOU, YING
Owner PRAXAIR TECH INC
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