Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Reexamination Certificate
1999-05-04
2001-04-17
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
C435S267000, C435S269000
Reexamination Certificate
active
06218101
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a method of removing chemical compounds which protect cells (cryoprotectants) during low temperature preservation (cryopreservation). More particularly, this invention is directed to the use of specific enzymes to convert cryoprotectant compounds into products which are not transported across the cell membrane. The enzymes convert cryoprotectants in solution and thereby work to shift the chemical potential of the cryoprotectant resulting in osmotic flow of cryoprotectants out of the cells and into the enzyme solution.
BACKGROUND OF INVENTION
Fresh biological cells such as sperm, red blood cells, platelets and the like, are typically viable for but a short period of time in vitro. Nevertheless, it is commercially and medically significant that such cells are available for use long after they have been collected from donors, sometimes several months or even years later. Various cryopreservation methods, have been developed to preserve biological cells for these relatively longer periods of time. For example, cryopreservation of sperm cells permits a domestic animal breeder to maintain stocks of valuable sperm cells for use when necessary; it enables the inexpensive transport of such stocks; and it ultimately permits genetically superior males to inseminate a larger number of females. Beyond livestock, artificial insemination is also used in human clinical medicine. As another example, cryopreservation of blood permits donated blood to be stored much longer that the typical 14 day storage period. Moreover, diseases carried in blood with a latency period longer than 14 days may not be discovered in the donor until the blood has been placed into a patient. Cryopreserved blood can be stored for a time sufficient to allow donors to be screened well beyond their date of donation.
The survivability of viable cells and tissues using prior art freezing methods is often quite low. Freezing conditions are relatively harsh and thermal shock or other phenomena such as ice crystal formation often destroy biological cells and tissues. Therefore, maximizing the viability of thawed cells and tissues has been the goal of many researchers.
The prior art discloses various methods for improving the survivability of frozen cells and tissues. In many cases, the cells are removed from their physiological milieu and suspended into artificial tissue culture media prior to preservation. U.S. Pat. No. 4,007,087 to Ericsson discloses a sperm fractionation and storage method which claims to increase the percentage of motile sperm that survive frozen storage. Ericsson discloses a method whereby motile sperm are separated from non-motile, defective or dead sperm. The fraction containing the motile sperm is then frozen. Ericsson reports that his method increases the fertility of a sperm sample by enhancing the environmental (the ratio of total sperm to motile sperm) and the viability (progressiveness of motility of the motile sperm) factors affecting the fertility of a sample, but his method does not improve the population (motile sperm count) factor which is possibly the most critical.
U.S. Pat. No. 3,791,384 to Richter et al. discloses a method for deep freezing and thawing boar sperm which includes inactivating the fresh sperm by means of an inactivating solution that includes dextrose, dihydrate of ethylenedinitrotetra-acetic acid, sodium hydrogencarbonate. Reicter reports that inactivation of the sperm gives them a greater power of resistance to freezing.
U.S. Pat. No. 4,429,542 to Sakao et al., U.S. Pat. No. 4,487,033 to Sakao et al., U.S. Pat. No. 3,893,308 to Barkay at al., and U.S. Pat. No. 4,480,682 to Kameta et al., all disclose different freezing methods which claim to improve the fertility of sperm samples. In all of these methods, the temperature of sperm in solution is lowered by various means which attempt to reduce the thermal shock and increase the survivability of the viable cells present. Most of these methods are, however, complex, cumbersome and expensive to utilize. Other freezing methods are also used including the method of rapidly freezing in liquid nitrogen vapors (Sherman J K, Improved Methods of Preservation of Humans Spermatozoa by Freezing and Freeze Drying, Fert. Steril. 14: 49-64, 1963), and the method of gradual freezing (Behrman et al., Heterologous and Homologous Insemination with Human Semen Frozen and Stored in a Liquid Nitrogen Refrigerator, Fert. Steril., 17: 457-466, 1966).
A disadvantage of the aforementioned methods resides in that low temperature preservation of the cells and tissues is accomplished by the ice crystallization process. As ice forms in the solution surrounding the cells or tissues, electrolytes and other solutes become progressively concentrated, quickly reaching concentrations which are damaging to the cells. This solute damage is attenuated by the addition of cryoprotectant chemicals such as glycerol, propylene glycol, ethylene glycol or dimethylsulfoxide. However, the cryoprotectants themselves can cause osmotic damage to the cell during their addition and removal. During cryoprotectant addition the cells and tissues undergo shrinkage and during removal they undergo swelling. The cryoprotectant removal process and associated cell swelling is particularly damaging. It is also the final step in the series of steps involved in the cryopreservation process and the one most often carried out in a clinical setting (e.g., operating room or emergency room). Therefore the process used for cryoprotectant removal must: (1) provide relatively rapid removal of the cryoprotectant, (2) provide a “closed” system to avoid potential contamination of the preparation, (3) be relatively simple to conduct and (4) require minimal specialized laboratory equipment. The present invention addresses each of these needs.
SUMMARY OF INVENTION
The present invention relates to a method of removing chemical compounds which protect cells (cryoprotectants) during low temperature preservation (cryopreservation). Typically such cryoprotectants are small molecular weight molecules such as glycerol, propylene glycol, ethylene glycol or dimethyl sulfoxide (DMSO). More particularly, this invention is directed to the use of specific enzymes to convert the cryoprotectant compounds into forms which cannot move across the cell membrane and enter the cell (are plasma membrane impermeable). Therefore, when applied to the cells, the enzymatic activity will shift the chemical potential of the cryoprotectants inside and outside the cells, causing the cryoprotectants to flow out of the cell, in turn become enzymatically modified, and thereby removed from the cells. The rate of conversion can be controlled.
This approach differs fundamentally from all previous methods in that it utilizes enzymes to effect cryoprotectant removal thereby minimize osmotic damage due to cell swelling and thus enhances the viability of cryopreserved cell populations.
REFERENCES:
patent: 3791384 (1974-02-01), Richter et al.
patent: 3893308 (1975-07-01), Barkay et al.
patent: 4007087 (1977-02-01), Ericsson
patent: 4429542 (1984-02-01), Sakao et al.
patent: 4480682 (1984-11-01), Kaneta et al.
patent: 4487033 (1984-12-01), Sakao et al.
patent: 5700632 (1997-12-01), Critser et al.
Thorner, J. and Paulus, H., “Catalytic and Allosteric Properties of Glycerol Kinase fromEscherichia coli,” Jun. 10, 1973,J. Biol. Chem., vol. 248, No. 11, pp. 3922-3932.
Sherman, J.K., “Improved Methods of Preservation of Human Spermatozoa by Freezing and Freezing-Drying,”Fertility&Sterility, vol. 14, No. 1, pp. 49-64, 1963.
Behrman, et al., “Heterologous and Homologous Inseminations with Human Semen Frozen and Stored in a Liquid-Nitrogen Freezer,”Fertility&Sterility, vol. 17, No. 4, pp. 457-466, 1966.
Willoughby, et al., “Osmotic Tolerance Limits and Properties of Murine Spermatozoa,”Biol. of Reproduction, vol. 55, pp. 715-727, 1996.
Gilmore, et al., “Determination of optimal cryoprotectants for their addition and removal from human spermatozoa,”Human Reproduction, vol. 12, No. 1, pp. 112-118, 19
Critser John K.
Woods Erik J.
Barnes & Thornburg
General Biotechnology, LLC
Saucier Sandra E.
LandOfFree
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