Chemistry: molecular biology and microbiology – Differentiated tissue or organ other than blood – per se – or... – Including freezing; composition therefor
Reexamination Certificate
1999-10-06
2001-10-16
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Differentiated tissue or organ other than blood, per se, or...
Including freezing; composition therefor
C435S002000, C435S283100
Reexamination Certificate
active
06303285
ABSTRACT:
The invention relates to an apparatus and method for freezing live cells, in particular sperm, according to the preamble of claim
1
. Such known apparatus is known from EP 0 117 037.
Apparatus for freezing live cells, in particular for freezing sperm, are used for storing cells in living condition for later use. To that end, a sample comprising for instance a number of sperm cells in a liquid solution is introduced into a container and cooled so that freezing occurs. In this specification, the apparatus and method will be described on the basis of sperm samples, yet the invention should not be construed as being limited thereto. Many other live cells can be treated in a like manner or with like means, with like advantages.
A known apparatus comprises a vessel into which liquid nitrogen can be introduced, for cooling the contents of the vessel. In samples of a slight volume, the sperm is introduced into a straw or a like relatively thin, tubular container, after which a large number of filled containers are simultaneously introduced into the vessel and cooled. When complete freezing of the contents has taken place, the containers are removed from the vessel and stored at a storing temperature, suitable for later use for, for instance, artificial insemination.
The temperature change occurring in the vessel and in the containers is substantially determined by the type and temperature of the containers when being introduced into the vessel, the number of containers and the temperature in the vessel when the containers are being introduced therein. Moreover, research by applicant has shown that the heat development in the containers resulting from the crystallization occurring therein contributes significantly to the change of the temperature in the containers. This change proves to be of particular importance for the result of the freezing of the sample, in particular for the chances of survival and the vitality of the cells after passing through the method and a subsequent thawing. The above research has demonstrated that in this respect, in particular the temperature change in the sample during the occurrence of crystallization is of great importance.
In the known apparatus, it is not possible to accurately control the change of the temperature in the separate containers. During the use of such apparatus, relatively great differences will occur in the change of temperature in the different containers, for instance due to the position of the containers relative to each other and to the vessel and due to differences in the presence of crystallization nuclei in the sample and, accordingly, differences in the phase in which crystallization occurs. Due to the fact that when a known apparatus is employed for freezing for instance one ejaculate, from which a very large number of containers can be filled, these influences cannot be removed or controlled, such apparatus is economically not very advantageous and the usability of each sample is adversely affected.
EP 0 117 037 discloses an apparatus for freezing live cells, such as fertilized ova, spermatozoa and the like, comprising a cylindrical outer wall connected to a disk shaped bottom plate through which cooling channels extend. Centrally, concentric within said cylindrical outer wall a cylindrical inner wall has been provided which is thermically isolated from said bottom plate by an insulating ring. The lower part of the inner cylinder is made of copper and comprises a second cooling channel, spiralling around a heating element. Between said cylindrical inner wall and said cylindrical outer wall a ring shaped space extends into which straw like tubes, containing the live cells to be frozen, can be positioned. Within the upper part of the inner cylindrical wall an open container has been provided containing liquid nitrogen for containing the holders with frozen samples. Control means have been provided for supplying and discharging a cooling liquid such as nitrogen to the specific cooling channels and for controlling said heating element. Temperature sensors have been provided in the bottom plate and the ring shaped holding space. The object of this known apparatus is to execute a method according to Japanese patent application No. 124996/1981 of the same applicant, in which method crystallization nuclei has to be achieved within a buffer solution, distanced from the living cells therein, such as to prevent thermical shock to said living cells.
This known apparatus can only contain a limited number of containers, which containers have to be prepared such that a space containing only buffer solution, no live cells, is provided near the lower end, for initiating freezing. With such known apparatus freezing of live cells will therefore be time consuming and costly.
The object of the invention is to provide an apparatus for freezing live cells, in particular sperm, in which the above drawbacks of the known apparatus are avoided, while the advantages thereof are maintained. To that end, an apparatus according to the invention is characterized by the features of claim
1
.
In this specification, temperature gradient should be interpreted as a spatial change of the temperature (°C./cm). Where a temperature change in time is meant, this change will be referred to as temporal gradient, cooling rate, variation in temperature or a like, time-related term (°C./min).
Because of the or each contact face to be cooled, by means of which the or each container, by resting thereon, can be cooled, the advantage is achieved that a proper control of the temperature of the or each container, and accordingly of the or each sample, can be realized regardless of the number of containers to be cooled. The control means provide the possibility of accurately controlling the temperature and the change thereof during the entire cooling or freezing path over which the containers are moved for providing for the desired temporal gradient within the containers. As a result of the accurate control of the temperature, in particular the change thereof during the freezing path, more cells survive freezing and the subsequent thawing, while, moreover, the surviving cells have a better vitality. An important additional advantage is that due to the greater percentage of surviving, more vital cells, fewer sperm cells have to be included into a sample, while the same sample quality is maintained for use in artificial insemination. This means that a greater dilution of an ejaculate can be carried out, as a result of which more containers can be filled per ejaculate. This means that a better economical efficiency is obtained and that, moreover, at least as far as sperm is concerned, more female animals can be fertilized from one ejaculate, which has for instance operational advantages to the owner or at least the holder of both the male and the female animal.
Moreover, the chance of a successful insemination with a sample from an ejaculate which in the starting situation has very few vital cells is greater, which is of substantial importance in particular with respect to, for instance, humans, particular species of animals, and the like.
During cooling, ice formation within the cells (intracellular ice formation) should be prevented in particular near the freezing point or path of the solution, because this is fatal to the cells in question. The cause of the prevention thereof is an efflux of water from the cell, i.e. water flowing away through the cell membrane to the environment. As a consequence, the water concentration in the cell decreases, i.e. the proportion of water in the cell volume becomes less, so that the freezing point falls.
In the case of unduly high cooling rates, the efflux of water will take place very rapidly, which in itself already seems unfavorable for the cells. Moreover, there is a substantial chance that the water efflux, relative to the cooling rate, does not take place rapidly enough, i.e. the freezing point falls less quickly than the temperature. As a consequence, the temperature falls far below the freezing point, so that intracellular ice is fo
Lankford , Jr. Leon B.
Stichting Dienst Landbouwkundig Onderzoek
TraskBritt
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