Electric heating – Heating devices – With heater-unit housing – casing – or support means
Reexamination Certificate
2002-03-19
2003-11-11
Wallberg, Teresa (Department: 3742)
Electric heating
Heating devices
With heater-unit housing, casing, or support means
C435S001300, C435S284100, C435S285200, C062S063000, C062S065000
Reexamination Certificate
active
06646238
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention concerns a method for sample picking on cryosubstrates, particularly a method for transferring samples in a cryopreserved or thawed condition from a cryosubstrate to a target substrate. The invention also concerns a device for implementing a method of this type and a cryosubstrate which is functionally textured for sample taking.
The operation of cryobanks for preserving biological cell material is generally known in cell biology, molecular biology, and genetic engineering. In a cryobank, the cell material is kept available for decades, with, for example, suspended cells being frozen in small-volume containers (volumes range from 0.1 ml to a few ml) filled with a cryoliquid. In order to ensure the viability of the cell material after thawing, numerous procedures have been developed which, for example, relate to the timing of the thawing, media additives, container shapes, and similar things. With conventional cryobanks, survival rates ranging from a few percent up to 90% are achieved during thawing. Although this is already a relatively good result and cryobanks have found worldwide distribution, the following disadvantages are connected with the cryopreservation procedures disseminated until now.
The position of individual cell material samples in the volume of the cryoliquid is unknown during both the freezing and the thawing procedures. The material samples are not accessible in the preserved, deep frozen state. However, there is interest in, for example, removing single cells from cryopreserved material, measuring, or changing them. However, in order to be able to remove cells, the entire sample must be thawed. This requires costly recultivation of the cell material to compensate for the thawing losses. Over the course of time, the cryopreserved material thus no longer contains only the originally preserved cells, but a mixture of daughter cells of greatly varying generations, which restricts the specificity and reproducibility of cell investigations. In order to subject all material samples in a cryocontainer to the same cooling progression, extremely slow freezing procedures must be provided, since the cooling proceeds from the container walls and all samples in the cryovolume are to experience approximately the same temperature progression over time. Finally, the suspension medium (cryoliquid) prevents or makes more difficult measurement and processing of single cells at low temperatures.
There is an interest in new cryopreservation technologies for overcoming the disadvantages mentioned and for opening new fields for cell preservation, particularly since researches in biotechnology, genetic engineering, and medicine are increasingly directed toward single cells, such as in hybridoma cell production in connection with tumor treatment, stem cell culture, and embryogenesis. The development of new cryopreservation technologies is based on the following knowledge and considerations.
From a physical and physiological viewpoint, a cell frozen at −196° C. is in a solid state. The metabolic processes have come to a complete standstill down to the molecular level. Cell changes only arise through slow restructuring (e.g. through the growth of ice crystals at temperatures above −80° C.) and through damage due to cosmic radiation. The latter, however, has a rate of approximately 90% damage after 30,000 years, which is non-critical for practical applications. In the deep frozen state, cells should therefore able to be measured, treated, changed, sorted and otherwise manipulated in a mechanically robust way without time pressure and with the highest precision. However, this assumes the ability to individually handle the cells in the cryomedium and the availability of tools for cell manipulation.
The physical and chemical procedures during the freezing or thawing of biological materials are, for example, described in the publication of F. Franks “Biophysics and biochemistry of low temperature and freezing”in “Effects of Low Temperatures on Biological Membranes”(Editor G. J. Morris et al., Academic Press, London, 1981) or P. Mazur in “Ann. N.Y. Acad. Sci.”, vol. 541, 1988, p. 514 et seq. The prevention of the formation of intracellular or extracellular ice crystals and excessive dehydration of the cells is decisive for freeze preservation over long periods of time and thawing with the greatest possible survival rate. In this case, the following characteristics are to be taken into consideration from a physical viewpoint during freezing and thawing. Producing so-called vitrified water, in which any type of ice crystal formation is suppressed, through extremely high freezing speeds is known. However, this cannot be used for careful and positionally defined freezing of cell material, since the size of the biological cells of interest and heat conduction restricts the freezing speeds to values below a few ten thousands of degrees per second. Therefore, at the microscopic scale and under physiological conditions, segregation, i.e. formation of eutectic phases, which also include domains of pure ice, can be observed. To minimize the segregation, cell-specific freezing programs have provided the best results, particularly at the beginning of cooling (down to −30° C.), (see also S. P. Leibo et al. in “Cryobiol.”, vol. 8, 1971, p. 447 et seq). In this temperature interval, cooling rates of a few degrees per minute have been shown to be more favorable than rapid temperature jumps. It is inferred from this that the cooling and thawing procedures should be performed with a biologically specific temperature profile over time.
As soon as temperatures at which ice formation begins have been reached, however, higher cooling rates are appropriate, since in this way the migratory growth of larger ice domains at the cost of smaller ones can be prevented. At temperatures below the range of −80° C., no further ice crystal growth occurs, so that cell storage over long periods of time is possible. The storage of the container with cell material which is suspended in a cryoliquid is typically performed in liquid nitrogen (at −196° C.). Since the sample container is closed, there is no direct contact with the liquid coolant phase. Comparable temperature sequences are used for thawing the cell material.
Cooling procedures are also known from preparation for electron microscope recordings (see D. G. Robinson et al. in “Präparationsmethodik in der Elektronenmikroskopie”, Springer-Verlag, Berlin, 1985). In contrast to cryopreservation, which has the goal of maintaining the vitality of the cells, in electron microscopy, the least possible change in the molecular position of the cell components plays the decisive role. Therefore, during this preparation, particularly rapid freezing technologies are realized, which include, for example, shooting the sample into liquid or undercooled gases or spraying drops into an undercooled atmosphere and liquids. In this case, cooling rates of more than 10,000 degrees per second are achieved, which, however, due to the cell volume, the finite thermal conductivity, and the wettability of the material, represent a limiting value.
A general problem in cryopreservation is that not all types of cells can be cryopreserved in the same way. In particular, larger objects (cell groups or the like) or cells containing large numbers of vacuoles, which particularly occur in plant sample material, can be revitalized only with difficulty or not all. The development of new microinjection and cell handling technologies, as well as new cryoprotectives, is directed toward these problems. A technology which is different from the preservation in containers described above is based on the freezing and/or thawing of the cell material to be preserved in adhered form on cooled surfaces (see, for example, T. Ohno et al in “Cryotechnol.”, vol. 5, 1991, p. 273 et seq).
Cryopreservation on cooled surfaces is more difficult to handle than the suspension principle, but has been shown to have advantages in the investigation of the processes
Fuhr Günter
Hagedorn Rolf
Caesar Rivise Bernstein Cohen & Pokotilow Ltd.
Evotec Oai AG
Fuqua Shawntina
Wallberg Teresa
LandOfFree
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