Chemistry: molecular biology and microbiology – Differentiated tissue or organ other than blood – per se – or... – Including freezing; composition therefor
Patent
1997-08-29
1999-07-20
Redding, David A.
Chemistry: molecular biology and microbiology
Differentiated tissue or organ other than blood, per se, or...
Including freezing; composition therefor
435 405, 4352841, 4352864, 4352873, 4352887, A01N 102
Patent
active
059255110
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method and an apparatus for cryopreserving microscopic objects, more particularly biological objects, single cells, cell formations or cell components or macromolecules fixated in accordance with predefinable temperature programs in two or three-dimensional patterns, changing these in the cryogenic condition and rethawing same with retention of cell vitality.
It has been common practice since decades in medical engineering, in setting up cell banks and in biological/pharmaceutical research to freeze cells and rethaw them according to given procedures without the living processes being irreversibly destroyed in at least part of the cryopreserved material. For this purpose the cells are mostly suspended in a solution and frozen in microtubes (R. Ian Freshney: "Tierische Zellkulturen", Walter de Gruyter 1990, page 221). The solutions are provided with substances which affect ice crystal formation (so-called cryopreservants such as e.g. dimethylsulfoxide, glycerine). These techniques have a proven record of success and result in survival rates of between a few percent and practically 100% when applied properly. Since in very many traditional applications the cell material after thawing first needs to be incorporated in a culture and strongly reproduced the survival rate following this kind of cryopreservation plays a minor role, this necessitating, however, that sufficient cells are available.
There is an increasing tendency for this no longer to be the case, however, in view of the methods in obtaining cells in medicine, genetics or molecular biology becoming more and more selective. One example is the designed genetic change of living cells which only produces the desired result in the case of few cells. By means of screening methods these cells are isolated and are available for further applications. One such application is hybridomas cell procurement (B. Gustafsson, "Cryopreservation of Hybridomas", in "Methods in Molecular Biology", Vol. 5, Eds. J. W. Pollard and J. M. Walker, 619-621) as is necessary and usual in the production of monoclonal antibodies e.g. for cancer therapy in which as a rule only very few cells from cell pairs (lymphocyte, myeloma cell) are formed and cryopreserved. It is especially in view of these low cell numbers as usual in this case that cryopreservation is possible only to a limited extent (R. Ian Freshney: "Tierische Zeilkulturen", Walter de Gruyter 1990, page 221) since major difficulties arise in handling the resulting small amounts of solution and reobtaining the cells. Furthermore, identifying cells individually by suspension techniques is not possible which, however, is necessitated by the aforementioned methods.
One particular drawback in freezing cells in cell suspensions is the gradient of the temperature distribution in the solution since the freezing conditions for the single cell are not known, or differ greatly due to their position in the suspension being undefined and failing to remain constant (M. J. Ashwood-Smith and J. Farrant, "Low Temperature Preservation in Medicine and Biology", Kent, England (1980)). Reproducing the conditions and thus standardization is, however, becoming more and more important for continuing work on cryopreserving single cells.
One modified kind of cryopreservation, especially for adhering cells is the preservation of cells grown on a surface (T. Ohno, "A simple method for in situ freezing of anchorage-dependent cells", in: A. Doyle, J. B. Griffiths, D. G. Newell, Cell and Tissue Culture, John Wiley & Sons, page 4C: 2.1). For this purpose, as a rule, thin cell layers, so-called cell monolayers are grown on suitable substrates (treated glass or plastic surfaces) and subsequently frozen and thawed as cell lawns. This method has the drawback that cell growth occurs randomly and a single cell in this case too, is difficult to be localized beyond the cryogenic time period. Since hitherto so-called microtiter plates have been used for growth, here too, no locally defined conditions can be achieved. On top of this th
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Fuhr Gunter
Hagedorn Rolf
Hofmann Ulrich
Hornung Jan
Howitz Steffen
Fraunhofer Gesellschaft Zur Fordereung Der Angeweandten Forschun
Kinsella N. Stephan
Murray William H.
Redding David A.
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