Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of storing cells in a viable state
Reexamination Certificate
1999-07-15
2002-06-04
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of storing cells in a viable state
C435S383000, C435S325000
Reexamination Certificate
active
06399375
ABSTRACT:
INTRODUCTION
The present invention relates to a method and an apparatus for cultivation of cells and tissues. In particular the invention relates to in vitro cultivation, e.g. maturation, fertilization, growing, propagation, production and/or maintenance of cells and tissues, especially sensitive cells and tissues such as e.g. oocytes and embryos and other sensitive cells and tissues derived from multicellular organisms as well as certain sensitive bacteria, yeasts, fungi, molds and mucors which can advantageously be produced or propagated by the method and the apparatus of the present invention. Besides, genetically modified cells and tissues of the above origin, in particular such stemming from multicellular organisms like mammals and other warm-blooded animals, can be cultured by the method and apparatus of the present invention. Furthermore, the use of such cells and tissues for producing particular desired compounds and materials can also be effected by the method and apparatus of the present invention.
DESCRIPTION OF THE PRIOR ART
In vitro culture, e.g. maturation, fertilization, growth, propagation, production and/or maintenance, of certain sensitive cells and tissues, in particular such stemming from multicellular organisms like oocytes and preimplantation embryos, but also certain bacteria, yeasts, fungi, molds and mucors, and genetically modified cells and tissues, in particular cells and tissues stemming from multicellular organisms like mammals and other warm-blooded animals, require highly stable and constant environments in order to be successful.
Thus, the culture medium must contain at least water, salts, nutrients, essential amino acids, vitamins, hormones and possibly proteins and growth factors in well defined proportions and levels as well as a buffer system establishing a strict defined narrow pH-range and a source of oxygen.
Usually, the buffer system is a CO
2
/bicarbonate buffer system, the bicarbonate being incorporated into the aqueous culture medium as from the start of the culturing period but may optionally be supplemented during the culture period from time to time, whereas the CO
2
(carbon dioxide) is provided from the atmosphere surrounding the culture medium during the culture period. The source of oxygen is usually gaseous free oxygen (O
2
) which is also supplied from the surrounding atmosphere to the culture medium. Besides, the culture temperature should be kept within rather narrow limits in order to obtain optimum and/or successful results.
Small scale in vitro production, i.e. maturation, fertilization, growth, propagation etc. as mentioned above, of sensitive cells and tissues is usually effected in receptacles like small culture flasks, petri- or well-dishes provided with the culture medium and the necessary initial cells or tissues. The receptacles are then placed in an incubator which provide for a selected constant temperature and an environing atmosphere containing the gases necessary for development and/or maintenance of the particular cells or tissues concerned. In particular the necessary gases comprise humidity (i.e. water vapor), free oxygen (O
2
) and carbon dioxide (CO
2
) in specific proportions and levels.
Up to now the incubators used for the above production purposes have been provided in the form of cases or cabinets provided with insulating thermostate jackets, optionally one or more shelves for supporting the culture receptacles and dividing partially the interior volume of the incubator in two or more compartments, a (usually) vertically hinged front door providing access to the interior of the incubator, and one or more inlets and outlets for the gas or gas mixture to be supplied to and released from (respectively) the interior of the incubator. Such incubators may have a work space, i.e. an interior volume, of from as small as about 30-50 liters (except for some special types mentioned below) to as high as about 350 liters and even more and may be provided with sensors and control equipments for maintaining the temperature, humidity, carbon dioxide, oxygen and nitrogen at preselected levels in the interior of the incubator. The insulating thermostate jacket may be filled with water in order to provide easy and even delivery of heat to all points of the walls of the incubator and to avoid the risk of overheating which can be very harmful to the cell cultures in the incubator.
However, each time the front door of the incubator is opened during a working-day in order to inspect or remove a culture receptacle already placed in the incubator or to place a new culture receptacle in the incubator, all the parameters, i.a. the above mentioned, of the interior of the incubator and of all the culture receptacles therein are disturbed. The sensors and control equipments may restore the parameters in the interior volume of the incubators at the preset levels within about 10 minutes or so, but in the culture receptacles and media the restoration of the preselected levels of the parameters concerned may take much longer time. Furthermore, in as much as the front door of the incubator may be opened and closed many times during a working-day the cumulative effect on the development, growth and propagation of the cultured cells and tissues in the culture receptacles may be rather significant and serious, i.a. result in decreasing development and viability of the cells.
In particular mammalian oocytes and preimplantation embryos are considered to be very sensitive to environmental changes. On the other hand, in vitro production and propagation of such embryos requires frequent openings of the incubator door for changing or supplementing the culture media and transferring embryos from one culture receptacle to another. Professional laboratories working with in vitro production of embryos start up at least one in vitro fertilization program per day and try to avoid the above problems by purchasing a number of expensive CO
2
incubators and/or regulating the number and duration of openings per day of each incubator strictly and/or try to keep the temperature of the whole laboratory containing the incubators at a level as near as possible to that of the interior of the incubators, i.e. at about 37-39° C. However, the above approach does not solve all the technical problems concerned and from an economical point of view it is a very costly approach which is hard to realize for most laboratories.
Minimizing the size of the incubators, in particular their interior working spaces, might be beneficial to embryo development and has been studied by Boone and Saphiro, 1990; Avery and Greve, 1992; and Gordon, 1994. Analyzing the possible factors affecting embryo development, Avery and Greve (1992) found no direct correlation between negative effects on embryo development and frequency of door openings of large volume incubators and therefore supposed other factors to be involved, i.e. toxic fumes in the laboratory air or toxic components originating from the inner cupper walls of the incubator. However, later observations made by the present inventor (Vajta, 1994) after a series of unsuccessful embryo culture experiments in the same type of incubator, but with inner stainless steel walls instead of copper walls, revealed that high temperature differences could occur in the partially divided inner space of large incubators and could persist for hours after frequent openings of doors.
Some researchers therefore prefer working with culture receptacle(s) containing culture medium/media and placed in commercially available airtight plastic boxes, for example “modular incubator chambers” manufactured by Flow Laboratories (circular shape, 30 cm in diameter×12 cm in depth), which are placed in the incubator after filling or refilling them with the desired gas mixture. However, this requires a more complicated preparation procedure and the results obtained may vary. The plastic boxes may eliminate small temperature differences caused by frequent opening of the incubator door, but the cumulative effects may even be enhanced.
Small size double wall
Demtek A/S
Ladas & Parry
Patten Patricia
Weber Jon P.
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