Method and apparatus for culturing cells and tissues

Abstract

Cells and tissues, in particular sensitive cells and tissues, such as oocytes, fertilized oocytes and preimplantation embryos, which require highly stable physical and chemical environment for in vitro development, are cultured in closed containers submerged or immersed in thermostatically controlled liquid baths, the containers being provided with an appropriate inner atmosphere containing, e.g. carbondioxide, oxygen and humidity in appropriate levels. The incubator containers may e.g. be gas and liquid impervious, flexible, sealable, preferably transparent bags which after sealing are submerged or immersed directly in the thermostatically controlled liquid bath. The liquid in the bath is preferably water. An incubator for submerse or immerse culture of cells and tissues in the above manner is also described. Also a transportable liquid incubator for culturing cells and tissues in the field is described.

Description

[0001] 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 th...

AI Technical Summary

Problems solved by technology

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.

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.

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.

However, this requires a more complicated preparation procedure and the results obtained may vary.

However, the high price of these K-system boxes prohibit most laboratories from purchasing the necessary optimum quantity of these boxes, i.e. at least one for maturation and fertilization experiment initiated and at least one for each continuing experiment.

Moreover, even though the boxes are supplied with pre-mixed gas at a constant flow rate, recovery rate of the required atmospheric gas composition is still low and this problem is only partially solved by the possible manual increase of the flow rate of the gas mixture.

However, as heat is transferred to the boxes by air-conduction, temperature recovery rates are not optimum and opening of the door of the incubator for working with one culture will disturb the physical parameters and environments of all the others in the incubator.

Moreover, a single wall box requires a system to establish the correct gas mixture in each box in the incubator if optimum conditions shall be maintained within the boxes, which means high expenses for gas mixture consumption, control and regulating equipments and provides a source of complications, errors and troubles.

However, there is no disclosure or even a contemplation of culturing or growing the cells in a vessel immersed or submerged in the water bath before harvesting the cells.

However, this document does not suggest or contemplate the culturing of sensitive cells and / or tissues like e.g. oocytes and embryos requiring highly stable and constant environments both as to pH-range and composition of the culture medium as well as the composition of the environing atmosphere.

Besides, the water bath is not sufficiently covered or sealed to secure an exact temperature control of the water in the water bath and thus neither in the culture trays.

Therefore, up to now no simple, reliable and inexpensive method of in vitro production of sensitive cells and tissues has been available, nor has any simple, reliable and inexpensive incubator system for such purpose.

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