WO2010124663A2

WO2010124663A2 - Method for adding, removing or exchange a liquid medium in culturing biological cells and culture device

Info

Publication number: WO2010124663A2
Application number: PCT/CZ2010/000054
Authority: WO
Inventors: Daniel Hlinka
Original assignee: Synga, S.R.O.
Priority date: 2009-04-27
Filing date: 2010-04-22
Publication date: 2010-11-04
Also published as: WO2010124663A3; CZ301758B6; CZ2009264A3

Abstract

A method for adding, removing or exchange a liquid medium in culturing biological cells without any contact with said cells and a culture device 1 for culturing biological cells and manipulating with said cells, preferably embryonic cells, comprising at least one elongated culture well 6 having V-shaped cross-section and at least one media exchange well 7, which allows culturing embryos in groups in close proximity to each other utilizing supporting effects of the growth factors released by the embryos, wherein full identification of each individual embryo throughout the entire culture period is ensured, and which simultaneously allows non-contact nutrient media exchange. The device 1 can be arranged on the common platform 11 in a combination with another device 12, such as for in-vitro fertilization, and covered by a lid 10.

Description

Method for adding, removing or exchange a liquid medium in culturing biological cells and culture device

FIELD OF THE INVENTION

The invention is related to a method for adding, removing or exchange a liquid medium in culturing biological cells and the culture device for culturing biological cells and manipulating with said cells, preferably embryonic cells.

BACKGROUND OF THE INVENTION

At present human and nonhuman embryos are cultured in-vitro in different culture devices or multiple-well plates.

Such a typical arrangement represents a conventional flat device, on the bottom of which an aqueous culture medium is placed in the form of individual drops. The drops are usually covered by mineral oil during culturing. Extreme care must be taken in order to prevent dislodgement of the drops during handling of the devices.

In the document US 5,891 ,712 an improved micro drop culture device is disclosed, in the flat bottom portion of which a plurality of circular wells is embedded having open upper surfaces, which wells may have markings identifying individual wells for tracking purposes. According to the specification, the culture device along with a detachable cover is able to provide a stable environment for the development of gametes/embryos and thus a shorter incubation time for the procedures, clear identification of the samples contained therein, and prevents dislodgement of the drops during handling of the devices.

Embryos can be grown up to 5-6 days and during this time the culture medium have to be refreshed to avoid toxic products accumulation and nutrient components depletion. In any of the systems used the oocytes/embryos are transferred to the next compartment (chamber, well etc.) or simply to another drop of fresh medium. During this handling the clusters of embryos are separated what diminish the supporting effect of autocrine/paracrine growth factors released by the embryos. Moreover, they must be handled by qualified personnel by glass/plastic devices and such a way the embryos are exposed to a possibility of the damage and/or loss. To cover the loss of embryos during handling the starting number of the embryos must always be higher than the number finally implanted into woman. The surplus embryos should be avoided for ethical reasons, however.

Accordingly it would be highly desirable to have an improved culture device as well as a corresponding improved method suitable for non- contact nutrient media exchange wherein the cultured embryos/cells are neither manipulated nor come into contact with replacement media devices (e.g. pipettes).

There are several embryo culture procedures in a routine use. In any of the procedures used the viability of the individual embryo is assessed according to its morphological features determined by the light microscopy observation. It requires the permanent identification of each embryo from the group during the whole culture period. The culture in clusters is unsuitable because the embryos can easily be disturbed during handling. To avoid this disadvantage the individual embryos are placed in the culturing dishes in spaced-apart locations. However, the culture of individual embryos diminishes the effect of the growth factors released by the embryos, which support their development.

Moreover, during the culture prolonged up to 5 days there is a need for nutrient media exchange and in such a case the embryos are to be transferred to a fresh medium and growth-by products are lost. If the embryos are not transferred, accumulation of the toxic metabolites in culture media can compromise the next development of the embryos.

US 6,448,069 teaches an embryo-culturing apparatus comprising at least one compartmentalized structure, wherein each individual compartment is sized for receiving a single embryo, and wherein intercompartmental fluid migration is allowed. In the preferred embodiment, each compartment is bounded by spaced-apart pickets projecting upwardly from the bottom wall, or by a fluid permeable membrane. After the embryos are placed in the compartments in each structure, each of the structures is covered with a drop of a nutrient and so the embryos in each compartmentalized structure can share by-products with other embryos in the same structure. But even in this apparatus the need for contact manipulation of the embryos during their replacement to a fresh medium still exists.

Therefore, there is still a need of a simple and inexpensive culture device, which would enable, without creating rather complicated microstructures such as spaced-apart pickets or a fluid permeable membrane, culturing embryos in groups in close proximity to each other utilizing supporting effects of the growth factors released by the embryos, wherein full identification of each individual embryo throughout the entire culture period is ensured, and which simultaneously would allow non- contact nutrient media exchange.

SUMMARY OF THE INVENTION

The above problems are solved by the first object of the present invention, which is a method of adding, removing or exchange a liquid medium in culturing and/or any other handling with biological cells, preferably embryonic cells disposed in one or more wells arranged in the bottom wall of a culture device, without any handling with the cells or without any contact of the media handling device such as a pipette with said cells. This method allows any handling with any liquid aqueous or non-aqueous media such as nutrient media as well as washing, storing, treating, buffering, equilibrating, protecting or covering media, which should be brought into contact with the cells.

In the inventive method any handling, such as for example transferring the cells or embryos into the fresh medium by a pipette is avoided, and thus the disruption of the embryo clusters can be excluded. The inventive method comprises two basic steps: the step of adding the medium into the culture device, in which step the level of the medium in said culture device is gradually increased above the level of the open ends of said wells, whereby a fluid communication between the added medium and the interior of said wells is established through the open ends of said wells, and/or the step of removing the medium from the culture device, in which step the level of the medium in said culture device is gradually decreased below the level of the open ends of said wells, whereby the fluid communication through the open ends of said wells between the added medium and said well interior is cut off.

Even if it is possible by the inventive method the medium to be added to or removed from any culture device having wells for culturing biological cells (culture wells), for example from any point of the bottom wall of the culture device, at which it does not come to a contact of the media handling device with the cells, preferably from the lowest point of the upper surface of the bottom wall of the culture device, more preferably a well arranged at the lowest point of the upper surface of the bottom wall of the culture device, which does not comprise any cells, even more preferably from the lowest point of the upper surface of the bottom wall of the culture device, which the upper surface of the bottom wall of said culture device is sloped toward, most preferably the medium is added to or removed from the media exchange well of the special culture device, which is the further subject-matter of the present invention.

In said most preferred embodiment the process of media exchange starts by aspirating medium, if present, from the media exchange well arranged at the lowest point of the upper surface of the bottom wall of the culture device. It has been surprisingly found by the inventors that after removal of the medium the said one or more wells in which cells/embryos are disposed (culture wells) not only remain filled with the medium, but the embryos are neither disturbed nor flotated and keep their original sequence.

When adding the fresh media, in preferred embodiment by displacing medium into the media exchange well arranged at the lowest point of the upper surface of the bottom wall of the culture device, the level of the medium gradually arises above the level of the open ends of said culture wells with cultured embryos/cells, and the original medium is extensively diluted by the fresh one.

The operation can additionally be done very easily and simultaneously in one step for all wells with cultured embryos/cells, is less time-consuming and less demanding for highly experienced laboratory personal than the methods of the art. Additionally no precise tools for manipulating with the embryos/cells are necessary.

The dilution ratio and thus the possibility of preserving the growth factors released by the embryos/cells in close neighborhood thereof may be influenced to some extent by the amount of the fresh medium added and thus by the level of the fresh medium above the open ends of said culture wells with cultured embryos/cells and by the time of incubation before the medium is removed. In the step of removing the medium from the culture device the level of the medium in said culture device is gradually decreased below the level of the open ends of said culture wells, whereby the fluid communication through the open ends of said culture wells between the added medium and said well interior is cut off and for example the cultivation of embryos can further be conducted as any standard micro-drop cultivation.

If desired or necessary, by omitting the above step of removing the medium from the culture device the level of the medium in said culture device remains above the level of the open ends of said wells, and the fluid communication among all wells of the culture device through the medium is preserved. If desired or necessary, the above steps of removing and adding the medium can be repeated several times for example to achieve washing of the embryos/cells.

The inventive method additionally enables adding or removing medium below the layer of the covering non-aqueous liquid such as a mineral oil as commonly used in the art.

In a preferred embodiment the medium is removed by aspiration, from the well arranged at the lowest point of the upper surface of the bottom wall of the culture device, under the layer of the covering non-aqueous liquid, such that substantially all the covering non-aqueous liquid remains in the device. The adding of the fresh media is preferably carried out in this case by displacing medium into the same well, likewise under the layer of the covering non-aqueous liquid. In this way, the non-aqueous liquid remains unchanged during the whole culturing. Most preferably the method is carried out on any embodiment of the culture device as described hereinbelow.

A further object of the present invention is represented by a culture device having a bottom wall and a side wall defining the internal space of said culture device, in which in the bottom wall at least one elongated culture well is embedded having V-shaped cross-section seen along its longer axis, facing with its upper open end into said internal space of the device, and at least one media exchange well is embedded, facing with its upper open end into the internal space of the device. It has been found that embryos cultured in a line in elongated culture wells having a substantially V-shaped cross-section, thus forming a groove- or a channel-like structure in the bottom wall of a culture device, maintain original positions throughout the entire culturing process.

The culture wells are designed to have a suitable profundity and the walls thereof to make a suitable angle to minimize embryo flotation during manipulation, with maintaining good accessibility to the embryos during manipulation. For non-contact medium adding, removing or exchange at least one media exchange well is embedded in the bottom wall of the culture device, facing with its upper open end into the internal space of the culture device. Preferably at least a part of the upper surface of the bottom wall of the culture device comprising one or more culture wells is sloped toward the media exchange well for easy collecting the medium in said media exchange well.

The bottom of the culture well is preferably horizontal. In two or more culture wells the bottoms are preferably at the same horizontal level.

The border of the upper open end of the culture well facing into the internal space of the culture device can be elevated above the upper surface of the bottom wall of the culture device to form a rim having the horizontal upper end, said rim encircling said upper open end of the culture well. This rim ensures constant profundity of the elongated culture well along its longer axis and further minimizes embryo flotation during media exchange and enables consistent accessibility by the operator to all embryos during manipulation. In case of two or more culture wells the horizontal upper ends of said rims are preferably at the same horizontal level.

One media exchange well may thus form "a unit" with one or more surrounding culture wells, wherein a part of the upper surface of the bottom wall comprising such "a unit" can be sloped toward said media exchange well. One culture device may comprise several such self standing units at any place of the bottom wall, wherein each unit can be separated from other units by side walls or wherein two or more units can be surrounded by a single common side wall. Such an arrangement can be useful for example in genetic analysis for individual embryo culturing.

Preferably longer axes of the culture wells surrounding one media exchange well are parallel with each other to enable easy access to all embryos by the pipette hold under the same angle with respect to the culture device for all culture wells as well as safe identification of the embryos.

The shape of the media exchange well is not critical. In a preferred embodiment the media exchange well has conical side walls with the broader end facing upward, to enable removing even the lowest amount of the remaining medium and for easy distinguishing the level of the nonaqueous covering liquid, if used. The media exchange well can be round- or flat-bottomed.

In order to ensure equal conditions for the medium exchange in the individual culture wells, the culture wells are preferably arranged around the central media exchange well, in substantially at the same distance from said central media exchange well.

Preferably the bottom wall of the culture device contains markings such as numbers or letters for identifying the individual culture wells. The corner at the connection place between the upper surface of the bottom wall and the side wall is preferably round or cut into the internal space of the cultivation device to minimize the amount of adhered liquid.

The culture device may further comprise a detachable lid covering the internal space of said culture device. It is possible for the culture device to be arranged on a common platform with one or more identical culture devices and/or one or more additional devices or structures such as replacement media reservoirs or other supporting structures, which may be separated from each other by additional side walls. Such an additional structure may be a compartment for in-vitro fertilization of oocytes known in the art including separate wells/chambers for spermatozoa and oocytes, respectively, connected by a narrow channel in their upper endings. After swim-up only living and highly motile spermatozoa can contact eggs to be fertilized. Any such structure can comprise a detachable lid. In case of more devices and/or structures of the same or of the different kind arranged on a common platform it may be preferred if the individual lids covering the internal space of each culture device and/or the internal space of any other device present on a common platform are replaced by a single common lid covering all devices and/or devices on the same common platform.

Preferably the common lid in its first position covers all the culture devices and/or the further devices arranged on the common platform, and in its second, with respect to the first position horizontally rotated position, allows access to the internal spaces of said devices arranged on the common platform, for example through suitable open segments or cut outs. This principle has already been used in the art for example in the well dish with a cover plate according to FI884551.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a side elevational cross-sectional view of one embodiment of the culture device 1 of the invention with one central media exchange well 7 and two culture wells 6; FIG. 2 illustrates a top view of the culture device 1 of FIG. 1 with one central media exchange well 7 and four culture wells 6 arranged around the central media exchange well 7;

FIG. 3 illustrates a top view of the culture device 1 with one central media exchange well 7 and four culture wells 6 arranged around the central media exchange well 7 containing markings A, B, C, and D on the bottom wall 2, identifying the culture wells 6;

FIG. 4 illustrates a side elevational cross-sectional view of the embodiment of FIG. 1 having the upper surface 8 of the bottom wall 2 of the culture device 1 sloped to the media exchange well 7, with an optional lid 4 covering the internal space 5; FIG. 5 A-C illustrate a side elevational cross-sectional view of several embodiments of the culture well 6 seen along the longer axis of the well 6, showing an embryo 9 present in the well 6 respectively, FIG. 5D illustrates a side elevational cross-sectional view of the culture well 6 seen along the longer axis of the well 6, showing a rim 13 encircling the upper open end of the culture well 6,

FIG. 5E illustrates a side elevational cross-sectional view of the culture well 6 seen perpendicular to the longer axis of the well 6, showing a rim 13 encircling the upper open end of the culture well 6,

FIG. 5F illustrates a detailed top view of the culture well 6,

FIG. 5G illustrates the same view as FIG. 5F, showing embryos 9 placed in the culture well 6; FIG. 6 illustrates a side elevational cross-sectional view of one embodiment of the media exchange well 7;

FIG. 7 illustrates a top view of an arrangement of two culture devices 1, each comprised of four wells 6 around the single central media exchange well 7, on a common platform 1 1 ; FIG. 8A-B illustrate two different side elevational views of one culture device 1 arranged on the common platform IJ. in a combination with one device 12 for in-vitro fertilization, with one possible embodiment of a single detachable lid 10;

FIG. 9A-B illustrate two different side elevational views of one culture device 1 arranged on the common platform H in a combination with one device 12 for in-vitro fertilization, with the lid 10 in its opened position (A) and rotated to its closed position (B). EXAMPLES

Some of the preferred embodiments of the present invention will now be described in detail and some of them are illustrated in the accompanying drawings. It should be apparent to persons skilled in the art that even if the embodiments of the present invention are described in the specification on the embryos, the invention is not limited to human or non-human embryonic cells only but can be useful for all sensitive and/or fragile biological materials such as oocytes and other animal or plant cells or cell parts such as protoplasts.

Additionally, the terms "culture well" or "culturing" should be interpreted to include all feasible processes such as receiving, holding, storing, washing, inoculating or treating biological materials.

The non-contact media exchange according to the first embodiment of the present invention can substantially be used for media adding to or removing from any culture device having wells for culturing biological cells (culture wells). However, the method is preferably carried out on the culture device according to the present invention. In the non-contact media exchange according to the first embodiment of the present invention the cultured embryos/cells are neither manipulated nor come into contact with replacement media devices (e.g. pipettes), i.e. the nutrient medium is preferably exchanged by its removing/adding into a media exchange well 7. In the typical culturing procedure the following steps are carried out:

Step 1 - the establishment of the nutrient media

In this step the media exchange well 7 is filled using for example a common pipette with exchangeable tips by the nutrient media. The level of the medium in the culture device 1 is thereby gradually increased above the level of the open ends of the culture wells 6 and finally the medium is overlaid by mineral oil Step 2 - the adding the embryos

In this step the embryos 9 are pipetted into one or more V-shaped culture wells 6. This manipulation may require special handling tools and trained personnel. Embryos/cells culturing is possible in this arrangement, i.e. with the medium level left above the open ends of the culture wells 6, or to continue further with step 3.

Step 3 - the removal of excess media

In this step the aspirating device (tip of the pipette) is inserted into the media exchange well 7 and the medium is removed. After this operation only the culture wells 6 containing embryos remain filled with the medium and the medium remains covered by mineral oil to avoid medium evaporation and the changes of the osmolarity.

Step 3 - the replacement of depleted media In this step the aspirating device (tip of the pipette) is inserted into the media exchange well 7 and new medium is expelled from the pipette until the level of the medium in said culture device 1 is increased above the level of the open ends of said wells 6, whereby a fluid communication between the added medium and the interior of said wells overlaid by fresh medium is established through the open ends of said wells 6 containing embryos 9. The depleted medium in the culture wells 6 is extensively diluted by a fresh one.

The medium can remain covered by the same mineral oil during the whole culture period to avoid medium evaporation and the changes of the osmolarity

One embodiment of a simple culture device 1 according to the second embodiment of the present invention, having a bottom wall 2 and a side wall 3 defining the internal space 5 of the culture device 1, consists of a single media exchange well 7 and two culture wells 6 embedded in the bottom wall 2 of said device λ_. The device as shown in FIG. 1 is comprised of a sufficiently rigid and transparent material such as polystyrene.

A perspective view of the similar culture device 1 is shown in FIG. 2. In this arrangement it is represented by a single central media exchange well 7 surrounded in substantially equal distances by four culture wells 6, the longer axes of all four culture wells 6 running in parallel with each other.

To enable easy access to the cells by a handling device such as a pipette, the diameter of the internal space 5 of the culture device 1 suitable for in-vitro embryo culture can (or the diameter of the parts of the upper surface 8 of the bottom wall 2 surrounding media exchange wells 7 and comprising one or more of culture wells 6) can be for example 15 to 30 mm. The depth of the internal space 5 of the culture device 1 can be 5 to 15 mm, without limitations to said dimensions.

A perspective view of the similar culture device 1 with letters identifying each of the culture wells 6 is illustrated in FIG. 3.

An embodiment showing the sloping of the upper surface 8 of the bottom wall 2 of the culture device 1 in the area surrounding the media exchange well 7 is shown in FIG. 4. The angle β of the slope toward the media exchange well 7 is preferably 0.5 to 2°, which is sufficient for gravity drainage the media into the media exchange well 7, but can range from 0° (flat upper surface 8) up to 10°, for example.

It should be noted that the media exchange well 7 need not be necessarily situated in the central area of the culture device. This embodiment is shown in Fig. 7 in which the combined culture device comprises two media exchange wells 7 on a common platform V\_, and thus two separate parts of the upper surface 8 of the bottom wall 2 surrounding said media exchange wells 7 and comprising one or more of culture wells 6 are sloped toward the two respective media exchange wells 7. If additionally said two separate parts of the upper surface 8 of the bottom wall 2 are encircled by side walls 3, two self-standing culture devices 1 are created on said common platform 1_1_. The elongated culture well 6 has a substantially V-shaped cross-section, seen along its longer axis, as shown in Fig. 5, thus forming a groove- or a channel-like structure in the bottom wall 2 of a culture device 1, in which the embryos 9 maintain original positions throughout the entire culturing process.

The term "substantially V-shaped cross-section" shall mean not only the embodiment shown in Fig. 5A, but also flat bottomed cross-section as shown in Fig. 5B, or any other cross-section with the broader end facing upward, as long as it keeps the embryos protected from floating and aligned in a stable permanent order to identify the embryos throughout the whole culture period. To this end, the flat bottom part of the "V" should not be broader than the diameter of the embryo or of any other cultured cell respectively, such as 0.02 to 0.08 mm, for example. The volume capacity of the culture wells 6 depends on the embryos/cells cultured and can be approx. 10 to 50 μl, for example.

Suitable angle α of the longitudinal walls of the elongated culture wells 6 to minimize embryo flotation during manipulation, with maintaining good accessibility to the cultured embryos from all directions during handling, is 40-60°, and the length of the well 6, preferably calculated for culturing 10 - 15 embryos in a line, is for example equal to 10 - 20 times the diameter of the embryo (typically 0.015 mm), i.e. preferably 3 mm. Embryos placed in the elongated culture wells 6 are shown in Fig. 5G.

Suitable angle of the transverse walls as shown in Fig. 5E of the elongated culture wells 6 is not critical and preferably is the same as the angle α of the longitudinal walls of the elongated culture wells 6.

As also seen in Fig. 5E, the bottom of the culture well 6 is preferably horizontal. In two or more culture wells the bottoms are preferably at the same horizontal level - see Figs. 1 and 4.

The border of the upper open end of the culture well 6 facing into the internal space 5 of the culture device 1 is preferably elevated above the upper surface 8 of the bottom wall 2 of the culture device to form a rim

13 as shown in Fig. D-G, having a horizontal upper end, said rim 13 encircling said upper open end of the culture well 6. The width of the rim 13 as well as its elevation above the upper surface 8 is preferably 0.2 to 2 mm.

In two or more culture wells 6 the horizontal upper ends of said rims 1_.3 are preferably at the same horizontal level.

In a preferred embodiment as shown in Figs. 1 to 4 the media exchange well 7 has a larger volume and higher profundity when compared to the culture wells 6 containing the embryos. The volume of the media exchange well 7 can be approximately 50 to 150 μl. The media exchange well 7 preferably has side walls of the conical shape with the broader end facing upward, and a flat bottom, to be easily accessible by a pipette and/or by a liquid handler in case of a possible automatic media exchange.

The angle γ of the side walls of the media exchange well 7 is shown in Fig. 6 and is selected in such a way that the level of the medium or the interface between the medium and the covering non-aqueous liquid is clearly seen on said side walls, and preferably is the same as the angle α of the longitudinal walls of the elongated culture wells 6.

The device 1 may be covered by a detachable lid 4 as shown in Fig. 4 allowing gas exchange between the internal space 5 of the device and the external environment.

A top view of an arrangement of two culture devices 1, each comprised of four wells 6 around the single central media exchange well 7, on a common platform H, is illustrated in FIG. 7. In preferred embodiment of the present invention, one culture device 1 is arranged on the common platform H in a combination with one device 12 for in-vitro fertilization, such as illustrated in FIG. 8A-B. In FIG. 8A a preferred embodiment of the culture device 1 is additionally shown having six culture wells 6 arranged around one media exchange well 7, the longer axes of all six culture wells 6 running in parallel with each other. The most preferred embodiment of the present invention comprises a single detachable lid 10, which is common for, and which in its first position as shown in FIG. 9A allows access to the internal spaces 5 of said devices 1 and/or 1_2 arranged on the common platform H, and in its second, with respect to the first position horizontally rotated position as shown in FIG. 9B covers all the culture devices 1 and/or the further one or more devices 12, arranged on the common platform H.

The specially designed shape of the lid 10 thus allows the access to the embryos/cells for routine manipulation avoiding the necessity of the lid displacement. The lid 10 covers said devices 1 and/or YZ arranged on the common platform H or opens them by simple rotation of the lid by 90°.

Claims

1. A method of adding, removing or exchange a liquid medium in the culturing and/or handling with biological cells such as embryonic cells, disposed in one or more wells embedded in the bottom wall of a culture device, each of the said wells facing with its upper open end into the internal space of said culture device, without any manipulation with said cells or contact of the media handling device with said cells, characterized in that said method comprises the step of adding the medium into the culture device, in which step the level of the medium in said culture device is gradually increased above the level of the open ends of said wells, whereby a fluid communication between the added medium and the interior of said wells is established through the open ends of said wells, and/or the step of removing the medium from the culture device, in which step the level of the medium in said culture device is gradually decreased below the level of the open ends of said wells, whereby the fluid communication through the open ends of said wells between the added medium and the interior of said wells is cut off and the interior of said wells remains filled with said medium.

2. The method of claim 1 , characterized in that the medium is added to or removed from a well embedded in the bottom wall of a culture device.

3. A culture device (1 ) having a bottom wall (2) and a side wall (3) defining the internal space (5) of the culture device (1 ), characterized in that in the bottom wall (2) at least one elongated culture well (6) having substantially V- shaped cross-section seen along its longer axis is embedded, facing with its upper open end into the internal space (5) of the device (1 ), and at least one media exchange well (7) is embedded, facing with its upper open end into the internal space (5) of the device (1 ).

4. The culture device of claim 3 characterized in that the longitudinal walls of the culture well (6) make an angle of 40-60°.

5. The culture device of claim 4 characterized in that the bottom of the culture well (6) is horizontal.

6. The culture device of any of claims 3 to 5, characterized in that the border of the upper open end of the culture well (6) facing into the internal space (5) of the culture device (1 ) is elevated above the upper surface (8) of the bottom wall (2) of the culture device (1 ) to form a rim (13) having the horizontal upper end, said rim (13) encircling said upper open end of the culture well (6).

7. The culture device of any of claims 3 to 6 characterized in that at least a part of the upper surface (8) of the bottom wall (2) surrounding any of the media exchange wells (7) and comprising one or more of said culture wells (6) is sloped toward said media exchange well (7).

8. The culture device of claim 7 characterized in that that the angle β of the slope of the upper surface (8) toward the media exchange well (7) is 0,5 to 2°.

9. The culture device of any of claims 3 to 8 characterized in that the media exchange well (7) has side walls of a conical shape with the broader end of the well (7) facing upward.

10. The culture device of any of claims 3 to 9 characterized in that the culture wells (6) are arranged in the bottom wall (2) of the culture device (1 ) with the longer axes of the culture wells (6) parallel with each other.

1 1 . The culture device of any of claims 3 to 10 characterized in that the bottom wall (2) of the culture device (1 ) contains identifying markings.

12. The culture device of any of claims 3 to 1 1 characterized in that said culture device (1 ) further comprises a detachable lid covering the internal space (5) of said culture device (1 ).

13. The culture device of any of claims 3 to 1 1 characterized in that said culture device (1 ) is arranged on a common platform (1 1 ) with one or more additional devices selected from the group of culture devices (1 ) and devices (12) for in-vitro fertilization.

14. The culture device of claim 13 characterized in that it comprises a single detachable lid (10), which is common for, and which in its first position covers all the culture devices (1 ) and/or the further one or more devices (12), arranged on the common platform (1 1 ), and in its second, with respect to the first position horizontally rotated position, allows access to the internal spaces of said devices (1 ) and/or (12) arranged on the common platform (1 1 ).

15. The culture device of any of claims 3 to 14 characterized in that the corner at the connection place between the upper surface (8) of the bottom wall (2) and the side wall (3) is round or cut into the internal space of the cultivation device (1 ).