RU2588376C2 - Plate configured for turning to form hanging droplets for cell culture - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions refers to cell cultivation. Disclosed is a plate configured for turning to form hanging droplets for cell cultivation, set of plates for transfer of three-dimensional cell aggregates and method of testing substance on toxicity in relation to cells. Plate has a given number of cells for drops, cell comprises circular microfluidic wetting barrier. Barrier is configured to enclose cavity of cell and to prevent spreading of drops beyond microfluidic wetting barrier. Cell for drops includes closed bottom and at least one additional circular microfluidic wetting barrier, and section to be wetted is located between two adjacent microfluidic wetting barriers. Method involves introduction of liquid drops in cells for drops, each drop contains a volume of substance for testing and a liquid nutrient medium. Method then includes turning and incubation of plate, an additional supply of liquid nutrient medium, as well as analysis of three-dimensional cell sets.

EFFECT: inventions provide stability of hanging drops and evaporation.

23 cl, 41 dwg

Description

The present invention relates to a plate made with the possibility of turning over to form hanging drops in accordance with an independent paragraph.

It is generally accepted that cells cultured in a three-dimensional configuration are physiologically more relevant than cells in classical two-dimensional (monolayer) cultures in most applications and samples, for example, when drugs are detected or in samples for toxicity. Drops of cell culture medium have been proposed for the formation of such three-dimensional cell aggregates. Drops of a cell culture medium with suspended cells are placed in a culture cell or well in such a plate, then the plate is turned over. Since there is no substrate to which the cells can adhere, the cells accumulate at the top of the hanging drop and form a three-dimensional cell aggregate. If germ stem cells are used, these stem cells descend to the top of the drop where they form a three-dimensional cell population called the embryoid body.

A plate with hanging drops, which is not necessary to be turned over, is shown in WO 2010/031194. The plate provided in this link contains a housing, a first and second coplanar surface and a plurality of channels permeating the housing perpendicularly from the first (upper) surface to the second (lower) surface. The channel contains a funnel-shaped inlet cell near the first (upper) surface, an inverted funnel-shaped culture cell near the second (lower) surface, and a capillary portion located between the inlet cell and the culture cell. Relief structures are provided in the form of separate rims protruding from the second (lower) surface of the body and surrounding each culture cell. These rims prevent liquid droplets from spreading.

The introduction of fluid containing cells into the culture cell is carried out through the inlet cell and the capillary part. As an example, a liquid nutrient medium containing stem cells can be introduced in this way into the culture cells, and then the plate with hanging drops can be kept in a thermostat for a predetermined time so that the cells combine to form three-dimensional embryoid bodies. If after some time it is necessary to supply fresh liquid nutrient medium, this is done by suctioning the "old" liquid nutrient medium through the inlet cell and the capillary part (for example, using a pipette) and then supplying the "fresh" liquid nutrient medium in the manner described above.

Although the plate described in WO 2010/031194 is generally suitable for culturing cells to form three-dimensional cell assemblies, there is room for improvement. For example, in order to suck out the old liquid nutrient medium through the inlet cell and the capillary part, it is necessary to have a suction device (for example, a pipette) that must fit snugly against the walls of the inlet cell for proper suction. The same applies to the subsequent supply of fresh liquid nutrient medium. However, it is more important that only the stability of the hanging drop in the corresponding culture cell is limited. Another disadvantage is that the liquid can evaporate through an open inlet cell at an uncontrolled speed.

Therefore, it is an object of the invention to provide a plate adapted to be turned over to form hanging droplets for cell culture, which solves all of the above problems related to the stability of hanging droplets and the evaporation of a liquid. Also, the additional supply or replacement of a liquid nutrient medium should be simple to perform.

In order to achieve the aforementioned goal (s) in accordance with the present invention, there is provided a plate configured to be turned over to form hanging drops, as it is characterized by features of an independent item aimed at such a plate made to be turned over to form hanging drops. Additional embodiments of the plate, made with the possibility of turning over to form hanging drops in accordance with the invention, are the subject of dependent clauses.

In particular, it is proposed a plate made with the possibility of turning over to form hanging drops, containing a given number of cells with drops, each of which can take a drop of liquid. The corresponding droplet cell comprises a circular microfluidic wetting barrier configured to surround the cavity of the droplet cell and preventing the droplet from spreading beyond the microfluidic wetting barrier. Moreover, the corresponding cell contains a closed bottom and at least one additional circular microfluidic wetting barrier, each additional circular microfluidic wetting barrier is configured to surround the previous circumferential microfluidic wetting barrier, while the wetted portion is located between two adjacent microfluidic wetting barriers.

The configuration of the closed bottom of the culture cells along with the circular wetting barrier serves to increase the stability of droplets hanging from the respective culture cells, in particular when compared with plates with hanging drops shown in WO 2010/031194, and this is believed to be due to the lack of air access in contrast to the plate with hanging drops shown in WO 2010/031194, in which there is air access in the inlet cell. Also, due to the lack of air access, the evaporation of the liquid is significantly reduced.

Additionally, although a plate made with the possibility of turning over to form hanging drops should be turned over again to additionally supply a drop of liquid nutrient medium to a drop already contained in the culture cell to form a larger drop, this can be done in a simple and convenient way. Firstly, this is due to the sufficient stability of the droplet and, secondly, due to the additional circular microfluidic wetting barrier and the wettable area between the additional microfluidic wetting barrier and the previous microfluidic wetting barrier, which makes it easy to introduce an additional drop of liquid nutrient medium. The larger droplet thus formed is protected against spreading by the mentioned additional microfluidic barrier so that the stability of the larger droplet is maintained after repeated flipping of the plate to obtain a hanging droplet pattern. Typically, the number of additional microfluidic wetting barriers is not limited, however, in practical embodiments, only one or two additional microfluidic wetting barriers can be used.

In one embodiment of a plate configured to be flipped to form hanging drops according to the invention, the respective culture cells are wells, and in another embodiment, the cells with drops may form a flat surface (surrounded by microfluidic wetting barriers, respectively).

In an additional embodiment of the plate, made with the possibility of turning over to form hanging drops in accordance with the invention, the circular microfluidic wetting barrier is a circular edge, and at least one additional circular microfluidic wetting barrier is at least one additional circular edge. Each additional circular edge is configured to surround the previous circular edge, wherein the wetted portion is between two adjacent circular edges. In an additional version of this embodiment, two adjacent circular edges are arranged in steps.

In an additional embodiment, the plate, made with the possibility of turning over to form hanging droplets in accordance with the invention, is made of a separately manufactured plate having a predetermined number of holes and a corresponding predetermined number of separately made holes forming a drop cell, each individually made hole being pressed into corresponding hole of a separately manufactured plate. This provides for the ease of manufacture of both the plate and the holes, which can be made of polystyrene or other suitable material. Separately made plates and wells can be easily assembled by pressing holes into holes. Although ultrasonic welding of the holes to the inner surface of the plate is also an option (in this case, the plate may not contain holes), press-fitting the holes into the holes is preferable, since ultrasonic welding can make it difficult to microscope the contents of the holes.

In an additional embodiment of the plate, made with the possibility of turning over to form hanging drops in accordance with the invention, separately made holes are pressed into the holes from the side of the plate forming the outer surface of the plate. This provides for easy assembly of the plate and wells, which can be done automatically. In a preferred embodiment, the plate, made with the possibility of turning over to form hanging drops in accordance with the invention, may contain 24 holes, 96 holes or 384 holes.

In another embodiment of the plate, made with the possibility of turning over to form hanging drops in accordance with the invention, the circular microfluidic wetting barrier is a circular rim, and at least one additional microfluidic wetting barrier is an additional circular rim, each additional circular rim surrounds the previous a circular rim, while the wetted portion is located between two adjacent circular rims. According to one version of this embodiment, the adjacent circular rims are arranged in steps, and according to another version of this embodiment, the circular rims are located on a flat surface.

An additional aspect of the invention relates to a set of plates for transferring three-dimensional cell assemblages cultured in hanging drops from a plate made with the possibility of turning over to form hanging drops in a receiving plate containing a plate made with the possibility of turning over to form hanging drops in accordance with the invention, as described above, and the receiving plate. A plate made with the possibility of turning over to form hanging drops has a predetermined number of cells or holes for drops, and a receiving plate has a number of holes corresponding to a given number of cells or holes for drops of a plate made with possibility of turning over for forming hanging drops. The plate made with the possibility of turning over to form hanging drops and the receiving plate are assembled so that, in the assembled state, the holes of the receiving plate are adapted to be combined with cells or holes for drops of the plate made with the possibility of turning over to form hanging drops. This kit allows you to easily transfer three-dimensional cell aggregates from a plate made with the possibility of turning over to form hanging drops in the wells of the receiving plate by centrifugation of the kit.

Another aspect of the invention relates to a method for testing a substance for its toxicity to cells. The method comprises the following steps:

a) introducing a predetermined number of liquid drops into the corresponding number of culture cells of the plate, made with the possibility of turning over to form hanging drops, each drop containing a predetermined volume of the substance to be tested and a liquid nutrient medium that ensures the growth of non-human embryonic stem cells, as well as many non-human embryonic stem cells;

b) turning and incubating a plate made to turn over to form hanging drops for a predetermined period of time, wherein the plate made to turn over to form hanging drops contains drops so that they hang from the respective culture cells to allow non-human embryonic stem cells to form three-dimensional cell aggregates representing embryoid bodies of embryonic stem cells non-human in appropriate drops;

c) the additional supply of a liquid nutrient medium that provides the growth of non-human embryonic stem cells into droplets in the culture cells in order to facilitate the growth of three-dimensional cell aggregates representing embryoid bodies of non-human embryonic stem cells;

d) analysis of three-dimensional cell assemblies, which are embryoid bodies of non-human embryonic stem cells, in order to evaluate whether the substance to be tested is toxic to three-dimensional cell assemblies, which are embryoid bodies of non-human embryonic stem cells.

In the method in accordance with the invention, a plate made with the possibility of turning over to form hanging drops in accordance with the invention is used as described above, and step c) is performed by repeatedly turning the plate made with the possibility of turning over to form hanging drops, by adding an additional drop of liquid nutritional environment that provides the growth of non-human embryonic stem cells into appropriate drops containing three-dimensional cell aggregates, Representing an embryoid body embryonic stem cells, non-human, to form respective droplet larger size in the corresponding culture cells. Then, a flip-up plate to form hanging droplets is flipped again to allow three-dimensional cell assemblies, which are non-human embryoid bodies of embryonic stem cells, to grow into corresponding larger droplets hanging from the culture cells. The advantages have already been mentioned above when discussing the advantages of a plate made with the possibility of turning over to form hanging drops in accordance with the invention. In addition, it is allowed to automate at least some process steps, for example, the additional supply of a liquid nutrient medium that allows the growth of non-human embryonic stem cells.

One embodiment of the method in accordance with the invention further comprises the steps of:

e) transferring the grown three-dimensional cell assemblies, which are embryoid bodies of non-human embryonic stem cells, from culture cells of a plate configured to be flipped to form hanging drops in the corresponding number of wells of the receiving plate;

f) incubation of the receiving plate with the wells containing three-dimensional cell aggregates representing the embryoid bodies of non-human embryonic stem cells for an additional predetermined period of time; and

g) after the incubation step, analysis of three-dimensional cell aggregates representing embryoid bodies of non-human embryonic stem cells in order to assess whether the substance to be tested is toxic to three-dimensional cell assemblies representing embryoid bodies of non-human embryonic stem cells.

In a preferred version of this embodiment of the method in accordance with the invention, step e) is performed by assembling a plate configured to be flipped to form hanging drops and a receiving plate so that the respective wells of the receiving plate are opposite the corresponding culture cells of the plate configured to be turned for the formation of hanging drops, followed by centrifugation of the collected plates. This allows you to conveniently and automatically perform the transfer of three-dimensional cell aggregates, which are embryoid bodies of non-human embryonic stem cells, from a plate made with the possibility of turning over to form hanging drops in the corresponding wells of the receiving plate.

In a further embodiment of the method of the invention, the step of analyzing three-dimensional cell assemblies representing non-human embryoid bodies of embryonic stem cells in order to assess whether the substance to be tested is toxic to three-dimensional cell assemblies representing embryoid bodies of embryonic stem cells non-human is performed by analyzing whether the embryonic bodies contain myocardial cells. This option is suitable because the presence of myocardial cells in three-dimensional cell populations can be easily detected since myocardial cells are pulsating.

Additional advantageous features of the invention will become apparent from the following detailed description of embodiments with reference to the accompanying drawings, in which:

FIG. 1 is a perspective view of a first embodiment of a wafer configured to turn over to form hanging drops in accordance with the invention, wherein one well comprises a drop and the other contains a drop to which an additional drop is added;

FIG. 2 shows a top view of an embodiment of a plate rotatable to form hanging drops according to FIG. one;

FIG. 3 shows a section along line III-III in FIG. 2, wherein the plate, capable of being turned over to form hanging drops, is inverted again;

FIG. 4 shows a section along line III-III in FIG. 2, wherein the plate configured to be turned over to form hanging drops is inverted;

FIG. 5 shows an enlarged image of part V in FIG. one;

FIG. 6 is an enlarged view of one well of an embodiment of a plate configured to be flipped to form hanging drops of FIG. one;

FIG. 7 shows a section through a well of a second embodiment of a plate made to be turned over to form hanging drops in accordance with the invention, wherein the well comprises a drop;

FIG. Figure 8 shows the well of a second embodiment of a plate that can be turned over to form hanging drops, with an additional drop added;

FIG. 9 shows a well of a second embodiment of a plate made to be turned over to form hanging drops, with another additional drop added;

FIG. 10 is a cross-sectional view of a third embodiment of a wafer configured to be flipped to form hanging drops in accordance with the invention, wherein the drop cell comprises a drop;

FIG. 11 shows a third embodiment of a plate made to be turned over to form hanging drops in accordance with the invention, with an additional drop added;

FIG. 12 shows a third embodiment of a plate that is rotatable to form hanging drops in accordance with the invention, with another additional drop added;

FIG. 13 shows a third embodiment of a plate that can be turned over to form hanging drops in accordance with the invention, with yet another additional drop added;

FIG. 14 shows a fourth embodiment of a plate that can be turned over to form hanging droplets in accordance with the invention, with another additional drop added;

FIG. 15 shows an embodiment of an embryonic stem cell sample using a plate that is invertible to form hanging drops in accordance with the invention;

FIG. 16 shows a perspective view of a separately manufactured hole of an embodiment of a plate configured to be turned over to form hanging drops with 24 wells in accordance with the invention;

FIG. 17 shows the hole in FIG. 16 in cross section;

FIG. 18 is a plan view of a separately manufactured plate of an embodiment of a plate configured to be turned over to form hanging drops with 24 wells in accordance with the invention, into the openings of which individually made holes can be inserted in FIG. 16;

FIG. 19 is a bottom view of the plate of FIG. eighteen;

FIG. 20 is an enlarged view of a detail of the plate of FIG. 18 and FIG. 19, including one of the holes into which the wells should be inserted;

FIG. 21 shows a top view of a plate made with the possibility of turning to form hanging droplets with 24 holes, while the wells of FIG. 16 are inserted into the holes of the plate of FIG. eighteen;

FIG. 22 shows a section through a plate made with the possibility of turning over to form hanging droplets with 24 wells. FIG. 21;

FIG. 23 is a bottom view of an invertible plate to form hanging droplets with 24 wells. FIG. 21;

FIG. 24 is a sectional view of the part of FIG. 22, including one well inserted in one hole;

FIG. 25 is a perspective view of an embodiment of an additional 24-well plate to which a plate configured to be turned over to form hanging drops with 24 wells can be attached, shown in FIG. 21 (similar to the kit shown in FIG. 15);

FIG. 26 is a sectional view of an embodiment of an additional 24-well plate in FIG. 25;

FIG. 27 is a perspective view of a plate configured to be turned over to form 24-well hanging droplets as shown in FIG. 21 attached to an additional plate with 24 holes;

FIG. 28 is a sectional view of the plate set shown in FIG. 27;

FIG. 29 shows a separately manufactured hole of an embodiment of a plate configured to be flipped to form hanging drops with 96 wells in accordance with the invention, in an enlarged perspective view;

FIG. 30 shows the well of FIG. 29 in cross section;

FIG. 31 shows a detail of the well of FIG. 29, including stepped circular rims forming microfluidic wetting barriers;

FIG. 32 is a plan view of a separately manufactured plate of an embodiment of a plate configured to be turned over to form hanging droplets with 96 wells in accordance with the invention, into the holes of which individually manufactured wells can be inserted. FIG. 29;

FIG. 33 shows a section through the plate of FIG. 32, including holes into which the wells of FIG. 29;

FIG. 34 is a bottom view of the plate of FIG. 32;

FIG. 35 shows a top view of a plate configured to be flipped to form hanging droplets with 96 wells, wherein the wells of FIG. 29 are inserted into the holes of the plate of FIG. 32;

FIG. 36 shows a section through a plate configured to be turned over to form hanging droplets with 96 wells. FIG. 35;

FIG. 37 shows a bottom view of a plate configured to be turned over to form hanging droplets with 96 wells. FIG. 35;

FIG. 38 is a sectional view of the part of FIG. 36, including one well inserted in one hole;

FIG. 39 is a plan view of an embodiment of an additional 96-well plate to which a plate configured to be turned over to form 96-well hanging droplets as shown in FIG. 35 (similar to the kit shown in FIG. 15);

FIG. 40 is a sectional view of an embodiment of an additional 96-well plate. FIG. 39, and

FIG. 41 is a perspective view of a plate configured to be turned over to form hanging droplets with 96 wells. FIG. 35 attached to an additional 96-well plate.

In FIG. 1 - FIG. 6 shows a first embodiment of a plate 1, which can be turned over to form hanging droplets in accordance with the invention, or details of a variant in various forms (see above). Plate 1 contains a predetermined number of cells with droplets in the form of holes 10. The embodiment shown is a plate with 24 holes, however, a different number of holes is acceptable. Preferred are plates having a standard number of wells that are arranged in accordance with standard micro-well plates, for example a 96-well plate or a 384-well plate.

Although various embodiments of the wells are acceptable, wells 10 of the embodiment of the plate 1 are provided, which are invertible to form hanging drops, as shown in the enlarged image of FIG. 6. The hole 10 contains a first circular wetting barrier in the form of a sharp circular edge 102 surrounding the first cavity 100 having a closed bottom 101. The first circular edge 102 is surrounded by a second sharp circular edge 104, while the wetted portion 103 is located between the first circular edge 102 and the second circular edge. In FIG. 6 shows that the circular edges are arranged in steps, although this is not necessary. There is an annular cutout surrounding the second circular edge 104.

The reason for using the geometric shape of the holes 10 becomes clearer from FIG. 3, FIG. 4 and FIG. 5, which shows the process of supplying droplets to the wells 10 and forcing them to hang from the wells 10. FIG. 3, a plate 1, invertible to form hanging droplets, is shown in an inverted position, i.e. with drops facing up. In this well 10, which is located in the leftmost position, a small drop of liquid 20 can be recognized. In the second well 10 on the left, a larger drop 21 can be recognized, which is obtained by feeding another drop of liquid to the drop 20 to form a larger drop 21. It is clear that the supply of drops to the wells 10 can be carried out, for example, using a pipette. In particular, dropping droplets into the wells 10 can be carried out using a pipette robot, but this can also be done manually. Also, if you need to pump liquid from a well (for example, when you should replace the "old" liquid nutrient medium with a "fresh" liquid nutrient medium), this can be easily done, since there is access to the drops.

In FIG. 4, a plate 1 is shown which is rotatable to form hanging droplets, where a smaller drop 20 and a larger drop 21 hang from respective wells 10, best shown in FIG. 5, in which part V of FIG. 4. A smaller drop 20 hangs from the leftmost hole 10, and a larger drop 21 hangs from the second hole 10 on the left.

From FIG. 5 and FIG. 6, the function of the first and second circular edges 102 and 104 becomes more understandable as microfluidic barriers preventing spreading beyond the edges of the droplet 20 and the larger droplet 21. Therefore, the first and second circular edges 102 and 104 help to stabilize the drooping of the droplet from the corresponding hole 10. As for the second circular edge 104, this action is enhanced by the annular cutout 105. The wetted portion 103 located between the first and second circular edges 102 and 104 (see Fig. 6) makes it easy to add an additional liquid drop to drop 20 to form a larger drop 21 (see Fig. 4 and Fig. 5). The closed bottom of 100 wells 10 prevents the evaporation of liquid through an opening or channel passing through the bottom, and also serves to stabilize droplets hanging from the holes, due to the fact that dropping the droplet with air "from underneath" is prevented.

A second embodiment of a plate configured to be flipped to form hanging drops in accordance with the invention is shown in FIG. 7 - FIG. 9, in which only one hole 30 of the plate 3 is shown, made with the possibility of turning to form hanging drops, respectively. This embodiment, one way or another, relates to the first embodiment, described in detail above, in that it also contains a stepwise configuration of microfluidic barriers that prevent the droplet from spreading outside the corresponding microfluidic barrier. However, the second embodiment differs from the first embodiment in that, instead of sharp edges, the holes 30 comprise a cavity 301 having a closed bottom 300, which is surrounded by a first circular rim 302 surrounding the cavity 301 and protruding beyond the cavity toward the open end of the hole 30. FIG. 7 shows a small drop of liquid 40 hanging from a well 30. Drop 40 is retained in the cavity 301 by a first circular rim 302 forming a microfluidic barrier.

FIG. 8 differs from FIG. 7 in that another drop of liquid is added to the liquid drop 40 to form a larger liquid drop 41. A larger liquid droplet 41 is held by a second circular rim 304 surrounding the first circular rim 302, the wettable portion 303 being located between the first circular rim 302 and the second circular rim 304. The second circular rim 304 again acts as a microfluidic barrier preventing the larger droplet 41 from spreading. beyond the rim 304.

FIG. 9 differs from FIG. 8 in that a further liquid drop is added to the larger liquid drop 41 to form an even larger liquid drop 42. The droplet 42 is held by a third circular rim 306 surrounding the second circular rim 304, while another wettable portion 305 is located between the second circular rim 304 and the third circular rim 306. The third circular rim 306 again acts as a microfluidic barrier preventing the droplet 42 from spreading even larger bezel limits 306.

In FIG. 10 - FIG. 14 shows one cell 50 for a drop of a third embodiment of a plate 5, which is invertible to form hanging drops in accordance with the invention. This embodiment of the plate 5, made with the possibility of turning over to form hanging droplets, differs from the first and second variants described in detail above in that the cell 50 contains microfluidic barriers not in a step configuration, but in the form of circular rims located on a flat lower surface plates 5. Although the cell 50 again contains a closed bottom 500, the cavity 501 is bounded by a first circular rim 502 surrounding the cavity 501 and protruding from the bottom surface of the plate 5. In FIG. 10 shows a small drop 64 of liquid hanging from the cell 50. Drop 64 is held in the cavity 501 by a first circular rim 502 forming a microfluidic barrier.

FIG. 11 differs from FIG. 10 in that another drop of liquid is added to the liquid drop 64 to form a larger liquid drop 65 (also a larger liquid drop 41 can be introduced into the drop cell 50 as a first step). A larger liquid droplet 65 is held by a second circular rim 504 surrounding the first circular rim 302, the wetting portion 503 being located between the first circular rim 502 and the second circular rim 504. The second circular rim 504 again acts as a microfluidic barrier preventing the larger droplet 65 from spreading. beyond the rim 504.

In FIG. 12, an even larger droplet 66 is shown which is held by a third circular rim 506 that surrounds the second circular rim 504, with an additional wettable portion 505 located between the second circular rim 604 and the third circular rim 506. The third circular rim 506 again acts as a microfluidic barrier, preventing the spreading of the droplet 66 beyond the rim 506.

In FIG. 13, an even larger droplet 67 is shown which is held by a fourth circular rim 508 that surrounds the third circular rim 506, with yet another additional wetted portion 507 located between the third circular rim 506 and the fourth circular rim 508. The fourth circular rim 508 again acts as microfluidic a barrier preventing the spreading of the droplet 67 beyond the rim 508.

And finally, in FIG. 14, an even larger droplet 68 is shown which is held by a fifth circular rim 510 that surrounds the fourth circular rim 508, with another wettable portion 509 located between the fourth circular rim 508 and the fifth circular rim 510. In the same manner as described above, the fifth circular the rim 510 acts as a microfluidic barrier that prevents the droplet 68 from spreading even larger outside the rim 510.

In FIG. 15 shows an embodiment of an embryonic stem cell sample representing a variant of the method in accordance with the invention in which any of the above embodiments of a plate configured to be turned upside down to form hanging drops can be used. For the following description of the sample, it is contemplated to use a first embodiment of a plate 1 that is invertible to form hanging drops in accordance with the invention. In this variant of the test, the toxicity of the substance with respect to non-human embryoid bodies of embryonic stem cells is checked. To this end, a predetermined number of drops of liquid is introduced into the corresponding number of cells or wells for drops. Each drop contains a predetermined volume of the substance to be tested, as well as a liquid nutrient medium that provides for the growth of embryoid bodies of non-human embryonic stem cells, as well as a plurality of stem cells 82. In the first embodiment, the plate 1, made with the possibility of turning over to form hanging drops , you can enter 24 drops 20 into the corresponding wells 10, while the plate 1 is again upside down (see Fig. 4). After droplets are introduced into the wells 10, the plate 1 is turned over and mounted with an additional plate 7 having a corresponding number of holes 70, which are located opposite the holes 10 of the plate 1, which are capable of being turned over to form hanging drops to form a closed set. Wells 70 may also contain culture media. The kit thus formed may be such that droplets 20 hang from the wells 10, as has been explained in detail above. The kit thus formed (see Fig. 15, top left) is incubated for a predetermined period of time, for example, for three days. During this incubation period, stem cells 82 contained in the corresponding drops precipitate and form a three-dimensional embryoid body 83 (more commonly a three-dimensional cell population) of non-human embryonic stem cells in a corresponding drop 20 at the top of the drop, i.e. at the lowest point of the drop 20 (see Fig. 15, top right).

After the first incubation period, it may be necessary to supply an additional liquid nutrient medium to drop 20 in order to promote additional growth of the embryoid bodies of 83 non-human embryonic stem cells. To this end, the kit opens again and plate 1 flips again. In this reverted position, the liquid nutrient medium is additionally supplied to the corresponding drops 20 in the corresponding wells 10 to form larger drops 21. Upon completion of the additional supply of liquid nutrient medium to form larger droplets 21, the plate 1 is again turned over and mounted again with the additional plate 7 (the wells 70 of which may also contain fresh liquid nutrient medium). The kit thus formed is incubated for an additional predetermined period of time, for example, for two days, to promote additional growth of embryoid bodies of non-human embryonic stem cells.

After an additional incubation period, the embryoid bodies 83 can be transferred to the wells 70 of the plate 7 (receiving plate) by centrifugation, which can be done using a centrifugation device well known in the art. After centrifuging the embryoid bodies 83 into the wells 70 of the plate 7 containing the liquid nutrient medium, the assembly can be incubated for another predetermined period of time, for example, for five or seven days. Embryoid bodies 83 are deposited on the flat bottom of the corresponding holes 70 of the plate 7.

During this incubation period, the kit can be reopened and the cell sets, which are embryoid bodies of non-human embryonic stem cells, can be checked to see if the test substance is toxic to stem cells. Although various types of assays are offered, one type of assay is for microscopic analysis of cell aggregates. Since the cell aggregates are located on the flat bottom of the wells of plate 7, microscopic analysis can be performed. For example, it is possible to test whether myocardial cells contain cellular aggregates, since this type of cells can be easily identified under a microscope, because these cells contract and expand (pulsate).

The above transfer of embryoid bodies or three-dimensional cell aggregates to the wells 70 of the additional plate 7 by centrifugation is optional. Alternatively, the kit can be reopened, and plate 1 can be attached to the centrifuge in such a way that during centrifugation the embryoid bodies or three-dimensional cell aggregates move from the top of the droplet towards the bottom of 101 wells 10 (see Fig. 6). Therefore, embryoid bodies can be analyzed in the holes 10 of the plate using a microscope, since they are located at the bottom 101 of the holes 10, which forms a stable background for microscopic analysis.

In FIG. 16 - FIG. 24 shows an additional embodiment of a plate 6, made with the possibility of turning over to form hanging drops (Fig. 21 - Fig. 23) in accordance with the invention. This embodiment comprises separately made holes 60 (see FIG. 16 - FIG. 17), which can be pressed into the holes 610 of the separately manufactured plate 6 (see FIG. 18 - FIG. 20). As can be seen from the section of the separately manufactured hole 60 shown in FIG. 17, the hole 60 comprises a first circular rim 602, a second circular rim 604, and a third circular rim 606. The structures and functions of the well and the first, second, and third circular rims are already described in detail using FIG. 7, FIG. 8 and FIG. 9, and therefore this applies to these parts of the description presented above. In the shown embodiment, the holes 60 are pressed into the holes 610 of the plate 61 from the side of the plate 61, which forms the outer surface 611 of the plate 61. As soon as all the holes 60 are pressed into the holes 610, they are rigidly attached to the plate 61, and the formation of the plate 6 with hanging drops ends (see Fig. 21 - Fig. 24).

Alternatively, it is possible that separately made holes 60 are attached to the inner surface 612 of the plate 61 (for example, by ultrasonic welding or by gluing). In this case, the plate 61 may not contain holes. However, the places where ultrasonic welding or gluing of the holes 60 to the inner surface 612 of the plate 61 was performed may be visible and may complicate microscopic analysis of the contents of the holes. Therefore, it is preferable to press holes 60 into the holes 610 of the plate 61.

In FIG. 25 and FIG. 26 shows an embodiment of an additional plate 8, which contains the number of holes corresponding to the number of holes 60 of the plate 6, which is invertible to form hanging drops. Also, the holes 80 of the additional plate 8 are located in accordance with the location of the holes 60 of the plate 6, made with the possibility of turning to form hanging drops.

In FIG. 27 and FIG. 28 you can see that the plate 6, made with the possibility of turning over to form hanging drops, can be stacked on an additional plate 8 so that each hole 60 of the plate 6, made with the possibility of turning over to form hanging drops, is located above the corresponding hole 80 of the additional plate 8. One purpose of such a kit has already been described in detail with reference to FIG. 15, and therefore this applies to these parts of the description presented above.

With reference to FIG. 16 - FIG. 28, only 24-well plates or 24-well plate sets are described, however, similar considerations apply to 96-well plates, which will become apparent from the following description.

In FIG. 29 - FIG. 38 shows yet another embodiment of a plate 9 turned over to form hanging drops (FIG. 35 - FIG. 37) in accordance with the invention, which is presented in the form of a plate made to turn over to form hanging drops with the 96th holes. Similar to the 24-well option, the 96-well option contains separately manufactured wells 90 (see FIG. 29 - FIG. 31) that are pressed into the holes 910 of the separately manufactured plate 91 (see FIG. 32 - FIG. 34 ) As can be seen from the cuts of the separately manufactured hole 90 shown in FIG. 30 and FIG. 31, hole 90 comprises a first circular rim 902, a second circular rim 904, and a third circular rim 906. The structures and functions of the hole and the first, second, and third circular rims are already described in detail using FIG. 7, FIG. 8 and FIG. 9, and therefore this applies to these parts of the description presented above. In the shown embodiment, the holes 90 are pressed into the holes 910 of the plate 91 on the side of the plate 91, which forms the outer surface 911 of the plate 91. As soon as all the holes 90 are pressed into the holes 910, they are rigidly attached to the plate 91, and the formation of the plate 9 is made with the possibility the flipping for the formation of hanging drops is completed (see Fig. 35 - Fig. 38).

In FIG. 39 - FIG. 41 shows a variant of the additional plate 92, which contains the number of holes 920 corresponding to the number of holes 90 of the plate 9, which is made with the possibility of turning over to form hanging drops. Also, the holes 920 of the additional plate 92 are located in accordance with the location of the holes 90 of the plate 9, made with the possibility of turning to form hanging drops.

In FIG. 41 you can see that the plate 9, made with the possibility of turning over to form hanging drops, can be stacked on an additional plate 92 so that each well 90 of the plate 9, made with the possibility of turning over to form hanging drops, is located above the corresponding hole 920 of the additional plate 92.

All of the above options are only examples of various fields of application of the method, the plate made with the possibility of turning over to form hanging drops, and the plate made with the possibility of turning over to form hanging drops in accordance with the invention, therefore, the invention is not limited to these described examples. Rather, the scope of protection is determined by the attached claims.

Claims (23)

1. A plate made with the possibility of turning over to form hanging drops for cell culture, containing a predetermined number of cells for a drop, each cell is capable of receiving a drop of liquid in the cavity of a corresponding cell for a drop, the corresponding cell for a drop contains a circular microfluidic wetting barrier configured to surround the cavity of the cell for the drop and preventing the spreading of the drop beyond the microfluidic wetting barrier, while the corresponding cell for the drop contains an open bottom and at least one additional circular microfluidic wetting barrier, each additional circular microfluidic wetting barrier is configured to surround the previous circular microfluidic wetting barrier, and the wettable portion is located between two adjacent microfluidic wetting barriers. 2. The plate according to claim 1, in which the corresponding cells for the drop are the wells (10; 30; 60; 90). 3. The plate according to claim 1 or 2, in which the circular microfluidic wetting barrier is a circular edge (102), wherein at least one additional circular microfluidic wetting barrier is at least one additional circular edge (104 ), each additional circular edge (104) is configured to surround the previous circular edge (102), and the wetted portion (103) is located between two adjacent circular edges (102; 104). 4. The plate according to claim 3, in which two adjacent circular edges (102; 104) are arranged in the form of steps. 5. The plate according to claim 1 or 2, in which the circular microfluidic wetting barrier is a circular rim (302; 502; 602; 902) wherein at least one additional circular microfluidic wetting barrier is an additional circular rim, each additional a circular rim (304; 306; 504; 506; 508; 510; 604; 606; 904; 906) surrounds the previous circular rim (302; 304; 502; 504; 506; 508; 602; 604; 902; 904), and the wetted area (303; 305; 503; 505; 507; 509) is located between two adjacent circular rims. 6. The plate according to claim 5, in which the adjacent circular rims (302; 304; 306; 602; 604; 606; 902; 904; 906) are located in the form of steps. 7. The plate according to claim 2, in which the plate is made of a separately manufactured plate (61; 91) having a predetermined number of holes (610; 910), and from a corresponding predetermined number of separately made holes (60; 90) forming a drop cell , each separately manufactured hole (60; 90) is pressed into the corresponding hole (610; 910) of the separately manufactured plate (61; 91). 8. The plate of claim 3, in which the plate is made of a separately manufactured plate (61; 91) having a predetermined number of holes (610; 910), and from a corresponding predetermined number of separately made holes (60; 90) forming a drop cell, each individually manufactured hole is pressed into the corresponding hole (610; 910) of the separately manufactured plate (61; 91). 9. The plate according to claim 4, in which the plate is made of a separately manufactured plate (61; 91) having a predetermined number of holes (610; 910), and from a corresponding predetermined number of separately made holes (60; 90) forming a drop cell , each separately manufactured hole is pressed into the corresponding hole (610; 910) of the separately manufactured plate (61; 91). 10. The plate according to claim 5, in which the plate is made of a separately manufactured plate (61; 91) having a predetermined number of holes (610; 910), and from a corresponding predetermined number of separately made holes (60; 90) forming a drop cell , each separately manufactured hole is pressed into the corresponding hole (610; 910) of the separately manufactured plate (61; 91). 11. The plate according to claim 6, in which the plate is made of a separately manufactured plate (61; 91) having a predetermined number of holes (610; 910), and from a corresponding predetermined number of separately made holes (60; 90) forming a drop cell , each separately manufactured hole is pressed into the corresponding hole (610; 910) of the separately manufactured plate (61; 91). 12. The plate according to claim 7, in which the separately made holes (60; 90) are pressed into the holes (610; 910) of the separately manufactured plate (61; 91) from the side of the plate forming the outer surface (611; 911) of the plate. 13. The plate according to claim 8, in which the separately made holes (60; 90) are pressed into the holes (610; 910) of the separately manufactured plate (61; 91) from the side of the plate forming the outer surface (611; 911) of the plate. 14. The plate according to claim 9, in which the separately made holes (60; 90) are pressed into the holes (610; 910) of the separately manufactured plate (61; 91) from the side of the plate forming the outer surface (611; 911) of the plate. 15. The plate according to claim 10, in which the separately made holes (60; 90) are pressed into the holes (610; 910) of the separately manufactured plate (61; 91) from the side of the plate forming the outer surface (611; 911) of the plate. 16. The plate according to claim 11, in which the separately made holes (60; 90) are pressed into the holes (610; 910) of the separately manufactured plate (61; 91) from the side of the plate forming the outer surface (611; 911) of the plate. 17. The plate according to claim 2, containing 24 holes, 96 holes or 384 holes. 18. The plate according to claim 5, in which the circular rims (503; 504; 506; 508; 510) are located on a flat surface. 19. A set of plates for transferring three-dimensional cell aggregates cultured in hanging drops from a plate according to any one of paragraphs. 1-18 in the receiving plate containing the plate, made with the possibility of turning over to form hanging drops according to any one of paragraphs. 1-18, and having a predetermined number of cells or holes for a drop, and a receiving plate (7) having a number of holes (70) corresponding to a predetermined number of cells or holes for a drop of a plate configured to be turned over to form hanging drops, the plate made with the possibility of turning over to form hanging drops and the receiving plate (7) are mounted so that in the assembled state the holes (70) of the receiving plate are aligned with the cells or holes for the drop of the plate, made with the possibility of vorachivaniya to form hanging drops. 20. A method for testing a substance for toxicity to cells, comprising the steps of:
a) a predetermined number of liquid droplets is introduced into the corresponding number of cells for a plate droplet that can be turned over to form hanging droplets, each drop contains a predetermined volume of the substance to be tested and a liquid nutrient medium that provides the growth of non-human embryonic stem cells, and also many non-human embryonic stem cells (82);
b) turn over and incubate the plate, made with the possibility of turning over to form hanging drops, for a predetermined period of time, while the plate contains drops (20; 40; 64) so that they hang from the corresponding cells for the drop to allow to form non-human embryonic stem cells to form three-dimensional cell aggregates representing the embryoid bodies of non-human embryonic stem cells in appropriate drops;
c) carry out an additional supply of a liquid nutrient medium that provides additional growth of the embryoid bodies of non-human embryonic stem cells into drops in the respective drop cells in order to facilitate the growth of three-dimensional cell aggregates representing non-human embryoid bodies of embryonic stem cells; and
d) analyze three-dimensional cell assemblies representing non-human embryoid stem cells, to evaluate whether the substance to be tested is toxic to three-dimensional cell assemblies representing non-human embryoid stem cells, wherein uses a plate made with the possibility of turning over to form hanging drops according to any one of paragraphs. 1-18; and
wherein step c) is carried out by repeatedly turning the plate over so as to flip over to form hanging drops, adding a drop of liquid nutrient medium that allows the growth of embryoid bodies of non-human embryonic stem cells into the corresponding drops containing three-dimensional cellular aggregates representing embryoid non-human embryonic stem cell bodies to form larger corresponding droplets in corresponding cells kah to drop, and then, again inverted plate to enable a three-dimensional cell aggregates, representing an embryoid body of embryonic stem cells, non-human, to grow into a corresponding drop larger, hanging from the cells to drop. 21. The method according to p. 20, additionally containing the following steps, in which:
e) transfer the grown three-dimensional cell aggregates (83), which are embryoid bodies of non-human embryonic stem cells, from cells for a drop of a plate configured to be turned over to form hanging drops in the corresponding number of holes (70) of the receiving plate (7) ;
f) incubate the receiving plate (7) with the wells (70) containing three-dimensional cell aggregates representing embryoid bodies of non-human embryonic stem cells for an additional predetermined period of time; and
g) after incubation, an analysis of three-dimensional cell assemblies, which are embryoid bodies of non-human embryonic stem cells, is carried out to assess whether the substance to be tested is toxic to three-dimensional cell assemblies, which are embryoid bodies of non-human embryonic stem cells human 22. The method according to p. 21, in which step e) is performed by assembling a plate configured to be flipped to form hanging drops and a receiving plate (7) so that the corresponding holes (70) of the receiving plate (7) are located opposite the corresponding cells for a drop of a plate made with the possibility of turning over to form hanging drops, and by means of subsequent centrifugation of the collected plates. 23. The method according to any one of paragraphs 21 or 22, wherein the step of analyzing three-dimensional cell assemblies, which are embryoid bodies of embryonic stem cells that are not human, for assessing whether the substance to be tested is toxic to three-dimensional cell assemblies representing The non-human embryoid bodies of embryonic stem cells are performed by analyzing whether the embryoid bodies contain myocardial cells.

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