Method for the culturing of cells

Abstract

The invention refers to a method for culturing cells in a substrate in which a chamber having at least one side wall, a bottom, and a top is formed, comprising introduction of cells into the chamber, tilting of the substrate such that the cells accumulate on a side wall of the chamber, and holding the substrate in the tilted orientation such that the cells form a three-dimensional cell aggregate.

Claims

1. A method for culturing cells in a substrate, the method comprising: introducing cells into a chamber of the substrate, the substrate having a microfluidic structure and comprising a first microfluidic channel and a second microfluidic channel connected to the chamber, the chamber having at least one side wall, a continuous planar bottom, and a top, wherein the top covers the chamber; tilting the substrate to a tilted orientation such that the cells accumulate in an arrangement on a side wall of the chamber; holding the substrate in the tilted orientation so that the cells form a three-dimensional cell aggregate, such that the cells grow together approximately in the arrangement in which they have accumulated on the side wall of the chamber; tilting the substrate back to a non-tilted orientation following formation of the three-dimensional cell aggregate; and perfusing the three-dimensional cell aggregate in the chamber by transporting liquid into and/or out of the chamber via the first microfluidic channel and/or the second microfluidic channel.

2. The method according to claim 1, wherein the substrate comprises a plurality of chambers and cells are introduced into each of the plurality of chambers before tilting, so that by the tilting and holding of the substrate, a distinct three-dimensional cell aggregate is formed simultaneously in each of the plurality of chambers.

3. The method according to claim 2, wherein the method further comprises perfusing a plurality or all of the cell aggregates.

4. The method according to claim 3, wherein the cell aggregates are perfused independently.

5. The method according to claim 1, wherein the substrate is tilted from an arrangement in which the continuous planar bottom of the chamber is arranged substantially horizontally by an angle of 20 to 45.

6. The method according to claim 5, wherein the bottom of the chamber is arranged substantially horizontally by an angle of 30 to 40.

7. The method according to claim 6, wherein the continuous planar bottom of the chamber is arranged substantially horizontally by an angle of 35.

8. The method according to claim 1, wherein the holding of the substrate is for 4 to 26 hours, 6 to 25 hours, 8 to 24 hours, 10 to 23 hours, 12 to 22 hours, 14 to 21 hours, 16 to 20 hours, 17 to 19 hours, or 18 hours.

9. The method according to claim 1, wherein the perfusing is performed by means of one or a plurality of pumps and/or passively transporting the liquid into and/or out of the chamber.

10. The method according to claim 9, wherein the passively transporting the liquid into and/or out of the chamber is by means of gravity.

11. The method according to claim 1, wherein the perfusing comprises passively transporting the liquid into and/or out of the chamber, wherein the substrate is tilted to transport the liquid.

12. The method according to claim 1, wherein the perfusing comprises the three-dimensional cell aggregate being placed in a liquid stream or superperfusion of the three-dimensional cell aggregate.

13. The method according to claim 1, further comprising: performing microscopic examination of the three-dimensional cell aggregate while the three-dimensional cell aggregate is disposed on the continuous planar bottom in the non-tilted orientation in the chamber.

14. The method according to claim 13, wherein the microscopic examination of the three-dimensional cell aggregate in the chamber is through the continuous planar bottom of the chamber.

15. The method according to claim 1, wherein: the continuous planar bottom is a continuous planar cell-repellent bottom; the substrate comprises an opening in the top above the chamber, wherein the opening is closed with a lid during the formation of the three-dimensional cell aggregate; and the method further comprises removing the cell aggregates from the substrate through the opening, wherein the lid is taken off for the removal and the cell aggregates are subsequently removed directly from the chamber through the opening.

16. The method according to claim 15, wherein the lid comprises a film.

17. The method according to claim 15, wherein the opening is closed with the lid during perfusion.

18. The method according to claim 1, wherein the perfusing of the three-dimensional cell aggregate in the chamber is performed during and/or after the substrate tilting back to the non-tilted orientation.

19. The method according to claim 18, wherein the tilting back is tilting back to an initial position from which the substrate was tilted into the tilted orientation.

Description

(1) Further features and advantages are explained below with reference to the exemplary Figures:

(2) FIGS. 1a to 1c show a schematic, not to scale side view of a substrate and top views of a substrate of a first embodiment with two alternative outlines of the chamber,

(3) FIG. 2 shows a schematic, not to scale side view of a substrate of a second embodiment,

(4) FIG. 3 shows a schematic, not to scale side view of a substrate of a third embodiment,

(5) FIG. 4 shows a schematic, not to scale side view of a substrate of a fourth embodiment,

(6) FIG. 5 shows a schematic, not to scale side view of a substrate of a fifth embodiment,

(7) FIGS. 6a and 6b show a schematic, not to scale top view of a substrate and side view of a substrate of a sixth embodiment having a plurality of chambers,

(8) FIGS. 7a and 7b show a schematic, not to scale top view of a substrate and side view of a substrate of a seventh embodiment having a plurality of chambers,

(9) FIGS. 8a to 8f show schematic, not to scale views of various outlines of the chamber,

(10) FIG. 9 shows a schematic, not to scale view of a system of an embodiment, and

(11) FIGS. 10a to 10h show a schematic view of various possible steps of a method of an embodiment.

(12) FIG. 1a shows a side view of a substrate 1 with a chamber 2, two fluid channels 3a and 3b, and two fluid connections 4a and 4b. One end of the fluid channels opens into the chamber and the other end opens into the respective fluid connection. At least one of the fluid connections can comprise a fluid reservoir, or a fluid reservoir can be mounted on the fluid connection.

(13) FIG. 1a shows an example of a substrate having an opening 5 above the chamber, which opening is closed with a removable lid 6, for example a film, during the formation of the cell aggregate. However, such an opening is optional.

(14) The substrate, when having such an opening, can be formed in multiple parts and comprise a bottom part in which the bottom and side wall or side walls of the chamber are formed and the lid, the lid being removably attached to the bottom part and, when attached, closing the opening and forming at least a portion of the top of the chamber.

(15) Alternatively, the substrate can be formed, for example, such that no openings are formed directly above the chamber. In particular, the substrate can be formed such that it has only openings directly connected to fluid connections. A substrate without the opening 5 is shown by way of example in FIG. 2, the substrate otherwise being formed as described in connection with FIG. 1.

(16) The chamber has a bottom 7a, which is in particular planar and parallel to the bottom side 1a, which in this example is also planar, and optionally, as in this example, also parallel to the top side 1b of the substrate. The top side can also be planar, optionally apart from the fluid connections and the opening. In particular, the lid 6 can be planar. The chamber has a top 7(b).

(17) The chamber can have different outlines, for example rectangular or round. FIG. 1b shows an example of a top view of the substrate with a rectangular outline and FIG. 1c shows a top view with a round outline.

(18) If the substrate has a round or elliptical outline, it has a circumferential side wall 8, which can optionally be interrupted by mouths of fluid channels. If the side wall has a rectangular outline, it has four side walls 8a, 8b, 8c, 8d, which can also optionally be interrupted by mouths of fluid channels. The side wall 8 or one or a plurality of the side walls 8a to 8d, in particular all of the side walls, can be arranged substantially perpendicular to the bottom of the chamber. However, this is optional, as will be explained in detail below.

(19) In particular, it can have any of the outlines described below in connection with FIGS. 8a to 8f. There, regions 9a and 9b of the chamber at which cells 16 located in the chamber accumulate and form a cell aggregate 10 by tilting of the substrate in a predetermined direction and by a predetermined angle are also indicated with a dashed line. In each case, the axis of rotation and direction in which the substrate is tilted for this purpose are an axis of rotation and direction that causes the tilting to cause the corresponding region of the chamber to be positioned further down relative to the rest of the chamber. Examples of how these regions of the chamber can be configured are explained below with reference to FIGS. 8a to 8f, wherein any of the configurations of the chamber shown there can be combined with the basic configuration of the substrate shown in FIG. 1a.

(20) The wall or walls and the bottom of the chamber can have a cell-repellent property, in particular a cell-repellent coating 2a, at least in the region or regions. In particular, the entire bottom and/or the entire side wall or side walls, can have a cell-repellent property.

(21) In particular, the substrate can be configured in the form of a microscopy carrier, especially, at least above and/or below the chamber, made of an optically high-quality material described in the general part.

(22) In the examples shown in FIGS. 1a to 1c and 2, both fluid channels open into the upper region of the chamber. In FIG. 2, as an example, the substrate is shown without an opening above the fluid channel. Such a substrate can in particular be configured in one piece.

(23) FIG. 3 shows a substrate that can be configured as the substrates in FIGS. 1 and 2 (i.e., with or without an opening above the chamber), with the difference that in the substrate in FIG. 3, the fluid channel 3a opens into the lower region of the chamber and the fluid channel 3b opens into the upper region of the chamber. Also in this example, the chamber can have any of the outlines described below in connection with FIGS. 8a through 8f.

(24) Optionally, a third fluid channel can also be provided, which runs above the fluid channel 3b, in particular laterally offset from it, and opens into the upper region of the chamber. The third fluid channel can be connected to its own fluid connection. In this way, flow around or a superperfusion can optionally take place with the same substrate.

(25) In FIG. 4, a substrate is shown that can be configured as the substrates in FIGS. 1 and 2 (i.e., with or without an opening above the chamber), with the difference that in the substrate in FIG. 4, the fluid channel 3a opens into the lower region of the chamber and the fluid channel 3b opens into the upper region of the chamber. In this example, one of the side walls or a section of the side wall of the chamber is inclined with respect to the bottom of the chamber, in particular such that the side wall and the bottom enclose an obtuse angle. In particular, in this example, the side wall or section of the side wall that is disposed between the bottom of the chamber and the mouth of the fluid channel 3b opening into the top of the chamber is inclined. Also in this example, the chamber can have any of the outlines described below in connection with FIGS. 8a through 8f, optionally with the limitation that the region where the cell aggregates are formed is not located where one of the fluid channels opens into the chamber at the bottom.

(26) In FIG. 5, a substrate is shown which can be configured in the same way as the substrates in FIGS. 1 and 2 (i.e. with or without an opening above the chamber), with the difference that in the substrate in FIG. 5 both fluid channels 3a and 3b open into the lower region of the chamber.

(27) FIGS. 6a and 6b show a top view and a side view of a substrate with a plurality of chambers 2. Here, all of the chambers can have the same shape or they can have different shapes. For example, each of the chambers can be configured as described above in connection with FIGS. 1 to 5. Each chamber is connected to two fluid connections 4a and 4b of its own.

(28) Alternatively, a group of a plurality of chambers can each be connected via a fluid channel with common fluid connections. The substrate can have exactly one or a plurality of such groups. An example with two such groups is shown in FIGS. 7a and 7b in side view and top view.

(29) FIGS. 6a, 6b, 7a and 7b show six chambers in the substrate as an example. However, this number can be varied as desired. In particular, for example, twelve or 24 chambers can be provided in the substrate and, if necessary, can be grouped as desired.

(30) FIGS. 8a to 8f show various possible outlines of a chamber that can be used, for example, for each of the substrates described above. The chamber can have at least one side wall such as 8, 8a, 8b, 8c, 8d, 8e, or 8f. Exemplary arrangements of the fluid channels are shown in dashed lines. Exemplary tilting axes and the regions 9a and 9b resulting from tilting about this axis, in which cells accumulate, are indicated in dashed lines in the Figures.

(31) FIG. 8a shows a chamber with a rectangular outline. FIG. 8b also shows a chamber with a rectangular outline, but here the edge between the fluid channel walls 8a and 8b is rounded. In FIG. 8c, a chamber with a polygonal outline is shown, here with six edges. In FIG. 8d, a chamber with a round outline is shown. FIG. 8e shows a chamber with an elliptical outline.

(32) FIG. 8f shows a chamber having a first region 9a where the side walls 8a and 8b meet perpendicularly, the edge optionally being rounded as shown here. Furthermore, the chamber has a second region 9b which is opposite the first region. In this region, the side wall is in the form of an arc of a circle or ellipse. In this example, the other side walls are planar. In the present example, the planar side walls are those into which the fluid channels open.

(33) Especially in the case of a non-rectangular and non-circular outline and/or an asymmetrical outline, a large degree of flexibility is possible with regard to the geometry of the forming cell aggregates. Depending on the direction in which the substrate is tilted, cell aggregates with different geometries can be generated.

(34) FIG. 9 shows a system 11 with a substrate, the substrate of FIG. 1 being shown here as an example, and a tilting device 12 which is configured in such a way that it tilts the substrate, in particular automatically, and holds it in a tilted position. The tilting device can also be configured for tilting back the substrate, in particular automatically. For example, the tilting device can comprise a motor 12a that tilts the substrate and an axle 12b to which the substrate is attached and which is rotated by the motor so that the substrate is tilted. The system includes a control device 13 that controls, for example, the motor. The tilting device 12 can be configured to tilt the substrate about one or a plurality of axes.

(35) The system can optionally also include one or a plurality of pumps 14 connected to the substrate in such a way as to be capable of pumping liquid from a reservoir 15 through the fluid connections, fluid channels, and optionally also the chamber. The pumps can be controlled by means of a control device, in particular by means of the control device 13. The pumps are not necessarily provided. Fluid transport can alternatively be accomplished by the tilting of the substrate, as explained below in connection with the method.

(36) In the following, a method for culturing cells in a three-dimensional cell aggregate in a substrate 1 in which a chamber is formed 2 is explained with reference to FIGS. 10a to 10g. It can be carried out, for example, using one of the substrates and, where appropriate, systems described above. Other suitable substrates or systems can also be used.

(37) The method comprises introducing cells 16 in the chamber 2. In the present example, the substrate is arranged such that the bottom of the chamber is substantially horizontal. Thereafter, the cells are distributed approximately uniformly, in particular in a monolayer, in the chamber, as shown in FIG. 10a.

(38) The substrate is then tilted to a tilted orientation such that the cells accumulate on a side wall of the chamber. For example, the substrate can be tilted by 20 to 45. This condition is shown in FIG. 10b. In FIG. 10b, the substrate is tilted about the transverse axis, which here is perpendicular to the drawing plane.

(39) In the next step, the substrate is held in the tilted orientation for a predetermined period of time, such as several hours, particularly between 17 and 19 hours, especially 18 hours. The cells and the substrate are configured such that cell aggregates 10 are formed during this period. In particular, the shape of the region of the chamber where the cells accumulate determines the shape of the cell aggregates. The cell-repellent property of the side wall or side walls and the bottom of the chamber in this region allow that the cells do not grow on there. The side wall or side walls, together with the bottom, form a boundary for the cell aggregate and thus influence the shape in which the cell aggregate grows. Due to, among other things, gravity and/or surface tension, as well as growth characteristics of the cells, it is not necessary to limit the cell aggregate from all sides in order to obtain the desired shape.

(40) Optionally, the substrate is then tilted back. Due to gravity and possibly the surface characteristics of the chamber, the cell aggregate 10 moves away from the side wall or side walls toward the center of the chamber, as shown in FIG. 10c. Optionally, the substrate can be tilted further through the initial position and then tilted back to the initial position (not shown here).

(41) A perfusion of the cell aggregate can be performed in the chamber. For this purpose, liquid, for example cell culture medium, is introduced into the substrate through a first fluid connection 4a and a first fluid channel 3a and is discharged from the substrate through a second fluid channel 3b and a second fluid connection 4b.

(42) When flowing through the substrate, the liquid can only flow along the edge of the chamber, as shown in FIG. 10d. This is the case, for example, when the two fluid channels open into the chamber in the upper region of the chamber. In this case, a superperfusion occurs, i.e., the liquid does not flow around the cell aggregate but flows past it above. Alternatively, the liquid can flow around the cell aggregate, as shown in FIG. 10e, for example, when one or both fluid channels open into the chamber at the bottom.

(43) The perfusion can be performed after the tilting back. Alternatively, the perfusion can already be performed during the tilting back.

(44) The method is described above using a substrate in which one chamber is formed. If a substrate with a plurality of chambers is used, for example as shown in FIGS. 6a, 6b, 7a, 7b, a plurality of cell aggregates can also be formed simultaneously by introducing cells into a plurality of chambers.

(45) The perfusion can then also be performed for a plurality of cell aggregates, in particular simultaneously. The perfusion of the individual cell aggregates can be performed independently for all cell aggregates if each chamber is connected to two separate fluid connections, for example as shown in FIGS. 6a and 6b. For this purpose, liquid is transported into the chamber via one of the two fluid connections for each of the chambers.

(46) Alternatively, liquid can be supplied to a group with a plurality of chambers via a split fluid connection, for example with a substrate as shown in FIGS. 7a and 7b. In this way, a uniform perfusion of the cell aggregates within a group can be achieved. The substrate can have a plurality of such groups. Then, for example, a perfusion with liquid in a different composition can be performed for cell aggregates of different groups.

(47) In particular, when using a substrate with a plurality of chambers, as shown for example in FIGS. 6a, 6b, 7a and 7b, parallel independent substance measurements can also be performed in this way.

(48) The perfusion can be accomplished by means of one or a plurality of pumps, such as in a system as shown in FIG. 9. Alternatively, the perfusion can be accomplished by the tilting of the substrate due to gravity, as shown in FIG. 10f. For example, the substrate can be tilted alternately in opposite directions to obtain flow.

(49) In FIG. 10b, the substrate is tilted about the transverse axis. Alternatively, it can also be tilted about the longitudinal axis. In particular, if the regions used to form the cell aggregates are offset from the longitudinal axis in the transverse direction. Alternatively or additionally, the substrate can be tilted about other axes, for example one of the diagonals.

(50) The method can optionally comprise performing a microscopic examination of the cell aggregate or cell aggregates in the chamber, particularly through the bottom of the substrate. In particular, a high resolution microscopy method can be performed, especially when the bottom of the chamber is planar. This examination is preferably performed when the substrate is oriented horizontally. It can be performed before, during and/or after the perfusion.

(51) The method can also optionally comprise removal of cells, in particular the cell aggregate or cell aggregates, from the substrate, in particular after the perfusion and, if applicable, microscopic examination. If the cell aggregates are not removed as a whole, the cells can optionally be lysed and the resulting cell suspension removed, for example sucked off. If the substrate, as shown in FIG. 1, has an opening closed with a lid, for example a lid in the form of a film, for example adhesive film, the lid can be taken off for removal so that cells can be removed directly from the chamber. In that case, the cell aggregate can optionally be removed as a whole, for example with a pipette. This is shown schematically in FIG. 10h.

(52) It is understood that features mentioned in the previously described embodiments are not limited to these particular combinations and are also possible in any other combinations.