Method and devices for the in vitro production of arrangements of cell layers

Abstract

The invention is directed to a method for the in vitro production of arrangements of cell layers in which a first well (2.1) which is closed off from its environment except for a first inlet opening (4.1) and a first outlet opening (5.1) and which has as a first cell substrate (6.1) a first wall (2.1.1) and as a second cell substrate (6.2) an opposite, second wall (2.1.2) which is separated from the first wall (2.1.1) by a first gap, a free surface of a cell substrate (6.1, 6.2) to be colonized with cells is oriented orthogonal to the Earth's gravitational force, and cells (9) are adhered to the cell substrate (6.1, 6.2) to be colonized. The invention is further directed to a method of maintaining the biological functionalities of the cell layers and semi-finished products of a device for the in vitro production and culturing of cell layers and a method for the production of the device.

Claims

1. Semi-finished products for a device, having one or at least two of said semi-finished products mounted one above the other, for in vitro production and culturing of cell layers, said semi-finished products having a first and a second well which are arranged one above the other in a single base plate which has an upper side and a lower side, said first well being formed in the upper side of the single base plate and the second well being formed in the lower side of the single base plate, said first and second wells being separated from one another by a single porous membrane with two lateral surfaces and sealed against the environment by a bonding foil on the upper side and a bonding foil on the lower side, wherein a first wall of the first well is formed as a first cell substrate by the bonding foil on the upper side and a first wall of the second well is formed as a fourth cell substrate by the bonding foil on the lower side and a second wall of the first well is formed as a second cell substrate through one of the lateral surfaces of the membrane and a second wall of the second well is formed as a third cell substrate through the other of the lateral surfaces of the membrane; the first well is formed so as to narrow to a point at its ends and to be rectangular in its middle part, and one of the ends terminating in a point leads into a first inlet opening for ingress of a first fluid flow into the first well and the other of the ends terminating in a point leads into a first outlet opening for egress of the first fluid flow out of the first well; the second well is rectangular and is connected to a second inlet opening for ingress of a second fluid flow into the second well and a second outlet opening for egress of the second fluid flow out of the second well through channel-like restrictions.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will be described more fully in the following with reference to illustrations and embodiment examples. The drawings show:

(2) FIG. 1 is a top view of a first embodiment example of a semi-finished product according to the invention with bonding foil partially shown;

(3) FIG. 2 is a semi-finished product according to the first embodiment example in a view from below with bonding foil;

(4) FIG. 3 is an overview of a liver sinusoid;

(5) FIG. 4 is an example of cell layers produced according to the invention in a semi-finished product with bonding foil;

(6) FIG. 5 is a schematic view of the hydraulic linking of four wells;

(7) FIG. 6 is a first embodiment example of a device according to the invention;

(8) FIG. 7 is a sectional view of the first embodiment example of the device according to the invention with cell layers;

(9) FIG. 8 is a second embodiment example of a device according to the invention with measuring points;

(10) FIG. 9 is a sectional view of the second embodiment example of the device according to the invention with cell layers;

(11) FIG. 10 is a schematic view of the cell layers in the wells of the second embodiment example of the device according to the invention; and

(12) FIG. 11 is a schematic view of a first electrode arrangement.

DESCRIPTION OF THE EMBODIMENTS

(13) In the following embodiment examples, the devices and elements are shown schematically and in a simplified manner. Identical reference numerals designate identical technical features unless otherwise stated. The association of cell layers with determined cell substrates are merely exemplary.

(14) As key elements of a first embodiment example of a semi-finished product according to the invention, FIG. 1 shows a base plate 1, a first well 2.1 and a first inlet opening 4.1 for ingress of a first fluid flow 12.1 (see FIG. 5) into the first well 2.1 and a first outlet opening 5.1 for egress of the first fluid flow 12.1. A first membrane 11.1 is provided between the first well 2.1 and a second well 2.2 (see FIG. 2).

(15) The first well 2.1 narrows to a point at its ends and is approximately rectangular in its middle part. The first inlet opening 4.1 leads into one of the ends terminating in a point, and the first outlet opening 5.1 leads into the other end terminating in a point. This shape results in an extensively laminar flow of the first fluid flow 12.1 through the first well 2.1.

(16) The second well 2.2 is provided on the underside of the base plate 1 (see FIG. 2) which is not visible in FIG. 1. A second inlet opening 4.2 for ingress of a second fluid flow 12.2 (see FIG. 5) into the second well 2.2 and a second outlet opening 5.2 for egress of the second fluid flow 12.2 are provided on the visible upper side.

(17) A view of a section along section plane A-A is shown in FIG. 4 and described.

(18) In FIG. 1, a third well 2.3 and a fourth well 2.4 (see FIG. 2) are provided on the base plate 1. Also, a third inlet opening 4.3 for ingress of a third fluid flow 12.3 (see FIG. 5) into the third well 2.3, and a third outlet opening 5.3 for egress of the third fluid flow 12.3, and a fourth inlet opening 4.4 for ingress of a fourth fluid flow 12.4 (see FIG. 5) into the fourth well 2.4, and a fourth outlet opening 5.4 for egress of the fourth fluid flow 12.4 are provided. All of the wells 2.1 to 2.4 are surrounded by edges 13 which are formed through the base plate 1.

(19) The semi-finished product is shown in FIG. 1 in a configuration in which the first well 2.1 and the third well 2.3 are sealed in a gas-tight and liquid-tight manner relative to the environment by a bonding foil 3 (shown only partially over well 2.3).

(20) A first wall 2.1.1 constituting a first cell substrate 6.1 is formed through the bonding foil 3 in the first well 2.1. The first membrane 11.1 is held in a second wall 2.1.2. The first membrane 11.1 is part of the second wall 2.1.2 and constitutes a second cell substrate 6.2.

(21) A second membrane 11.2 is arranged between the third well 2.3 and fourth well 2.4 in the same way as that just described.

(22) For simplicity, the first well 2.1 and second well 2.2 (section plane A-A) and associated elements will be referred to in the following. The technical elements of the third well 2.3 and fourth well 2.4 are shown and designated where applicable but are referred to expressly only when required for the explanation.

(23) The underside of the base plate 1 is shown in FIG. 2. The second well 2.2 and the underside of the first membrane 11.1 which serves as third cell substrate 6.3 are visible. This perspective shows a second wall 2.2.2 of the second well 2.2 in which the first membrane 11.1 is held and formed by the back of the second wall 2.1.2 of the first well 2.1 shown in FIG. 1.

(24) The second well 2.2 is approximately rectangular in its middle part, while it is connected to the second inlet opening 4.2 and to the second outlet opening 5.2 through channel-like restrictions.

(25) In further embodiments of the semi-finished product according to the invention, the shapes and dimensions of the wells may be identical to one another.

(26) FIG. 3 schematically shows a liver sinusoid. Cells 9 of different cell types and layered arrangements of the cell types can be seen. Further, flow ratios in the liver sinusoid are shown by arrows. Shown approximately in the middle is an area with a dashed border whose sequence of cell layers is mimicked by a device shown in FIG. 4.

(27) The device according to FIG. 4 is a semi-finished product according to the invention (see FIGS. 1 and 2) which has a bonding foil 3 on its upper side and its lower side, the first well 2.1 and second well 2.2 being sealed against the environment by the bonding foil 3. A first wall 2.1.1 of the first well 2.1, which serves as a first cell substrate 6.1, is formed by the bonding foil 3 on the upper side. A first wall 2.2.1 of the second well 2.2 which serves as a fourth cell substrate 6.4 is formed by the bonding foil 3 on the underside. A first gap 7.1 is provided between the first wall 2.1.1 of the first well 2.1 and the second wall 2.1.2 of the first well 2.1; a second gap 7.2 is provided between the first wall 2.2.1 of the second well 2.2 and the second wall 2.2.2 of the second well 2.2.

(28) In the section shown in enlarged manner, a first to third cell layer 10.1 to 10.3 is shown, these cell layers 10.1 to 10.3 being provided on the second cell substrate 6.2, third cell substrate 6.3 and fourth cell substrate 6.4. An arrangement of cell layers with cells 9 such as is shown in the bordered area in FIG. 3 is replicated by the first to third cell layer 10.1 to 10.3, the first gap 7.1 and the second gap 7.2.

(29) In one possibility for generating the arrangement of cell layers, a cell suspension 8 with cells 9 of the cell type of the first cell layer 10.1 is inserted into the first well 2.1. The second wall 2.1.2 of the first well 2.1, which second wall 2.1.2 is to be colonized, is oriented horizontally and located in a lower position so that the cells 9 are sedimented on the second wall 2.1.2 of the first well 2.1, which second wall 2.1.2 is to be colonized, by the action of gravity. Cells 9 adhere to the upper side of the first membrane 11.1 serving as second cell substrate 6.2 and form the first cell layer 10.1.

(30) Subsequently, a cell suspension 8 with cells of the cell type of the third cell layer 10.3 is inserted into the second well 2.2. Its position remains unchanged. The first wall 2.2.1 of the second well 2.2, which first wall 2.2.1 is to be colonized, is oriented horizontally and located in a lower position so that the cells 9 sediment on the first wall 2.2.1 of the second well 2.2, which first wall 2.2.1 is to be colonized, by the action of gravity. Cells 9 adhere to the first wall 2.2.1 of the second well 2.2 serving as fourth cell substrate 6.4 and form the third cell layer 10.3.

(31) Subsequently, the device is rotated by 180 degrees so that the second well 2.2 is now the upper well. The second wall 2.2.2 of the second well 2.2, which second wall 2.2.2 is to be colonized, is oriented horizontally and is now located in the lower position so that the cells 9 sediment on the second wall 2.2.2 of the second well 2.2, which second wall 2.2.2 is to be colonized, by the action of gravity. Cells 9 adhere to the side of the first membrane 11.1 serving as third cell substrate 6.3 and form the second cell layer 10.2.

(32) To maintain the already existing first cell layer 10.1, the latter is supplied with a suitable fluid, for example, a nutrient medium, in the first well 2.1.

(33) After producing the arrangement of the three cell layers 10.1 to 10.3, the arrangement of the cell layers 10.1 to 10.3 corresponding to the marked area in FIG. 3 is reproduced and an organoid is created.

(34) The four wells 2.1 to 2.4 of a base plate 1 can be hydraulically linked to one another as is shown by way of example schematically in FIG. 5. In this regard, the first outlet opening 5.1 is connected to the third inlet opening 4.3 and the second outlet opening 5.2 is connected to the fourth inlet opening 4.4 so that a first fluid flow 12.1 flows out of the first well 2.1 into the third well 2.3 and flows through the latter as a third fluid flow 12.3. A second fluid flow 12.2 flows through the second well 2.2 and arrives at the fourth inlet opening 4.4 via the second outlet opening 5.2 and into the fourth well 2.4 as fourth fluid flow 12.4.

(35) In further embodiments of the invention, the principle just described can also be used between wells of different base plates 1 or devices (see, for example, FIGS. 6 to 9). Further, the fluid flows 12.1 to 12.4 can be guided differently. Accordingly, the first fluid flow 12.1 can be guided into the fourth well 2.4 and the second fluid flow 12.2 can be guided into the third well 2.3 in a further embodiment of the invention.

(36) It is further possible that the fluid flows 12.1 to 12.4 are guided into one another or are divided and guided into different wells. It is also possible that only portions of the fluid flows 12.1 to 12.4 are guided into subsequent wells. Accordingly, portions of the fluid flows 12.1 to 12.4 can be guided back into the wells that they have just flowed through and can flow through them again.

(37) FIG. 6 shows a first embodiment example of a device according to the invention. A first base plate 1.1 and a second base plate 1.2 are placed against one another with their respective undersides so that the edges 13 of the first base plate 1.1 and second base plate 1.2 touch (the illustration shows a state during assembly). The two base plates 1.1 and 1.2 are sealed at their respective upper sides by bonding foils 3 as has already been described. The second base plate 1.2 likewise has wells which, following the logic described above, are the fifth to eighth wells 2.5 to 2.8 (only the fifth well 2.5 and the seventh well 2.7 are shown; the sixth well 2.6 and the eighth well 2.8 are only referenced by arrows in FIG. 6). The second base plate 1.2 further has a third membrane 11.3 and fourth membrane 11.4. Both base plates 1.1 and 1.2 are connected, e.g., glued, to one another in a liquid-tight manner. The second well 2.2 (see also FIG. 1) and the fourth well 2.4 (see also FIG. 1) of the base plate 1.1 are moved over the fifth well 2.5 or over the seventh well 2.7 of the base plate 11.2 and in each instance form a common well, namely, the fifth well 2.5 and seventh well 2.7, respectively.

(38) In FIG. 7, the construction of the device according to the invention provided in this way is shown as a sectional diagram along section line A-A (FIG. 6). A first well 2.1 is formed by the first base plate 1.1, shown above. This first well 2.1 is separated from the fifth well 2.5 by the first membrane 11.1. The fifth well 2.5 is in turn separated from the sixth well 2.6 by the third membrane 11.3. A sixth fluid flow 12.6 flows through the sixth well 2.6 via the sixth inlet opening 4.6 and the sixth outlet opening 5.6.

(39) The first well 2.1, fifth well 2.5, sixth well 2.6, first membrane 11.1 and third membrane 11.3 are shown in the section enlargement. A cell layer 10.1 to 10.4 is provided on the two membranes 11.1 and 11.3 on both sides.

(40) A second embodiment example of a device according to the invention with measuring points 14 is shown in FIG. 8. Measurement points 14 for acquiring measured values based on fluorescing characteristics of constituents of the fluid flows 12.1 to 12.4 (see, e.g., FIG. 5) are arranged on the base plate 1 configured according to FIG. 1 in every well 2.1 to 2.4 (only the first well 2.1 and the third well 2.3 are shown). They are data-communicatively connected to an evaluating and controlling unit 17 by means of which the composition, the pressure and the temperature of the fluid flows 12.1 to 12.4 can be adjusted so as to be controlled on the basis of the acquired measurement data. Further, measurement data for further evaluations are stored for later recall. The measurement points 14 serve for contactless measurement of glucose and partial oxygen consumption of the cells 9 of the cell layers.

(41) An organoid of the liver (liver organoid I) is provided according to the described method in the first well 2.1 and second well 2.2, while an organoid of the intestine (intestinal organoid II) is provided in the third well 2.3 and fourth well 2.4. The first outlet opening 5.1 is hydraulically connected to the third inlet opening 4.3. Through the second inlet opening 4.2, a second fluid flow 12.2 is fed to the second well 2.2 for supplying the cell layers in the second well 2.2 and the cell layers on the first membrane 11.1 in the first well 2.1. The second fluid flow 12.2 is guided out via the second outlet opening 5.2 and is mixed in portions with fresh fluid before this mixture arrives in the second well 2.2 again via the second inlet opening 4.2. The same applies for the fourth well 2.4. Through the first inlet opening 4.1, a first fluid flow 12.1 is guided through the first well 2.1 to the first outlet opening 5.1 and, from there, travels via the third inlet opening 4.3 into the third well 2.3, flows through the latter as third fluid flow 12.3 and is guided out via the third outlet opening 5.3. Through this configuration, organoids and their physiological effects on one another are simulated in vitro in an experimental environment and can be acquired and evaluated.

(42) In FIG. 9, the placement of the measurement points 14 in the first well 2.1 and in the second well 2.2 is shown in a sectional view along section plane B-B (see FIG. 8). Areas of the liver organoid or intestinal organoid are designated by Roman numerals I and II and are shown enlarged in FIG. 10.

(43) The liver organoid I is shown in the section enlargement on the left-hand side in FIG. 10. On the second wall 2.1.2 of the first well 2.1 on the first membrane 11.1, there is a first cell layer 10.1 as a HIMEC (Human Intestinal Microvascular Endothelial Cells) layer over a second cell layer 10.2 FB (fibroblasts) and a third cell layer 10.3 ECM (extracellular matrix). On the second wall 2.2.2 of the second well 2.2 on the first membrane 11.1, there is a fourth cell layer 10.4 HCEC (Human Colon Epithelial Cells, human intestinal epithelial cells).

(44) The intestinal organoid II is shown in the section enlargement on the right-hand side in FIG. 10. On the second wall 2.3.2 of the third well 2.3 on the second membrane 11.2, there is a fifth cell layer 10.5 as a HSEC (Human Sinusoidal Endothelial Cell) layer. This is interspersed with moM cells (primary human monocyte derived macrophages) which were colonized as co-culture. On the second wall 2.4.2 of the fourth well 2.4 on the second membrane 11.2, there is a sixth cell layer 10.6 HSC (human stellate cells). On the first wall 2.4.1 of the fourth well 2.4, which first wall 2.4.1 serves as eighth cell substrate 6.8, there is a HHC (human differentiated hepatocytes) layer as seventh cell layer 10.7, and this is interspersed with co-cultured BC (bile canaliculi).

(45) In further embodiments, there may be more cell layers 10.1, 10.2, . . . which, in addition, can have cells 9 of other cell types, other sequences of cell layers and/or other arrangements of cell layers 10.1, 10.2, . . . on the various cell substrates 6.1, 6.2. . . .

(46) One possibility for acquiring measurement data at semi-finished products according to the invention and at devices according to the invention is shown schematically in FIG. 11. A first current-supplying electrode 15.1 and a first measurement electrode 15.2 are arranged at the upper side of the base plate 1. A second current-supplying electrode 15.3 and a second measurement electrode 15.4 are arranged at the underside of the base plate 1. These electrodes 15.1, 15.2 and 15.3, 15.4 are printed on a bonding foil 3. In further embodiments, they can also be integrated in or arranged, for example, printed, on the base plate 1. Each electrode 15.1 to 15.4 has a contacting surface 16 for electrical and/or measuring contact.

(47) By applying voltage to the electrodes 15.1 to 15.4, measurement data of acquired electric voltages, electric currents, ohmic resistances and/or impedances can be obtained and evaluated.

(48) The electrodes 15.1 to 15.4, e.g., for impedance measurement, are arranged, for example, sintered or pressed, on the bonding foils 3 directly above and below the first membrane 11.1 and/or second membrane 11.2. The supply of alternating current for building up an electrical field is carried out on the contacting surfaces 16. The two current-supplying electrodes 15.1 and 15.3 lie opposite one another, for example, in the first well 2.1 on the liquid-supplying side and in the second well 2.2 on the liquid-removal side.

(49) While the invention has been illustrated and described in connection with currently preferred embodiments shown and described in detail, it is not intended to be limited to the details shown since various modifications and structural changes may be made without departing in any way from the spirit of the present invention. The embodiments were chosen and described in order to best explain the principles of the invention and practical application to thereby enable a person skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.

LIST OF REFERENCE CHARACTERS

(50) 1 base plate

(51) 1.1 first base plate

(52) 1.2 second base plate

(53) 2.1 first well

(54) 2.1.1 first wall (of the first well 2.1)

(55) 2.1.2 second wall (of the first well 2.1)

(56) 2.2 second well

(57) 2.2.1 first wall (of the second well 2.2)

(58) 2.2.2 second wall (of the second well 2.2)

(59) 2.3 third well

(60) 2.3.2 second wall (of the third well 2.3)

(61) 2.4 fourth well

(62) 2.4.1 first wall (of the fourth well 2.4)

(63) 2.4.2 second wall (of the fourth well 2.4)

(64) 2.5 fifth well

(65) 2.6 sixth well

(66) 2.7 seventh well

(67) 2.8 eighth well

(68) 3 bonding foil

(69) 4.1 first inlet opening

(70) 4.2 second inlet opening

(71) 4.3 third inlet opening

(72) 4.4 fourth inlet opening

(73) 4.6 sixth inlet opening

(74) 5.1 first outlet opening

(75) 5.2 second outlet opening

(76) 5.3 third outlet opening

(77) 5.4 fourth outlet opening

(78) 5.6 sixth outlet opening

(79) 6.1 first cell substrate

(80) 6.2 second cell substrate

(81) 6.3 third cell substrate

(82) 6.4 fourth cell substrate

(83) 6.8 eighth cell substrate

(84) 7.1 first gap

(85) 7.2 second gap

(86) 8 cell suspension

(87) 9 cells

(88) 10.1 first cell layer

(89) 10.2 second cell layer

(90) 10.3 third cell layer

(91) 10.4 fourth cell layer

(92) 10.5 fifth cell layer

(93) 10.6 sixth cell layer

(94) 10.7 seventh cell layer

(95) 11.1 first membrane

(96) 11.2 second membrane

(97) 11.3 third membrane

(98) 11.4 fourth membrane

(99) 12.1 first fluid flow

(100) 12.2 second fluid flow

(101) 12.3 third fluid flow

(102) 12.4 fourth fluid flow

(103) 12.6 sixth fluid flow

(104) 13 edges

(105) 14 measurement points

(106) 15.1 first current-supplying electrode

(107) 15.2 first measurement electrode

(108) 15.3 second current-supplying electrode

(109) 15.4 second measurement electrode

(110) 16 contacting surface

(111) 17 evaluating and controlling unit

(112) I liver organoid

(113) II intestinal organoid