MICROPHYSIOLOGICAL CHOROID MODEL

Abstract

The invention relates to the field of cultivating biological cells and tissues having an organ-like function on a microphysiological scale and provides a microphysiological reproduction of the choroid and the blood-retinal barrier as an in vitro test system.

Claims

1. An in vitro tissue culture arrangement comprising: a first chamber (120) in a bioreactor (100), a 3D melanocyte culture (200) arranged in the first chamber (120), in which isolated melanocytes (220) are embedded in a hydrogel (240), a second chamber (140) in the bioreactor (100) which adjoins the first chamber (120) of the bioreactor (100), a first semipermeable membrane (130) which separates the second chamber (140) of the bioreactor (100) from the first chamber (120) of the bioreactor (100), wherein the membrane side (132) of the first semipermeable membrane (130) facing the first chamber (120) rests against the 3D melanocyte culture (200), and, a confluent first 2D endothelial cell layer (310) of isolated endothelial cells which is arranged in the second chamber (140) and rests against the membrane side (134) of the first semipermeable membrane (130) facing the second chamber (140).

2. The in vitro tissue culture arrangement according to claim 1 further comprising: a third chamber (160) in the bioreactor (100) which adjoins the second chamber (140) of the bioreactor (100), a second semipermeable membrane (150) which separates the third chamber (160) of the bioreactor (100) from the second chamber (140) of the bioreactor (100), and, a confluent second 2D endothelial cell layer (320) of isolated endothelial cells which is arranged in the second chamber (140) of the bioreactor (100) and rests against the membrane side (152) of the second semipermeable membrane (150) facing the second chamber (140).

3. The in vitro tissue culture arrangement according to claim 2, further comprising: a confluent third 2D epithelial cell layer (400) of isolated epithelial cells which is arranged in the third chamber (160) of the bioreactor (100) and rests against the membrane side (154) of the second semipermeable membrane (150) facing the third chamber (160).

4. The in vitro tissue culture arrangement according to claim 1, further comprising: a fourth chamber (180) in the bioreactor (100) which adjoins the first chamber (120) of the bioreactor (100), a third semipermeable membrane (170) which separates the fourth chamber (140) of the bioreactor (100) from the first chamber (120) of the bioreactor (100), wherein the membrane side (172) of the third semipermeable membrane (170) facing the first chamber (120) rests against the 3D melanocyte culture (200), and, a confluent third 2D endothelial cell layer (330) of isolated endothelial cells which is arranged in the fourth chamber (180) of the bioreactor (100) and rests against the membrane side (174) of the third semipermeable membrane (170) facing the fourth chamber (180).

5. The in vitro tissue culture arrangement of claim 4, wherein the 3D melanocyte culture (200) is embedded between the first semipermeable membrane (130) and the third semipermeable membrane (170).

6. The in vitro tissue culture arrangement according to claim 1, wherein the chambers (120, 140, 160, 180) in the bioreactor (100) are arranged layered directly one above the other.

7. The in vitro tissue culture arrangement according to claim 6, wherein the bioreactor (100) is designed as a microphysiological bioreactor and the chambers (120, 140, 160, 180) are designed as channel structures with a chamber volume of less than 10 μL each.

8. A method for producing an in vitro tissue culture arrangement according to claim 1, comprising the steps: c) Seeding isolated endothelial cells into a second chamber (140) of a bioreactor (100), with such an orientation of the bioreactor (100) in relation to the gravity vector that endothelial cells sink onto a membrane side (134) of a first semipermeable membrane (130) facing the second chamber (140), which membrane separates the second chamber (140) from a first chamber (120) of the bioreactor (100), d) Cultivating the endothelial cells that have sunk onto this membrane side (134) of the first semipermeable membrane (130) so that endothelial cells adhere to this membrane side (134) and grow there to form a confluent first 2D endothelial cell layer (310), and, g) Adding a suspension of isolated melanocytes (220) in liquid hydrogel precursor to the first chamber (120) of the bioreactor (100), and, h) Allowing the hydrogel precursor to harden to form a hydrogel (240), so that a 3D melanocyte culture (200) in which isolated melanocytes (220) are embedded in the hydrogel (240) is formed in the first chamber (120).

9. The method according to claim 8, further comprising the steps: e) Seeding isolated endothelial cells into a second chamber (140) of the bioreactor (100), with such an orientation of the bioreactor (100) in relation to the gravity vector that endothelial cells sink onto a membrane side (152) of a second semipermeable membrane (150) facing the second chamber (140), which membrane separates the second chamber (140) from a third chamber (160) of the bioreactor (100), and, f) Cultivating the endothelial cells that have sunk onto this membrane side (152) of the second semipermeable membrane (150) so that endothelial cells adhere to this membrane side (152) and grow there to form a confluent second 2D endothelial cell layer (320).

10. The method according to claim 9, wherein steps (c)-(f) are carried out temporally before steps (g)-(h).

11. The method according to claim 9, further including the steps: a) Seeding isolated epithelial cells into the third chamber (160) of the bioreactor (100), with such an orientation of the bioreactor (100) in relation to the gravity vector that epithelial cells sink onto the membrane side (154) of the second semipermeable membrane (150) facing the third chamber (160), which membrane separates the second chamber (140) from a third chamber (160) of the bioreactor (100), and, b) Cultivating the epithelial cells that have sunk onto this membrane side (154) of the second semipermeable membrane (150) so that epithelial cells adhere to this membrane side (154) and grow there to form a confluent first 2D epithelial cell layer (400).

12. The method according to claim 11, wherein steps (a)-(b) are carried out temporally before steps (c)-(h).

13. The method for in vitro testing of the modulatory effect of a substance on the function of the blood/retinal barrier, comprising the steps: Providing the in vitro tissue culture arrangement according to claim 1, Adding the substance to at least one chamber (120, 140, 160, 180) of this in vitro tissue culture arrangement, Registering and detecting changes in the function of the blood/retinal barrier after the substance has been added compared to the state before the substance was added, wherein the characteristic value determined for the function of the blood/retinal barrier is selected from: macromolecule Transport Rate and Electrical Impedance (TEER).

14. The method for in vitro testing of the modulatory effect of a substance on the immune reaction in the choroid, comprising the steps: Providing the in vitro tissue culture arrangement according to claim 1, Adding immune cells to a chamber (140, 180) of this in vitro tissue culture arrangement that carries endothelial cells, Adding the substance to at least one chamber (120, 140, 160, 180) of this in vitro tissue culture arrangement, Registering and detecting the immune reaction after the substance has been added, the immune reaction being selected from: Migration of the immune cells from the endothelial cell layer into the neighboring 3D melanocyte culture and proliferation of the immune cells in the 3D melanocyte culture.

Description

[0048] The invention is described in more detail using the following figures and examples without these being limiting.

[0049] FIG. 1 shows a schematic sectional view of a first embodiment of the inventive in vitro tissue culture arrangement with at least two chambers (120, 140), in which arranged in a first chamber (120) in a bioreactor (100) is a 3D melanocyte culture (200), in which isolated melanocytes (220) are embedded in a hydrogel (240). A second chamber (140) of the bioreactor (100) directly adjoins the first chamber (120). A first semipermeable membrane (130) separates the second chamber (140) from the first chamber (120). It is particularly provided that the membrane side (132) of the first semipermeable membrane (130) facing the first chamber (120) rests against the 3D melanocyte culture (200). A first 2D endothelial cell layer (310) is arranged in the second chamber (140) and rests against the membrane side (134) of the first semipermeable membrane (130) facing the second chamber (140). As a result, the first 2D endothelial cell layer (310) is separated from the 3D melanocyte culture (200) only by the first semipermeable membrane (130), but is connected in a semipermeable manner.

[0050] FIG. 2 shows a schematic sectional view of a further embodiment of the inventive in vitro tissue culture arrangement with four chambers (120, 140, 160, 180), in which arranged in the bioreactor (100) in a first chamber (120) is a 3D melanocyte culture (200) in which isolated melanocytes (220) are embedded in a hydrogel (240). A second chamber (140) of the bioreactor (100) directly adjoins the first chamber (120). A first semipermeable membrane (130) separates the second chamber (140) from the first chamber (120). The membrane side (132) of the first semipermeable membrane (130) facing the first chamber (120) rests against the 3D melanocyte culture (200). A first 2D endothelial cell layer (310) is arranged in the second chamber (140) and rests against the membrane side (134) of the first semipermeable membrane (130) facing the second chamber (140). A third chamber (160) of the bioreactor (100) directly adjoins the second chamber (140), specifically on a side of the second chamber (140) opposite the adjoining first chamber (120). A second semipermeable membrane (130) separates the third chamber (160) from the second chamber (140). In this embodiment, a second 2D endothelial cell layer (320) is arranged in the second chamber (140) and rests against the membrane side (152) of the second semipermeable membrane (150) facing the second chamber (140). A 2D epithelial cell layer (400) is also arranged in the third chamber (160) of the bioreactor (100) and rests against the membrane side (154) of the second semipermeable membrane (150) facing the third chamber (160). As a result, the second semipermeable membrane (150) is colonized on both sides and the second 2D endothelial cell layer (320) is separated from the 2D epithelial cell layer (400) by this membrane (150), but connected in a semipermeable manner. In the embodiment shown here with four chambers, in particular a fourth chamber (180) is also formed on the opposite side of the first chamber (120), which is separated from the first chamber (120) by a third semipermeable membrane (170). It is particularly provided that the membrane side (172) of the third semipermeable membrane (170) facing the first chamber (120) rests against the 3D melanocyte culture (200). Arranged in the fourth chamber (180) of the bioreactor (100) is in particular a third 2D endothelial cell layer (330) which rests against the membrane side (174) of the third semipermeable membrane (174) facing the fourth chamber (180). As a result, the third 2D endothelial cell layer (330) is also separated from the 3D melanocyte culture (200) only by the third semipermeable membrane (170), but is connected in a semipermeable manner.

[0051] FIG. 3 shows a schematic top view of a typical practical embodiment of the in vitro test system (100) with three channel structures, particularly according to FIG. 4 with one channel (160) for seeding retinal pigment cells, a further channel (140) for seeding endothelial cells, and one channel (130) for loading a hydrogel with melanocytes that is provided there.

[0052] FIG. 4 shows a schematic sectional view of one embodiment of the in vitro test system with three channel structures (120, 140, 160) which are separated from one another by two semipermeable membranes (130, 150). In the uppermost channel structure (160), a 2D monolayer of epithelial cells (400), preferably retinal pigment epithelial cells, is applied to the uppermost semipermeable membrane (150). In the case of the channel structure (140) arranged in the center, a 2D monolayer of endothelial cells (310, 320), preferably microvascular endothelial cells, is applied to the underside (152) of the uppermost semipermeable membrane (150) and to the upper side (134) of the lower semi-permeable membrane (130). A hydrogel (240) with melanocytes (220), which forms a 3D melanocyte culture (200), is added to the lowermost channel structure (120) on the underside (132) of the lower semipermeable membrane (130).

[0053] FIG. 5 shows schematic top views of the embodiment of the in vitro test system with three channel structures according to FIG. 3 which are partially closed (FIG. 5A) or opened (FIG. 5B) for the different cell types used or can be washed with a constant flow of nutrient medium (FIG. 5C).

[0054] FIG. 6 shows a schematic sectional view of one embodiment of the in vitro test system and the introduction of the various cell types; A: Seeding retinal pigment epithelial cells into the uppermost channel to create a 2D RPE monolayer (400) on top side of the semipermeable membrane; B: Seeding endothelial cells in the center channel, in vitro test system is turned upside down to create a 2D endothelial cell monolayer (320) on the underside of the semipermeable membrane; C: In vitro test system is rotated back to create a second 2D endothelial cell monolayer (310) on the top side of the semipermeable membrane; D: A hydrogel is added to the lower canal and colonized with melanocytes to form the 3D melanocyte culture; E: In the test mode, substances and/or immune cells (500) are applied to the center channel occupied by endothelial cells.

[0055] FIG. 7 shows cell densities of melanocytes in a hydrogel in the inventive in vitro arrangement: A: Hydrogel+melanocytes in a cell density that corresponds to that of the human choroid; B: Hydrogel+melanocytes in a cell density that corresponds to the choroid of a primate.

[0056] FIG. 8 shows the three-dimensional distribution of hydrogel+melanocytes of the inventive in vitro arrangement, determined and represented by means of the autofluorescence of the melanin formed by the melanocytes.

[0057] FIG. 9 shows the schematic sectional view of a further embodiment of the in vitro test system with two channel structures (120, 160) which are separated from one another by a semipermeable membrane (150). Endothelial cells or epithelial cells (320) are added to the upper channel (160) as a 2D monolayer. A hydrogel (240) with melanocytes (220) is added to the lower channel (120); endothelial cells (310) are also added to the lower channel (120) and attach to the outside of the hardened hydrogel.

[0058] FIG. 10 shows melanocytes embedded in a collagen hydrogel in the inventive in vitro arrangement; the melanocytes have been stained by means of a living/dead stain (vital=fluorescein diacetate, non-vital=propidium iodide (PI)): A: at a concentration of 3 mg/mL; B: at a concentration of 2 mg/mL; C: at a concentration of 1 mg/mL; D shows a bar graph for the cells that are positive (dead) for propidium iodide (PI): The number of PI-positive cells drops significantly as the gel concentration goes down and, conversely, leads to higher vitality.

PRODUCTION OF A MICROPHYSIOLOGICAL IN VITRO CHOROID TEST SYSTEM

[0059] To produce the test system, melanocytes, endothelial cells, and epithelial cells are seeded into a microphysiological bioreactor. The steps are as follows:

[0060] 1) Seeding of epithelial cells, preferably retinal pigment epithelial cells, in the uppermost channel structure of the bioreactor, said cells forming a 2D monolayer there: For this purpose, the outlet of the endothelial channel of the bioreactor is closed, the outlet of the retinal pigment epithelial channel is closed, the outlet of the melanocyte+hydrogel channel is closed. Cell solution with retinal pigment epithelial cells is flushed into the inlet of the retinal pigment epithelial channel and flushed out via the outlet of the endothelial cell channel.

[0061] 2) Seeding of endothelial cells, preferably microvascular endothelial cells, in the center channel structure, wherein: a) a first 2D monolayer of said endothelial cells is created on the upper side of the second semipermeable membrane, and b) a second 2D monolayer of said endothelial cells is produced on the lower side of the first semipermeable membrane. For this, the inlet of the endothelial channel is closed, the outlet and inlet of the retinal pigment epithelial channel are closed, and the outlet of the hydrogel+melanocyte channel is closed. Cell solution is flushed into the outlet of the endothelial channel and flushed out via the outlet of the melanocyte channel. A first 2D monolayer is thus created in that a cell solution is flushed into said channel and the in vitro test system is turned upside down to allow the endothelial cells to sink onto the underside of the first semipermeable membrane. The second 2D monolayer is created on the upper side of the second semipermeable membrane by rotating the in vitro test system back after a certain time (15 minutes).

[0062] 3) Addition of a solution of melanocytes and hydrogel to the lowermost channel structure. The ratio of melanocytes to hydrogel can reproduce the melanocyte cell density of the choroid of humans or primates. The hydrogel can be native ECMs such as collagen, fibronectin, or synthetic hydrogels such as those based on dextran. For this purpose, the inlet and outlet for the retinal pigment epithelial channel are closed. Nutrient medium is flushed into the inlet of the endothelial channel at a constant flow rate (5 μL/hour) and flushed out via the outlet thereof. At the same time, a liquid solution of hydrogel+melanocytes is flushed into the inlet of the melanocyte channel and the outlet thereof is rinsed out. The hydrogel then hardens/solidifies in the channel.

[0063] Microphysiological 3D Melanocyte Culture Based on Collagen Hydrogel

[0064] Modification of the collagen density/porosity with regard to optimal vitality of the melanocytes as well as the possibility that said cells can adhere to the hydrogel. At a higher collagen concentration (3 mg/ml), vitality decreases sharply, and only a small number of melanocytes can adhere to the gel. This is shown by the fact that these cells are spherical in shape. At lower concentrations (2 mg/ml-1 mg/ml), vitality increases significantly and a greater proportion of the cells can adhere to the hydrogel. An optimal collagen concentration was found at 1 mg/ml, which also has a better viscosity in terms of handling for later flushing into the chip. Higher concentrations of collagen (3 and 2 mg/ml) are difficult to pipette and are therefore flushed into the reactor together with the cells.