CELL CULTURE CHAMBER AND METHOD FOR CULTURING CELLS AND FOR THE IN VITRO PRODUCTION OF CELL LAYERS AND ORGAN MODELS

Abstract

A cell culture chamber is for the in vitro production and cultivation of cell layers and organ models with two first and second channels arranged one above the other and separated from one another by a porous membrane with two side surfaces and through which flow can pass, wherein a cell substrate is formed in each case by the side surfaces of the membrane. The cell culture chamber is characterized in that at least the inner walls of the first and the second channels consist of polybutylene terephthalate. Further, a method is for cultivating human or animal cells, and in particular liver sinusoidal endothelial cells, alone and in co-culture with hepatocytes and immune cells.

Claims

1. A cell culture chamber, comprising: a first channel comprising an inner wall, through which a first flow can pass; a second channel comprising an inner wall, through which a second flow can pass; and a porous membrane; wherein the first and second channels are arranged one above the other and are separated from one another by the membrane, wherein the membrane has a first side surface oriented towards the first channel and a second side surface oriented towards the second channel, wherein the first and second side surfaces form a cell substrate, and wherein the inner walls of the first channel and the second channel consist of polybutylene terephthalate (PBT).

2. The cell culture chamber according to claim 1, wherein the membrane has a thickness of 10 m to 75 m.

3. The cell culture chamber according to claim 1, wherein the membrane has a thickness of 10 m to 50 m.

4. The cell culture chamber according to claim 1, wherein the membrane has a thickness of 10 m to 13 m.

5. The cell culture chamber according to claim 1, wherein the membrane consists of polyethylene terephthalate (PET), or a thermoplastic elastomer (TPE), or an elastic polyurethane (TPU).

6. The cell culture chamber according to claim 1, wherein the at least one side surface of the membrane has an additional surface structure.

7. The cell culture chamber according to claim 1, wherein at least one of the first and second side surfaces of the membrane is plasma-treated, and/or wherein at least one of the first or the second channels is plasma-treated.

8. The cell culture chamber according to claim 1, wherein at least one of the first and second side surface of the membrane is coated with a mixture comprising at least one of: 0.5 g/mL to 5 g/mL fibronectin, 100 g/mL to 300 g/mL collagen I, and 100 g/mL to 300 g/mL collagen IV.

9. The cell culture chamber according to claim 1, wherein at least one of the first and second side surfaces of the membrane is coupled to dextran chains to which fibronectin peptides, arginine-glycine-aspartic acid (RGD) tripeptides, vitronectin peptides, or bone sialoprotein peptides are bound with an RGD sequence.

10. The cell culture chamber according to claim 1, wherein at least one of the first and second side surfaces of the membrane is coupled to heparin chains to which fibronectin peptides or fibroblast growth factor (FGF) peptides are bound with an arginine-glycine-aspartic acid (RGD) sequence, RGD peptides alone, vitronectin peptides with an RGD sequence, or bone sialoprotein peptides with an RGD sequence.

11. The cell culture chamber according to claim 1, wherein human collagen I and/or human collagen IV with a concentration of more than 100 g/mL is coupled to at least one of the first or second side surfaces of the membrane.

12. A method for culturing human or animal cells, which comprises: providing the cell culture chamber according to claim 1, providing the human or animal cells, adding the human or animal cells to the culture chamber through one of the channels of the culture chamber, and incubating the cells in the culture chamber under culturing conditions.

13. The method according to claim 12, further comprising conveying a culture medium through the first channel and/or the second channel into the culture chamber, wherein the cells are cultured on an apical side of the membrane.

14. The method according to claim 12, further comprising conveying a culture medium through the first channel and/or the second channel into the culture chamber, wherein the cells are cultured on a basolateral side of the membrane.

15. The method according to claim 12, wherein the cultured cells are liver sinusoidal endothelial cells.

16. The method according to claim 12, wherein vascular conditions are simulated in the first channel and hepatic conditions are simulated in the second channel, by: conveying through the first channel an apical culture medium possessing a shear rate of more than 0.2 dyn/cm.sup.2 to 1 dyn/cm.sup.2, and conveying through the second channel a basolateral culture medium possessing a shear rate of greater than zero up to and including 0.2 dyn/cm.sup.2.

17. The method according to claim 16, wherein the apical culture medium possesses a shear rate of 0.5 dyn/cm.sup.2 to 0.8 dyn/cm.sup.2.

18. The method according to claim 12, wherein vascular conditions are simulated in the second channel and hepatic conditions are simulated in the first channel, by: conveying through the first channel a basolateral culture medium possessing a shear rate of greater than zero up to and including 0.2 dyn/cm.sup.2, and conveying through the second channel an apical culture medium possessing a shear rate of more than 0.2 dyn/cm.sup.2 to 1 dyn/cm.sup.2.

19. The method according to claim 18, wherein the apical culture medium possesses a shear rate of 0.5 dyn/cm.sup.2 to 0.8 dyn/cm.sup.2.

20. The method according to claim 12, in which a culture medium is conveyed through at least one of the first and second channels, wherein the oxygen content in the culture medium is adjusted at an inlet of the channel to a value of 15%, and wherein the oxygen content in the culture medium is adjusted at an outlet of the channel to a value of between 3% and 5%.

21. The method according to claim 15, in which the liver sinusoidal endothelial cells are co-cultured with one or more of Kupffer cells, hepatocytes, and stellate cells.

22. The method according to claim 12, wherein the cultured cells are one or more of intestinal endothelial cells, intestinal epithelial cells, and intestinal smooth muscle cells.

23. The method according to claim 12, wherein the cultured cells are pulmonary epithelial cells and/or pulmonary endothelial cells.

24. The method according to claim 12, further comprising flushing an active substance and/or a signal molecule into the cell culture chamber.

25. The method according to claim 12, further comprising flushing immune cells and/or microorganisms into the cell culture chamber.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0044] The invention will now be described with reference to the drawings and graphics of performed experiments wherein:

[0045] FIG. 1 shows a schematic representation of a cell culture chamber according to the disclosure with two channels and a membrane colonized in co-culture;

[0046] FIG. 2 shows a schematic representation of a second embodiment of a cell culture chamber according to the disclosure with four channels in an exploded view;

[0047] FIG. 3A shows a graphical representation of the results of an experiment on the adsorption effect of PBT in two cell culture chambers according to the disclosure;

[0048] FIG. 3B shows a graphical representation of the results of another experiment on the adsorption effect of PBT in two cell culture chambers according to the disclosure;

[0049] FIG. 4 shows a graphical representation of the results of an experiment on the release of LDH (lactate dehydrogenase) over a period of 10 or 14 days compared with a cell culture chamber according to the disclosure and the conditions of the cell lysis;

[0050] FIG. 5 shows a graphical representation of the results of an experiment on the release of ASAT (aspartate aminotransferase) over a period of 10 or 14 days compared with a cell culture chamber according to the disclosure and the conditions of the cell lysis;

[0051] FIG. 6 shows a graphical representation of the results of an experiment on the detection of the synthesis and release of albumin over a period of 10 or 14 days compared to a cell culture chamber according to the disclosure and the conditions of the cell lysis;

[0052] FIG. 7 shows a graphical representation of the results of an experiment on the detection of the synthesis and release of urea over a period of 10 or 14 days compared to a cell culture chamber according to the disclosure and the conditions of the cell lysis;

[0053] FIG. 8 shows a graphical representation of the results of an experiment on the detection of interleukin-1 beta over a period of 10 or 14 days compared to culturing in a cell culture chamber according to the disclosure and with the addition of LPS;

[0054] FIG. 9 shows a graphical representation of the results of an experiment on the detection of interleukin-10 over a period of 10 or 14 days compared to culturing in a cell culture chamber according to the disclosure and with addition of LPS; and,

[0055] FIG. 10 shows a graphical representation of the results of an experiment on the detection of interleukin-6 over a period of 10 or 14 days compared to culturing in a cell culture chamber according to the disclosure and with addition of LPS.

DETAILED DESCRIPTION

[0056] A cell culture chamber 1 according to the disclosure according to FIG. 1 includes a housing 2 which is made of polybutylene terephthalate (PBT) at least in the regions of a first channel 3 and a second channel 4. Each of the channels 3, 4 has an inlet 5 and an outlet 6 which serve for the inflow and discharge, respectively, of a culture medium 7 into and out of the particular channels 3, 4. The first channel 3 and the second channel 4 are separated from one another by a porous membrane 8. In the first channel 3, which at the same time represents an apical region of the cell culture chamber 1, a cell culture 9, for example, of liver sinusoidal endothelial cells, is located on a side surface, facing the first channel 3, of the membrane 8, which side surface is referred to as apical side 8.1 of the membrane.

[0057] An optionally present co-culture 10 containing hepatocytes is located on a side surface, facing the second channel 4, of the membrane 8, which side surface is referred to as the basolateral side 8.2 of the membrane. The membrane 8 is, for example, 12 m thick and made of PET.

[0058] The channels 3, 4 each have a width B, a height H transverse thereto, and a depth (not designated) perpendicular to the plane of the drawing.

[0059] In another embodiment of the disclosure, vascular conditions of, for example, a blood vessel are simulated in the first channel 3 arranged at the top relative to the gravitational effect, while in the second channel 4, hepatic conditions are simulated.

[0060] The shear rate in the model is adjusted for the vascular side and the hepatic side in that an apical culture medium 7a with a shear rate of 0.7 dyn/cm.sup.2 is conveyed through the first channel 3, and a basolateral culture medium 7b with a shear rate of >0 to 0.2 dyn/cm.sup.2 is conveyed through the second channel 4.

[0061] In the embodiment, the coating of the membrane 8 consists of a mixture of fibronectin/collagen I and collagen IV (fibronectin 5 g/mL, collagen I 0.3 mg/mL, collagen IV 100 g/mL). The cell cultures 9, 10 are cultivated in cell-type-specific and species-specific media with specific formulations. The apical culture medium 7a of the first channel 4 (ECM) contains 10% human serum and growth factors, antioxidants, and ITS in suitable concentrations familiar to a person skilled in the art. The basolateral medium 7b of the second channel 4 contains growth factors, a collagen IV/I mixture, and ITS in suitable concentrations familiar to a person skilled in the art.

[0062] The addition of specific media from the basolateral side can be replaced by the culture of hepatocytes and, optionally, stellate cells in cell-specific medium 7b in the second channel 4. In addition, macrophages have a positive influence on the properties/functionality of the cell culture 9 on the apical side (first channel 3).

[0063] A pump unit for each channel 3, 4 for controlled conveying of the particular culture medium 7, 7a, 7b and a controller for controlling the pump units are not shown in more detail. In addition, various sensors (for example, for oxygen, pH, lactate, TEER) can be present, via whose measured values the cell cultures 9 and 10 can be investigated. The sensors can be connected to the control unit in order, for example, to regulate the composition and/or the shear rate or flow rate of the particular culture medium 7, 7a, 7b as a function of the detected measured values.

[0064] A second embodiment of a cell culture chamber 1 according to the disclosure with two first channels 3 and two second channels 4 (not visible) is shown in FIG. 2 in an exploded view. The housing 2 is formed from an upper cover 2.1, a lower cover 2.2, and a central piece 2.3 in the form of an injection-molded piece. The center piece 2.3 contains the formations of the channels 3 and 4, which are each separated from one another by an inserted membrane 8.

[0065] FIGS. 3A and 3B show results of experiments on the adsorption effect of PBT in two cell culture chambers 1 according to the disclosure of different designs. The designation, Chip PBT-BC1, denotes a first embodiment of the cell culture chamber 1, and Chip PBT-BC2 a second embodiment. The first embodiment has a width B of 34.6 mm, a depth T of 6.56 mm, and a height H of 0.7 mm for the first channel 3. The second channel 4 has the dimensions of 44.8 mm, 3.6 mm, and 0.8 mm (W/D/H). The corresponding dimensions for the second embodiment, Chip PBT-BC2, of the cell culture chamber 1 are 34.6 mm, 8.06 mm, and 0.7 mm (W/D/H) for the first channel 3 and 49.4 mm, 5.61 mm, and 1.0 mm (B/T/H) for the second channel 4.

[0066] The graph in FIG. 3A shows the percentage (RTCratio to control) of propiconazole remaining and detectable in the culture medium 7 (log P 3.72) after an incubation time of 4 hours and after 24 hours. In comparison, a control in the form of a stock solution (100 M) with propiconazole is given. The graph in FIG. 3B shows the percentage fraction of the troglitazone remaining in the culture medium 7 (log P 3.60). Also in the example of FIG. 3B, samples were taken after 4 hours and after 24 hours incubation time, which were compared with a control in the form of a troglitazone stock solution (64 M). In each case, it can be seen from the two graphics that, in both embodiments of the cell culture chamber 1 according to the disclosure, more than 85% of the propiconazole (FIG. 3A) and more than 80% of the troglitazone (FIG. 3B) in the culture medium 7 could be detected even after 24 hours.

[0067] FIGS. 4 and 5 show the time profiles of the release of LDH (lactate dehydrogenases; FIG. 4) or of ASAT (aspartate aminotransferase; FIG. 5) of liver sinusoidal endothelial cells cultured in a cell culture chamber 1 according to the disclosure over a period of 10 or 14 days in co-culture with macrophages and hepatocytes compared to cell lysis. LDH and ASAT are enzymes that are released when there is cell damage and are therefore suitable for assessing the vital condition of an organ or tissues and cells. A significantly lower release of LDH and ASAT by the cultured cells compared to cell lysis can be seen even after 14 days.

[0068] FIGS. 6 and 7 show the concentrations of albumin (FIG. 6) and urea (FIG. 7) of liver sinusoidal endothelial cells cultured in a cell culture chamber 1 according to the disclosure over a period of 10 or 14 days in co-culture with macrophages and hepatocytes compared to cell lysis. Albumin and urea can be regarded as an expression of existing and intact synthesis processes in liver tissue or hepatocytes. It can be seen that, both in the 10-day cultures and in the 14-day cultures, concentrations above 1,300 g/L albumin or more than 0.7 mmol/L urea can also be detected after 10 or 14 days.

[0069] FIG. 8 shows the results of an experiment on the detection of interleukin-1 beta (IL-1b) over a period of 10 or 14 days. In addition, lipopolysaccharide (LPS) was added to some samples at the tenth day and the fourteenth day to check the activatability of immune cells (macrophages) contained in the liver model, and the respective reactions were shown. The addition of LPS as a so-called pyrogenic substance represents a test for endotoxins that is customary in the art. Immune cells such as macrophages respond to this stimulus with increased release of cytokines. The results show a distinct increase in the concentration of IL-1beta after the addition of LPS. It can be deduced from this that macrophages in co-culture with the investigated liver sinusoidal endothelial cells and hepatocytes were capable of reacting with an immune response to an endotoxin even after 10 or after 14 days. This is important for being able to detect immune cell-mediated side effects of drugs.

[0070] In principle, the same results are obtained for interleukin-10 (IL-10; FIG. 9) and interleukin-6 (IL-6; FIG. 10).

[0071] It is understood that the foregoing description is that of the preferred embodiments of the invention and that various changes and modifications may be made thereto without departing from the spirit and scope of the invention as defined in the appended claims. [0072] 1 Cell culture chamber [0073] 2 Housing [0074] 2.1 Upper cover [0075] 2.2 Lower cover [0076] 2.3 Center piece [0077] 3 First channel [0078] 4 Second channel [0079] 5 Channel inlet [0080] 6 Channel outlet [0081] 7 Culture medium [0082] 7a Apical culture medium [0083] 7b Basolateral culture medium [0084] 8 Membrane [0085] 8.1 Apical side of the membrane [0086] 8.2 Basolateral side of the membrane [0087] 9 Cell culture [0088] 10 Co-culture [0089] B Width [0090] H Height