DISPOSABLE HOUSINGS WITH CELLS AND / OR CO-CULTURES FOR IN-SITU TESTING

Description

[0001] The invention relates to the field of biological toxicity and efficacy tests, in particular housings, holders containing the housings, devices containing the holder and housing, and methods using the housing.

[0002] Biological purity testing is required in the manufacture of cosmetics, medical devices, and pharmaceuticals, or in areas where people are present. For example, tests using skin models are required under the new Cosmetics or REACH Regulations. Skin models consisting of small plastic cups with porous bottoms, measuring 0.5 to 3 cm in diameter, are commercially available according to OECD specifications. They are transported loose in culture vessels and must be filled with externally obtained and prepared sample material under aseptic conditions in the cell biology laboratory. These skin models are then further cultivated in special incubators and evaluated after one to two days using optical readers for laboratory culture vessels or histologically. The use of such skin models is therefore tied to a specialized cell biology laboratory. Accordingly, corresponding tests cannot currently be carried out directly at the production site.

[0003] Furthermore, state-of-the-art concepts for biological air testing are known, such as the lung-on-a-chip concept. DE 10 2006 020 494 A1 discloses an artificial lung system suitable for producing a lung model system as well as a replacement for animal model systems in toxicological studies. US 2016 / 0145554 A1 also discloses an organ-on-chip system suitable for culturing both lung and skin tissue. Lung-on-a-chip devices are used as miniaturized lung models in research laboratories to study the fundamental effects of toxic substances on the lungs. They are optimized for investigating complex cellular interactions under mechanical stress similar to that found in lung tissue. However, they are not suitable for measurements outside of cell biology laboratories, and in particular for on-site measurements or for testing large air volumes.Their production is also complex and therefore only suitable for series production to a limited extent. Culture containers for cultivating and differentiating cells and tissues are also known in many different forms. For example, DE 19952847 A1 discloses a culture container with closed chambers containing a cultivation area and an inlet and an outlet for a culture and / or treatment medium. However, the culture container is not suitable for lung- or skin-specific co-cultures. DE 10 2007 007 718 describes a disposable bioreactor in which a chamber is formed from a lower housing half and an upper housing half, which are connected to each other on one side by a foil joint.

[0004] The present invention was therefore based on the object of providing a disposable device for transportable co-cultures that can be used for sampling outside of biological laboratories.

[0005] The object is achieved by a disposable housing as claimed for enclosing cells and / or co-cultures for in vitro tests, with a plurality of separate chambers, each having at least one inlet opening and one outlet opening for a culture and / or treatment medium, wherein the housing is formed from a first housing part and a second housing part, and wherein a carrier layer permeable to the culture and / or treatment medium is fixed in the chambers in the first housing part, whereby a first and a second inner lumen are formed in each of the chambers.

[0006] The disposable housing according to the invention enables the use of a prefabricated device with test cells and nutrient reservoirs for testing purposes outside of biological laboratories without the need for biological technicians. This enables measurements on skin models in the production environment, particularly those required for approval procedures. Furthermore, it is possible to combine air sampling and on-site biological testing directly in living and working areas in order to characterize these areas with regard to the desired level of safety. This allows air pollutants to be biologically detected directly at the sampling site without sample tampering. A further major advantage is that the disposable device, in combination with a mobile measuring and / or reading device at the site of use, eliminates the need for the equipment and personnel required in a cell biology laboratory.Another advantage is its suitability for series production, particularly compared to lung-on-a-chip methods.

[0007] The permeable carrier layer forms a first and a second inner lumen in each of the chambers, particularly when closed. This advantageously allows biochemical conditions to be created in the first inner lumen that ensure the survival or revival of cells in and around the inner lumen after the disposable housings have been separated from their cell-biological supply. The first inner lumen can be designed as a nutrient reservoir, and the second inner lumen as a test substance chamber.

[0008] In preferred embodiments, the two housing parts each have a relief structure and are hinge-connected, with the relief structures of the two housing parts interlocking when closed, forming the chambers. Because the two housing parts are hinge-connected, they are foldable or "collapsible." For example, a fold or double fold between the two housing parts allows them to be closed and opened again using a folding mechanism. The disposable housing can be designed, in particular, as a plastic folding relief. The housing parts can be manufactured from plastic films by deep-drawing, for example, using a blister process. The possibility of manufacturing the disposable housing is therefore also advantageous, since plastic reliefs can be produced cost-effectively using deep-drawing processes. Low manufacturing costs are particularly advantageous for disposable devices.

[0009] The relief structures of the two housing parts interlock when closed, forming separate chambers. In this case, an opposing chamber part, in particular a chamber half, can be formed in each housing part, which, when closed or when the plastic reliefs are folded, each forms a chamber with a carrier layer within it. The carrier layer divides the cavity of the chambers into two inner lumens, even when the housing is folded. The individual chambers are separated from each other. The two housing parts can also be referred to below as the "upper" and "lower part." The first housing part, in which the carrier layer is fixed, is referred to as the "lower part," and the partial lumen of the hollow chamber body formed thereby is referred to as the "lower" inner lumen. Accordingly, the second housing part is referred to as the "upper part," and its lumen is referred to as the upper inner lumen.

[0010] In preferred embodiments, the chambers are closed by a fitting of interlocking relief structures in the two housing parts, preferably by means of ribbing or a tongue and groove connection. A closed plastic relief can provide a gas-tight and / or liquid-tight separation of the individual chambers by suitable fitting with opposing relief structures. It is advantageous that in particular the lower inner lumens, which can be filled with culture or nutrient media for test cells, are sealed leak-proof during transport and can be covered with the upper inner lumen. In this case, the lower inner lumen of the disposable housing can serve as a nutrient reservoir for supplying cells while the media supply from the inlet and outlet openings is separated. Furthermore, by providing a hydrophobic surface coating in the area of ​​the relief structures orIn the tongue and groove region, protection against capillary fluid loss should also be provided.

[0011] The carrier layer can be sealed after forming a plastic housing and cut along the grooves separating the chambers. Alternatively, the grooves can be used, after sealing the parallel outer edges with springs in a sealing stamp, to tension the carrier layer during the complete sealing of the remaining support areas in a second step. The carrier layer can be made of biodegradable or non-degradable, durable material or mixtures thereof. Preferred biodegradable materials are poly-L-lactide, polyhydroxybutyric acid, polyglycolic acid, hyaluronic acid, or mixtures thereof. Preferred durable materials are polyethylene, polyamide, polyester, polyurethane, polypropylene, Goretex, or mixtures thereof. The carrier layer is preferably porous and thus permeable to the culture and / or treatment medium. The carrier layer can be designed as a filter membrane.The carrier layer can be coated with adsorption-promoting substances such as collagen before sealing. This can improve the subsequent settlement of the cells. The hinge-like connection and the interlocking relief structures in the upper and lower sections further enable the upper housing section to be opened over the entire carrier layer surface even after test cells have been fixed to the carrier layer, allowing contact of sample material such as air, cosmetic compositions, or substances contained in a medium with the cell-bearing carrier layer. The carrier layer is preferably permeable to culture and / or treatment media.A media-permeable carrier layer allows the supply of test cells seeded on the carrier layer from an underlying inner lumen, as well as the transport of test cell metabolites into the inner lumen for subsequent detection. Permeability to air volumes of more than 10 L can also be useful before the test cells come into contact with the carrier layer, allowing for subsequent characterization of the air particles retained from the air volume.

[0012] For the purposes of this application, "medium" refers to a liquid suitable for culturing cells and which can be contained in the disposable housing. This is preferably a conventional cell culture medium such as DMEM (Dulbecco's Modified Eagle Medium), RPMI (Roswell Park Memorial Institute) medium, or another medium particularly suitable for culturing human cells, which may optionally be supplemented with additives such as fetal calf serum, growth factors, antibiotics, or suitable substrate analogues. Substrate analogues are understood here to be those that are physically or electrochemically detectable in the spent medium after their conversion. Medium also includes other liquids generally compatible with cell culture, such as PBS (phosphate buffered saline). Solutions used for detaching cells, such as trypsin / EDTA, are also referred to as medium for the purposes of this invention.If a culture medium contains substances that are not intended to supply the cells but rather to influence them or to be examined, the medium is not referred to as a culture medium but as a treatment medium.

[0013] In embodiments, the chambers in the first and / or second housing part each have an inlet opening and an outlet opening for a culture and / or treatment medium. The chambers in the first, or "lower," housing part can each have a closable inlet opening and a closable outlet opening for a culture and / or treatment medium. The inlet opening and outlet opening allow the lower inner lumens to be perfused with culture or nutrient media for the test cells. Preferably, the chambers in both housing parts each have a closable inlet opening and a closable outlet opening. The carrier layer can divide the chambers into an upper and a lower permeable inner lumen, each with a closable inlet and an outlet opening.This allows a carrier layer populated by cells on both sides to be contacted on both sides by the same or different liquid culture or treatment media or gaseous media.

[0014] The inlet and outlet openings can be closed gas-tight and / or liquid-tight. Preferably, the inlet and outlet openings can be closed by a septum, in particular in a nozzle. It is particularly advantageous if the disposable housings can be closed leak-proof during transport. A septum is understood to be a sealing membrane made of an elastic material, which reseals automatically, for example, after being punctured with a cannula. Such a septum can be formed, for example, from silicone foam. A septum is preferably formed in a nozzle, which can be easily manufactured by deep drawing during the production of the plastic relief of the disposable housing. Such a nozzle can be sealed alternatively or additionally with adhesive film after removal of a pierced hollow body.A pointed hollow body with a hose connection can be inserted through a septum in a nozzle, thus connecting the chambers of the disposable housing to external gas or liquid reservoirs.

[0015] In embodiments, the chambers of the disposable housing may have an opening in the second housing part, i.e., the upper part. Such an opening may allow air or ambient contact of the upper inner lumen. This is advantageous, for example, for air sampling using a lung model.

[0016] For use in in vitro testing, the carrier layer can be seeded with cells. The disposable housing, which can be sealed with septa, allows cells to be seeded under sterile and pyrogen-free conditions, such as those found in a cell culture laboratory, and the pre-cultured cells are transportable in the disposable housing. Pre-cultured cells can be fixed in the disposable housing as co-culters, for example, in the form of a skin or lung model, and shipped ready for immediate in vitro testing. Furthermore, cells in the lower inner lumen can be freeze-dried and, after storage at room temperature, reconstituted with medium for subsequent testing.

[0017] In The disposable housing contains a plurality of separate chambers, allowing for parallel experiments, including controls. Providing four chambers, for example, enables FDA-compliant sampling in a 2+2 format, with two test and two control samples simultaneously from one sample. Furthermore, readable information about the cells can be added to the disposable housing. A code or barcode can be embossed or affixed, allowing data from cells used in batches to be read and used in the calculation of results.

[0018] Depending on the intended type of in vitro testing, the carrier layer can be colonized with cells on one or both sides. Colonization of the carrier layer with test cells under aseptic conditions is preferred. In particular, the disposable housing is also suitable for in vitro testing using co-cultures. For this purpose, the carrier layer, which is permeable to the culture and / or treatment medium, is colonized with first cells on the side facing the first, or "lower," inner lumen, and with second and optionally further third cells on the side facing the second, or "upper," inner lumen. The cells can, in particular, be human cells. Through the colonization on both sides, these cells can model the organotypic cell layers in a healthy or diseased organism.

[0019] The first and second cells are preferably cell types used in co-culture models, for example, fibroblasts and keratinocytes in skin models or endothelial cells and lung epithelial cells in lung models. These models can be constructed with or without additional monocytes, which form the third cells. Cells such as epithelial cells, fibroblasts, or monocytes can be obtained as primary cultures using known methods or are commercially available. In In preferred embodiments, the carrier layer is populated with fibroblasts on the side facing the first, or "lower," inner lumen, and with keratinocytes on the side facing the second, or "upper," inner lumen. In application examples in which fibroblasts are cultivated on the lower carrier side and keratinocytes on the upper, air-facing side of the carrier layer, three-dimensional carrier-stabilized skin models are created in the disposable housing in several separate chambers. These models can be used, for example, to test cosmetics or pharmaceuticals. For this purpose, the disposable housing populated with fibroblasts and keratinocytes can be opened, and the cosmetics to be tested can be applied to some of the skin models, while the skin models in the remaining chambers can serve as controls in parallel.This allows the detection of metabolic products of the second cells, or especially the first cells, and the effects of cosmetics can be characterized based on the differences in the signals measured on the skin models. By additionally co-culturing third cells such as monocytes, immunological effects can also be investigated.

[0020] In equally preferred embodiments, the carrier layer is populated with endothelial cells on the side facing the first, or "lower," inner lumen and with lung epithelial cells on the side facing the second, or "upper," inner lumen. The first inner lumen can be filled with nutrient medium or perfused with it. In such an application example, endothelial cells are populated on the liquid-facing side of the carrier layer and lung epithelial cells on the air-facing side. Additionally, monocytes can be mixed with the endothelial or epithelial cells to promote cytokine-related interactions. A controlled vacuum can be applied via the inlet and outlet openings of the upper inner lumen, allowing ambient air to be passed past the test cells with a defined and, if necessary, intermittent air flow through the openings in the upper housing section.A continuous nutrient flow through the lower inner lumen can be intermittently superimposed with pressure fluctuations that simulate the pressure fluctuations during lung respiration. For this purpose, the disposable housing can be connected to a suitable, preferably mobile, test device. This allows the disposable housing to be used as a multi-chamber lung model to test the air in vehicles, in polluted indoor spaces, at traffic points exposed to particulate matter, or in workplaces. By connecting a controlled piezo nebulizer of pyrogen-free water to the inlet openings and blending the air sample and the humidified mist, it is possible to ensure that the epithelial cells do not suffer any drying damage during the test.

[0021] To conduct in vitro tests, it is preferred to connect the transportable housings with pre-cultured cells to mobile measurement and control technology. A further subject matter therefore relates to a holder for a disposable housing according to the invention, wherein the holder forms a negative relief for each of the two housing parts, thereby clamping the disposable housing flat. In embodiments, cannulas can be attached, for example screwed, to the holder in such a way that the cannulas pierce the septa during clamping without touching the carrier layer. The cannulas are preferably connected to pumps in a supply unit so that treatment medium or reaction liquids and / or gases can be guided through the chambers. The supply and discharge of the reaction liquids and / or gases can be provided, for example, by suitable control software.Another subject matter relates to a device for analyzing substances, comprising a disposable housing according to the invention and a holder according to the invention for the disposable housing. Preferably, this device further comprises suitable control, measuring, input, and display electronics. Depending on the design, biochemical or physical measurements can be performed in the holder or the supply unit on reaction fluids drained from the chambers. These measurements can be used to determine metabolic products of the first cells after contact of the second cells with a substance to be examined, or to characterize the reaction of the cells on the carrier layer to the substances. Furthermore, the supply unit can also contain reservoirs for media and reagents, as well as tubing and pump systems.

[0022] The disposable housing is particularly suitable for in vitro tests such as purity tests, biocompatibility tests, toxicity tests, or efficacy tests. The invention therefore also relates to the use of the disposable housing for in vitro tests, as well as to a method for detecting substances using the disposable housing. Such methods for the biological characterization of samples can advantageously be carried out by incubating them with test cells in the disposable housing. The cell response can be characterized by measuring metabolic products of the test cells, which can be measured molecularly, immunologically, electrochemically, or optically in the cell-surrounding media.

[0023] A further aspect of the invention relates to a method for detecting substances, comprising the steps: a) providing a disposable housing according to the invention and optionally a holder according to the invention for the disposable housing, b) loading the disposable housing with first, second and / or third cells, c) optionally storing and / or transporting the disposable housing, d) bringing the first, second and / or third cells into contact with the substance to be examined, and e) determining metabolic products of the first, second and / or third cells after contact of the first, second and / or third cells with the substance to be examined.

[0024] The disposable housing can preferably be loaded with first and second cells, and optionally with third cells. In these embodiments, the second and / or third cells are brought into contact with the substance to be tested, and the metabolites of the first, second, and / or third cells are determined after contact of the second and / or third cells with the substance. Alternatively, the disposable housing can be loaded only with monocytes.

[0025] The disposable housing can be stored before use and / or transported to the site of use.

[0026] The detection method is carried out using a disposable housing as described above. The method can therefore be used, in particular, for biological sample characterization using matrix-bound cells in flow-through plastic spheroids. The substance to be analyzed can be solid, liquid, or gaseous. The determination of metabolites can be performed using molecular biological, immunological, electrochemical, or optical methods in the cell-surrounding media. The determination can be performed, for example, photometrically or using IR spectroscopic measurements.

[0027] Preferably, the determination is carried out using electrochemical measurements. In preferred embodiments, the determination of metabolic products is carried out according to the principle of the electrochemical three-electrode measuring arrangement, wherein the carrier layer is designed as an electrically conductive working electrode and the counter and reference electrodes are introduced through the septa into the lower inner lumen or are represented as electrically conductive paths applied by screen printing. For this purpose, a connection of the screen-printed electrodes to the outside of the disposable housing can be made possible by through-plating the housing parts. Furthermore, the working electrode can be made of redox-reactive plastic or coated with it. Specific oxidoreductases for the detection of cellular metabolic products are preferably immobilized on the redox-reactive working electrode.

[0028] A particular advantage is that, for example, skin models do not need to be analyzed histologically or biochemically after cell lysis, but can be read electrochemically on the intact model. Alternatively, one or two electrodes for potentiometric measurements can be connected to the carrier layer using the principle of the electrochemical three-electrode measuring arrangement. For this purpose, the working electrode can be created by coating the carrier layer with redox-reactive materials. Furthermore, the electrodes can be connected to the outside of the disposable housing by through-plating the sealing surface of the carrier layer.

[0029] The invention is explained in more detail below with reference to drawings and exemplary embodiments. Fig. 1a schematic representation of a disposable housing in cross section in Fig. 1A, 1B und 1C , under supervision in Fig. 1D , as well as in longitudinal section in Fig. 1E und 1F .

[0030] The Figuren 1A und 1B show in cross-section a second or upper housing part 3 and a first or lower housing part 2, which form a disposable housing 1. The housing parts 2 and 3 each have a relief structure. The relief structures of the two housing parts 2, 3 engage in the Fig. 1C shown closed or folded state into each other, whereby four separate chambers 5 are formed. Fig. 1B shows that in the first housing part 2, a carrier layer 4 permeable to a culture and / or treatment medium is fixed in the chambers 5. As a result, a first or lower inner lumen 6 and a second or upper inner lumen 7 are formed in the chambers 5. The Fig. 1D shows the disposable housing 1 with the second or upper housing part 3 as well as the first or lower housing part 2 and the four chambers 5 in plan view. By folding the disposable housing 1, the three springs 8 can be inserted into the precisely manufactured grooves 9, whose relief structures 8 and 9 in cross section in Fig. 1A und 1B shown, snap into place and thus, as shown in the cross section, Fig. 1C four chambers 5 are formed and sealed. The chambers 5 each have an inlet opening 10 and an outlet opening 11 for a culture and / or treatment medium in the upper and lower housing parts 3 and 2. The inlet openings 10 and outlet openings 11 are sealed with a septum 12. Such a septum can be formed, for example, by silicone foam. Fig. 1E In longitudinal section, shaped nozzles 13 are shown, in which the septa 12 are mounted.

[0031] The disposable housing 1 can be deep-drawn from plastic foil using the blister process. Fig. 1E A longitudinal section of a disposable housing is shown, still in relief after the deep-drawing process. One half of the housing is drawn in millings below the aluminum tool surface 17, while the other half is formed by molded parts placed on the tool during the deep-drawing process. After forming, a carrier layer 4 is sealed in the size of the outline of the Fig. 1D The carrier layer can be coated with adsorption-promoting substances such as collagen before or after sealing. After sealing the carrier layer 4, it is cut into four parts along the three chamber-separating grooves 9. Now, when folding the plastic housing according to the folding direction of the dashed arrow in Fig. 1E along the double fold 16, the three springs 8 snap into the precisely manufactured grooves 9 and thus as shown in the cross section of the Fig. 1C four chambers 5 with upper inner lumen 7 and lower inner lumen 6 are formed, which are separated by the four carrier layers 4.

[0032] To colonize the carrier layer 4 with cells, the first inner lumen 6 can be filled with a cell culture medium containing living cells such as fibroblasts or endothelial cells via the puncture ports 12 and with venting at the puncture ports 11. These cells can release characteristic substances into the environment in response to environmental stimuli. To colonize the carrier layer 4, the disposable housing 1 is turned over on the side facing the first inner lumen 6, whereby the culture medium containing the cells is on the carrier layer. During a subsequent first incubation under state-of-the-art culture conditions, the cells will sediment and adhere to the side of the carrier layer 4 facing the inner lumen. During cell culture incubation, the media can be changed in the four chambers 4 via the inlet openings 10 and outlet openings 11.When a multilayer cell layer has formed on the side of the carrier layer facing the first inner lumen 6, the folded housing 1 is opened again and positioned with the populated carrier side facing downwards.

[0033] In a subsequent step, the side of the carrier layer 4 facing the second inner lumen 7 can be populated with second cells. For this purpose, the opened housing can be filled with a cell culture medium up to a level above the carrier layer, in which second living cells are suspended, in particular those that are characteristic of contact with air in the original organism, such as keratinocytes, epithelial cells, or monocytes. These cells can later, stimulated by environmental stimuli, release characteristic substances into the environment. During a subsequent second cell culture incubation, the cells sediment onto the other side of the carrier layer opposite the previously formed multilayered first cell layer. The cells are thus located on the carrier layer side that, when folded together, borders the second or upper inner lumen 7.To protect the cells during the second incubation, the housing can be closed with a cover. The first cells also continue to be supplied with medium during the second incubation, with the culture medium being renewed in the lower inner lumen 6 via the inlet and outlet openings 10 and 11, as bubble-free as possible. As soon as a second sufficiently thick cell layer has formed on the surface of the carrier layers 4, the culture medium above the carrier layers is removed and the plastic housing 1 is closed again as shown in cross-section C. The culture fluid in the lower inner lumen 6 is then changed again. The multilayer, carrier-stabilized second test cell layer now has contact with air through the four openings 14 in the second or upper housing part 3 to the four chambers 5. The supply of culture medium to the folded plastic housing 1 can then be disconnected.The septa 12 can tightly seal the chambers 4. Additionally, the nozzles 13 can be sealed with adhesive foil. Determination of air pollutants

[0034] The disposable housing populated with co-cultures of first, second, and third cells can be sterilely packaged in this form and delivered, for example, by temperature-controlled mail to the site of in vitro testing. At the site of use, the disposable housing can be connected to a holder. The holder suitably has a negative relief for each of the two housing parts 2 and 3, whereby the disposable housing 1 can be clamped flat. The holder can be a mobile measurement and control device that controls the flow rate and temperature of the continuously renewed culture medium and optimizes the temperature in the four chambers for the test cells. For this purpose, the septa 12 in the inlet and outlet openings 10 and 11 can be pierced using cannulas. For this purpose, cannulas can be attached to the holder in such a way that they pierce the septa when clamped without touching the carrier layer 4.The cannulas can in particular be connected to pumps in a supply unit so that treatment media and / or gases for in vitro tests can be passed through the chambers 5.

[0035] Such a device, comprising a disposable housing and holder, is equipped for in-vitro testing or biological tests for analyzing substances at the point of use. For this purpose, the second cells, such as epithelial cells, and the third cells, such as monocytes, are brought into contact with the substance to be tested. For this purpose, an air stream can be directed through the openings 14 in the second or upper housing part 3 over the layer of epithelial cells. Epithelial cells and monocytes, stimulated by environmental stimuli, can release characteristic substances into the environment, to which the endothelial cells react and, in turn, can release characteristic substances into the surrounding medium. Metabolic products of the endothelial cells can then be determined. For this purpose, the mobile device can be equipped with suitable electronic or optical sensors with which metabolic products of the second test cells can be detected directly or indirectly.Biochemical, molecular biological, immunological or electrochemical reactions can be involved both in the cell layers and in the culture medium to generate the electronic or optical signals.

[0036] To ensure the quality of the tests, two of the four chambers can be loaded with the analytes to be tested as positive controls, while two chambers can be used for the actual measurement. The mobile device for measurement and control technology can also be used to calculate averages of the measured signals and to verify the validity of the measurements using the positive controls. The mobile device can also be equipped with a results display, an internet connection, and / or a storage option for test results and batch data in the disposable housing. Example 1 Production of disposable housings

[0037] For the production of the Fig. 1 For the disposable housing shown, a colorless transparent PET-GAG film with a thickness of 0.4 mm (PET-GAG 340, glossy, 400 x 0.4 mm, Folien Werk Wolfen) was used and the two housing parts were manufactured as a blister cassette in the deep drawing process at 540°C on a deep drawing machine SKA3550T (Zappe GmbH).

[0038] A Versapore membrane (Versapore, 100W / WA 10'x3M, pore size 1.2 µm, Pall Corporation) was cut to 1.6 × 8.5 cm and stored in Anosteralyt® (diluted 1:3, WABOSAN Arzneimittelvertriebs GmbH) for 72 hours for decontamination. Before sealing, the Versapore membrane was then placed on a paper towel for 2 minutes. Silicone baking paper was placed around the cavities of the blister cassettes as a base. The membrane was sealed on a QUICK blister sealing machine (Zappe GmbH) at 120°C for 20 seconds and at a pressure of 5 bar.

[0039] To seal the inlet and outlet openings of the blister cassettes, 2 µl of Loctite 4304 LC adhesive (Henkel) was added to the nozzles, which were then filled with foam cord (Coltene). Curing was performed at room temperature for 3 hours in a shaking incubator (Shaking Incubator 1000, Heidolph) at 750 rpm.

[0040] For decontamination, the blister cassettes were finally treated at 5×10 -4< bar, 150 W, for 45 seconds with 99% Ar (Argon C, 99.996 vol.%, Linde) and 1% O 2 (Oxygen C, 99.5 vol.%, Linde) in a microwave plasma (100-E Plasma Systems, Technisch Plasma GmbH). Example 2 Production of a skin model in a disposable housing

[0041] a) Preparation of a collagen type I gel To produce a skin model in a disposable housing, a collagen type I gel containing fibroblasts was first prepared. The collagen type I gel was prepared at 4°C. The required materials and solutions were stored in the refrigerator, and a frozen plastic box filled with water served as a cooling station. In a first step, solution B was prepared with 720 µl 10 × DMEM (Dulbecco's Modified Eagle Medium with 3.7 g / l NaHCO 3 , 4.5 g / l glucose, 0.5% L-glutamine, Biochrom), 360 µl 1M HEPES buffer (Ultra Saline A, HEPES Buffered Saline, Lonza), and 250 µl NaOH (0.7 mol / l, VWR), which was then stored in the refrigerator. To prepare 1 ml of collagen type I gel, 100 µl of solution B and 800 µl of collagen G (4 mg / ml, Biochrom) were pipetted into a centrifuge tube and mixed on a test tube shaker (vortexer).Subsequently, 100 µl of cell suspension of 5 × 10 5< cells / ml in FKS (Fetal Bovine Serum, Biochrome) of the fibroblast lines NIH-3T3 (Laboratory for Biomaterials Iserlohn) or Hs-68 (Sigma-Aldrich Prod. No.: 89051701) were added and also mixed on the test tube shaker. The collagen type I gel was stored in the refrigerator for 10 minutes until the resulting air bubbles had diffused out of the gel. For four wells of a blister cassette prepared according to Example 1, the required volume of collagen type I gel was 3.2 ml at 800 µl collagen type I gel per well. b) Application of the collagen type I gel into the disposable housing A silicone mask was placed on the Versapore membrane. 800 µl of collagen type I gel containing fibroblasts was pipetted into sections of the mask. The blister cassettes containing the gels were shaken in a shaking incubator for 7 minutes at 600 rpm and 37°C to ensure even distribution of the gel.The blisters were then stored in an incubator (XC, Binder) for 2 hours at 37°C and 5% CO2 to allow the collagen type I gels to harden. c) Application of the keratinocytes After hardening of the collagen type I gels, 3 ml of cell suspension containing 5.3 × 105< cells / ml of the keratinocyte cell line HaCaT (Laboratory for Biomaterials Iserlohn) in DMEM was added to each well. The disposable housings were then incubated in an incubator at 37°C and 5% CO2 for 24 hours. The medium was then aspirated, and new medium was added to the individual wells on the non-growth side to allow the HaCaT keratinocytes to grow exposed to air. The skin models were cultured in an incubator at 37°C and 5% CO2 for 14 days, with the medium being changed every 24 hours. . Example 3 Determination of the storage stability of human keratinocytes by detecting metabolic activity

[0042] HaCaT keratinocytes were seeded into 24-well cell culture dishes (growth area: 1.82 × 10.32 cm ) at a cell density of 4–10 cells / cm and incubated for 48 h at 37°C and 5% CO in an incubator. Six of the cell culture dishes were then stored for a further 24 h at 37°C and 5% CO in the incubator, while six other cell culture dishes were stored for 24 h at 6°C in an undefined atmosphere. The cell culture dishes were then incubated for a further 24 h at 37°C and 5% CO before the metabolic activity of the cells was determined using the MTT assay according to the manufacturer's instructions. The viability of cells stored for 24 hours at 6°C was 9% compared to cells incubated in an incubator at 37°C and 5% CO2, which had a viability of 100%. This is considering that the cells were mitotically active and could therefore continue to proliferate at 37°C under optimal conditions.If postmitotic cells were tested for storage stability, it is assumed that the viability would approach that of the reference sample when stored at 37°C.

[0043] The determination of vitality shows that the human keratinocytes could be successfully stored for 24 h at 6°C. This shows that a disposable housing populated with human test cells of a skin model can be transported cooled to a location outside of a cell culture laboratory and there for in-vitro- can be used for exams. Example 4 Manufacturing process of a lung model in a disposable housing

[0044] To create a lung model, 2.4 10 5 cells / cm 2 of the endothelial cell line EA.HY-926 were placed per well on the Versapore membrane (growth area 10.32 cm 2 ). Each well was filled with 3 ml of cell suspension and then incubated in an incubator at 37°C and 5% CO 2 for 4 h until the EA.HY-926 cells had adhered. Subsequently, 1.2 10 5 cells / cm 2 of the epithelial cells A549 were added to the other side of the membrane. This lung model in a disposable housing was incubated for two days at 37°C and 5% CO 2 in an incubator. On day 3, THP-1 monocyte cells were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA, Biochrom). PMA was added to THP-1 cells at a density of 4 × 10 5 cells / ml, resulting in a final concentration of 20 ng / ml PMA in the medium containing the THP-1 cells. The cells were then incubated at 37 °C and 5% CO 2 in an incubator for 24 hours.

[0045] The adhered THP-1 cells, which had differentiated into macrophages, were then detached from the bottom according to a standard protocol (PANBiotech), except that no trypsin was used for lysis. Instead, 0.5 mM EDTA (PANBiotech) was used to lyse the THP-1 cells. The THP-1 cells were added to the side of the membrane containing the A549 epithelial cells at a concentration of 2.4 10 5 cells / cm 2 . The lung models were incubated for a further 4 hours in an incubator at 37 °C and 5% CO 2 until the THP-1 cells were adherent. The medium was then removed for air-exposed cultivation of the A549 and THP-1 cells. On day 5, the substance to be tested could be applied to the lung model.

[0046] Overall, the examples demonstrate that disposable housings can be manufactured as blister cassettes using the deep-drawing process, and that a carrier layer for test cells can be sealed onto them. The disposable housings were successfully populated with human fibroblasts and keratinocytes for a skin model, as well as with endothelial, epithelial, and monocytes for a lung model. These can be transported at a temperature-controlled location outside of a cell culture laboratory and stored there for in-vitro- exams can be used. Example 5 Manufacturing process of a freeze-dried monocyte activation test in a disposable container

[0047] To produce a test mixture with activated and subsequently lyophilized monocytes, which can be stored for several months at room temperature, freezing medium was first prepared. For this purpose, 40 ml of Ca 2+< - and Mg 2+< -free PBS were added, 34.9 mg of EGCG were weighed and dissolved in the PBS. Then, 3.02 g of trehalose dihydrate (0.1 M) was added and made up to 80 ml with Ca 2+< - and Mg 2+< -free PBS. Following this preparation, two heparin monovettes were obtained from each of five blood donors and, after gentle inversion 10 times in a blood bag system, mixed on a tilting mixer at 30 cycles per minute. 3 ml of Histopaque was then dispensed into each of 30 centrifuge tubes, carefully layered with 3 ml of the mixed human blood, and centrifuged for 30 minutes at 1000g.Monocytes were removed by carefully pipetting off the corresponding band and dissolved in 10 ml each of Ca 2+< - and Mg 2+< -free PBS. 50 mg of AgNP solution (50 µg / ml final concentration) and 1 mg of LPS solution (1 µg / ml final concentration) were added. Pre-activated for 1 h at 37 °C, and then centrifuged at 180 g for 10 min at 4 °C to stop pre-activation. The pellets were then resuspended in centrifuge tubes in 10 ml each of Ca 2+< - and Mg 2+< -free PBS. The centrifuge tubes were centrifuged for 10 min at 250 g, and four pellets were then resuspended in 2.5 ml of freezing solution each. After cell counting (e.g., using a CasyCounter), the pellets were adjusted to approximately 10 7< cells / ml with freezing solution. The samples were injected with cannulas in 0.5 ml portions onto the bottom of the inner lumen 6 of blister cassettes with filter membranes manufactured according to Example 1 and cooled to -70 °C at approximately 5 °C / min, e.g.,with liquid nitrogen in a suitable device. The blister cassettes containing the deep-frozen monocytes were freeze-dried in a lyophilizer under vacuum (0.5 µbar) at -70 °C.

[0048] The blister cassettes containing lyophilized monocytes prepared in this way were initially used for air filtration through a sealed filter membrane using a commercially available air sampling pump. Following subsequent resuspension of the monocytes and fluid contact of this suspension with the air particles retained on the filter membrane, their content of pyrogenic substances could be measured using the monocyte activation test according to European Pharmacopoeia 2.6.30. List of reference symbols:

[0049] 1 Disposable housing 2 First housing part 3 Second housing part 4 Carrier layer 5 Chamber 6 First inner lumen 7 Second inner lumen 8 Tongue 9 Groove 10 Inlet opening 11 Outlet opening 12 Septum 13 Nozzle 14 Opening 15 Sealing surface 16 Double fold 17 Aluminum tool

Claims

1. Disposable housing for enclosing cells and / or co-cultures for in vitro tests, having a plurality of chambers (5) separated from one another which each have at least one inlet opening (10) and one outlet opening (11) for a culture medium and / or treatment medium, wherein the housing (1) is formed from a first housing part (2) and a second housing part (3), and a carrier layer (4) that is permeable to the culture medium and / or treatment medium is fixed in the chambers (5) in the first housing part (2), whereby a first inner lumen (6) and a second inner lumen (7) are formed in each of the chambers (5), and wherein the two housing parts (2, 3) each have a relief structure and are connected in a hinge-like manner, wherein the relief structures of the two housing parts (2, 3) interlock in the closed state, thereby forming the chambers (5), and wherein the inlet openings (10) and outlet openings (11) are closable in a gas-tight and / or liquid-tight manner.

2. The disposable housing according to claim 1, characterised in that the chambers (5) are closed particularly by fitting interlocking relief structures (8, 9), preferably by means of ribbing or tongue and groove connection (8, 9).

3. The disposable housing according to claim 1 or 2, characterised in that the chambers (5), preferably in each housing parts (2, 3), have a closable inlet opening (10) and a closable outlet opening (11) for a culture and / or treatment medium.

4. The disposable housing according to one of the preceding claims, characterised in that the inlet openings (10) and the outlet openings (11) are closable by a septum (12), in particular in a nozzle (13).

5. The disposable housing according to any one of the preceding claims, characterised in that the carrier layer (4) that is permeable to the culture medium and / or treatment medium is populated with first cells on the side facing the first inner lumen (6) and is populated with second cells, and optionally furthermore third cells, on the side facing the second inner lumen (7).

6. The disposable housing according to claim 5, characterised in that the carrier layer (4) is populated with fibroblasts on the side facing the first inner lumen (6) and with keratinocytes on the side facing the second inner lumen (7), or is populated with endothelial cells on the side facing the first inner lumen (6) and with lung epithelial cells on the side facing the second inner lumen (7).

7. A holder containing a disposable housing according to any one of claims 1 to 6, characterised in that the holder forms a negative relief for each of the two housing parts (2, 3), whereby the disposable housing (1) is clamped over its surface.

8. The holder according to claim 7, characterised in that cannulae are mounted in the holder in such a way that the cannulae pierce the septa (12) during clamping without touching the carrier layer (4), whereby the cannulae preferably are connected to pumps in a supply unit so that treatment media and / or gases can be guided through the chambers (5).

9. A device for analysing substances, comprising a holder according to any one of claims 7 or 8 containing a disposable housing.

10. A method for the detection of substances, comprising the steps of: a) providing a disposable housing according to any one of claims 1 to 6 or a holder according to any one of claims 7 or 8 containing a disposable housing, b) loading the disposable housing with first, second and / or third cells, c) optionally storing and / or transporting the disposable housing, d) bringing the first, second and / or third cells into contact with the substance to be analysed, and e) determining metabolic products of the first, second and / or third cells after contact of the first, second and / or third cells with the substance to be analysed.

11. The method according to claim 10, characterised in that the determination of metabolic products is carried out according to the principle of the electrochemical three-electrode measuring arrangement, wherein the carrier layer (4) is designed as an electrically conductive working electrode and the counter electrode and reference electrode are introduced through the septa (12) into the lower inner lumen (6) or are represented as electrically conductive paths applied by means of screen printing.