EP1290144A2 - Infection model - Google Patents

Abstract

The invention relates to agents and methods for analyzing and diagnosing human or animal infections caused by pathogenic and/or parasitic microorganisms, to agents and methods for analyzing and diagnosing degenerate or genetically modified human or animal cells, to agents and methods for examining and testing antiinfectives and medicaments for combating tumors and to three dimensional <i>in vitro</i> organ and tissue test models, especially for tissues which are susceptible to infections, such as intestine, skin, cornea, trachea and mucosa tissues.

Description

infection models

description

The invention relates to means and methods for the analysis and diagnosis of infections caused by pathogenic and / or parasitic microorganisms and / or; Diseases of the human or animal body, means and methods for analyzing and diagnosing degenerate human and animal cells, means and methods for analyzing and diagnosing genetically modified human and animal cells, and means and methods for examining and testing anti-infectives and pharmaceuticals against Tumors, in particular cytostatics, and three-dimensional animal in vitro organ and tissue models, in particular tissues susceptible to infection, such as the intestine, skin, cornea, trachea and mucous membranes.

Macroorganisms, for example humans, can be infected by a large number of microorganisms, which include both prokaryotic organisms, such as bacteria, and eukaryotic organisms, such as fungi and protozoa, and also viruses in the broader sense. This can lead to very different consequences for an affected macroorganism. For example, infection by microorganisms can lead to the development of an infectious disease. Other microorganisms live in or on the host organism, which means that they live at the expense of theirs Host organism without seriously becoming ill. On the other hand, some viruses, especially oncogenic viruses, are known to be able to neoplastic transform human or animal cells in vivo, some being associated with the formation of degenerate cells and tumor pathogenesis.

The infection of a human or animal organism by a microorganism comprises several steps, such as the transfer of the microorganism to its host organism, the adherence (adhesion) of the microorganism to cells or tissues of the host organism, the penetration of the microorganism (invasion or penetration) into spe - specific cells or tissues of the host organism and the multiplication of the microorganism therein. Infection is determined by the infectious properties, for example the transferability, contagiosity, adherence, penetration and reproductive ability, and the pathogenic properties of the microorganism. The development and course of an infectious disease is also determined by the susceptibility and immunity of the infected host organism. Depending on the microorganism, for example whether it is a prokaryotic or a eukaryotic organism, and depending on the host organism concerned, for example which organs or tissues are affected, the mechanisms of an infection are very different. In addition to the properties of the virus, the development and course of a tumor disease also depend on the susceptibility and immunity of the host organism concerned. It is known that viruses preferentially lead to the formation of neoplasias in immunocompetent organisms.

The drugs used to combat either infectious diseases or tumors also have a correspondingly different effect. Anti-infectives used against infectious diseases that damage or kill microorganisms in the body of the affected organism are aimed at inhibiting the cell wall synthesis of the microorganism, disturbing the permeability of its cytoplasmic membrane, blocking its protein synthesis and / or suppressing its nucleic acid synthesis without, however, harming the host organism itself. In the case of cancer tumors caused by oncogenic viruses, on the other hand, the actual causative agent, the oncogenic virus, cannot currently be targeted, but the degenerated cell is destroyed or its growth is inhibited, for example by using cytostatic agents.

To investigate the complex interactions between a human or animal host and a microorganism that lead to the development of an infectious disease, as well as to analyze the complex cellular and / or viral mechanisms leading to the development of degenerate animal cells, experiments in the past have primarily been used in animals Service. Also for Development and testing of anti-infectives or cytostatics have mainly been carried out on animals, for example as part of preclinical tests. However, it has been shown that results obtained from animals can only be transferred to humans to a limited extent.

With the development of cell culture techniques, tests were also carried out on two-dimensional in vitro cell systems in order to complete or replace animal experiments. For example, studies of the cell pathogen Candida albicans, which causes Candida mycoses in humans and can lead to life-threatening infections in immunosuppressed patients, were also carried out on such cell systems (Mitchell, Curr. Opin. Microbiol., 1 (1998), 687- 692). Among other things, an epidermis model consisting of a cell type and consisting of a cell monolayer was developed for examining the adhesion and penetration of Candida (Körting et al., J. Infect., 36 (1998), 259-267; zinc et al., Infect. Immun., 64: 5085-5091 (1996).

However, the disadvantage of such two-dimensional in vitro cell systems is that no statements can be made about a further infection mechanism or the exact course of a tumor pathogenesis. Due to their structure, these systems do not allow interactions between different cell types, as is the case, for example, in whole organs in vivo. In addition, these systems do not contain a connective tissue-like matrix, but are based on synthetic see membranes built up. As a result, in the case of an infectious disease, for example, these systems do not allow a more detailed analysis of the complex interactions between a pathogen, the cell types that build the organs or tissues, and the solid connective tissue matrix. Such systems can therefore only be used to a very limited extent for the development and testing of anti-infectives and cytostatics.

The technical problem on which the present invention is based is therefore means and methods for analyzing and diagnosing infections caused by pathogenic or parasitic microorganisms in human or animal host organisms, means and methods for analyzing and diagnosing degenerate or genetically modified human or to develop animal cells and means and methods for examining and testing diagnostic and therapeutic agents, in particular anti-infectives and anti-tumor drugs, in particular cytostatics, which overcome the above-described disadvantages in the prior art and which are particularly suitable for To investigate the development of infection-causing interactions between pathogenic or parasitic microorganisms and their target organs or tissues and the cellular and / or viral mechanisms leading to tumor formation, and the development and testing of these mechanisms allow specific diagnostic and therapeutic agents, such as anti-infectives or cytostatics. The invention solves the technical problem on which it is based by providing in vitro methods for differentiating and / or multiplying isolated animal and human cells, in the course of which three-dimensional animal or human in vitro organ or tissue models are produced. For this purpose, according to the invention, in particular primary cells or other cells from tissues or organs susceptible to infections, such as the intestine, skin, cornea, trachea or mucous membranes, but also degenerate cells to be checked, in particular the organs and tissues mentioned above, or genetically modified cells to be checked , in particular the organs and tissues mentioned above, are used. Building on this and using the aforementioned methods, the present invention provides in vitro methods for cocultivating the differentiated and / or increased cells with pathogenic or parasitic microorganisms and in vitro methods for investigating the interaction of substances with the in vitro co - Cultured differentiated and / or propagated cells and microorganisms ready. These methods allow the analysis of infection processes and the provision of diagnostic and therapeutic agents useful in the field of infection medicine.

The results obtained with the organ test systems according to the invention can be more meaningful than the results determined with animal experiments and can ensure better transferability to humans. The cells described above are cultivated according to the invention in a three-dimensional, gel-like, connective tissue-like biomatrix and can multiply there. In addition to the cells to be cultivated, this biomatrix contains a framework of collagen constituted by a collagen solution, i.e. tissue-typical matrix proteins. Depending on the desired tissue type, other cell types, preferably other primary cells, can be applied to this cell-containing collagen gel. Using specific culture conditions and specific culture media, the cells contained in the biomatrix and the other cell types applied to the biomatrix can undergo a differentiation into a multi-layered three-dimensional animal tissue or organ test model.

With the aid of the method according to the invention, three-dimensional animal or human tissue or organ test models are thus obtained which consist of several tissue-typical cell layers and largely correspond histologically and functionally to the native organs or tissues. These organ or tissue test models are therefore much better suited for the natural modeling of infection courses in animals and humans than the usual in vitro systems made up of only one cell type and can therefore be used for the targeted analysis of the infection and resistance mechanisms of bacteria, Fungi, viruses and protozoa can be used. Thus, according to the invention, the in vitro organ or tissue test models according to the invention can be inoculated with a parasitic or pathogenic microorganism and, together with this, under suitable ones Conditions are cultivated. By using different primary cells from different organs to build up the animal in vitro organ or tissue test systems, the behavior of a pathogen in different tissue systems can also be studied.

The cocultivation of the animal in vitro tissue or organ test system according to the invention with a parasitic or pathogenic microorganism offers the possibility of studying both the infection process itself and the defense reaction of the corresponding organoid cell systems. For example, larger amounts of an infected cell material and the pathogen itself can be obtained. The material obtained can then be further analyzed using conventional histological, biochemical, molecular biological or immunological methods, for example to determine morphological changes in infected cells, the release of specific substances by the pathogen, such as toxins or proteins relevant to resistance, or the release of specific substances by infested cells To study cells, such as interleukins, more closely as a defense reaction or to create transcription and / or expression profiles, on the basis of which, for example, virulence factors can be identified as targets for the development of anti-infectives.

The above-mentioned procedure according to the invention preferably allows the screening and analysis of potential diagnostics, with the help of which, for example, the presence of symptoms of infection can be demonstrated. The invention therefore also relates to methods for identifying diagnostic agents or analyzing their specificity, with a potential or testable diagnostic agent for its being included in the co-cultivation of the single- or multi-layered in vitro tissue or organ test systems according to the invention with infectious agents Ability to recognize infections or infection processes is tested. In the context of these methods, the diagnostics to be tested can be added to the system according to the invention, it being possible to determine to what extent there is a correlation between the state of infection and the marking or detection by the diagnostics on the basis of morphological, physiological or other parameters.

With the aid of the animal and human in vitro tissue or organ test systems according to the invention and the cocultivation method according to the invention, in particular the effectiveness or the mechanism of action and / or the side effects of therapeutic agents, such as anti-infectives, can be analyzed much more precisely than in conventional test systems, for example on the basis of them Effects on gene expression, metabolism, proliferation, differentiation and reorganization of the cells of an in vitro organ or tissue test system. These tests of active substances, therapeutic agents and diagnostic agents as well as of interactions between infectious agent and cultured cells on the animal or human in vitro tissue test systems according to the invention can be both conventional morphological or histological methods as well as conventional biochemical, toxicological, immunological and / or molecular biological methods.

According to the invention, the methods according to the invention and the agents used in them, that is to say the single-layer and multi-layer in vitro tissue or organ systems, can be used to screen for potential active substances and to investigate properties such as specificities and interactions of active substances with target cells. According to the invention, the term “active substance” means any substance, but also other agents, including physical parameters such as electromagnetic radiation, radioactivity, heat, sound or the like, which can influence or recognize biological cells or parts thereof, in particular cell organelles , Such active substances can be, in particular, of a chemical nature, for example diagnostic or therapeutic agents. In connection with the present invention, diagnostic agents are understood to mean any substances which can specifically recognize the presence of conditions, processes or substances or their absence and in particular can provide conclusions about disease states. Diagnostics often have recognizing and marking functions. The term therapeutic agents is understood to mean in particular those substances which can be used either prophylactically or accompanying the disease in order to avoid, alleviate or eliminate disease states. In the context of the present invention, diseases also understood states such as unnatural states of mind, pregnancies, signs of aging, developmental disorders or the like. In connection with the present invention, therapeutic agents are also understood to mean those substances which have only or also cosmetic effects.

The methods according to the invention are also suitable for examining the mechanisms of tumor pathogenesis and / or for examining substances for their suitability as medicaments, for example against a specific tumor. For example, an in vitro organ or tissue test system constructed from degenerate cells, in particular the organs or tissues mentioned above, can be used to obtain larger amounts of a degenerate cell material. The material obtained can then be further analyzed using conventional methods, for example histological, biochemical, molecular biological or immunological methods, in order, for example, to examine morphological changes in degenerate cells or the release of specific substances more precisely or to create transcription and / or expression profiles. The effect of drugs or other substances with regard to their ability to inhibit cell division can also be studied on an in vitro organ or tissue test system constructed from degenerate cells. On the other hand, an in vitro organ or tissue test system constructed from non-degenerate primary cells can be co-cultivated with oncogenic viruses. With the help of this method, the propagation and / or The spread of oncogenic viruses in the cells of the in vitro test system in the presence and absence of specific substances that can inhibit specific functions of the virus are examined.

The methods according to the invention can also be used to check genetically modified cells, in particular the above-mentioned tissues and organs. For example, cells can be tested that have been genetically modified with a view to gene therapy to eliminate gene-related malfunctions or to restore normal gene functions in diseases of the above-mentioned organs.

A preferred embodiment of the invention comprises the cultivation of animal or human cells in a three-dimensional gel-like biomatrix to multiply these cells and to produce a three-dimensional animal or human in vitro organ or tissue test system.

In a particularly preferred embodiment, the invention comprises the cultivation of human dermal fibroblasts in the biomatrix to produce a three-dimensional human in vitro skin equivalent consisting of dermis equivalent and epidermis equivalent.

In connection with the present invention, the term “culturing cells” means maintaining the vital functions of cells, for example fibroblasts, in a suitable environment, preferably in vitro, for example with the addition and removal of metabolic educts and products, in particular also an increase in the cells. In connection with the present invention, dermal fibroblasts are understood to mean naturally occurring fibroblasts, particularly those found in the dermis, or genetically modified fibroblasts or their precursors. Fibroblasts are the precursors of dermal fibrocytes. The fibroblasts can be of animal or human origin.

The biomatrix provided for the cultivation of the fibroblasts contains the fibroblasts to be cultivated and a collagen scaffold newly constituted from a, preferably fresh, collagen solution at a concentration of preferably 3.5 to 4.5 mg collagen per ml biomatrix. The collagen structure is obtained from a, preferably cell-free, acidic solution of collagen I, the protein concentration of the collagen solution preferably being 5 to 7 mg / ml. The pH of the collagen solution is preferably 3.8. To produce the fibroblast-containing biomatrix according to the invention, the collagen solution is preferably at 4 ° C. with a solution containing a, preferably five times concentrated, cell culture medium, buffer, preferably Hepes buffer, serum, preferably fetal calf serum (FCS), and chondroitin (4 / 6) sulfate, and preferably 1.5 x 10 ^s / ml fibroblasts, especially precultivated fibroblasts, added and mixed well. This mixture is gelled by raising the temperature to room temperature or 37 °. After the gels have gelled, fibronectin is added to the gels. The function of fibronectin in vivo consists in binding to other macromolecules, for example collagen, and in attaching cells to neighboring cells. The subsequent cultivation of the fibroblasts in the collagen gel is preferably carried out in submerged culture. In connection with the present invention, a “submerged culture” is understood to mean a method for culturing cells, the cells being covered with a nutrient solution. The biomatrix containing fibroblasts is therefore covered with a layer of cell culture medium and incubated at 37 ° C.

One to three days, preferably two days, after the incubation of the gels described above, keratinocytes are sown on the gel. In connection with the present invention, “keratinocytes” are understood to mean cells of the epidermis that form keratinized squamous epithelium, or genetically modified keratinocytes or their precursors, which can be of animal or human origin. The keratinocytes sown on the collagen gel are preferably as possible Pre-cultivated, undifferentiated keratinocyte stem cells from human biopsy tissue, ie cytokeratin 19- or integrin ßl-positive basal stem cells.The keratinocytes are sown on the biomatrix in a cell culture medium, preferably in KBM medium (clonetics), which is 5% fetal The biomatrix is then filled with KBM medium containing human epidermal growth factor (0.1 μg / 500 ml medium) (hEGF), BPE (bovine pituitary gland extract) (15 mg protein / 500 ml medium) and 0.8 mM CaCl ₂ , layered and a 1 - to 3-day Submersed to cultivation. A complete differentiation of the keratinocyte layers is achieved by an airlift culture with KBM medium containing 1.8 mM CaCl ₂ without hEGF and BPE. In the context of the present invention, an “airlift culture” is understood to mean a culture in which the height of the nutrient medium level is matched exactly to the height of the biomatrix, while the keratinocytes or the cell layers formed by the keratinocytes lie above the nutrient medium level and not from the nutrient medium that is, the cultivation takes place at the boundary layer air-nutrient medium, whereby the cultures are supplied with nutrients from below. After a “preferably 12- to 14-day airlift culture, a skin-typical, dermis equivalent and epidermis equivalent develops existing in vitro full skin model which can be used advantageously for the test methods according to the invention.

The invention therefore also relates to a skin-typical in vitro whole skin test model, in particular animal or human in vitro whole skin test model, which was produced by the method according to the invention and which comprises at least 2 to 4 proliferative, some differentiating and at least 4 to 5 horny cell layers, wherein the epidermis equivalent stratum basale, stratum spinosum, stratum granulosum and stratum corneu, and wherein between the dermis equivalent and the epidermis equivalent there is a functional basement membrane made of matrix proteins. This model is ideal as a test system for the investigation of potential or actual active substances. such as therapeutics, diagnostics or for investigations into the course of infection processes.

Another particularly preferred embodiment of the invention comprises the cultivation of intestinal fibroblasts in the biomatrix for producing a three-dimensional human in vitro intestinal test system consisting of preferably Caco2 cells or also intestinal epithelial cells or other human cell lines.

In connection with the present invention, intestinal fibroblasts are understood to mean naturally occurring fibroblasts, in particular intestinal tissue, or genetically modified fibroblasts or their precursors. The intestinal fibroblasts can be of animal or human origin.

In connection with the present invention, intestinal epithelial cells are understood to mean naturally occurring epithelial cells, in particular in the intestinal tissue, or genetically modified epithelial cells or their precursors. The intestinal epithelial cells can be of animal or human origin.

To produce the biomatrix containing intestinal fibroblasts according to the invention, the collagen solution is mixed in a volume ratio of 1: 1, preferably at 4 ° C., with a solution also referred to as a gel solution, containing a, preferably 2-fold, cell culture medium, buffer, preferably He pes buffer and serum, preferably 10% serum, and preferably 1.5 x 10 ⁵ / ml intestinal fibroblasts, especially pre-cultivated intestinal fibroblasts, mixed and mixed well. If a gel solution with a concentration of x is present, the collagen solution is preferably mixed in a volume ratio (xl): 1 with the gel solution, where x is the concentration factor. This mixture is gelled by raising the temperature to room temperature or 37 °. The subsequent cultivation of the intestinal fibroblasts in the collagen gel is preferably carried out in submerged culture. The biomatrix containing fibroblasts is incubated at 37 ° C.

Intestinal epithelial cells are seeded onto the gel, preferably 1 to 3 days after incubation of the gels.

The intestinal epithelial cells sown on the collagen gel are preferably pre-cultivated, undifferentiated intestinal epithelial cells. The intestinal epithelial cells are sown on the biomatrix in a cell culture medium, preferably in DMEM medium (Dulbecco's Modified Eagle Medium, Life Technologies, Cat. No. 41966 or 52100), the 10% FCS and glutamine (2 mM) and 1% non-essential amino acids (MEM, Life Technologies, Cat. No. 11140). Then the biomatrix is overlaid with DMEM medium containing 10% FCS and glutamine (2 mM) and 1% nonessential amino acids and subjected to a 10 to 20 day submerged cultivation until the epithelial layer or layers has been completely differentiated which can be used advantageously for the test method according to the invention. A further advantageous embodiment of the invention comprises the cocultivation of a three-dimensional in vitro organ or tissue test system produced according to the invention with a pathogenic or parasitic microorganism. In connection with the present invention, “pathogenic or parasitic microorganisms”, here also referred to as infectious agents, are understood to mean both eukaryotic and prokaryotic microorganisms, such as bacteria, fungi, protozoa, vitoids, but also prions or viruses, which attack a macroorganism, in particular a human or animal organism, and which live in or on tissues of this organism and can lead to an infection of this organism, but need not necessarily lead to it. In the context of the invention, the term "cocultivation" means a, preferably In vitro simultaneous maintenance of the vital functions of animal cells and microorganisms in the same environment suitable for both, for example with the addition and removal of metabolic products and products, in particular also a simultaneous multiplication of the cells and the microorganisms.

In a preferred embodiment, the human pathogenic fungus Candida albicans is cocultivated with the human in vitro skin test system produced in accordance with the invention in order to investigate the infection process of Candida on human skin tissue and with the human in vitro intestinal test system produced in accordance with the invention around the Infection process of Candida on human intestinal tissue to investigate. The results achieved with Candida, in particular the detailed description of the infection process, can also be transferred to other pathogens.

In a particularly preferred embodiment, the present invention relates to the cocultivation of the human pathogenic microorganism Candida albicans with the human in vitro skin test system or the human in vitro intestinal test system in order to increase the first stage of the infection process, namely the adhesion of the pathogen to skin or intestinal cells investigate. The adhesion of the pathogen is examined using the virulent Candida strain Sc5314 and the avirulent Candida strain Can34, which have already been investigated in a mouse-macrophage model (Lo et al., Cell, 90 (1997), 939- 949). The in vitro skin test system or the in vitro intestinal test system is inoculated in submerged culture with about 10 ³ pathogenic organisms in each case and cultivated with shaking. At defined points in time, for example every 30 minutes (up to a maximum of 4 hours), aliquots are removed and plated on Petri dishes with the appropriate nutrient media, for example YPD full medium (Difco). After an appropriate incubation period, the number of colonies on the Petri dishes is determined. The adhesion of the pathogens to the in vitro organ test systems can be determined on the basis of the comparison between the determined number of colonies and the originally inoculated number of pathogens. With the help of this method it could be shown that the virulent strain has the ability to probably has skin as well as intestinal cells, whereas only slight adhesion could be detected in the avirulent stem.

In a further particularly preferred exemplary form, the present invention ^'the co-cultivation of human pathogenic microorganism Candida albicans with the inventively produced human in vitro skin test system relates respectively to the inventively produced human in vitro intestinal test system to a further stage of the infection process, namely the penetration / Invasion of the pathogen into cells. For this purpose, the organoid tissue test systems are co-cultivated in the airlift process with the avirulent and virulent pathogen strains described above. The pathogen is preferably fixed at 10 ³ / ml in 1% agar and agar pieces with a diameter of 4 mm are placed on the organoid tissue test systems for a maximum of 98 hours. The penetration of the pathogen into the organoid structures is examined after 16 hours, 24 hours, 72 hours, 86 hours and 98 hours by means of histological methods on thin sections, using the PAS staining method (Mc Manaus, Romeis, 17th edition, page 393) , Using the histological sections, the invasion process of the virulent Candida strain down to the deeper layers of the connective tissue-like matrix can be documented.

In a further advantageous embodiment of the invention it is provided, using an in vitro organ organ or manufactured according to the invention Tissue test system and the co-cultivation method according to the invention to investigate the effect of chemical substances, in particular anti-infectives, or of agents on the infection process or the growth of a pathogenic microorganism. In connection with the invention, the term “agent” is intended in particular to include chemical, biological or physical agents, such as light or heat, which can have a potential effect on living cells. The studies on the action of anti-infectives have also been carried out with Candida there are two classes of substances, namely azoles and polyenes, which are preferably used as antifungals against systemic infections. Both classes of substances have disadvantages. The polyenes have strong side effects and azoles develop more and more resistance (DiDomenico, Curr. Opin. Microbiol. , 2 (1999), 509-515; Georgopapadakou, Curr. Opin. Microbiol., 1 (1999), 547-557. For this reason, the targeted development of new antifungals is urgently required.

In a preferred embodiment, the method for examining the adhesion of Candida to the in vitro intestinal test system and the in vitro skin test system according to the invention is modified in such a way that the cocultivation approach contains an antifungal, in particular amphothericin B or fluconazole. With the help of this method it could be shown that both antifungals had no influence on the adhesion of the pathogen, but on its growth rates. In a further preferred embodiment, the method for examining the penetration / invasion of Candida on the in vitro intestinal test system and the in vitro skin test system is modified such that the cocultivation approach contains amphothericin B or fluconazole. With the help of this method it could be shown that the invasion of the virulent pathogen strain could only be prevented by a complete inhibition of growth, whereby new active substances were also tested according to the invention.

A particularly preferred embodiment of the invention comprises the analysis of degenerate cells. In connection with the invention, the term “degenerate” encompasses all changes in a normal cell, for example cell polymorphism, anisocytosis, nuclear polymorphism, polychromasia, disturbed nuclear plasma relation and aneuploidy, which lead to an impaired differentiation or to a dedifferentiation and to a deregulated growth of the Cell and malignant tumors are particularly affected, and degenerate cells, particularly the organs or tissues mentioned above, are used to build an in vitro organ or tissue test system to recover large amounts of the degenerate cell material, using conventional methods , for example histological, biochemical, molecular biological or immunological methods, are further analyzed in order to investigate the release of specific substances and to create transcription and expression profiles. The in vitro organ or tissue test system built up from degenerate cells is used for d he effect of drugs and of substances potentially suitable as drugs, in particular with regard to their ability to inhibit cell division.

In a particularly preferred embodiment of the invention, patient-specific degenerate cells are used to establish an in vitro organ test system in order to investigate therapeutic options for the specific tumor disease of the patient.

In a further preferred embodiment of the invention, the checking of genetically modified cells, in particular the above-mentioned tissues and organs, is provided. In connection with the present invention, the term “genetically modified cells” encompasses all cells which have been manipulated with the aid of genetic engineering methods, whereby either foreign DNA has been introduced into the cell or the own DNA has been modified, for example by deletions, inversions and attachments In a particularly preferred embodiment, it is provided to test genetically modified cells in vitro with a view to gene therapy of patient-specific diseases, in particular for their functionality, an in vitro organ test system being established using such genetically modified cells.

The invention also relates to a, preferably gel-like, biomatrix in which the aforementioned cultivation processes can be carried out, specifically a biomatrix with cells of a tissue type. The combination of biomatrix and cells cultured therein provided according to the invention can, as described above, be used to produce an in vitro organ or tissue test system.

A biomatrix is understood to mean a gel structure which contains collagen, cell culture medium, serum and buffer, for example Hepes buffer. The collagen solution which is used for the preparation of the biomatrix is a solution which contains a high proportion of undenatured, native collagen in an acidic, aqueous medium, preferably with a pH of 3.8, for example in acetic acid. preferably in 0.1% acetic acid solution. A high proportion of undenatured collagen means a proportion of the total collagen in solution of

> 50%, in particular> 60%,> 70%,> 80%, -> 90% or

> 95%, preferably> 99%. In a preferred embodiment, no lyophilized collagen is used. The collagen content of the solution is advantageously 3 mg collagen per ml solution to 8 mg collagen per ml solution, more preferably 5 mg collagen per ml solution to 7 mg collagen per ml solution, most preferably 6 mg collagen per ml solution. While collagen is preferably used, the strength after isolation, for example from rats tails in 0.1 ^"% acetic acid was incubated for three to fourteen days at 4 ° C under stirring and wherein non-dissolved collagen parts were removed by centrifugation. Preferred cell culture media DMEM (Dulbecco's Modified Eagle Medium) and M199, however, any other cell culture medium can be used which cultivates cell len enables. Fetal calf serum (FCS) or human antologous serum is preferably used as the serum and, for example, Hepes buffer as the buffer. In a preferred embodiment, the pH of the solution of cell culture medium, buffer and serum is 7.5 to 8.5, for example 7.6 to 8.2, in particular 7.8. Of course, the biomatrix can contain further factors, for example growth factors, adhesive agents, antibiotics, selection agents and the like.

The invention therefore also relates to methods for producing a biomatrix containing cells, wherein in a first step fresh collagen, for example from rat tails, is produced by collagen fibers isolated from collagen-containing tissue being collected in buffer solution, disinfected superficially in alcohol and then washed in buffer solution and then in an acidic solution with a pH of 0.1 to 6.9, preferably 2.0 to 5.0, particularly preferably 3.0 to 4.0, in particular 3.3, for example a 0.1% solution Acetic acid solution to be transferred. Then, in a further step, the collagen in the solution is stirred at 2 to 10 ° C, in particular 4 ° C, for a few days, for example 3 to 14 days, the undissolved collagen components are centrifuged off and a collagen solution with a collagen content of 3 mg / ml to 8 mg / ml at 2 to 10 ° C, for example 4 ° C. It is of course possible to temporarily store the solution in a frozen state, for example at -10 ° C to -80 ° C, in particular -20 ° C. For the production of the biomatrix containing cells according to the invention in a third step, a solution, also referred to as a gel solution, of preferably multi-fold (x-fold) concentrated cell culture medium, serum and buffer mixed with precultivated and centrifuged cells, preferably 1 × 10 ⁵ to 2 × 10 ⁵ cells per ml, preferably 1 , 5 x 10 ⁵ cells per ml, can be used. This solution or suspension with a pH of 7.5 to 8.5, preferably 7.6 to 8.2, in particular 7.8, is then, for example in a ratio of 1: 2, with the aforementioned collagen solution at 2 to 10 ° C, especially 4 ° C, mixed. The mixing ratio (volume) of collagen solution to gel solution (buffer, serum, cells, culture medium) is preferably 1: 1, with a volume ratio of (x-1): 1 collagen solution to gel solution being preferred for x-fold concentrated gel solution. The gel solution is then pipetted into culture vessels and, after gelling, overlaid with medium at 37 ° C. The biomatrix is then cultivated for at least 2 days and then cells of other tissue types, for example also immune system cells, can be applied to it.

Further advantageous embodiments of the invention result from the description.

The invention is illustrated by the following figures and examples.

FIG. 1 shows a longitudinal section of the in vitro intestinal system produced according to the invention. FIG. 2 shows a longitudinal section of the in vitro intestinal system produced according to the invention with adherent cells of the Candida albicans fungus (adhesion of a virulent strain).

FIG. 3 shows a longitudinal section of the in vitro intestinal system produced according to the invention with penetrated cells of the Candida albicans fungus (invasion of a virulent strain).

Example 1:

Production of a three-dimensional human in vitro skin test system

Preparation of a gel solution

20 parts of five-fold concentrated M 199 cell culture medium (Life Technologies, 1999, Cat. No. 42966 or

52100; DMEM), 10 parts HEPES buffer (4.76 g in 100 ml PBS solution, pH 7.3) and 1 part chondroitin

(4,6) sulfate (5 mg / ml in PBS) are mixed and the pH of the mixture is adjusted to 7.8. The mixture is sterile filtered and then mixed with 10 parts of fetal calf serum.

Preparation of a collagen solution

Tissue containing collagen, such as rat tail tails, is used to prepare a collagen solution. All work is carried out under sterile conditions with sterile materials. The rat tails are surface disinfected at -20 ° C with 70% alcohol. The skin of the rat tails is pulled off and the individual collagen fibers are pulled out. If other starting tissues are used, existing cells can be gently removed by mechanical, enzymatic or chemical treatment. The collagen fibers are collected in phosphate buffered saline (PBS) (pH 7.2), surface disinfected in 70% alcohol for 10 min and then washed thoroughly with PBS. The weight of the fibers is determined and the fibers are transferred to a 0.1% acetic acid solution (final concentration about 8 to 12 mg / ml). This approach is about 3 to 14 Ta ge at 4 ° C and then stirred ^• the non-dissolved collagen parts by means of centrifugation (1000 rpm, 1 hour, 8 ° C). As a result, the collagen is in solution and not in fiber, framework or matrix form.

Preparation of the dermal fibroblast-containing collagen gels (preparation for 24 inserts)

16 ml of collagen solution are placed in a 50 ml centrifuge tube and placed on ice. Pre-cultivated dermal fibroblasts are harvested and counted. 1.2 x 10 ⁶ fibroblasts are taken up in 8 ml of ice-cold gel solution, well suspended and added to the collagen solution without air bubbles. Gel solution and fibroblasts are mixed well. 600 μl of the mixture are carefully poured into the well of a microtiter plate with 24 wells (diameter per well 10 mm). By incubation at 37 ° C for two minutes - 2 ^Q -

gelation of the mixture. After the mixture has gelled, 50 μl fibronectin (5 μg / ml) are added to each insert. After a 10-minute incubation at 37 ° C. or a 30-minute incubation at room temperature, 1 ml of M199 medium is added to each well, the inserts being overlaid with the medium. The fibroblasts contained in the gel are subjected to this submerged cultivation at 37 ° C. for about 1 to 2 days, the medium being exchanged for fresh medium after every 12 hours.

Sowing keratinocytes and cultivation of the in vitro skin test system

Before the keratinocytes are sown, the medium in the wells of the microtiter plate and the gels is carefully sucked off. Then ^'per well 500 ul KBM medium (clone tics) containing 5% FCS was added. The gels are coated with 50 μl fibronectin solution and incubated for 1 hour at 37 ° C. Then 100,000 keratinocytes per gel are sown in 50-100 μl of KBM medium containing 5% FCS and incubated at 37 ° C. for 1 to 2 hours. Then 500 μl of KBM medium containing 5% FCS, 8 mM CaCl ₂ , hEGF (0.1 μg / 500 ml medium) and BPE (15 mg / 500 ml medium) are added and the gels become 1 to 3 days Submersed cultivation, the medium being exchanged daily for fresh medium. Then the gels are further 2 to 3 days in 1 to 1.5 ml KBM medium containing 2% FCS, 8 mM CaCl ₂ , hEGF (0.1 μg / 500 ml medium) and BPE (15 mg / 500 ml medium), a submerged Subjected to cultivation. The gels with the developing skin equivalent are then subjected to an airlift cultivation. For this purpose, the gels are transferred to a 6-well plate and 1.5 to 2 ml of KBM medium with a CaCl ₂ content of 1.88 mM without hEGF and BPE are added to each well, the level of the medium being exactly at the level of the gel is adjusted, while the keratinocytes or the layers formed by keratinocytes are not covered by the medium. Airlift cultivation will continue for at least 12 to 14 days.

Example 2:

Production of a three-dimensional human in vitro intestinal test system

Preparation of a gel solution

77.5 parts of 2-fold concentrated DMEM cell culture medium (Life Technologies, Cat. No. 41966 or 52100, 1999), 20 parts of FCS, 2.5 parts of HEPES buffer (71.49 g in 100 ml of PBS solution, pH Value 7.8) are mixed and the pH of the mixture is adjusted to 7.4. The mixture is sterile filtered.

Production of collagen gels containing fibroblasts (preparation for 24 inserts)

7.5 ml of collagen solution are placed in a 50 l centrifuge tube and placed on ice. Pre-cultivated fibroblasts are harvested and counted. 1.2 x 10 ⁶ fibroblasts are in 7.5 ml ice-cold gel solution taken up, well suspended and added to the collagen solution without air bubbles. Collagen solution and gel solution with fibroblasts are mixed well. 300 μl of the mixture are carefully poured into the recess of an insert. The inserts are in a 24-well microtiter plate. The mixture is gelled by incubation at 37 ° C. for two minutes. After the mixture has gelled, 1 ml of medium are placed on and next to each insert. The fibroblasts contained in the gel are subjected to this submerged cultivation at 37 ° C. for about 1 to 3 days, the medium being exchanged for fresh medium after every 48 hours.

Sowing the intestinal epithelial cells and cultivating the intestinal equivalents

Before sowing the Caco2 cells, the medium in the wells of the microtiter plate and the gels is carefully sucked off. Then 200,000 epithelial cells are sown in 200 μl DMEM medium (compare above) containing 10% FCS per gel, about 600 μl medium are added to the inserts and cultured at 37 ° C. for 10 to 20 days. The medium is changed every 48 hours.

An intestinal equivalent produced in this way is shown in FIG. 1. Example 3:

Co-cultivation of Candida albicans with the in vitro skin test system or the in vitro intestinal test system for determining the adhesion of the pathogen to cells

To determine the adhesion of the pathogen to cells, 12 inserts of the human in vitro skin system kept in submersed culture or 12 inserts of the in vitro intestinal system kept in submersed culture each with 10 ³ pathogenic organisms of the virulent Candida strain were used Sc5314 or the avirulent Candida strain Can34 (Lo et al., Cell 90 (1997), 937-949). The inserts were then incubated with shaking at 37 ° C. for 30, 60, 90, 120, 150 or 180 minutes.

At the times indicated, the supernatant was removed and plated on petri dishes with YPD culture media. After an incubation period of 2 days, the colonies on the Petri dishes were counted. Based on the comparison between the determined number of colonies and the originally inoculated number of pathogens, it was determined that about 95% of the virulent strain adhered to the in vitro skin model after 2 hours and 10% of the avirulent strain. In the in vitro intestinal model, approximately 95% of the virulent strain (FIG. 2) and 10% of the avirulent strain showed adhesion. Example 4:

Co-cultivation of Candida albicans with the in vitro skin test system or the in vitro intestine test system for determining the penetration of cells by the pathogen

To determine the penetration of the pathogen, 12 inserts of the in vitro skin system kept in airlift culture and 12 inserts of the in vitro intestinal system kept in airlift culture each with 10 ³ pathogenic organisms of the virulent Candida strain Sc5314 or the avirulent Can - Dida strain Can34 infected. The inserts were then incubated at 37 ° C. for up to 3 days.

The penetration of the pathogen into the organoid structures is examined after about 18 to 24 hours by means of histological methods on thin sections, the PAS staining method being used both in the in vitro skin system and in the in vitro intestinal system. The invasion process of the virulent Candida strain down to the deeper layers of the connective tissue-like matrix was documented using the histological sections (FIG. 3). It was shown that the virulent Candida strain spreads in a star shape from the infection site into the connective tissue, whereas the avirulent strain could not penetrate the epithelial cells and showed no adhesion. Example 5:

Influence of antifungal agents on the adhesion of Candida albicans to skin and intestinal cells in vitro

To determine the effect of antifungal agents on the adhesion of Candida albicans to skin and intestinal cells were respectively 12 insert of the in vitro skin test system and 12 inserts each of the pathogenic in vitro intestinal test system with 10 ³ organisms of the virulent ^'Candida held in submerged culture Strain Sc5314 or the avirulent Candida strain Can34. Amphotericin B was added to 5 inserts in a concentration of 0.1; 0.5; 1.0 and 2.0 μg / μl and 5 inserts were added to fluconazole in a concentration of 0.1; 0.5; Given 1.0 and 2.0 μg / μl. The inserts were then incubated with shaking at 37 ° C. for up to 3 days.

After 16, 24, 72, 86 and 98 hours, aliquots were removed and plated on petri dishes with YPD culture media. After an incubation period of 2 days, the colonies on the Petri dishes were counted. Based on the comparison between the determined number of colonies of the samples without the addition of an antifungal agent and the number of colonies of samples with the addition of an antifungal agent, it was determined that the adhesion of the virulent Candida strain could only be prevented by adding amphotericin B and fluconazole and by inhibiting growth , Example 6:

Co-cultivation of Candida albicans with the in vitro skin test system or the in vitro intestinal test system for determining the penetration of the pathogen

To determine the influence of antifungal agents on the penetration of Candida albicans in skin and intestinal cells, 12 inserts of the in vitro skin system and 12 inserts of the in vitro intestinal system, each with 10 ³ pathogenic organisms, were kept in airlift culture of the virulent Candida strain Sc5314 or the avirulent Candida strain Can34. Amphotericin B was added to 5 inserts in a concentration of 0.1; 0.5; 1.0 and 2.0 μg / μl and 5 inserts were added to fluconazole in a concentration of 0.1; 0.5; Given 1.0 and 2.0 μg / μl. The inserts were then incubated with shaking at 37 ° C. for up to 3 days.

The penetration of the pathogen into the organoid structures is examined after about 18 to 24 hours using histological methods on thin sections according to Example 4. It could be shown that the addition of amphotericin B and fluconazole apparently prevents the invasion of the virulant Candida strain by inhibiting its growth.

Claims

Expectations 1. Method for producing a single-layer human or animal in vitro tissue test system, whereby human or animal cells are embedded in a three-dimensional, gel-like biomatrix containing at least 3 mg/ml collagen in buffered serum-containing cell culture medium and cultured in this way that a single-layer in vitro tissue system is obtained. 2. The method according to claim 1, wherein the cultivation of the human or animal cells comprises a submerged culture lasting at least one to two days. 3. The method according to claim 1 or 2, wherein the human or animal cells can be non-degenerate cells, degenerate cells or a mixture thereof. 4. The method according to claim 1 or 2, wherein the human or animal cells can be genetically modified cells or a mixture of genetically modified cells and genetically modified cells. 5. A method for producing a single-layer human or animal in vitro tissue test system according to one of claims 1 to 5, comprising isolating collagen-containing tissue, transferring the collagen-containing tissue into acidic solution, incubating the collagen tissue transferred into the acidic solution at 2 to 10 ° C, in particular 4 ^° C, centrifuging off undissolved collagen components, mixing the collagen solution obtained 2 to 10 ° C, preferably 4 ° C, with a solution containing the human or animal cells to be cultivated, cell culture medium, serum and buffer, and gelling of the mixed solution by increasing the temperature. 6. The method according to any one of claims 1 to 5, wherein the human or animal cells after cultivation in the. three-dimensional, gel-like biomatrix is removed from it and further cultivated at a higher cell density, so that a single-layer in vitro tissue system is obtained. 7. Method for producing a three-dimensional multilayer human or animal in vitro organ or tissue test system, wherein cells of a first human or animal tissue type are embedded in a three-dimensional, gel-like biomatrix containing at least 3 mg/ml collagen in buffered serum-containing cell culture medium and in this are subjected to a culture of at least one to two days and then cells of a second human or animal tissue type are sown in a cell culture medium on the biomatrix and then further cultured and then, if necessary, in an optional further step, cells of other human or animal tissue types in cell culture medium sown and then further cultured to obtain a three-dimensional multilayer in vitro organ or tissue testing system. 8. The method according to claim 7, wherein the cultivation of the cells of the first human or animal tissue type comprises at least a one to two day submerged culture. 9. The method according to claim 7 or 8, wherein the cultivation of ^the cells of the second human or animal tissue type comprises at least a 2 to 6 day submerged culture and at least a 10 to 14 day airlift culture. 10. The method according to any one of claims 7 to 9, wherein the cells of the second human or animal tissue type and optionally the cells of the further human or animal tissue types have a high proportion of undifferentiated stem cells and/or immune system cells. 11. The method according to any one of claims 7 to 10, wherein the cells of the first human or animal tissue type and the cells of the second human or animal tissue type as well as the cells of the additional human or animal tissue types that may be used are non-degenerate, degenerate cells or a mixture thereof. 12. The method according to any one of claims 7 to 10, wherein the cells of the first human or animal tissue type and the cells of the second human or animal tissue type are so- such as the cells of the other human or animal tissue types that may be used, can be genetically modified cells or a mixture of genetically modified cells and genetically modified cells. 13. The method according to any one of claims 7 to 12, wherein the cells of the first human or animal tissue type are dermal fibroblasts and the cells of the second human or animal tissue type are keratinocytes, so that after cultivation a three-dimensional in vitro skin testing system is produced. 14. The method according to any one of claims 7 to 12, wherein the cells of the first human or animal tissue type are connective tissue cells of the intestine and the cells of the second human or animal tissue type are enterocytes or intestinal epithelial cells, so that Cultivation of a three-dimensional in vitro intestinal test system is produced. 15. The method according to any one of claims 7 to 12, wherein the cells of the first human or animal tissue type are corneal endothelial cells, the cells of the second human or animal tissue type are corneal keratinocytes and the cells of a further, The third human or animal tissue type is corneal epithelial cells, so that after cultivation a three-dimensional in vitro corneal test system is produced. 16. The method according to any one of claims 7 to 12, wherein the cells of the first human or animal tissue type are fibroblasts, in particular tracheal fibroblasts, and the cells of the second human or animal tissue type are tracheal epithelial cells, so that After cultivation, a three-dimensional in vitro trachea test system is produced. 17. The method according to any one of claims 7 to 12, wherein the cells of the first human or animal tissue type are fibroblasts, in particular mucous membrane fibroblasts, and the cells of the second human or animal tissue type are mucous membrane epithelial cells, so that after cultivation a three-dimensional ^' in vitro mucosa test system is produced. 18. A method for producing a three-dimensional multilayer in vitro organ or tissue test system according to one of claims 7 to 17, comprising isolating collagen-containing tissue, transferring the collagen-containing tissue into acidic solution, incubating the transferred into the acidic solution Collagen tissue at 2 to 10 ° C, in particular 4 ° C, centrifuging off undissolved collagen components, mixing the collagen solution obtained at 2 to 10 ° C, preferably 4 ° C, with a solution containing the cells to be cultivated from a first human or animal Tissue type, cell culture medium, serum and buffer, gelling the mixed solution by increasing the temperature, incubating the gelled mixture at 37 ° C and seeding the cells of the second human or animal tissue system and, if necessary, seeding the cells of other human or animal tissue types onto the incubated, gelled mixture. 19. The method according to any one of claims 1 to 18, wherein the embedded biomatrix containing human or animal cells is produced by collagen fibers isolated from a tissue in acidic solution for 3 to 14 days at 2 to 10 ° C, preferably 4 ° C , stirred, undissolved collagen components centrifuged off and the finished collagen solution obtained with a collagen content of 3 mg/ml to 8 mg/ml with a solution containing the human or animal cells to be embedded, cell culture medium, serum and buffer, at 2 to 10 ° C, preferably 4 ° C, mixed and then gelled at a higher temperature, preferably at room temperature to 37 ° C. 20. The method according to claim 19, wherein the acidic solution is acetic acid solution, in particular 0.1% acetic acid solution. 21. The method according to claim 19 or 20, wherein the solution containing the cells, cell culture medium, serum and buffer is mixed with the collagen-containing solution in a volume ratio of 1:1. 22. Single-layer in-vitro tissue test system produced by a method according to one of claims 1 to 6 or 19 to 21. 23. Three-dimensional multilayer in vitro skin test system, produced by a method according to one of claims 7 to 13 or 18 to 21. 24. Three-dimensional multilayer in vitro intestinal test system, produced by a method according to one of claims 7 to 12, 14 or 18 to 21. 25. Three-dimensional multilayer in vitro corneal test system, produced by a method according to one of claims 7 to 12, 15 or 18 to 21. 26. Three-dimensional multilayer in vitro trachea test system, produced by a method according to one of claims 7 to 12, 16 or 18 to 21. 27. Three-dimensional multilayer in vitro mucous membrane test system, produced by a method according to one of claims 7 to 12 or 17 to 21. 28. A method for examining the infection process of a pathogenic or parasitic microorganism on a human or animal tissue, comprising bringing into contact with a single-layer in vitro tissue test system according to claim 22 or a three-dimensional multi-layer in vitro organ or tissue test system according to one of claims 23 to 27 a pathogenic or parasitic microorganism and the co-cultivation of the microorganism with the single or multilayer in vitro organ or tissue test system under conditions ments that allow subsequent or simultaneous examination of the infectious process. 29. The method according to claim 28, wherein the co-cultivation takes place in a submerged culture and the adhesion of the microorganism to cells of the single- or multi-layer in vitro organ or tissue test system is determined. 30. The method according to claim 28, wherein the co-cultivation takes place in an airlift culture and the penetration of the microorganism into cells of the single- or multi-layer in vitro organ or tissue test system is determined. 31. A method for determining the effect of a chemical substance or an agent on the infection process of a pathogenic or parasitic microorganism on a human or animal tissue, comprising bringing into contact a single-layer in vitro tissue test system according to claim 22 or a three-dimensional in vitro organ or tissue test system according to one of claims 23 to 27 with a pathogenic or parasitic microorganism, the co-cultivation of the microorganism with the single or multi-layer in vitro organ or tissue test system in the absence and presence of the substance or agent to be examined and the determination of the interaction between the single or multilayer in vitro organ or tissue test system, the microorganism and the substance or agent to be tested. 32. The method according to claim 31, wherein the influence of the substance to be tested or the agent to be tested on the adhesion of the microorganism is determined. 33. The method according to claim 31, wherein the influence of the substance to be tested or the agent to be tested on the penetration of the microorganism is determined. 34. The method according to claim 31, wherein ^■ the influence of the substance to be tested or the agent to be tested on the growth rate of the microorganism is determined. 35. Method for determining the effect of a chemical substance or agent on non-degenerate or degenerate human or animal cells, comprising bringing the chemical substance or agent into contact with a monolayer composed of degenerate and/or non-degenerate human or animal cells Vitro tissue test system according to claim 22 or a three-dimensional in vitro organ or tissue test system constructed from degenerate and / or non-degenerate human or animal cells according to one of claims 23 to 27 and determining the interaction between the single or multilayer in vitro organ - or tissue testing system and the chemical substance or agent.