EP1747459A1

EP1747459A1 - Multi-cellular test systems

Info

Publication number: EP1747459A1
Application number: EP05707686A
Authority: EP
Inventors: Charli Kruse; Günter FUHR
Original assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Current assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Priority date: 2004-05-21
Filing date: 2005-03-02
Publication date: 2007-01-31
Also published as: WO2005114178A1; IL179319A0; US20080064034A1

Abstract

The invention relates to methods for testing substances using multi-cellular, in vitro test systems, in particular systems that resemble organs and to devices and kits for carrying out said methods.

Description

Multicellular test systems

The invention relates to methods for testing substances using multicellular, in particular organ-like, in vitro test systems and devices and kits for carrying out these methods.

Carrying out animal experiments for pharmaceutical and cosmetic tests is an enormous cost factor and is often ethically problematic. For the cosmetics industry they are even in many countries, e.g. completely banned in Europe. The development of multicellular, in particular human, in vitro test systems is a good alternative for many examinations and such human test systems can even come closer to natural human tissues / organs in their properties than animal models.

In the prior art, tissue cultures from explanted tissue samples (see, for example, US 5726009 and US 2002/192638) and cultures of differentiated cells obtained from stem cells have been used in vitrσ test systems. The former approach has the disadvantage that it is difficult to maintain the cultures over longer periods of time and to produce larger amounts of cells without changing the properties of the cells or of the tissue. In the second approach, the stem cells are conventionally induced to differentiate into individual cell types, for example nerve cells, fibroblasts etc., by adding special factors and then the effect of various chemicals, for example active pharmaceutical ingredients, on these special cells is investigated. To study a wide range of differentiated cells Various special cell cultures have to be prepared and tested. When using conventional adult stem cells with a limited differentiation spectrum, this usually requires the cultivation of the differentiated cell cultures from different starting stem cells with often very different demands on the growth conditions. This is usually associated with additional time and costs. This disadvantage can be partially avoided by using pluripotent embryonic stem cells, which can differentiate into different cell types of all three cotyledons. However, the use of human embryonic stem cells is also questionable for ethical reasons and their availability is limited. Furthermore, even when using pluripotent e-bryonic stem cells in these conventional test systems, several different and spatially separated cell cultures are required. Another essential disadvantage of these cell monocultures is that the effect of a substance on a combination of different cells, such as that which is present in a natural tissue or organ, cannot be investigated.

Accordingly, the object of the invention is to provide improved multicellular, in particular human, in vitro test systems and methods with which the action of substances on different cell types and in particular on a combination of different cell types, as is present in natural tissues and organs, can be determined quickly and easily.

The present invention is based on the finding that multipotent or pluripotent adult stem cells, as can be obtained from exocrine glandular tissue (PCT 2004/003810), with simple means for aggregation and differentiation in three-dimensional cell aggregates, so-called organoid bodies or organoid bodies, which contain a spectrum of at least two cell types without the addition of special differentiation factors. With an adequate supply of nutrients, these organic bodies continue to grow and develop tissue or organ-like structures. At this stage they are also referred to as tissue bodies. If these organoid bodies are exposed to chemical substances, their effect, if present, can be determined by a morphological or otherwise detectable change in these organoid bodies or the cell types contained therein. In this way, different cell types can be tested simultaneously or one after the other, and in particular tissue or organ-like cell groups can also be examined.

The above-mentioned problems are thus solved according to the invention by providing a method for testing substances according to claim 1 and devices and kits for carrying out this method according to claims 23, 24 and 28. Advantageous embodiments of the invention are the subject of the dependent claims.

Multipotent or pluripotent adult stem cells are used to form the organoid bodies or organoid bodies used according to the invention. These pluripotent stem cells are preferably isolated from exocrine gland tissue.

The exocrine glandular tissue can come from an adult individual or juvenile individual. The term "adult" as used in the present application thus refers to the developmental stage of the starting tissue and not to that of the donor from which the tissue comes. "Adult" stem cells are non-embryonic stem cells.

Preferably, the exocrine gland tissue is isolated from a salivary gland, lacrimal gland, sebum gland, sweat gland, from genital tract glands including prostate, or from gastrointestinal tissue including pancreas or secretory tissue from the liver. In a highly preferred embodiment, it is acinar tissue. The acinar tissue very particularly preferably originates from the pancreas, the parotid gland or the submaxillary gland.

The adult stem cells obtained from such sources are easy to isolate and to be kept in a largely undifferentiated state in a stable long-term culture without feeder cell layer or special additives. The term feeder cells, as used here, encompasses all cells which promote the growth of the cells which are actually to be cultivated by releasing growth factors and / or providing an extracellular matrix or preventing the differentiation of the stem cell culture.

These adult stem cells can be easily stimulated to differentiate without the addition of special growth or differentiation factors - by culturing them under spatial conditions which ensure three-dimensional contact of the cells. In a preferred embodiment, these conditions are cultivation in hanging drops, as has already been described for embryonic stem cells (Wobus et al., Biomed. Biochim. Acta 47: 965-973 (1988). This method is described in more detail below in the examples However, it goes without saying that alternative cultivation methods which are suitable for the desired three-dimensional contact of the cells and are known and available to the person skilled in the art, can also be used. Examples of such alternative methods are cultivation in agitated suspension culture, cultivation in an electromagnetic field cage or laser tweezer, cultivation on surfaces to which the cells do not adhere or adhere poorly, or sowing non-resuspended cells of the primary culture. Such surfaces can be, for example, glass, polystyrene or surfaces treated with an anti-adhesion layer, for example PTFE or poly-HEMA-coated surfaces.

Under these conditions, three-dimensional cell assemblies or cell aggregates develop spontaneously, which, based on the "embryoid bodies" already described for embryonic stem cells, have been referred to as "organoid bodies" or organoid bodies. These organoid bodies or organoid bodies can be transferred to suspension cultures or adhesion cultures and further cultivated. With an adequate supply of nutrients, these organoid bodies continue to grow and can reach diameters of a few millimeters or more. These large organoid bodies have a tissue-like structure and are also called "tissue bodies" at this stage to distinguish them from simple cell aggregates.

If the organoid bodies are brought back into surface culture, a single cell layer emerges from growing individual cells, from which multilayer areas emerge, from which secondary organoid bodies with properties comparable to those of the primary organoid bodies are spontaneously formed. The organoid bodies according to the invention can, for example, be stored frozen at the temperature of the liquid nitrogen without their viability, ability to reproduce. loss of ability to grow and differentiate.

The organoid bodies contain different cell types of all three cotyledons. No differentiation factors are necessary for differentiation and the cells do not have to be transplanted to differentiate. However, it can be advantageous to use such differentiation factors in order to produce larger quantities of a certain cell type in a targeted manner or to produce organoid bodies with a certain cell type composition.

Differentiation factors are known in the art and include e.g. bFGF ("basic fibroblast growth factor") for the increased formation of heart cells and fibroblasts, VEGF

("vascular endothelial growth factor"), DMSO and isoproterenol, fibroblast growth actuator 4 (FGF4), hepatocyte

Growth factor (HGF) for the increased formation of heart and

Liver cells, TGF-beta ("transforming growth factor betal") for the increased formation of heart cells, EGF ("epidermal growth factor") for the increased formation of skin and heart cells, KGF ("keratinocyte growth factor") (sometimes together with cortisone) for the formation of keratinocytes, retinoic acid for the increased formation of nerve, heart and kidney cells, beta-NGF ("beta nerve growth factor") for the increased formation of brain, liver, pancreas and kidney cells, BMP-4 ( "bone morphogenic protein 4") and Activin-A for the formation of mesodermal cells in general, but are not limited to this.

Differentiated cells that can be contained in the organoid bodies include bone cells (osteoblasts and osteoclasts), chondrocytes, adipocytes, fibroblasts (eg skin and tendon fibroblasts), muscle cells, endothelial cells, However, epithelial cells, hematopoietic cells, sensory cells, endocrine and exocrine gland cells, glial cells, neuronal cells, oligodendrocytes, blood cells, intestinal cells, heart, lung, liver, kidney or pancreatic cells are not limited to this.

In the test method according to the invention, the substance to be tested is brought into contact with the organoid bodies and their effect is determined, if appropriate, by a morphological or otherwise detectable change in these organoid bodies or the cell types contained therein. This test method is, for example, a method for analyzing the effect of known or potential active substances or toxins or mutagens on all or special cell types of the organoid body. In a more specific embodiment, it is a method for screening active pharmaceutical ingredients or cosmetics.

The test substance can be of the most varied chemical nature, e.g. a protein, lipid, a nucleic acid, e.g. RNA, DNA or a derivative thereof, a low molecular or high molecular chemical compound, a chemical element or a mixture of these substances. Two or more test substances can also be tested in succession or simultaneously with the same organoid bodies, for example to determine interactions between the test substances.

The contacting of the test substance (s) with the organoid bodies can take place in any suitable manner, depending on the type of the test substance. Suitable methods are known to the person skilled in the art. If the test substance is a nucleic acid, for example RNA, DNA or a derivative thereof, it can be (s) by any known method of genetic engineering. technology, including the use of vectors, viruses, electroporation etc., into which cells of the organoid body are introduced. A test substance that is soluble or solubilizable in the culture medium is preferably simply added to the cell culture medium in which the organoid bodies are located, and the organoids are incubated with the test substance in suitable concentrations for various desired periods of time. If desired, used cell culture medium can be replaced by fresh medium with the desired concentration of test substance during the incubation, for example in order to keep the active substance concentration in the medium approximately constant. Depending on the type of active substance, the effective concentrations of the test substance can vary to a large extent, but can easily be determined by the person skilled in the art through routine tests. The contact period can vary from a few minutes to a few hours and several days and weeks. Contact periods of several weeks are not uncommon. Suitable contact periods will also depend on the type of active ingredient and can be determined by the person skilled in the art through routine tests. The treated organoids and a control without test substance, which was incubated for the same length of time, are then subjected to a detection method.

The detection method will depend on the type of active substance and the type of change to be observed in the organoid body or the differentiated cells contained therein.

A number of methods for demonstrating the action of drugs or toxins on mammalian cells are described by A. Vickers in "In Vitro Methods in Pharmaceutical Research", Academic Press, 1997. Basic methods for drug screening are described by Smith , CG., "The process of New Drug Development, CRC Press, 1992, and in "Advances in Drug Discovery Techniques," ed. Alan L. Harvey, John Wiley & Sons, 1998.

In specific embodiments of the present invention, the detection of the action of a substance comprises the use of one or more methods, which or those from the group consisting of protein assays, unoassays, enzymatic assays, receptor binding assays, ELISA assays, RIA assays , electrophoretic and chromatographic assays, including HPLC, Northern blots, Southern blots, Western blots, colorimetric assays, immunohistochemical, electrophysiological methods (ie current, voltage and impedance measurements), microscopic and spectroscopic detection methods are selected.

The effect of the substance can e.g. a change in the morphology or proliferation capacity, growth capacity or viability of all cell types or special cell types of the organoid body. In this case e.g. direct visual and optical detection methods, including cytometric, microscopic and colorimetric methods, can be used favorably.

Alternatively or simultaneously, the effect can be a change in the activity of specific or all cell types of the organoid body. For example, this change in activity can manifest itself in an increased or reduced or first-time occurrence of a detectable marker substance in or on the affected cells. In this case, the proof of the effect of the chemical substance is preferably carried out by proof of the presence or absence or quantity a marker substance that is formed by special or all cell types of the organoid body.

This marker substance can e.g. A protein, including, but not limited to, antibody, receptor, enzyme, hormone, ion channel, neurotransmitter, surface marker, RNA, DNA, a proteoglycan, a lectin or other suitable substance. Some cell type-specific examples of marker substances are mentioned in the following, but are in no way to be regarded as restrictive: PGP 9.5 and NF for nerve cells, S 100 and GFAP for glial cells, SMA for muscle cells (or myofibroblasts), collagen type II for cartilage cells, amylase and Trypsin for exocrine gland cells, insulin for endocrine gland cells, vigilin for highly translational cells and cytokeratin for epidermal cells, collagen type II for chondrocytes, osteonectin for osteoblasts and progenitor cells, osteocalcin for mature osteoblasts, CD45, CD34, CD13 cells for hematoplastos cardiac troponin I "), cTNT (" cardiac troponin T ") and ANF (" atrial natriuretic factor ") for cardiomyocytes, collagenase 1 and TIMP-1 (" tissue inhibitor of metalloproteinase I ") for fibroblasts, skeletal alpha Actin and tropomyosin for striped muscle cells, lipoprotein lipase (LPL) for adipocytes, alpha-fetoprotein for liver cells. A very large number of such marker substances are known in the prior art.

These marker substances can be detected, for example, by binding to a specific binding partner that is conjugated to a detectable group. The detectable group can be, for example, a dye, fluorescent dye, a radioactive label, enzyme label, luminescent label, magnetic resonance label or another label known in the art. If the marker substance is a protein in, the binding partner will preferably be a labeled or labelable antibody. Such labeled antibodies are already known for a large number of marker substances and can either be obtained commercially or can be easily produced by known processes. Optionally, labeled or labelable secondary antibodies can also be used. The specific binding partner can also be re-labeled, but bound to an affinity column or other carrier. In these cases, cells that have the marker substances on their surface can be detected by the specific binding to these carriers.

In some cases, the marker substance can also be detected by its own activity, e.g. if it is an ion channel or neurotransmitter or an enzyme that can be reacted with a detectable substrate. If the marker substance is a DNA or RNA, it can either be detected directly by complementary and possibly labeled probes or indirectly by the detection of a gene product if it is a complete coding sequence or a regulatory sequence. Another possibility is an increased DNA or RNA synthesis per se or an increased DNA repair activity. Such activities can e.g. can be determined by incorporating radioactive or otherwise labeled nucleotides.

The detection can be carried out while maintaining the cell structure, for example in microscopic and unhistochemical methods, or with destruction of the cell structure, for example in an electrophoresis method such as Southern blots, Northern blots or Western blots. In an advanced stage of differentiation, the organoid bodies have tissue-like or organ-like structures which are formed by two or more different cell types (cf. FIGS. 2-4). Such structures are, for example, neuromuscular structures or glia-nerve cell structures or skin cell structures. The formation of desired structures can be promoted by cultivating the organoid bodies in a medium with special differentiation factors.

In one embodiment of the method according to the invention, the test substance effects a change in these structures. This change can be, for example, a destruction of the structures, inhibition or stimulation of the development of the structures or a change in the activity of one or more cell types, from which these structures are composed. Accordingly, the detection of the effect of the test substance can consist of the change in these structures. Suitable detection methods can e.g. include direct observation of the morphological changes, possibly coupled with immunological, immunohistochemical or other detection methods.

Since, as already mentioned above, the cell type composition of the organoid bodies can be determined by culturing them in the presence of specific differentiation factors, this means that the cell type composition of the organoid bodies can be matched in more specific embodiments to the putative cell type-specific effect of the test substance. Specifically, this means that, for example, organoid bodies with a high proportion of nerve cells or organoid bodies, which predominantly or exclusively have neuromuscular structures or nerve-glial cell structures, in a test system are used in which test substances with a presumed effect on the nervous system are examined. Analogous to this, organoid bodies with a high proportion of epithelial cells or with skin-like structures can be used in a test of test substances which presumably act via the surface. Other such special test systems will be readily apparent to those skilled in the art and can be implemented.

In further more specific embodiments, the organoid bodies are located in cavities, matrices or other carrier and / or shaping systems. The organoids can be introduced, for example, by waxing. The cavities can be, for example, microchannels or capillaries for impedance measurement. The proof of the effect of the test substance can then e.g. consist in an impedance change. Alternatively, a gradient (e.g. pH, electrochemical, signal factors, etc.) can be generated over an organoid body and the effect of the test substance can be displayed by changing this gradient.

Another aspect of the invention relates to a device for carrying out the method according to the invention, comprising the organoid bodies in a suitable container, carrier or shaping system, means for contacting the organoid bodies with a chemical substance to be tested and means for detecting a morphological or other change thereof organoid body or the cell types it contains. In a more specific embodiment of this aspect, the organoid bodies are located in cavities, for example microchannels or capillaries, or in matrices. In one embodiment of the invention, the means for carrying out the method according to the invention are provided in the form of a kit. Such a kit comprises adult stem cells or the organoid bodies derived therefrom or differentiated cells in a suitable culture medium for maintaining the cells or the organoid bodies, optionally cryopreserved. Furthermore, the kit can contain further aids, for example reagents for cultivating and differentiating the stem cells into organoid bodies of a desired cell type composition, means for contacting the organoid bodies with a chemical substance to be tested, means for detecting a morphological or other change in these organoid bodies or the cell types contained therein.

DESCRIPTION OF THE FIGURES

Figure 1: shows schematically the cultivation of the stem cells in surface culture and in hanging drops as well as the formation and further cultivation of organoid bodies.

2 shows the expression of markers of neuronal cells, glial cells and smooth muscle cells as well as of amylase and insulin in the differentiated cells of the organoid bodies. a, b: PGP 9.5-labeled nerve cells show multipolar extensions, which show numerous varicosities. c, d: the neurofilament system (bright arrows, marked green in the original photo) extends through the pericaryon into the cytoplasmic foothills. GFAP-immunoreactive glial cells are located in close proximity (dark arrows, marked red in the original photo). e, f: -SMA-labeled cells (dark arrows, in the original red) and NF-marked nerve cells (bright arrows, in the original green) form a primitive neuro-muscular network (e), whereby contacts are established over considerably long distances (f). g: Immunostaining of GFAP (dark arrows, originally red) and NF (bright arrows, originally green) in 3-week-old OB with concentrations of nerve and glial cells. h: Immunostaining of α-SMA (dark arrows, originally red) and NF (bright arrows, originally green) in 3-week-old OBs in an advanced stage of the formation of a neuromuscular network. i, j: Cross sections of 8-week-old OBs were found for NF immunoreactive cells in the immediate vicinity of cells that were immunoreactive for α-SMA, similar to native tissues, k: a subset of cells showed a positive staining for Amylase (bright arrows, originally green). 1: Another cell subgroup contains granular vesicles with an inactivity for insulin. The cores are contrast-dyed with DAPI (originally blue).

3 shows the expression of extracellular matrix components and cytokeratins. a, b: Globular (a) and fibrillary (b) stores of proteoglycans resulted in staining with Alcian blue. ce: The globular (c) and fibrillary (d) areas are immunoreactive for the cartilage matrix protein collagen II. Two individual cells (e) show cytoplasmic labeling of collagen II. f: Cells that are immunoreactive for cytokeratins are arranged in clusters, g: confocal laser canning microscopy of an OB. The collagen II immunoreactivity (dark arrows, originally red marked) increases towards the middle of the OB. Vigilin im unreactive cells (bright arrows, marked green in the original) are mainly located on the outer edge of the OB, which indicates their high translational activity. The cores are contrast colored with DAPI.

Fig. 4 shows the transmission electron microscopy of differentiated OB. a-c: smooth muscle cells with myofilaments. The myofilament system extends across the cytoplasm in disseminated bundles (a) and shows typical dense bodies (arrows) (b). The myoblasts show star-shaped cell extensions that form a connecting network (c). d: Cell extension with a cluster of numerous small-sized vesicles, which most likely correspond to nerve fiber varicosities. e: collagen and reticular fibers. f-h: Secretory cells show electro-tight vesicles. f). Secretory cells often contact each other to form acinar structures (g). A subset of secretory cells contains vesicles (arrow) that correspond to ultrastructural features of endocrine granules (h), e.g. Beta granules from insulin-producing cells. 1: Beginning of the formation of an epithelial surface (arrow) in eight-week-old OBs. j: typical cell contacts between keratinocytes and desmosomes (arrows)

FIG. 5A: shows microscopic comparative images of an untreated and an organoid body treated with puromycin.

FIG. 5B: shows Western blots of protein separations of the homogenates of the organoids shown in FIG. 5A with the detection of the translation marker vigiline. FIG. 6: shows Western blots of protein separations of the homogenates of retinoic acid-treated and untreated organoid bodies with the detection of the protein marker α-SMA (FIG. 6A) or the detection of neurofilaments (NF) (FIG. 6B).

FIG. 7: shows western blots of protein separations of the homogenates of HGFR-treated and untreated organoid bodies with the detection of the liver protein α-fetoprotein. FIG. 8: shows Western blots of protein separations of the homogenates of organoid bodies treated and treated with conditioned medium from chondrocyte primary cultures with the detection of the cartilage protein collagen-II.

FIG. 9 shows a basic structure and test sequence for testing substances using the methods and systems according to the invention.

According to the scheme shown in Figure 1, exocrine gland tissue, e.g. Acinary tissue - preferably a salivary gland or the pancreas (pancreas) - mechanically and enzymatically comminuted in culture (step 10 in Figure 1). Contrary to the information given by Bachern et al., Gastroenterol. 115: 421-432 (1998) and Grosfils et al., Res. Co m. Chem. Pathol. Pharmacol. 79: 99-115 (1993), tissue blocks are not cultivated from which cells should grow, but the tissue is shredded more under the condition that the cell groups of the acini remain largely intact.

These cells and cell assemblies are cultivated in culture vessels for several weeks. The medium is used every 2 to 3 days changed, removing all differentiated cells. The cells that persist in culture are undifferentiated cells with unlimited ability to divide.

Similar cells have been isolated and described from the pancreas under the same conditions and have been referred to as a type of myofibroblast or pancreatic star cells (Bachern et al., 1998). In contrast to the cells of the present invention, however, an unlimited ability to divide could not be observed. Furthermore, these cells could not be passed through indefinitely without losing vitality.

In a second step (12), approximately 400 to 800 cells are cultivated in 20 μl medium in hanging drops. For this purpose, the drops are placed on the lid of bacteriological petri dishes, turned over and placed over the petri dish filled with medium, so that the drops hang down.

As a result of this type of cultivation, the cell aggregates (14) known as organoid bodies are formed within 48 hours, which are converted into a suspension culture for about 6 days (16). The partial view (18) from FIG. 1 shows a microscopic image of such an organoid body.

The organoid bodies growing in suspension culture form new organoid bodies, which also induce the formation of new organoid bodies in single cells. The cells can be frozen both as organoid bodies and as single cells while maintaining their vitality and differentiation potential. FIGS. 2-4 show microscopic and electron microscopic images of differentiated cells which were obtained from such organoid bodies or organoid bodies.

For example, the formation of a neuromuscular network can be observed:

Cells obtained from OBs strongly expressed α-SMA ("sooth muscle actin") (Fig. 2e-f). The presence of widespread bundles of myofilaments extending across the cytoplasm was confirmed by electron microscopy (Fig. 4a-c). Furthermore, cells were identified which were immunoreactive for the pan neuron marker PGP 9.5 and for neurofilaments (NF). The neurofilament system extended from the pericaryon into the radial cytoplasmic extensions (Fig. 2c, d). PGP 9.5 immunoreactive cells showed numerous varicosities along their branched extensions (Fig. 2a, b, 4d) and thus corresponded to typical morphological features of autonomous nerve fibers. Cells that were immunoreactive for GFAP ("glial fibrillary acidic protein") were in close proximity to cells that expressed neuronal markers (FIGS. 2c, d). Often the filamentous proteins did not extend through the entire cytoplasm, but were limited to areas next to the nerve cells. Furthermore, smooth muscle cells and nerve cells were not scattered arbitrarily, but instead formed connected networks with easily distinguishable connections (Fig. 2e, f). Nerve fiber stretches spanned considerable distances to contact neighboring smooth muscle cells as their putative targets. Because of their topographical arrangement, the two cell types thus showed features of a primitive neuromuscular network. In 3-week-old OBs, the beginning formation of tissue-like structures was found (Fig. 2g-j). Here a cluster of fibrous nerve cells found in contact with glial cells (Fig. 2g) or developed into a three-dimensional neuromuscular network (Fig. 2h), which was confirmed in cross-sections of 8-week-old OBs (Fig. 2i, j).

Detection of the Expression of Exocrine and Endocrine Pancreatic Proteins:

Immunohistochemical staining showed that cellular subgroups were positive for amylase (Fig. 2k). The immune response signal was limited to clearly distinguishable vesicles within the apical cytoplasm. In addition, most of the cell clusters that were immunoreactive for amylase were arranged in circles, with the secretory vesicles having a position towards the center, which is a morphological arrangement similar to that of exocrine pancreatic acini. Other cellular subsets showed immunoreactivity for insulin (Fig. 21). Similar to the amylase-positive cell clusters, the secretory product was stored in vesicular structures that were concentrated at one cell pole. The presence of secretory cells was confirmed by electron microscopy, which showed densely distributed electrodense particles, which are characteristic of excretory or incretoric functions (Fig. 4f-h).

A differentiation into chondrogenic cells and epithelial cells was also observed:

After a growth period of two months, OBs showed chondrogenic properties. Alcian blue staining revealed areas with high concentrations of proteoglycans (chondroitin sulfate), which were either globular (Fig. 3a) or fibrillar (Fig. 3b) deposits. Immunohistochemical staining with antibodies that were directed against the cartilage matrix protein collagen II also demonstrated The chondrogenic activity was within these globular (Fig. 3c) and fibrillar (Fig. 3d) areas. The immunoreactivity was highest in the middle of the cellular aggregates, which were most likely to correspond to areas of developing extracellular cartilage matrix. This observation was confirmed by confocal microscopy (Fig. 3g): while the amount of collagen storage increased towards the center of the cellular aggregates, the marginal areas were characterized by actively translating cells, as demonstrated by their high vigilin expression, which is common in cells with active translation machinery, for example collagen-synthesizing chondrocytes or in fibroblasts during chondroinduction. Typical single collagen II-translating chondrocytes were also observed in growing cells of OBs that produced a collagen II-containing matrix that surrounded the single cells (Fig. 3e). An ultrastructural examination of these areas clearly showed a network of reticular fibers and collagen fibers, the latter being identified by their characteristic band pattern (FIG. 4e). In addition to esenchymal markers, some cells also expressed several cytokeratins, indicating their potential for differentiation in epithelial cells. However, cells that were immunoreactive for cytokeratins were found less frequently than cells that expressed smooth muscle cell and neuron markers. Typically, they were arranged in clusters that were disseminated within the OBs (Fig. 3f). Typical cell contacts between keratinocytes were found by electron microscopic examinations (FIG. 4j) and epithelial cells were found on the surface of 8-week-old OBs, which grew from the cell culture medium into the air. In total, the following markers, for example, have been tested positive for specific cells: PGP 9.5 and NF for nerve cells, S 100 and GFAP for glial cells, SMA for muscle cells (or myofibroblasts), collagen type II for cartilage cells, amylase and trypsin for exocrine gland cells, insulin for endocrine gland cells, vigilin for strongly translating cells and cytokeratin for epidermal cells. In addition to light microscopic examinations, different types of cells could be morphologically characterized by electron microscopy, and cell-cell contacts could be found as a sign of cellular interactions.

So far, smooth muscle cells, neurons, glial cells, epithelial cells, fat cells, heart cells, kidney cells, fibroblasts (e.g. skin and tendon fibroblasts), chondrocytes, endocrine and exocrine gland cells and thus cell types of all three cotyledons in these organoid bodies morphologically / histologically and / or immunochemically detected ,

The present invention is intended to be explained in more detail in the following, non-limiting examples.

The general working instructions as they are used for methods for the cultivation of mammalian cells, in particular human cells, must be observed. A sterile environment in which the method is to be carried out must be observed in any case, even if no further description is given. The following buffers and media were used: HEPES stock solution (pH 7.6) 2.3 83 g HEPES per 100 ml A. bidist. HEPES Eagle Medium (pH 7.4) 90 ml Modified Eagle Medium (MEM) 10 ml HEPES stock solution

Isolation medium (pH 7.4) 32 ml HEPES-Eagle medium 8 ml 5% BSA in A. bidest. 300 μl 0.1 M CaC12 100 μl trasylol (200,000 KIE)

Digestion medium (pH 7.4) 20 ml isolation medium 4 ml collagenase (Collagenase NB 8 from Serva) Incubation medium Dulbecco's Modified Eagle Medium (DMEM)

Dulbecco's Modified Eagle Medium (DMEM) DMEM + 4500 mg / 1 glucose + L-glutamine - pyruvate + 20% FCS (inactivated) + 1 ml / 100 ml pen / strep (10000 U / l 0000 μg / ml) or DMEM + 10% own plasma + 1 ml / 100 ml pen / strep, warm to 37 ° C before use

Differentiation medium 380 ml DMEM 95 ml 30 min at 54 ° C inactivated FCS 5 ml glutamine (GIBCO BRL) 5 ml (3.5 μl ß-mercaptoethanol on 5 ml PBS) 5 ml non-essential amino acids (GIBCO BRL) 5 ml penicillin / Streptomycin (GIBCO BRL) (10000 U / 10000 μg / ml)

Instead of fetal calf serum (FCS) in the nutrient medium and differentiation medium, it is also possible, if appropriate, to use plasma or serum from another suitable species, in particular human plasma or, less preferably, human serum. Instead of the DMEM medium used, the nutrient medium can also contain another suitable base medium known for the cultivation of eukaryotic cells, in particular mammalian cells, in which the differentiated cells die and the desired stem cells multiply. Isolation medium, incubation medium and differentiation medium can also contain another customary and suitable base medium.

The following examples 1 to 3 describe working protocols for the isolation and cultivation of adult pluripotent stem cells from acinar tissue of the pancreas or from acinar and tubular tissue of the salivary gland. EXAMPLE 1

For the isolation and cultivation of human adult stem cells, human tissue was obtained from adult patients immediately after a surgical operation and processed immediately. Healthy tissue was separated from the surgically removed tissue, for example pancreatic tissue, and in digestion medium containing HEPES-Eagle medium (pH 7.4), 0.1 mM HEPES buffer (pH, 7.6), 70% (vol. / Vol.) Modified Eagle's medium, 0.5% (vol. / Vol.) Trasylol (Bayer AG, Leverkusen, Germany), 1% (wt. / Vol.) Bovine serum albumin), 2.4 mM CaCl ₂ and collagenase (0.63 P / mg, Serva, Heidelberg, Germany), added (at 20 ° C, lower metabolism). The pancreatic tissue was shredded very finely with scissors, fatty tissue floating at the top was sucked off and the tissue suspension was gassed with carbogen (Messer, Krefeld, Germany) without the nozzle getting into the medium with the cells (reduction of mechanical stress) and thus to pH 7 , 4 set. The suspension was then placed in a 25 ml Erlenmeyer flask (covered with aluminum foil) with constant shaking (150-200 cycles per minute) at 37 ° C in 10 ml digestion medium. After 15-20 minutes, the floating fat and medium were suctioned off and the tissue was again crushed and rinsed with medium without collagenase (repeat the process at least twice, preferably until the cell fraction is transparent), whereupon digestion medium was added and again with carbogen for about 1 minute was fumigated. Digestion with collagenase followed again for 15 minutes at 37 ^° C. in a shaker using the same buffer. After digestion, the acini were dissociated by successively pulling up and pushing out through 10 ml, 5 ml and 2 ml glass pipettes with narrow openings and through a single-layer nylon sieve (Polymon PES-200/45, Angst & Pfister AG, Zurich, Switzerland) with a mesh size of around 250 μm. The acini were centrifuged (at 37 ° C. and 600-800 rpm in a Beckman GPR centrifuge, corresponds to approximately 50-100 g) and further purified by washing in incubation medium containing 24.5 mM HEPES (pH 7.5), 96 mM NaCl, 6 mM KC1, 1 mM MgCl ₂ , 2.5 mM NaH ₂ PO ₄ , 0, mM CaCl ₂ , 11.5 mM glucose, 5 mM sodium pyruvate, 5 mM sodium glutamate, 5 mM sodium fumarate, 1% (Vol. / Vol.) Modified Eagle medium, 1% (wt. / Vol.) Bovine serum albumin, equilibrated with carbogen and adjusted to pH 7.4. The washing procedure (centrifugation, suction, suspension) was repeated five times. Unless otherwise stated, the above insulation works at about 20 ° C.

The acini were resuspended in incubation medium and cultivated at 37 ° C. in a humidified atmosphere with 5% CO ₂ . The acinar tissue died quickly (within two days) and the dying differentiated cells detached from the neighboring cells without damaging them (gentle isolation), the non-dying stem cells. len sank to the ground and attached themselves. The differentiated acini sizes are not able to do this. The incubation medium was changed for the first time on the second or third day after sowing, with a large part of the free-floating A-5 cini and acinar cells being removed. At this point, the first stem cells or their precursors had settled on the ground and began to divide. The medium change was then repeated every third day and differentiated acinar pancreatic cells were removed with each medium change.

On the seventh day in culture, the cells were passaged with a solution consisting of 2 ml PBS, 1 ml trypsin (+ 0.05% EDTA) and 2 ml incubation medium. The cells detach themselves from it

15 Bottom of the culture dish. The cell suspension was centrifuged for 5 minutes at about 1000 rpm (Beckmann GPR centrifuge), the supernatant was suctioned off and the cells were resuspended in 2 ml of incubation medium, transferred to a medium cell culture bottle and 10 ml of incubation medium were added.

On the fourteenth day in culture, the cells were passaged again, but this time with 6 ml PBS, 3 ml trypsin / EDTA and 6 ml incubation medium. The cell suspension is centrifuged at 1000 rpm for 5 minutes, the supernatant is suctioned off and the cell

2.5 ml resuspended in 6 ml incubation medium, transferred to 3 medium cell culture bottles and 10 ml incubation medium added.

On day 17 there was a third passenger on a total of 6 30 medium cell culture bottles and on day 24 a fourth passenger on a total of 12 medium cell culture bottles. At the latest now, all primary cells except the stem cells were removed from the cell culture. The stem cells can be further cultivated and passaged and sown as often as desired. The sowing is preferably carried out in a density of 2-4 × 10 ⁵ cells / cm ² in incubation medium.

EXAMPLE 2 Pancreatic acini were obtained from male Sprague-Dawley rats (20-300 g) who had been anesthetized (CO ₂ ) and bled through the dorsal aorta. A cannula was inserted into the pancreatic duct, and 10 ml of digestion medium containing HEPES-Eagle medium (pH 7.4), 0.1 mM HEPES buffer (pH, 7.6), 70% was introduced into the pancreas from behind. (Vol. / Vol.) Modified Eagle medium, 0.5% (Vol. / Vol.) Trasylol (Bayer AG, Leverkusen, Germany), 1% (wt. / Vol.) Bovine serum albumin), 2.4 mM CaCl ₂ and collagenase (0.63 P / mg, Serva, Heidelberg, Germany) were injected.

Adherent solid tissue, lymph nodes and blood vessels were partially removed from the pancreas before removal. Then healthy pancreatic tissue was collected in digestion medium (at 20 ° C., lower metabolism) and the pancreatic tissue was very finely shredded and processed as described in Example 1. EXAMPLE 3 The isolation and cultivation from exocrine tissue of the parotid gland was carried out analogously to the pancreas protocol with the following differences:

1. The exocrine tissue of the parotid gland was a mixture of acinar tissue and tubular tissue. 2. Since salivary glands contain fewer proteases and amylases than pancreas, it is possible to store the salivary gland tissue in the refrigerator for some time at about 4 ° C without damaging the tissue too much. In the specific example, the retention time was 15 hours and had no adverse consequences for the isolation of the desired stem cells.

The following examples 4 and 5 describe in detail two working protocols for the production of organoid bodies (organoid bodies) and differentiated cells.

EXAMPLE 4 The undifferentiated cells are trypsinized with a solution of 10 ml PBS, 4 ml trypsin, 8 ml differentiation medium and centrifuged for 5 minutes. The resulting pellet is resuspended in differentiation medium so that a dilution of 3000 cells per 100 μl medium is obtained. The cells are then suspended again well with a 3 ml pipette.

The lid is removed from bacteriological Petri dishes, which have previously been coated with 15 ml PBS (37 ° C) per plate, and turned over. With the help of an automatic pipette, about fifty 20 μl drops are placed on a lid. The lid is then quickly turned over and placed on the Petri dish filled with differentiation medium so that the drops hang down. The Petri dishes are then carefully placed in the incubator and incubated for 48 h.

The cells aggregated in the hanging drops, which are to be called organoid bodies (OB) here, are then transferred from four lids into a bacteriological petri dish with 5 ml of incubation medium with 20% FCS and cultured for a further 96 h. The organoid bodies are now carefully collected with a pipette and transferred into cell culture vessels coated with 0.1% gelatin with differentiation medium. In a particularly preferred embodiment of the method, 6 cm petri dishes coated with 0.1% gelatin are used as the culture vessel, into which 4 ml of differentiation medium has been placed and which are then loaded with 6 organoid bodies each. Another preferred culture vessel are Chamber Südes coated with 0.1% gelatin, into which 3 ml of different

10 medium was submitted and then each loaded with 3-8 organoid bodies. In addition, it is also possible to use 24-well microtiter plates which have been coated with 0.1% gelatin and into which 1.5 ml of differentiation medium per well has been placed and then with

15 4 organoid bodies each can be loaded.

Cultivated in this way, the differentiation ability of the cells in the organoid bodies is activated and the cells differentiate into cells of the three cotyledons Mesoderm, Entoderm 20 and Ektoderm. The cells can be stored and cultivated both as organoid bodies and as individual cells and retain their pluripotency.

EXAMPLE 5

25 _.. For the induction of differentiation were preferably stem cells after the 42nd day of cultivation used. The use of stem cells after the 3rd or 4th passage or cells which had been stored at the temperature of liquid nitrogen for 12-18 months was also tried.

30 possible without a seat.

First, the cells were transferred to differentiation medium with the composition given above and placed on a sealing set at about 3 x 10 ⁴ cells / ml; eg by trypsin treatment of a stem cell culture in nutrient medium, centrifugation at 1000 rpm for 5 minutes and resuspending the pellet in differentiation medium and dilution as necessary.

Then, using a 20 μl pipette, approx. 50 20 μl drops (600 cells / 20 μl) were placed on the inside of the lid of a bacteriological Petri dish (stuffed tips) and the lids were carefully placed on the Petri dishes filled with PBS so that the drops hang down. A new tip was used for each lid. The petri dishes were then carefully placed in the incubator and incubated at 37 ° C for 48 hours.

The cells aggregated in the hanging drops, the organoid bodies (OB), were each transferred from four lids into a bacteriological petri dish with 5 ml incubation medium with 20% FCS (hold the lid at an angle and rinse the OBs with about 2.5 ml nutrient medium ) and cultivated for a further 5-9 days, preferably 96 h.

The organoid bodies were then carefully collected with a pipette and transferred to cell culture vessels coated with 0.1% gelatin with differentiation medium. The OBs now multiplied and in some cases grew into individual cell colonies that could be multiplied, separated and multiplied again. In a particularly preferred embodiment of the method, 6 cm petri dishes coated with 0.1% gelatin, into which 4 ml of differentiation medium had been placed, and which were each loaded with 6 organoid bodies, were used as the culture vessel. Another preferred culture vessel were Chamber Südes, coated with 0.1% gelatin 3 ml of differentiation medium was placed in front of each of which were subsequently loaded with 3-8 organoid bodies, and Therma nox plates (Nalge Nonc International, USA) for electron microscopic studies. Another alternative was 24-well microtiter plates, which were coated with 0.1% gelatin and into which 1.5 ml of differentiation medium was placed in each well and which were then loaded with 4 organoid bodies each.

In a preferred embodiment of the method, OBs were cultured in the gelatin-coated 6 cm Petri dishes for about 7 weeks and then individual OBs were cut out with the microdissector (Eppendorf, Hamburg, Germany) according to the manufacturer's instructions and then e.g. transferred to fresh 6 cm Petri dishes, Chamber Südes or Thermanox plates. In a further preferred embodiment, individual OBs with pipette tips were detached and transferred by gentle suction, then e.g. Observation under the inverse microscope.

EXAMPLE 6 Characterization of Differentiated Cells in the Organoid Bodies

1. Immunohistochemical organoid bodies that had been cultured on Chamber Südes for at least 3 weeks, and cross sections of "long-term" OBs were rinsed twice in PBS for five minutes with methanol: acetone (7: 3) containing 1 g / ml DAPI (Röche, Switzerland) fixed at -20 ° C and washed three times in PBS. After incubation in 10% normal goat serum at room temperature for 15 minutes, the samples were incubated overnight with primary antibodies at 4 ° C in a humidification chamber. The primary antibodies were against the protein gene product 9.5 (PGP 9.5, polyclonal rabbit antibody, 1: 400, Ultraclone, Insel Wight), neurofilaments (NF-Pan-Cocktail, polyclonal rabbit antibody, 1: 200, Biotrend, Germany), α- smooth muscle actin (α-SMA , mouse monoclonal antibody, 1: 100, DAKO, Denmark), güales fibrillar acidic protein (GFAP, mouse monoclonal antibody, 1: 100, DAKO, Denmark), collagen II (mouse monoclonal antibody II-II-6B3, 1:20, de- velopmental Studies Hybridoma Bank, University of Iowa, USA), Vigiün FP3 (1: 200, Kügler et al., 1996), Cytokeratine (Pan Cytokeratin, monoclonal mouse antibody, 1: 100, Sigma, USA), Alpha-Amylase (polyclonal rabbit antibody, 1: 100, Calbiochem, Germany) and insulin (mouse monoclonal antibody, 0.5 g / ml, Dianova, Germany). After rinsing three times with PBS, slides were incubated for 45 minutes at 37 ° C with either Cy3-labeled anti-mouse IgG or FITC-labeled anti-rabbit IgG (Dianova), both diluted 1: 200. The slides were washed three times in PBS, coated with Vectashield Mounting Medium (Vector, USA) and analyzed with a fluorescence microscope (Axiosop Zeiss, Germany) or with a confocal laser scanning microscope (LSM 510 Zeiss, Germany). Alcian blue staining was performed using standard procedures.

2. Transmission electron microscopy OBs were cultured on Thermanox plates (Nalge Nonc International, USA) for at least 3 weeks. Samples adhering to the Thermanox plates were incubated at pH 7.4 for 24 hours by immersion in 0.1 M cocodylate buffer containing 2.5% glutaraladehyde and 2% paraformaldehyde. After post-fixation in 1% Os0 ₄ , "en bloc" staining with 2% uranyl acetate and dehydration in pure alcohols, the samples were embedded in Araldite. After removing the Thermanox plate, Semithin cuts were made either tangentially or vertically made for embedded cell culture and cut with methylene blue and Azure II. Ultrathin sections were cut out from the regions of interest, stained with lead citrate and examined under a transmission electron microscope (Philips, EM 109).

The following Examples 7 to 10 describe the contacting of organoid bodies, which were produced as described above, with various active substances and the detection of a change in the organoid body and / or the differentiated cells contained therein by direct visual detection or the detection of marker proteins. EXAMPLE 7

In this and the following examples, several organoid bodies of a batch which have been grown together (e.g. produced by separating suitable organoids and re-enlarging to the desired number), usually at least 6 in a group, are used for one experiment.

In this example, organoid bodies were exposed to various micromolar concentrations of puromycin, an agent that inhibits translation, for a period of 1 to 2 days. The size of the treated organoid bodies was then compared to that of an untreated control (see microscopic comparison picture in FIG. 5A). In a second detection method, the organoids were taken up in 10 ml lysis buffer containing 7.5 ml PBS, 2.5 ml NP-40 and 1 mM PEFA block, stored overnight in the refrigerator at 4 ° C. and then in mini homogenizers for Eppendorf tubes homogenized. The homogenate was centrifuged, the supernatant removed and electroplated using standard methods. separated phoretically and the gel subjected to a Western immunoblot. 5B shows the results of the treatment via the detection of the translation marker Vigiün. The first four bands result from the different amounts of puromycin, the last two show the amount of Vigiün in the untreated organoids; the same amount of total protein was always applied to each lane.

EXAMPLE 8 The organoid bodies were incubated with 2 x 10 ^{~ 6} M retinoic acid or without retinoic acid for 7, 11, 14 and 17 days. The organoid bodies were then homogenized as described in Example 7 and subjected to a Western blot assay. The markers α-SMA (α-smooth muscle actin) and a mixture of neurofilaments (NF) were stained (FIG. 6). While the amount of actin during the entire treatment period is higher than in the control, the amount of NF detected in the Western blot changes only on the 7th and 11th day of treatment.

EXAMPLE 9

The organoid bodies were incubated with 40 ng / ml HGF (“hepatocyte growth factor”) or without HGF for 7, 11, 14 and 17 days. The organoid bodies were then homogenized as described in Example 7 and subjected to a Western blot assay The formation of the liver protein α-fetoprotein was examined (Fig. 7) After 7 and 11 days of incubation, a clear synthesis of the fetoprotein can be observed, while a longer exposure time rather inhibits the formation. EXAMPLE 10

Organoid bodies were incubated with or without conditioned medium from chondrocyte primary cultures and the presence of collagen-II as a typical cartilage protein was again examined with a Western blot as above (FIG. 8). Both approaches with chondrocyte culture supernatants show a slight increase in collagen II compared to the control with simple medium.

The examples above only demonstrate a basic procedure for carrying out the present invention with a small selection of chemical active substances and detection methods. However, the invention is in no way limited to these exemplary embodiments. Alternatives, in particular alternative detection methods, are known to the person skilled in the art or are easy to find on the basis of the detailed disclosure of this application in conjunction with the cited prior art.

The features of the invention disclosed in the above description, the claims and the drawings can be of importance both individually or in combination for the implementation of the invention in its various configurations.

Claims

1. A method for testing substances with regard to their effect on biological cells using multicellular cell aggregates, referred to as organoid bodies, characterized in that the substance to be tested is brought into contact with the organoid bodies and their effect, if present, by a morphological or otherwise detectable change in these organoid bodies or the cell types contained therein is determined, the organoid bodies being formed by aggregation and differentiation of multipotent or pluripotent adult stem cells.

2. The method according to claim 1, characterized in that it is a method for analyzing the effect of known or potential active substances or mutagens on all or special cell types of the organoid body.

3. The method according to claim 1 or 2, characterized in that it is a method for screening active pharmaceutical ingredients or cosmetics.

4. The method according to any one of claims 1-3, characterized in that the cell aggregates are mammalian cell aggregates, in particular human cell aggregates.

5. The method according to any one of claims 1-4, characterized in that the substance to be tested is a protein, peptide, a nucleic acid, such as DNA, RNA or a derivative of it, or a low molecular weight chemical substance.

6. The method according to any one of claims 1-5, characterized in that the multipotent or pluripotent adult stem cells have been isolated from exocrine gland tissue.

7. The method according to claim 6, characterized in that the exocrine glandular tissue is acinar tissue.

8. The method according to any one of the preceding claims 1-7, characterized in that the effect of the substance is a change in the morphology or the Proüferations- ability, growth ability or viability or another activity of all cell types or special cell types of the organoid body.

9. The method according to any one of the preceding claims 1-8, characterized in that the detection of the effect of the chemical substance on the detection of the presence or absence or amount of a marker substance which is formed by special or all cell types of the organoid body takes place ,

10. The method according to claim 9, characterized in that the detection of the action of the chemical substance is carried out via the detection of the presence or absence or amount of a marker protein.

11. The method according to claim 10, characterized in that the marker protein by binding dyes, anti bodies or receptors, is detected by a Western blot, by enzymatic or other activity of the marker protein.

12. The method according to claim 9, characterized in that the detection of the action of the chemical substance via the detection of the presence or absence or amount of a nucleic acid, e.g. DNA or RNA or a derivative thereof.

13. The method according to any one of claims 1-12, characterized in that the detection of the effect of the chemical substance, the use of one or more methods selected from the group consisting of protein assays, immunoassays, enzymatic assays, receptor binding assays, E- LISA assays, RIA assays, electrophoretic and chromatographic assays, including HPLC, Northern blots, Southern blots, Western blots, colorimetric assays, immunohistochemical, electrophysiological, microscopic and spectroscopic detection methods.

14. The method according to any one of claims 1-13, characterized in that the cell types of the organoid body from the group consisting of osteoblasts, osteoclasts, chondrocytes, adipocytes, fibroblasts, muscle cells, endothelial cells, epithelial cells, hematopoietic cells, sensory cells, endocrine and exocrine gland cells , Glial cells, neuronal cells, ougodendrocytes, blood cells, intestinal cells, heart, lung, liver, kidney or pancreatic cells are selected.

15. The method according to any one of the preceding claims 1-14, characterized in that the cell type composition of the organoid body has been determined by cultivation in a medium which contains cell type-specific differentiation and / or growth factors.

16. The method according to claim 15, characterized in that the growth and / or differentiation factors from the group consisting of bFGF, VEGF, DMSO and isoproterenol, fibroblast growth factor 4 (FGF4), hepatocyte growth factor (HGF), TGF-beta, EGF, KGF, retinoic acid, beta NGF, BMP-4 and Activin-A are selected.

17. The method according to any one of claims 1-16, characterized in that two or more of the different cell types of the organoid body form tissue or organ-like structures.

18. The method according to claim 17, characterized in that two or more of the different cell types of the organoid body form neuromuscular structures or glia-nerve cell structures or skin cell structures.

19. The method according to claim 17 or 18, characterized in that the test substance causes a change in these structures.

20. The method according to claim 19, characterized in that the change is a destruction of the structures, inhibition or stimulation of the development of the structures and / or a change in the activity of one or more cell types from which these structures are composed.

21. The method according to any one of the preceding claims 1-20, characterized in that the organoid bodies are in cavities, matrices or other carrier and / or shaping systems.

22. The method according to any one of the preceding claims 1-21, characterized in that two or more test substances are tested simultaneously or in succession with the same organoid bodies.

23. Device for carrying out the method according to any one of claims 1-22, comprising the organoid bodies in a suitable container, carrier or shaping system, means for contacting the organoid bodies with a substance to be tested and means for detecting a morphological or other change thereof organoid body or the cell types it contains.

24. Kit for carrying out the method according to any one of claims 1-22, comprising the organoid bodies as defined in claim 1 in a suitable culture medium for maintaining the organoid bodies.

25. Kit according to claim 24, characterized in that the organoid bodies are in cavities, matrices or other carrier and / or shaping systems.

26. Kit according to claim 24 or 25, further comprising means for contacting the organoid body with a substance to be tested.

27. Kit according to one of claims 24-26, further comprising means for detecting a morphological or other change in these organoid bodies or the cell types contained therein, and optionally further reagents and auxiliaries.

28. Kit for carrying out the method according to any one of claims 1-22, comprising multipotent or pluripotent adult stem cells, from which the organoid bodies can be produced as defined in claim 1, in a suitable culture medium for maintaining these adult stem cells, and reagents, including, if appropriate Differentiation factors, and culture media to produce organoid bodies of a desired cell type composition.

29. The kit of claim 28, further comprising means for contacting the organoid body with a substance to be tested.

30. Kit according to claim 28 or 29, further comprising means for detecting a morphological or other change in these organoid bodies or the cell types contained therein, and optionally further reagents and auxiliary substances.