DE102004025080A1 - Method for testing substances for their effects on cells, useful e.g. in screening for pharmaceuticals or cosmetics, using organoid bodies derived from adult stem cells - Google Patents

Abstract

Method for testing substances (A) for their action on biological cells, using multicellular cell aggregates (organoid bodies; OB), comprises contacting OB with (A) and detecting any effect from a morphological or other visible change in OB, or its component cells. OB are formed by aggregation and differentiation of multi- or pluri-potent adult stem cells (aSC). Independent claims are also included for the following: (1) device for the new method comprising OB in a suitable container, carrier or shaping system; system for contacting OB with test compounds and a system for detecting any change in OB or its component cells; (2) kit for the new method comprising OB and a nutrient (maintenance) medium; and (3) kit for the new process comprising aSC and a nutrient (maintenance) medium, also reagents (including optionally differentiation factors) and culture media for proliferation of OB with the required cellular composition.

Description

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

The execution from animal experiments for Pharmaceutical and cosmetic examinations represents a tremendous amount Cost factor and is often ethically problematic. For the cosmetics industry they are even in many countries, e.g. in Europe, completely forbidden. The development of multicellular, especially humane, in vitro test systems provides for many studies represent a good alternative and such humane ones Test systems can natural human tissues / organs in their properties even closer than Animal models come.

In the prior art, tissue cultures from explanted tissue samples have hitherto been used as in vitro test systems (see, for example, US Pat US 5726009 and US 2002/192638) as well as cultures of differentiated cells obtained from stem cells. The former approach has the disadvantage that it is difficult to maintain the cultures for extended periods of time and to produce larger quantities of cells without altering the properties of the cells or tissue. In the second approach, the stem cells are conventionally by adding special factors targeted for differentiation into individual cell types, such as nerve cells, fibroblasts, etc., caused, and then the effect of various chemicals, such as drugs, examined for these special cells. To study a wide range of differentiated cells, different specialized cell cultures need to be prepared and tested. Using conventional adult stem cells with a limited spectrum of differentiation usually requires culturing the differentiated cell cultures from different parent stem cells, often with very different growth conditions. This is usually associated with extra time and expense. This disadvantage can be partially avoided by using pluripotent embryonic stem cells, which can differentiate into a wide variety of 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. Moreover, even with the use of pluripotent embryonic stem cells in these conventional test systems, several different and spatially separate cell cultures are required. Another major disadvantage of these cell monocultures is that the effect of a substance on a composite of different cells, such as is present in a natural tissue or organ, can not be studied.

Accordingly, there is the object of the invention is to improve multicellular, in particular to provide human, in vitro test systems and methods, with which the effect of substances on different cell types and in particular to a composite of different cell types, as in natural Tissues and organs, can be detected quickly and easily can.

The 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 of aggregation and differentiation in three-dimensional cell aggregates, so-called organoid body or Organoid Bodies, which can be induced, which already without addition special differentiation factors a spectrum of at least contain two cell types. These organoid bodies grow at sufficient levels nutrients constantly continue and develop tissue or organ-like structures, in this Stage they are also referred to as tissue body. You put these organoid bodies chemical substances, their effect, if any, may be by a morphological or otherwise demonstrable change this organoid body or the cell types contained therein. To this Way you can different cell types tested simultaneously or sequentially quickly and, in particular, also tissue or organ-like cell aggregates are investigated become.

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

To form the organoid body or organoid bodies used in the present invention, mul tipotente or pluripotent adult stem cells used. Preferably, these pluripotent stem cells are isolated from exocrine glandular tissue.

The exocrine glandular tissue may be from an adult individual or a juvenile individual. The term "adult", as in the present Application thus refers to the stage of development of the initial tissue and not on that of the donor, from the the tissue comes from. "Adult" stem cells are non-embryonic stem cells.

Preferably becomes the exocrine glandular tissue from a salivary gland, Lacrimal gland, sebaceous gland, sweat gland, out glands of the genital tract, including Prostate, or from gastrointestinal tissue, including pancreas, or secretory tissue of the liver isolated. In a highly preferred embodiment this is acinar Tissue. Most preferably, the acinar tissue is from the pancreas, the parotid gland or submandibular gland.

The adult stem cells obtained from such sources are easily too isolate and in a stable long-term culture without Feederzellschicht or special additives to keep in the largely undifferentiated state. The term feeder cells, As used here, all cells that actually grow to cultivate cells by promoting growth factors release and / or provide an extracellular matrix, or prevent the differentiation of stem cell culture.

These adult stem cells can without the addition of special growth or differentiation factors in a simple way to differentiation be stimulated by under spatial Conditions are cultivated, which for a three-dimensional contact the cells provide. In a preferred embodiment, these conditions the cultivation in hanging Drops, as they are for embryonic stem cells has already been described (Wobus et al., Biomed. Biochim. Acta 47: 965-973 (1988). This process will be described below in the examples described in more detail. However, it is understood that alternative cultivation methods, the for the wished provide three-dimensional contact of the cells and known in the art and available are, can be used as well. examples for such alternative methods are cultivation in agitated Suspension culture, cultivation in an electromagnetic field cage or laser tweezer, cultivating on surfaces where the cells are not or poorly seeding, or sowing non-resuspended cells the primary culture. Such surfaces can e.g. Glass, polystyrene or surfaces treated with an anti-adhesion layer, e.g. PTFE or poly-HEMA coated surfaces.

Under These conditions spontaneously develop three-dimensional cell aggregates or Cell aggregates, which refers to "embryoid bodies" already described for embryonic stem cells as "organoid bodies" or organoid bodies were. These organoid bodies or organoid bodies can transferred into suspension cultures or adhesion cultures and be cultivated further. With sufficient nutrient supply they grow organoid body continue and can Reach a diameter of a few millimeters or more. This big organoid Bodies show a fabric-like structure and become at this stage also referred to as "tissue body" to distinguish it from simple cell aggregates.

bring When the organoid bodies are returned to surface culture, they grow out of growing ones single cells a cellular Monolayer, emerge from the multilayer areas, from which spontaneously secondary Organoid Bodies with comparable properties as those the primary Organoid Bodies are formed. The organoid according to the invention Bodies can e.g. at the temperature of the liquid Nitrogen stored frozen, without their viability, Propagation ability, growth potential and to lose differentiability.

The organoid body contain different cell types of all three cotyledons. to Differentiation, no differentiation factors are necessary and the cells have to also can not be transplanted to differentiate. It can be advantageous to use such differentiation factors, to specifically larger quantities of a particular cell type or to organoid body with of a particular cell type composition.

Differentiation factors are known in the art and include, for example, basic fibroblast growth factor (bFGF) for increased production of cardiac and fibroblast cells, vascular endothelial growth factor (VEGF), DMSO and isoproterenol, fibroblast growth factor 4 (FGF4), hepatocyte growth factor (HGF) to increase the formation of heart and liver cells, TGF-beta1 (transforming growth factor beta1) for the increased formation of cardiac cells, epidermal growth factor (EGF) for increased formation of cardiac cells Skin and heart cells, keratinocyte growth factor (sometimes called keratinocyte growth factor) to form keratinocytes, retinoic acid to enhance the formation of nerve, heart and kidney cells, beta-NGF (beta nerve growth factor) However, formation of brain, liver, pancreas and kidney cells, BMP-4 ("bone morphogenic protein 4") and activin-A to form mesodermal cells in general are not limited thereto.

differentiated Cells that are in the organoid bodies can be included include bone cells (osteoblasts and osteoclasts), chondrocytes, Adipocytes, fibroblasts (e.g., skin and tendon fibroblasts), muscle cells, Endothelial cells, epithelial cells, hematopoietic cells, sensory Cells, endocrine and exocrine glandular cells, glial cells, neuronal Cells, oligodendrocytes, blood cells, intestinal cells, heart, lung, Liver, kidney or pancreas cells, but are not limited thereto.

at the test method according to the invention the substance to be tested is brought into contact with the organoid bodies and their effect optionally by a morphological or otherwise detectable change this organoid body or the cell types contained therein. In this test procedure For example, this is a method of analyzing the Effect of known or potential agents or toxins or Mutagenes on all or specific cell types of organoid bodies. In a more specific embodiment is a method of screening drugs or cosmetics.

The Test substance can be of a very different chemical nature, for example, 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. It can also two or more test substances in succession or simultaneously with the same organoid bodies be tested, for example, interactions of the test substances determine.

The Contacting the test substance (s) with the organoid bodies, dependent of the type of the test substance, in any suitable manner. Suitable methods are known to the person skilled in the art. If it is the test substance is a nucleic acid, e.g. RNA, DNA or a Derivative thereof, this can (s) with any known Techniques of genetic engineering, including the use of vectors, Viruses, electroporation, etc., into which cells of the organoid body are introduced. A soluble in culture medium or Solubilisable test substance is preferably simply the cell culture medium, in which are the organoid bodies are added, and the organoids are mixed with the test substance in suitable concentrations for different desired periods incubated. If desired, can consume spent cell culture medium during incubation fresh medium with the desired Be replaced concentration of test substance, e.g. to the drug concentration to be approximately constant in the medium. Depending on the nature of the drug, the effective concentrations of the test substance on a large scale can vary However, it will not be difficult for a person skilled in the art to determine it by routine experimentation. The contact period can be from a few minutes to several hours and several days and weeks vary. Contact periods of several weeks are not uncommon. Suitable contact periods are may also depend on the nature of the drug and can be determined by the skilled person through routine experimentation be determined. Subsequently the treated organoids and a control without test substance, which was incubated for the same length, subjected to a detection method.

The Detection method will depend on the type of active ingredient and type of observational change of organoid body or the differentiated cells contained therein.

A Range of methods for detecting the effect of drugs or Toxins on mammalian cells is written by A. Vickers in "In Methods in Pharmaceutical Research ", Academic Press, 1997. Basic methods for Drug screening is described by Smith, C.G., "The process of New Drug Development, CRC Press, 1992, and Advances in Drug Discovery Tchniques ", ed. Alan L. Harvey, John Wiley & Sons, 1998, described.

In special embodiments of the present invention the proof of the effect of a substance the application of one or several methods, that of the group of protein assays, immunoassays, 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 (i.e. Voltage and impedance measurements), microscopic and spectroscopic Detection methods is selected or are.

The Effect of the substance may e.g. a change in morphology or of proliferation, growth assets or viability all cell types or specific cell types of organoid bodies. In this case, you can e.g. direct visual and optical detection methods, including cytometric, microscopic and colorimetric method, used favorably become.

alternative or at the same time the effect may be a change of activity more specific or all cell types of organoid bodies. For example this activity change in an increased or decreased or first appearance express a detectable marker in or on the affected cells. In In this case the proof of the effect of the chemical substance takes place preferably over the detection of the presence or absence or amount of a marker substance, which are formed by special or all cell types of the organoid body becomes.

These Markersubstance can z. A protein, including, but not limited to, Antibody, receptor, Enzyme, hormone, ion channel, neurotransmitter, surface marker, RNA, DNA, a proteoglycan, a lectin or another suitable one Be substance. Some cell type-specific examples of marker substances are mentioned below, but are not to be considered as limiting: 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 strong translating cells and cytokeratin for epidermal cells, collagen Type II for Chondrocytes, osteonectin for Osteoblasts and progenitor cells, Osteocalcin for mature osteoblasts, CD45, CD34, CD13 for hematopoietic cells, cTNI ( "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 big one Number of such marker substances is known in the art.

These Marker substances can For example, by binding to a specific binding partner, with a detectable group is conjugated to be detected. The detectable Group can e.g. a dye, fluorescent dye, a radioactive Labeling, Enzyme Labeling, Luminescent Labeling, Magnetic Resonance Labeling or any other label known in the art. If the marker substance is a protein, the binding partner becomes preferably a labeled or labelable antibody. Such labeled antibodies are already for a variety of marker substances are known and can either be obtained commercially or easily prepared by known methods. If necessary, too labeled or labelable secondary antibodies can be used. Of the specific binding partner can also be labeled, but to a Affinity column or another carrier be bound. In these cases can Cells having the markers on their surface, be detected by the specific binding to these carriers.

In some cases The marker substance can also be detected by its own activity be, e.g. if it is an ion channel or neurotransmitter or an enzyme that has a detectable substrate can be implemented. If the marker substance is a DNA or RNA is, it can either directly by complementary and optionally marked Probes are detected or indirectly by the detection of a Gene product, if it is a complete coding sequence or is a regulatory sequence. Another possibility is an enhanced DNA or RNA synthesis itself or enhanced DNA repair activity. Such activities may e.g. by the incorporation of radioactively or otherwise labeled nucleotides be determined.

Of the Detection may be done to obtain the cell structure, e.g. in microscopic and immunohistochemical procedures, or destruction of the Cell structure, e.g. in an electrophoresis method such as Southernblots, Northernblots or Westernblots.

At an advanced stage of differentiation, the organoid bodies have tissue or organ-like structures formed by two or more different cell types (cf. 2 - 4 ). Such structures are, for example, neuromuscular structures or glial-nerve cell structures or skin cell structures. By culturing the organoid bodies in a medium with specific differentiation factors, the formation of desired structures can be promoted.

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

After this, as mentioned above, the cell-type composition of organoid bodies by their cultivation in the presence of specific differentiation factors can, this means that the Cell type composition of organoid bodies in more specific embodiments on the putative cell type specific Effect of the test substance can be adjusted. This means, in concrete terms, that e.g. organoid body with a high percentage of nerve cells or organoid bodies that predominantly or exclusively neuromuscular Having structures or nerve glial cell structures to be used in a test system in the test substances with a suspected effect on the nervous system to be examined. Analogous to this organoid body with a high proportion of epithelial cells or with skin-like ones Structures in a test of test substances presumed over the surface act, be used. Other such special test systems be for the skilled person readily apparent and feasible.

In further more specific embodiments are the organoid bodies in cavities, Matrices or other carrier and / or shaping systems. The introduction of the organoids can for example, by waxing done. The cavities can, for example microchannels or capillaries for impedance measurement. The proof of effect the test substance can then be e.g. in an impedance change consist. Alternatively, a gradient (e.g., pH, electrochemical, Signal factors, etc.) via a organoid body be produced and the effect of the test substance by a change this gradient will be displayed.

One Another aspect of the invention relates to a device for carrying out the inventive method, comprising the organoid body in a suitable container, Carrier or shaping system, Means for contacting the organoid body with a test to be tested chemical substance as well as means of proof of a morphological or otherwise this organoid body or the cell types contained therein. In a more specific embodiment In this aspect, the organoid bodies are in cavities, e.g. microchannels or capillaries, or in matrices.

In an embodiment The invention relates to the means for carrying out the method according to the invention provided in the form of a kit. Such a kit includes adults Stem cells or the derived organoid body or differentiated cells in a suitable culture medium for maintenance the cells or the organoid body, optionally cryopreserved. Furthermore, the kit can be further aids, e.g. Reagents for culturing and differentiating stem cells to organoid bodies a wish Cell type composition, means for contacting the organoids body with a chemical substance to be tested, means of detection morphological or otherwise altering these organoid bodies or the cell types contained therein.

DESCRIPTION OF THE FIGURES

1 : shows schematically the cultivation of stem cells in surface culture and in hanging drops and 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 body.
a, b: PGP 9.5-labeled neurons show multipolar shoots showing numerous varicosities. c, d: the neurofilament system (bright arrows, marked green in the original photo) passes through the pericaryon into the cytoplasmic extensions. GFAP-immunoreactive glial cells are in close proximity (dark arrows, marked red in the original photo). e, f: α-SMA-labeled cells (dark arrows, red in the original) and NF-labeled neurons (bright arrows, in the original green) form a primitive neuro-muscular network (e), establishing contacts over long distances (f). g: immunostaining of GFAP (dark arrows, red in the original) and NF (bright arrows, green in the original) in 3-week old OB with concentrations of nerve and glial cells. h: Immunostaining of α-SMA (dark arrows, red in the original) and NF (bright arrows, green in the original) in 3 week old OBs at an advanced stage of neuromuscular network formation. i, j: in cross sections of 8-week-old OBs, immunoreactive cells were found to be NF in the immediate vicinity of cells immunoreactive for α-SMA, similar to native tissues. k: a subset of cells shows a positive staining for amylase (bright arrows, green in original). 1: Another cell subset contains granular vesicles with immunoreactivity for insulin. The cores are contrast-dyed with DAPI (original blue).

3 shows the expression of extracellular matrix components and cytokeratins.
a, b: Globular (a) and fibrillar (b) stores of proteoglycans revealed staining with Alcian blue.
c-e: The globular (c) and fibrillar (d) regions 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 clustered. g: confocal laser scanning microscopy of a OB. The collagen II immunoreactivity (dark arrows, marked red in the original) increases toward the center of the OB. Vigilin-immunoreactive cells (bright arrows, marked green in the original) are located mainly at the outer border of the OB, indicating their high translational activity. The cores are contrast-dyed with DRPI.

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 spurs that form a connecting network (c). d: Cell shoots with an accumulation of numerous small-sized vesicles that most likely correspond to nerve fiber rarities. e: collagen and reticulum fibers.
f-h: secretory cells show electro-tight vesicles. f). Frequently, secretory cells contact each other to form acinous structures (g). A subgroup of secretory cells contains vesicles (arrow) corresponding to ultrastructural features of endocrine granules (h), eg, beta granules of insulin-producing cells. l: Start of the formation of an epithelial surface (arrow) in eight week old OBs. j: typical cell contacts between keratinocytes and desmosomes (arrows)

5A Figure 1: shows microscopic comparative photographs of an untreated and a puromycin-treated organoid body.

5B : shows Western blots of protein separations of the homogenates of 5A shown organoids with the detection of the translation marker vigilin.

6 : 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 ( 6A ) or the detection of neurofilaments (NF) ( 6B ).

7 Figure 3 shows Western blots of protein separations of the homogenates of HGFR-treated and untreated organoid bodies with the detection of the hepatic protein α-fetoprotein.

8th Figure 3 shows Western blots of protein separations of the homogenates of conditioned medium from chondrocyte primary cultures treated and untreated organoid bodies with the detection of the cartilage protein collagen II.

9 : shows a basic structure and experimental procedure for the testing of substances using the methods and systems according to the invention.

According to the in 1 Scheme shown is used to obtain the stem cells exocrine glandular tissue, eg acinar tissue - preferably a salivary gland or the pancreas (pancreas) - mechanically and enzymatically comminuted into culture (step 10 in 1 ). Contrary to the information given by Bachem et al., Gastroenterol. 115: 421-432 (1998), and Grosfils et al., Res. Comm. Chem. Pathol. Pharmacol. 79: 99-115 (1993), no tissue blocks are cultured from which to grow out cells, but on the condition that the cell clusters of acini remain largely intact, the tissue is comminuted more.

Over several Weeks, these cells and cell aggregates are cultured in culture vessels. Every 2 to 3 days, the medium is changed, all differentiated Cells are removed. In cells persisting in culture these are undifferentiated cells with unrestricted ability to divide.

Similar cells have been isolated and described under the same conditions from the pancreas and have been designated as a type of myofibroblasts or pancreatic stellate cells (Bachem et al., 1998). However, unlike the cells of the present invention, unrestricted partitioning ability could not be observed. Furthermore, these cells could not be indefinitely passaged without vitality to lose.

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

Through this type of cultivation, the cell aggregates called organoid bodies are formed within 48 hours ( 14 ) which are converted into a suspension culture for approximately 6 days ( 16 ). The partial view ( 18 ) out 1 shows a micrograph of such an organoid body.

The organoid bodies growing in suspension culture form new organoids Bodies, which also in single cells the formation of new Organoid Induce bodies. The cells are called organoid bodies as well also freezable as single cells while retaining their vitality and theirs Differentiation potential.

The 2 - 4 show microscopic and electron micrographs of differentiated cells obtained from such organoid bodies or organoid bodies.

For example, the formation of a neuromuscular network was observed:
Cells obtained from OBs strongly expressed α-SMA (smooth muscle actin) ( 2e -f). The presence of common bundles of myofilaments extending across the cytoplasm was confirmed by electron microscopy ( 4a c). Furthermore, cells were identified which were immunoreactive for the pan neuron marker PGP 9.5 and for neurofilaments (NF). The neurofilament system ranged from the pericaryon into the radial cytoplasmic shoots ( 2c , d). PGP 9.5 immunoreactive cells showed numerous varicosities along their branched extensions ( 2a , b, 4d ) and thus corresponded to typical morphological features of autonomic nerve fibers. Cells that were immunoreactive for glial fibrillary acidic protein (GFAP) were in close proximity to cells that expressed neuronal markers ( 2c , d). Frequently, the filamentous proteins did not extend through the entire cytoplasm but were confined to areas adjacent to the nerve cells. Furthermore, smooth muscle cells and nerve cells were not randomly distributed, but formed contiguous networks with easily distinguishable connections ( 2e , f). Nerve fiber spurs extended over considerable distances to contact adjacent smooth muscle cells as their putative targets. Thus, due to their topographical arrangement, the two cell types showed features of a primitive neuromuscular network. In 3-week-old OBs, incipient formation of tissue-like structures was noted ( 2g j). Here, a cluster of fibrous nerve cells was found in contact with glial cells ( 2g ) or further developed into a three-dimensional neuromuscular network ( 2h ), which was confirmed in cross-sections of 8 week old OBs ( 2i , j).

Proof of expression exocrine and endocrine pancreatic proteins:

Immunohistochemical staining showed that cellular subgroups were positive for amylase ( 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 located in circles, with the secretory vesicles having a position toward the center which is a morphological arrangement similar to that of exocrine pancreatic acini. Other cellular subgroups showed immunoreactivity for insulin ( 2l ). Similar to the amylase-positive cell clusters, the secretory product was stored in vesicular structures that were concentrated at a cell pole. The presence of secretory cells was confirmed by electron microscopy showing densely distributed electrodeless particles characteristic of excretory or incretory functions ( 4f -H).

Was also observed a differentiation into chondrogenic cells and epithelial cells:

After a growth period of two months, OBs showed chondrogenic properties. Alcianblue staining revealed areas of high concentrations of proteoglycans (chondroitin sulfate), either as globular ( 3a ) or fibrillar ( 3b ) Deposits occurred. Immunohistochemical staining with antibodies directed against the cartilage matrix protein collagen II additionally demonstrated the chondrogenic activity within this globular 3c ) and fibrillar ( 3d ) Areas. The immunoreactivity was highest in the middle of the cellular aggregates most likely to correspond to areas of developing extracellular cartilage matrix. This observation was confirmed by confocal microscopy ( 3g ): As the amount of collagen storage increased toward the center of the cellular aggregates, the margins were characterized by actively translating cells, as by their high vigi lin expression found commonly in cells with active translation machinery, eg collagen-synthesizing chondrocytes or in fibroblasts during chondroinduction. Typical single collagen II trans-latent chondrocytes were also observed in outgrowing cells of OBs that produced a collagen II-containing matrix surrounding the single cells ( 3e ). An ultrastructural study of these areas clearly showed a network of reticulum fibers and collagen fibers, the latter being identified by their characteristic banding pattern ( 4e ). Some cells expressed mesenchymal markers as well as several cytokeratins, indicating their potential for differentiation into epithelial cells. However, cells immunoreactive for cytokeratins were found less frequently than cells expressing smooth muscle cell markers and neurons. Typically, they were located in clusters disseminated within the OBs ( 3f ). Electron microscopy revealed typical cell contacts between keratinocytes ( 4y ) and in 8 week old OBs, epithelial cells were found on the surface, which grew from the cell culture medium into the air.

All in all could hitherto be e.g. following markers for specific cells positive 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 strong translating cells and cytokeratin for epidermal cells. Next The light microscopic investigations were also electron microscopically different cell types are morphologically characterized, as well Cell-cell contacts as a sign of cellular Interactions are found.

It were previously u.a. smooth muscle cells, neurons, glial cells, epithelial cells, Fat cells, cardiac cells, kidney cells, fibroblasts (e.g. Tendon fibroblasts), chondrocytes, endocrine and exocrine glandular cells and thus cell types of all three cotyledons in these organoid bodies morphologically / histologically and / or immunochemically detected.

In the following, non-limiting Examples, the present invention will be explained in more detail.

The general working instructions, as they are used for methods for culturing mammalian cells, in particular human cells, are to be observed. A sterile environment in which the procedure is to be carried out is to be adhered to in every case, even if no further description is given. The following buffers and media were used:

Instead of fetal Calf serum (FCS) in the nutrient medium and differentiation medium may optionally also plasma or Serum of another suitable species, in particular human plasma or, less preferably, human serum.

The broth may also be used as the basic medium instead of the DMEM medium used other for the Cultivation of eukaryotic cells, in particular mammalian cells, contain known suitable base medium in which the differentiated Dying cells and multiply the desired stem cells. Also Isolation medium, incubation medium and differentiation medium can be other usual and suitable base medium.

The Examples 1 to 3 below describe working protocols for the isolation and culturing adult pluripotent stem cells from acinar tissue of the pancreas or acinar and tubulosic 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 surgery and repaired immediately. From the surgically removed tissue, eg pancreatic tissue, healthy tissue was separated and placed 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% (v / v) Trasylol (Bayer AG, Leverkusen, Germany), 1% (w / v) bovine serum albumin), 2.4 mM CaCl ₂ and collagenase (0.63 P / mg, Serva, Heidelberg, Germany), taken (at 20 ° C, less metabolism). The pancreatic tissue was minced very finely with a pair of scissors, upper floating adipose tissue was aspirated and the tissue suspension was fumigated with carbogen (Messer, Krefeld, Germany) without the nozzle entering the medium containing the cells (reduction of mechanical stress) and thus to pH 7 , 4 set. Thereafter, the suspension was incubated in a 25 ml Erlenmeyer flask (covered with alufoil) with constant shaking (150-200 cycles per minute) at 37 ° C in 10 ml of digestion medium. After 15-20 minutes, the supernatant fat and medium were aspirated and the tissue was reshuffled and rinsed with collagenase-free medium (repeat at least twice, preferably until cell fraction transparent), followed by digestion medium and again with carbogen for about 1 minute was fumigated. Again, collagenase digestion followed for 15 minutes at 37 ° C in a shaker using the same buffer. After digestion, the acini were dissociated by successive pulling and ejection through 10 ml, 5 ml and 2 ml narrow-bore glass pipettes through a single-layer nylon mesh (Polymon PES-200/45, Angst & Pfister AG, Zurich, Switzerland). filtered with a mesh size of about 250 microns. The acini were centrifuged (at 37 ° C and 600-800 rpm in a Beckman GPR centrifuge, equivalent to about 50-100 g) and further purified by washing in incubation medium containing 24.5 mM HEPES (pH 7.5), 96mM NaCl, 6mM KCl, 1mM MgCl ₂ , 2.5mM NaH ₂ PO ₄ , 0mM CaCl ₂ , 11, 5mM Glucose, 5mM Sodium pyruvate, 5mM Sodium glutamate, 5mM sodium fumarate, 1 (Vol. / Vol.) Modified Eagle's medium, 1% (w / v) bovine serum albumin, equilibrated with carbogen and adjusted to pH 7.4. The washing procedure (centrifugation, aspiration, resuspension) was repeated five times. Unless otherwise stated, the above insulation is operated at about 20 ° C.

The acini were resuspended in incubation medium and cultured at 37 ° C in a humidified atmosphere with 5% CO ₂ . The acinar tissue died rapidly (within two days) and. the dying, differentiated cells detached themselves from the neighboring cells without damaging them (gentle isolation), the non-dying stem cells sank to the ground and became attached. The differentiated acinic cells are not able to do this. The incubation medium was first changed on the second or third day after seeding, with much of the free-floating acini and acinar cells removed. By that time, 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.

 At the seventh day in culture, the cells were made up with a solution from 2 ml PBS, 1 ml trypsin (+ 0.05% EDTA) and 2 ml incubation medium passaged. Solve it the cells from the bottom of the culture dish. The cell suspension was Centrifuged for 5 minutes at about 1000 rpm (Beckmann GPR centrifuge), the supernatant aspirated and the cells are resuspended in 2 ml of incubation medium, transferred to a middle cell culture flask and 10 ml incubation medium added.

At the fourteenth day in culture, the cells were re-but this time with 6 ml PBS, 3 ml trypsin / EDTA and 6 ml incubation medium, passaged. The cell suspension is centrifuged for 5 minutes at 1000 rpm, the supernatant aspirated and the cells resuspended in 6 ml incubation medium, transferred to 3 medium cell culture flasks and 10 ml each of incubation medium added.

At the Day 17 was a third passenger to a total of 6 middle Cell culture bottles and on day 24 a fourth passenger on total 12 medium cell culture bottles. By now all were primary cells removed from the cell culture except for the stem cells.

The stem cells can be further cultured and passaged and seeded as often as desired. The sowing is preferably carried out in each case at 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) that had been anaesthetized (CO ₂ ) and bled through the dorsal aorta. One cannula was transduodenally inserted into the pancreatic duct and 10 ml of digestion medium containing HEPES Eagle's medium (pH 7.4), 0.1 mM HEPES buffer (pH, 7.6), 70% (vol ./Vol.) Modified Eagle's medium, 0.5% (v / v) Trasylol (Bayer AG, Leverkusen, Germany), 1% (w / v) bovine serum albumin), 2.4 mM CaCl ₂ and Collagenase (0.63 P / mg, Serva, Heidelberg, Germany) injected.

In front the pancreas was removed by adhering festive tissue, lymph nodes and blood vessels partially freed. Then, healthy pancreatic tissue became digestive medium picked up (at 20 ° C, lesser metabolism) and the pancreatic tissue with scissors very finely crushed and processed as described in Example 1.

EXAMPLE 3

• 1. Das exokrine Gewebe der Ohrspeicheldrüse war eine Mischung von acinärem Gewebe und tubulösem Gewebe.
• 2. Nachdem Speicheldrüsen weniger Proteasen und Amylasen als Pankreas enthalten, ist es möglich, das Speicheldrüsengewebe vor der Aufarbeitung einige Zeit im Kühlschrank bei etwa 4°C aufzubewahren, ohne dass das Gewebe zu sehr geschädigt wird.

The isolation and cultivation from exocrine tissue of the parotid gland took place analogously to the pancreatic protocol with the following deviations:

• 1. The exocrine tissue of the parotid gland was a mixture of acinar tissue and tubular tissue.
• 2. After salivary glands contain less proteases and amylases than pancreas, it is possible to store the salivary gland tissue in the refrigerator at about 4 ° C for some time before working up, without damaging the tissue too much.

in the concrete example, the storage time was 15 h and brought no adverse consequences for the isolation of the desired Stem cells with them.

The Examples 4 and 5 below describe in detail two working protocols for the production of organoid bodies (Organoid Bodies) and differentiated cells.

EXAMPLE 4

The undifferentiated cells are washed with a solution of 10 ml PBS, 4 ml trypsin, 8 ml of differentiation medium trypsinized and centrifuged for 5 minutes. The resulting pellet is resuspended in differentiation medium, that is a dilution of 3000 cells per 100 μl Medium adjusts. Then be The cells are resuspended well with a 3 ml pipette.

From bacteriological Petri dishes, previously with 15 ml PBS each (37 ° C) have been coated per plate, the lid is removed and turned around. With the help of an automatic pipette to one Cover about fifty 20 μl drops given. The lid is then turned over quickly and with the Differentiation medium filled Petri dish given so that the drops hang down. The Petri dishes will follow Place carefully in the incubator and incubate for 48 h.

thereupon be in the hanging Drop aggregated cells, here called Organoid Bodies (OB) should be made of four lids each in a bacteriological Petri dish with 5 ml incubation medium with 20% FCS transferred and for further Cultivated for 96 h.

The Organoid Bodies are now collected carefully with a pipette, and transferred to 0.1% gelatin-coated cell culture vessels with differentiation medium. In a particularly preferred embodiment of the method used as a culture vessel with 0.1 % Gelatin coated 6 cm Petri dishes, into the 4 ml differentiation medium was submitted and then with 6 organoid bodies be charged. Another preferred culture vessel are with 0.1% gelatin coated chamber slides, into the 3 ml differentiation medium was submitted and then with 3-8 organoid Bodies be charged. In addition, 24-well microtiter plates can also be used used, which were coated with 0.1 gelatin and in which was submitted to 1.5 ml per well differentiation medium and subsequently be charged with 4 Organoid Bodies.

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, endoderm and ectoderm. The cells can be stored and cultured both as organoid bodies and as individual cells and retain their plu ripotenz.

EXAMPLE 5

For 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 at the temperature of liquid Nitrogen 12-18 Was stored for months, was also easily possible.

First, the cells were transferred to differentiation medium having the composition given above and adjusted to a density of about 3 × 10 ⁴ cells / ml; eg by trypsin treatment of a stem cell culture in nutrient medium, centrifugation at 1000 rpm for 5 minutes and resuspension of the pellet in differentiation medium and dilution as required.

Subsequently were with a 20 μl pipette Approx. 50 20 μl drops (600 cells / 20 μl) on the Inside the lid of a bacteriological Petri dish given (stuffed tips) and the lids carefully onto the PBS-filled Petri dishes slipped, so that the drops hang down. For each Lid was used a new tip. The Petri dishes were subsequently Place carefully in the incubator and incubate for 48 h at 37 ° C.

After that were those in the hanging Drops of aggregated cells, the Organoid Bodies (OB), from each four lids each in a bacteriological Petri dish with 5 ml incubation medium transferred with 20% FCS (cover aslant and rinse the OBs with about 2.5 ml of nutrient medium) and for further 5-9 days, preferably 96 h, cultured.

The Organoid bodies were now collected carefully with a pipette and transferred to 0.1% gelatin-coated cell culture vessels with differentiation medium. The OB now multiplied and grew in some individual cell colonies, which could be increased again, isolated and multiplied. In a particularly preferred embodiment of the method have been as a culture vessel with 0.1 % Gelatin coated 6 cm Petri dishes used in the 4 ml Differentiation medium was presented, and this with 6 organoid Bodies loaded. Another preferred culture vessel was with 0.1% gelatin-coated chamber slides, into the 3 ml differentiation medium and subsequently with 3-8 organoid Bodies and Thermanox plates (Nalge Nonc International, USA) for Electron microscopic studies. Another alternative was 24-well microtiter plates coated with 0.1% gelatin were submitted and in each 1.5 ml per well of differentiation medium and subsequently were each loaded with 4 Organoid Bodies.

In In a preferred embodiment of the method, OBs were about 7 Were cultured for weeks in the gelatin-coated 6 cm Petri dishes and then individual OBs with the Microdissector (Eppendorf, Hamburg, Germany) cut out according to the manufacturer's instructions and then transferred to fresh 6 cm Petri dishes, chamber slides or Thermanox plates. In In another preferred embodiment, individual OBs were included Pipette tips detached by light suction and transferred, then, for. Observation under the inverted microscope.

EXAMPLE 6

Characterization more differentiated Cells in the organoid bodies

1. Immunohistochemistry

Organoid bodies cultured for at least 3 weeks on chamber slides 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 (Roche, 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, rabbit polyclonal antibody, 1: 400, Ultraclone, Isle of Wight), neurofilaments (NF-Pan cocktail, rabbit polyclonal antibody, 1: 200, Biotrend, Germany), α-smooth muscle -actin (α-SMA, mouse monoclonal antibody, 1: 100, DAKO, Denmark), glial fibrillary acidic protein (GFAP, mouse monoclonal antibody, 1: 100, DAKO, Denmark), collagen II (mouse monoclonal antibody II-II-6B3, 1 : 20, Developmental Studies Hybridoma Bank, University of Iowa, USA), Vigilin FP3 (1: 200, Kügler et al., 1996), cytokeratins (Pan Cytokeratin, mouse monoclonal 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). Alcianblue staining was done by 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 by immersion in 0.1 M cacodylate buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde at pH 7.4 for 24 hours. After post-fixation in 1% OsO ₄ , en bloc staining with 2% uranyl acetate and dehydration in pure alcohols, the samples were embedded in Araldite. After removal of the Thermanox plate, semithin sections were made either tangentially or perpendicular to the embedded cell culture and cut with methylene blue and azure II. Ultrathin sections were excised from the regions of interest stained with lead citrate and examined under a transmission electron microscope (Phillips, EM 109).

The Examples 7 to 10 below describe contacting organoids bodies which were prepared as described above, with various active ingredients and the determination of a caused by the respective active ingredient change the organoid body and / or the differentiated cells contained therein by direct visual detection or detection of marker proteins.

EXAMPLE 7

at In this and the following examples, several are multiplied together organoid body of a Charge (e.g., by separation of suitable organoids and re-enlargement to to the desired Number generated), usually at least 6 in a group, for used a try.

In this example, organoid bodies were exposed to various micromolar concentrations of puromycin, a drug that inhibits translation, for a period of 1 to 2 days. Thereafter, the size of the treated organoid bodies was compared with that of an untreated control (see comparative microscopic 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 homogenized in mini homogenizers for Eppendorf tubes , The homogenate was centrifuged, the supernatant removed and electrophoretically separated by standard methods and the gel subjected to a Western immunoblot. 5B shows the results of the treatment by detecting the translation marker vigilin. The first four bands are due to the different effective amounts of puromycin, the last two show the amount of vigilin in the untreated organoids; the same amount of total protein was always applied per lane.

EXAMPLE 8

The organoid bodies were incubated with 2 × 10 ^-6 M retinoic acid or without retinoic acid for 7, 11, 14 and 17 days. Thereafter, the organoid bodies were 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 ( 6 ). While the amount of actin is higher during the entire treatment time than in the control, the amount of synthesized NF detectable in the Western blot only changes on the 7th and 11th day of treatment.

EXAMPLE 9

The organoid bodies were incubated with 40 ng / ml hepatocyte growth factor (HGF) or without HGF for 7, 11, 14 and 17 days, after which the organoid bodies were homogenized as described in Example 7 and subjected to a Western blot assay the formation of the hepatic protein α-fetoprotein was investigated ( 7 ). After 7 and 11 days of incubation, a clear synthesis of the fetoprotein can be observed, while a longer duration of action tends to inhibit the formation again.

EXAMPLE 10

Organoid bodies were incubated with or without conditioned medium from primary chondrocyte cultures and the presence of collagen II as a typical cartilage protein re-assayed with a Western blot as above ( 8th ). Both chondrocyte culture supernatant approaches show a slight increase in collagen II over simple medium control.

The The above examples demonstrate only a basic procedure in the execution of the present invention with a small selection of chemical Active substances and detection methods. However, the invention is by no means to these embodiments limited. Alternatives, in particular alternative detection methods, are the One skilled in the art or by the detailed disclosure of this Application in connection with the cited prior art easily find.

The in the foregoing description, claims and drawings Features of the invention can either individually or in combination for the realization of the invention be significant in their various embodiments.

Claims (30)

A method of testing substances for their action on biological cells using multicellular cell aggregates, called organoid bodies, characterized in that the substance to be tested is brought into contact with the organoid bodies and their action, if any, by a morphological or otherwise detectable one Change of these organoid bodies or the cell types contained therein is detected, wherein the organoid bodies were formed by aggregation and differentiation of multipotent or pluripotent adult stem cells. Method according to claim 1, characterized in that that it is a process for analyzing the effects of known or potential Agents or mutagens on all or specific cell types of organoid bodies. Method according to claim 1 or 2, characterized that it is a process for screening drugs or cosmetics is. Method according to one of claims 1-3, characterized that the Cell aggregates mammalian cell aggregates, especially human cell aggregates. Method according to one of Claims 14, characterized that the substance to be tested is a protein, peptide, nucleic acid, e.g. DNA, RNA or a derivative thereof, or a low-molecular chemical Substance is. Method according to one of claims 1-5, characterized in that that the multipotent or pluripotent adult stem cells from exocrine glandular tissue were isolated. Method according to Claim 6, characterized that it around the exocrine glandular tissue acinar Tissue acts. Method according to one of the preceding claims 1-7, characterized characterized in that Effect of the substance a change morphology or proliferative capacity, capacity for growth or the viability or another activity of all cell types or specific cell types of organoid bodies. Method according to one of the preceding claims 1-8, characterized characterized in that Evidence of the effect of the chemical substance on the presence of evidence or absence or amount of a marker substance derived from specific or all cell types of the organoid body is formed. Method according to claim 9, characterized in that that the Evidence of the effect of the chemical substance on the presence of evidence or absence or amount of a marker protein. A method according to claim 10, characterized in that the marker protein by binding of dyes, antibodies or receptors, by a Western blot, by enzymatic or other Aktivi the marker protein is detected. Method according to claim 9, characterized in that that the Evidence of the effect of the chemical substance on the presence of evidence or absence or amount of a nucleic acid, e.g. DNA or RNA or a derivative thereof. Method according to one of Claims 1-12, characterized that the Proof of the effect of the chemical substance the application of a or more than one method from the group of protein assays, immunoassays, 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, microscopic and spectroscopic detection methods includes. Method according to one of claims 1-13, characterized that the Cell types of organoid bodies from the group consisting of osteoblasts, osteoclasts, chondrocytes, adipocytes, Fibroblasts, muscle cells, endothelial cells, epithelial cells, hematopoietic Cells, sensory cells, endocrine and exocrine glandular cells, Glial cells, neuronal cells, oligodendrocytes, blood cells, intestinal cells, Heart, lung, liver, kidney or pancreas cells are selected. Method according to one of the preceding claims 1-14, characterized characterized in that Cell type composition of organoid bodies by culturing in a medium which cell type-specific differentiation and / or Contains growth factors, was determined. Method according to claim 15, characterized in that that the Growth and / or differentiation factors consisting of the group from bFGF, VEGF, DMSO and isoproterenol, fibroblast growth factor 4 (FGF4), hepatocyte growth factor (HGF), TGF-beta1, EGF, KGF, retinoic acid, beta-NGF, BMP-4 and Activin-A selected are. Method according to one of claims 1-16, characterized that two or more of the various cell types of the organoid body tissues. or organ-like Form structures. Method according to claim 17, characterized in that that two or more of the different cell types of organoid body neuromuscular structures or forming glial nerve cell structures or skin cell structures. Method according to claim 17 or 18, characterized that the Test substance a change causes these structures. Method according to claim 19, characterized that the change a destruction structures, inhibition or stimulation of the development of structures and / or a change the activity one or more cell types that make up these structures, is. Method according to one of the preceding claims 1-20, characterized characterized in that the organoid body in cavities, Matrices or other carrier and / or shaping systems. Method according to one of the preceding claims 1-21, characterized characterized in that two or more test substances at the same time or in succession with the same organoid bodies be tested. Apparatus for carrying out the method according to one the claims 1-22, comprising the organoid body in a suitable container, Carrier- or shaping system, means for contacting the organoid body with a substance to be tested and means for detecting a morphological or otherwise altering these organoid bodies or the cell types contained therein. Kit to carry out of the method according to any one of claims 1-22, comprising the organoids body as defined in claim 1 in a suitable culture medium for Maintaining the organoid body. Kit according to claim 24, characterized in that the organoid body in cavities, Matrices or other carrier and / or shaping systems. A kit according to claim 24 or 25, further comprising means for contacting the organoid body with a test to be tested Substance. A kit according to any of claims 24-26, further comprising means to prove a morphological or other change this organoid body or the cell types contained therein and optionally further Reagents and excipients. Kit to carry out of the method according to any one of claims 1-22, comprising multipotent or pluripotent adult stem cells that make up the organoid body in claim 1 can be produced in a suitable culture medium for the maintenance of these adult stem cells, as well as reagents, including optionally differentiation factors, and culture media to organoid body a desired one Produce cell type composition. A kit according to claim 28, further comprising means for Contacting the organoid body with a substance to be tested. A kit according to claim 28 or 29, further comprising means to prove a morphological or other change this organoid body or the cell types contained therein and optionally further Reagents and excipients.