EP1747459A1 - Systemes de test multicellulaires - Google Patents

Systemes de test multicellulaires

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Publication number
EP1747459A1
EP1747459A1 EP05707686A EP05707686A EP1747459A1 EP 1747459 A1 EP1747459 A1 EP 1747459A1 EP 05707686 A EP05707686 A EP 05707686A EP 05707686 A EP05707686 A EP 05707686A EP 1747459 A1 EP1747459 A1 EP 1747459A1
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EP
European Patent Office
Prior art keywords
cells
organoid
bodies
cell
organoid bodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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EP05707686A
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German (de)
English (en)
Inventor
Charli Kruse
Günter FUHR
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Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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Priority claimed from DE102004025080A external-priority patent/DE102004025080B4/de
Application filed by Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV filed Critical Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Publication of EP1747459A1 publication Critical patent/EP1747459A1/fr
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0677—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/04—Screening or testing on artificial tissues

Definitions

  • 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.
  • tissue cultures from explanted tissue samples see, for example, US 5726009 and US 2002/192638
  • cultures of differentiated cells obtained from stem cells have been used in vitr ⁇ 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.
  • 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.
  • various special cell cultures have to be prepared and tested.
  • 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.
  • 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.
  • 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.
  • it is acinar tissue.
  • the acinar tissue very particularly preferably originates from the pancreas, the parotid gland or the submaxillary gland.
  • feeder cells 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.
  • 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.
  • organoid bodies or organoid bodies.
  • organoid bodies or organoid bodies.
  • 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.
  • tissue bodies are also called “tissue bodies” at this stage to distinguish them from simple cell aggregates.
  • 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.
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • vascular endothelial growth factor (“vascular endothelial growth factor”), DMSO and isoproterenol, fibroblast growth actuator 4 (FGF4), hepatocyte
  • HGF Growth factor
  • TGF-beta transforming growth factor betal
  • EGF epidermal growth factor
  • KGF keratinocyte growth factor
  • cortisone keratinocyte growth factor
  • retinoic acid for the increased formation of nerve, heart and kidney cells
  • beta-NGF beta nerve growth factor
  • BMP-4 "bone morphogenic protein 4”
  • 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,
  • bone cells osteoblasts and osteoclasts
  • chondrocytes eg skin and tendon fibroblasts
  • fibroblasts eg skin and tendon fibroblasts
  • muscle cells eg skin and tendon fibroblasts
  • endothelial cells e.g 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.
  • 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.
  • 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.
  • test substance 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • direct visual and optical detection methods including cytometric, microscopic and colorimetric methods, can be used favorably.
  • the effect can be a change in the activity of specific or all cell types of the organoid body.
  • 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.
  • 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 A
  • 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.
  • the binding partner will preferably be a labeled or labelable antibody.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the organoid bodies 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a gradient e.g. pH, electrochemical, signal factors, etc.
  • 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.
  • the organoid bodies are located in cavities, for example microchannels or capillaries, or in matrices.
  • the means for carrying out the method according to the invention are provided in the form of a kit.
  • kits 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.
  • 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.
  • 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.
  • 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 ⁇ -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 ⁇ -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).
  • 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 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
  • 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.
  • h endocrine granules
  • 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 ⁇ -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 ⁇ -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.
  • 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).
  • 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.
  • a second step (12) approximately 400 to 800 cells are cultivated in 20 ⁇ l medium in hanging drops.
  • 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.
  • 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.
  • FIGS. 2-4 show microscopic and electron microscopic images of differentiated cells which were obtained from such organoid bodies or organoid bodies.
  • a neuromuscular network For example, the formation of a neuromuscular network can be observed:
  • glial fibrillary acidic protein glial fibrillary acidic protein
  • glial fibrillary acidic protein glial fibrillary acidic protein
  • the filamentous proteins did not extend through the entire cytoplasm, but were limited to areas next to the nerve cells.
  • 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).
  • 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.
  • cytokeratins 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.
  • 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 SMA for muscle cells (or myofibroblasts)
  • amylase and trypsin for exocrine gland cells insulin for endocrine gland cells
  • vigilin for strongly translating cells
  • cytokeratin for epidermal cells.
  • different types of cells could be morphologically characterized by electron microscopy, and cell-cell contacts could be found as a sign of cellular interactions.
  • 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 ,
  • HEPES stock solution pH 7.6
  • HEPES Eagle Medium pH 7.4
  • MEM Modified Eagle Medium
  • Isolation medium (pH 7.4) 32 ml HEPES-Eagle medium 8 ml 5% BSA in A. bidest. 300 ⁇ l 0.1 M CaC12 100 ⁇ l 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)
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS inactivated
  • 1 ml / 100 ml pen / strep 10000 U / l 0000 ⁇ g / ml
  • 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 ⁇ l ß-mercaptoethanol on 5 ml PBS) 5 ml non-essential amino acids (GIBCO BRL) 5 ml penicillin / Streptomycin (GIBCO BRL) (10000 U / 10000 ⁇ g / ml)
  • the nutrient medium and differentiation 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.
  • FCS fetal calf serum
  • 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).
  • 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.
  • carbogen Merase, Krefeld, Germany
  • 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.
  • 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.
  • 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 ⁇ m.
  • 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.
  • 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
  • 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
  • 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.
  • HEPES-Eagle medium pH 7.4
  • 0.1 mM HEPES buffer pH, 7.6
  • 70% was introduced into the pancre
  • 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.
  • organoid bodies organoid bodies
  • differentiated cells 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 ⁇ l 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 ⁇ l 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.
  • organoid bodies 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.
  • 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
  • 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.
  • stem cells after the 42nd day of cultivation were preferably stem cells after the 42nd day of cultivation used.
  • 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.
  • 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.
  • 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.
  • 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.
  • individual OBs with pipette tips were detached and transferred by gentle suction, then e.g. Observation under the inverse microscope.
  • the primary antibodies were against the protein gene product 9.5 (PGP 9.5, polyclonal rabbit antibody, 1: 400, Ultraclone, Drei Wight), neurofilaments (NF-Pan-Cocktail, polyclonal rabbit antibody, 1: 200, Biotrend, Germany), ⁇ - smooth muscle actin ( ⁇ -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, kugler 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 monoclo
  • 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).
  • 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.
  • organoid bodies of a batch which have been grown together e.g. produced by separating suitable organoids and re-enlarging to the desired number
  • organoids usually at least 6 in a group
  • 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).
  • 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 ⁇ -SMA ( ⁇ -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.
  • ⁇ -SMA smooth muscle actin
  • NF neurofilaments
  • 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 ⁇ -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.
  • HGF hepatocyte growth factor

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Abstract

L'invention concerne des procédés servant à tester des substances en utilisant des systèmes de test in vitro multicellulaires, notamment de type organes. L'invention concerne également des dispositifs et des nécessaires pour exécuter ces procédés.
EP05707686A 2004-05-21 2005-03-02 Systemes de test multicellulaires Ceased EP1747459A1 (fr)

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DE102007028423A1 (de) 2007-06-20 2008-12-24 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren und Vorrichtung zur Bildung von Aggregaten biologischer Zellen
DE102016217174A1 (de) * 2016-09-09 2018-03-15 Henkel Ag & Co. Kgaa In-vitro Verfahren zur Identifizierung und Analyse von Sekretionsproteinen unter Verwendung eines dreidimensionalen Zellkulturmodells der Schweißdrüse

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DE10362002B4 (de) * 2003-06-23 2006-10-12 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Adulte pluripotente Stammzellen

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