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DE102007006736A1 - In-vitro test kit for animal experiment-free determination of sensitizing potential of substance, has coproculture of human keratinocyte and human monocyte - Google Patents

In-vitro test kit for animal experiment-free determination of sensitizing potential of substance, has coproculture of human keratinocyte and human monocyte Download PDF

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DE102007006736A1
DE102007006736A1 DE200710006736 DE102007006736A DE102007006736A1 DE 102007006736 A1 DE102007006736 A1 DE 102007006736A1 DE 200710006736 DE200710006736 DE 200710006736 DE 102007006736 A DE102007006736 A DE 102007006736A DE 102007006736 A1 DE102007006736 A1 DE 102007006736A1
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Reinhard Wanner
Dagmar Briechle
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    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The in-vitro test kit has a coproculture of human keratinocyte and human monocytes. The monocyte is generated during the coproculture of cytokine interleukin-4 (IL-4), granulocyte monocyte colony stimulating factor (GM-CSF) and transforming growth factor (TGF)beta by dendritic cells. The coprocultured arborescent cells locker overlie on the adherent keratinocyte or float in the cell culture medium. An independent claim is also included for a method for manufacturing an in-vitro test kit for animal experiment-free determination of the sensitizing potential of a substance.

Description

Die vorliegende Erfindung betrifft ein in-vitro Testkit zur tierversuchsfreien Bestimmung des sensibilisiernden Potentials einer Substanz. Das Testkit besteht aus einer lockeren Kokultur aus aktivierten humanen basalen Keratinozyten und aus beweglichen humanen Dendritischen Zellen. Die vorliegende Erfindung umfasst des weiteren das Herstellungsverfahren dieses Testkits.The The present invention relates to an in vitro test kit for animal-free Determination of the sensitizing potential of a substance. The Test Kit consists of a loose co-culture of activated human basal keratinocytes and from mobile human dendritic Cells. The present invention further includes the manufacturing method this test kit.

Stand der TechnikState of the art

Die Kontaktdermatitis ist ein weit verbreitetes berufsbezogenes oder umweltbedingtes Gesundheitsproblem.The Contact dermatitis is a widespread occupation-related or environmental health problem.

Die Begriffe Kontaktdermatitis und Kontaktekzem (contact hypersensitivity) beschreiben Hautreaktionen nach Kontakt mit niedermolekularen Substanzen (typisch < 500 kDa). Ist diese Hautreaktion durch Aktivitäten des Immunsystems vermittelt, handelt es sich um ein allergisches Kontaktekzem/eine allergische Kontaktdermatitis. Die auslösenden Chemikalien werden als Allergene, Haptene oder als sensibilisierende Stoffe bezeichnet. Ist das Immunsystem nicht an der Ausbildung der Hautreaktionen beteiligt, werden die auslösenden Stoffe als Irritantien oder Toxine bezeichnet (Nomenklatur: Johansson et al., 2001 ).The terms contact dermatitis and contact dermatitis (contact hypersensitivity) describe skin reactions after contact with low-molecular substances (typically <500 kDa). If this skin reaction is mediated by activities of the immune system, it is an allergic contact eczema / allergic contact dermatitis. The triggering chemicals are referred to as allergens, haptens or as sensitizing substances. If the immune system is not involved in the formation of skin reactions, the triggering substances are referred to as irritants or toxins (Nomenclature: Johansson et al., 2001 ).

Das Potential einer Chemikalie, eine allergische Kontaktdermatitis auslösen zu können, wird als sensibilisierendes Potential (sensitization potential) bezeichnet. Der derzeit gängige und von der OECD anerkannte Test, mit dem ein solches Potential ermittelt wird, ist der lokale Lymphknotentest an der Maus (mouse local lymph node assay, LLNA) ( Kimber and Weisenberger 1989 ; OECD. Guideline 429, 2002 ). Durch die Einführung dieses Tests konnte gegenüber seinen Vorgängern die Anzahl der benötigten Tiere reduziert und auch deren Belastung vermindert werden. Ethisch vertretbarer wäre jedoch ein tierversuchsfreies Testverfahren (in-vitro assay). Außerdem liefert LLNA teilweise falsche Ergebnisse, wie beispielweise bezüglich der Substanz Benzocaine ( Basketter et al., 1995 ), so dass auch die Sicherheit der Testaussage betreffend weitere Fortschritte notwendig sind.The potential of a chemical to induce allergic contact dermatitis is called a sensitization potential. The current and accepted by the OECD test to determine such a potential is the mouse local lymph node assay (LLNA) ( Kimber and Weisenberger 1989 ; OECD. Guideline 429, 2002 ). The introduction of this test compared to its predecessors, the number of animals required reduced and their burden can be reduced. Ethically more acceptable, however, would be an animal-free test method (in vitro assay). In addition, LLNA provides partially incorrect results, such as the substance benzocaine ( Basketter et al., 1995 ), so that the safety of the test statement regarding further progress is necessary.

Langerhanszellen sind unreife Dendritische Zellen in der Haut ( Banchereau and Steiman, 1998 ). Dendritische Zellen besitzen die Fähigkeit, eine primäre Immunreaktion zu initiieren und sind damit auch an der Initiation der allergischen Kontaktdermatitis zentral beteiligt. Sie können erkennen, ob sich in ihrer Umgebung Gefahr-signalisierende Moleküle befinden. Kontakt mit Gefahr-signalisierenden Molekülen induziert die Reifung der Dendritischen Zelle zur Antigen-präsentierenden Zelle, die Allergene in passender Form dem spezifischen Immunsystem zuführen kann. In der Folge werden damit auch T- und B-Lymphozyten sowie weitere Zellen in die Reaktion auf Allergenkontakt einbezogen (Übersicht in: Reis e Sousa, 2006 ). Es läge daher nahe, in einem alternativen in-vitro Testverfahren Langerhanszellen zu kultivieren und geeignete Reaktionen der Zellen auf Allergenkontakt zu messen (Übersicht in: Ryan et al., 2005 ). Dies ist derzeit nicht möglich, da kein Zellkulturverfahren beschrieben ist, mit dem aus der menschlichen Haut isolierte Langerhanszellen ausreichend lang reaktiv gehalten werden können ( Peiser et al., 2003 ).Langerhans cells are immature dendritic cells in the skin ( Banchereau and Steiman, 1998 ). Dendritic cells have the ability to initiate a primary immune response and are thus centrally involved in the initiation of allergic contact dermatitis. They can see if there are danger-signaling molecules in their environment. Contact with danger-signaling molecules induces the maturation of the dendritic cell to the antigen-presenting cell, which can deliver allergens in appropriate form to the specific immune system. As a result, T and B lymphocytes and other cells are also involved in the reaction to allergen contact (overview in: Rice e Sousa, 2006 ). It would therefore be natural to cultivate Langerhans cells in an alternative in-vitro test method and to measure suitable reactions of the cells to allergen contact (overview in: Ryan et al., 2005 ). This is currently not possible, since no cell culture process is described with which Langerhans cells isolated from human skin can be kept sufficiently reactive ( Peiser et al., 2003 ).

Es liegen keine menschlichen Zelllinien vor, die authentische Dendritische Zellen ersetzen könnten. Versuche mit den Zelllinien. THP-1, KG-1 oder MUTZ-3 zeigten, dass diese Zellen nur auf extrem starke Allergene reagieren und das in einem Konzentrationsbereich, der an der Grenze zu toxischer Aktivität liegt (borderline concentrations) ( Yoshida et al., 2003 ; Azam et al., 2006 ). Außerdem neigen Zelllinien zu genetischer Instabilität.There are no human cell lines that could replace authentic dendritic cells. Trials with the cell lines. THP-1, KG-1 or MUTZ-3 showed that these cells react only to extremely strong allergens and in a concentration range that is borderline to toxic activity (borderline concentrations) ( Yoshida et al., 2003 ; Azam et al., 2006 ). In addition, cell lines are prone to genetic instability.

Es wurden Verfahren entwickelt, mit denen sich aus Vorläufern Dendritische Zellen generieren lassen, die in ihren Leistungen authentischen Dendritischen Zelle nahe kommen. Vorläuferzellen sind dabei Monozyten, die aus adultem peripherem Blut oder aus Leukozytenkonzentrat (buffy coat) gewonnen werden oder CD34+-Blutstammzellen aus Nabelschnurblut oder Knochenmark. Die Vorläuferzellen differenzieren, durch Cytokine stimuliert, zu unreifen Dendritischen Zellen. Eingesetzt wurden die Cytokine IL-4 zusammen mit GM-CSF, teilweise auch zusätzlich mit TNF-α ( Sallusto and Lanzavecchia, 1994 ; Schuler et al., 1995 ; Steckel et al., 1995 ; Chapius et al., 1997 ; Caux et al., 1996 ). Der Begriff unreife Dendritische Zelle bedeutet hier, dass es sich bei diesen Zellen um differenzierte Zellen handelt, die auf einen Reifestimulus durch pathogenassoziierte Moleküle oder Allergene warten und die nach Kontakt damit zur Antigen-präsentierenden Zelle reifen können. Dieser Typ Dendritischer Zellen wurde ausgiebig auf seine Fähigkeit untersucht, sensibilisierende Substanzen in Testverfahren detektieren zu können. Zusammenfassend lässt sich sagen, dass diese Dendritische Zellen, wenn sie solitär kultiviert werden, als Testsystem für Allergene ungeeignet sind. Detektierbare Konzentrationen von Kontaktallergenen liegen im subtoxischen bis bereits toxischen Bereich, und eine Unterscheidung zwischen Allergenen und Irritantien ist kaum möglich ( Hulette et al., 2002 ; Straube et al., 2005 ; Übersicht in: Ryan et al., 2005 ). Außerdem zeigte sich, insbesondere für aus CD14+ Monozyten gewonnenen Dendritischen Zellen, eine erhebliche Spendervarianz bezüglich der Testergebnisse, so dass Testzellen verschiedener Spender gepoolt werden mussten ( Pichowski et al., 2000 ; Aeby et al., 2004 ).Methods have been developed to generate precursor dendritic cells that are close in their performance to authentic dendritic cells. Precursor cells are monocytes derived from adult peripheral blood or buffy coat or CD34 + blood stem cells from umbilical cord or bone marrow. The precursor cells differentiate, stimulated by cytokines, to immature dendritic cells. The cytokines IL-4 were used together with GM-CSF, in some cases additionally with TNF-α ( Sallusto and Lanzavecchia, 1994 ; Schuler et al., 1995 ; Steckel et al., 1995 ; Chapius et al., 1997 ; Caux et al., 1996 ). The term immature dendritic cell means here that these cells are differentiated cells that are waiting for a mature stimulus by pathogen-associated molecules or allergens and that can mature after contact therewith to the antigen-presenting cell. This type of dendritic cell has been extensively studied for its ability to detect sensitizing substances in test procedures. In summary, these dendritic cells, when solitary cultured, are unsuitable as a test system for allergens. Detectable concentrations of contact allergens are in the subtoxic to already toxic range, and a distinction between allergens and irritants is hardly possible ( Hulette et al., 2002 ; Straube et al., 2005 ; Overview in: Ryan et al., 2005 ). In addition, especially for CD14 + monocytes derived dendritic cells, a significant donor variance with respect to the test results, so that test cells from different donors had to be pooled ( Pichowski et al., 2000 ; Aeby et al., 2004 ).

Ein weiteres Problem bezüglich einer Anwendung generierter Dendritischer Zellen in einem Testsystem auf sensibilisierende Substanzen ist, dass diese Zellen in Kultur zu einer spontanen, von Stimulation von außen unabhängigen Reifung neigen. Detektiert werden soll aber gerade eine potentielle allergeninduzierte Reifung. Ein Zellkulturverfahren, aus dem Dendritische Zellen mit typischen Merkmalen authentischer Langerhanszellen resultieren, benutzt für die Generierung aus CD14+ Zellen eine Kombination der Cytokine IL-4, GM-CSF und TGF-β. So generierte Zellen neigen nicht zu spontaner Reifung, aber solitär kultiviert bleiben auch bei diesem System überzeugende Reaktionen der Zellen auf Allergene aus ( Geissmann et al., 1998 ; Geissmann et al., 1999 ; Aiba et al., 2000 ; Peiser et al., 2004 ).Another problem with using sensitized dendritic cells in a test system for sensitizing substances is that these cells in culture are prone to spontaneous, external stimulation-independent maturation. But to be detected is currently a potential allergen-induced maturation. A cell culture procedure that yields dendritic cells with typical characteristics of authentic Langerhans cells uses a combination of cytokines IL-4, GM-CSF and TGF-β for the generation of CD14 + cells. So generated cells do not tend to spontaneous maturation, but solitary cultivated remain in this system convincing reactions of the cells to allergens ( Geissmann et al., 1998 ; Geissmann et al., 1999 ; Aiba et al., 2000 ; Peiser et al., 2004 ).

Niedermolekulare Allergene sind Haptene, die an Proteine gebunden werden müssen, bevor sie Immunreaktionen auslösen können. An dieser Proteinbindung sind Keratinozyten beteiligt. Keratinozyten könnten außerdem zur Reifung Dendritischer Zellen nach Allergenkontakt beitragen, indem sie Gefahrsignale an die Dendritischen Zellen senden ( Vandebriel et al., 2005 ). In einem zellbasierten Testsystem für sensibilisierende Substanzen sollten daher Dendritische Zellen und Keratinozyten kombiniert benutzt werden.Low molecular weight allergens are haptens that need to be bound to proteins before they can trigger immune responses. Keratinocytes are involved in this protein binding. Keratinocytes may also contribute to the maturation of dendritic cells after allergen exposure by sending danger signals to the dendritic cells ( Vandebriel et al., 2005 ). Dendritic cells and keratinocytes should therefore be used in combination in a cell-based sensitizer test system.

Primäre humane Keratinozyten können in-vitro zu einer dreidimensionalen Zellkultur herangezüchtet werden, die Zellschichten verschiedener Differenzierungsstufen aufweist. Einige Modelle beinhalten alle epidermalen Schichten, von der basalen- bis zur Hornschicht. Die basalen Keratinozyten liegen meist auf einer Schicht von Fibroblasten, die ihrerseits auf eine Kollagenmatrix aufgebracht wurden. Solche Kulturen werden als Hautäquivalent, Epidermis-Äquivalent, organtypisches Hautmodell oder rekonstruierte Epidermis bezeichnet ( Bell et al., 1981 ; Limat and Hunziker, 2002 ). Epidermis-Äquivalente unter anderem zur Testung auf Irrttantien und Toxine werden beispielsweise angeboten von den Firmen SkinEthic (Nizza, Frankreich), CellSystems (St. Katharinen, Deutschland), Euroderm (Leipzig, Deutschland) oder MatTek Inc. (Massachussets, USA).Primary human keratinocytes can be cultured in vitro to form a three-dimensional cell culture which has cell layers of different differentiation levels. Some models include all epidermal layers, from the basal to the horny layer. The basal keratinocytes are usually located on a layer of fibroblasts, which in turn were applied to a collagen matrix. Such cultures are referred to as skin equivalent, epidermis equivalent, organtypic skin model or reconstructed epidermis ( Bell et al., 1981 ; Limat and Hunziker, 2002 ). Epidermis equivalents for, inter alia, testing for irritants and toxins are offered, for example, by the companies SkinEthic (Nice, France), CellSystems (St. Katharinen, Germany), Euroderm (Leipzig, Germany) or MatTek Inc. (Massachussets, USA).

Ein in-vitro Testsystem zur Bestimmung des sensibilisierenden Potentials einer Substanz, das auf derart rekonstruierter Epidermis basiert, sollte zusätzlich Dendritische Zellen enthalten. Wird allerdings Epidermis in organische Kultur genommen, wandern die Langerhanszellen spontan aus der Epidermis in das Kulturmedium aus ( Larsen et al., 1990 ). Es ist nicht bekannt, ob in der Epidermis zwischen suprabasalen Keratinozyten und Langerhanszellen tatsächlich feste Zellkontakte bestehen oder ob schon unreife Langerhanszellen beweglich sind. Vereinzelt wurde von mehrschichtigen Hautäquivalenten berichtet, in die sich Vorläufer Dendritischer Zellen kohärent integrieren und dann differenzieren ließen ( Facy et al., 2004 ; Schaerli et al., 2005 ). Im Patent EP 0789074 ist beschrieben, wie CD34+ Vorläuferzellen in ein Epidermisäquivalent eingebracht werden, differenzieren und sich dann suprabasal in das Epidermisäquivalent integrieren. Die Offenlegungsschrift DE 102004061289 A1 beschreibt die Integration von Immunzellen in ein Testkit, das aus einem geschichteten Epidermisäquivalent besteht. Unklar ist, wieso, anders als in-vivo, die Dendritischen Zellen im Hautäquivalent gebunden werden und es bleiben sollen, bis ihre Reifung induziert wird. Es gibt kein anerkanntes Testkit zur in-vitro Bestimmung des sensibilisierenden Potentials einer Substanz, das auf geschichteten Hautäquivalenten mit integrierten Dendritischen Zellen basiert. Probleme bereiten eine hohe Spendervarianz bezüglich der Testergebnisse, schlechte Kohärenz der sich im Modell befindlichen Zellen sowie der Einfluss der Fibroblasten auf die Testergebnisse.An in vitro test system for determining the sensitizing potential of a substance based on such reconstructed epidermis should additionally contain dendritic cells. However, if epidermis is taken into organic culture, the Langerhans cells migrate spontaneously from the epidermis into the culture medium ( Larsen et al., 1990 ). It is not known whether there are any solid cell contacts in the epidermis between suprabasal keratinocytes and Langerhans cells, or whether immature Langerhans cells are mobile. There have been isolated reports of multi-layered skin equivalents, in which precursors of dendritic cells can be coherently integrated and then differentiated ( Facy et al., 2004 ; Schaerli et al., 2005 ). In the patent EP 0789074 describes how CD34 + progenitor cells are introduced into an epidermis equivalent, differentiate and then integrate suprabasally into the epidermis equivalent. The publication DE 102004061289 A1 describes the integration of immune cells into a test kit consisting of a layered epidermis equivalent. It is unclear why, unlike in vivo, the dendritic cells are bound in the skin equivalent and should remain until their maturation is induced. There is no recognized test kit for in vitro determination of the sensitizing potential of a substance based on stratified skin equivalents with integrated dendritic cells. There are problems with high donor variance in terms of test results, poor coherence of the cells in the model and the influence of the fibroblasts on the test results.

Vor diesem Hintergrund präsentiert die vorliegende Erfindung ein Testkit zur Bestimmung des sensibilisierenden Potentials einer Substanz, bestehend aus einer lockeren Kokultur einer einschichtigen Lage von humanen nicht-differenzierenden Keratinozyten und aus beweglichen Dendritischen Zellen, die mittels IL-4, GM-CSF und TGF-β aus humanen allogenen CD14+ Monozyten in Anwesenheit der Keratinozyten generiert werden.Against this background, the present invention presents a test kit for determining the sensitizing potential of a substance consisting of a loose co-culture of a monolayer of human non-differentiating keratinocytes and of mobile dendritic cells produced by IL-4, GM-CSF and TGF-β are generated from human allogeneic CD14 + monocytes in the presence of keratinocytes.

Die im Testkit eingesetzten Keratinozyten sind humane normale Keratinozyten, die während ihrer ersten Kultur durch „Antrypsinieren" hinsichtlich starker Adhärenz an Zellkultur-Plastikmaterial selektiert worden sein können.The keratinocytes used in the kit are human normal keratinocytes, who during their first culture by "Antrypsinieren" for strong adherence to cell culture plastic material may have been selected.

Die kokultivierten Keratinozyten erreichen ein aktiviertes Stadium, das vergleichbar ist mit dem Stadium von an der Wundheilung beteiligten Keratinozyten. Dies ist z. B. an der hohen Sekretionsrate der Metalloproteinase MMP-9 erkennbar.The cocultivated keratinocytes reach an activated stage, comparable to the stage of wound healing Keratinocytes. This is z. For example, the high secretion rate of metalloproteinase MMP-9 recognizable.

Das Zellkulturmedium muss eine Differenzierung der Keratinozyten nicht unterstützen, z. B. durch eine niedrige Calciumkonzentration.The Cell culture medium does not need differentiation of keratinocytes support, z. B. by a low calcium concentration.

Das vorgeschlagene Testkit kann sensibilisierende Substanzen anhand der Erhöhung von Reifungsmarkern Dendritischer Zellen detektieren. Bevorzugt wird die CD86-Expression auf den Dendritischen Zellen per FACS-Analyse gemessen (verwendeter Antikörper z. B. R-Phycoerythrin mouse anti-human CD86 (z. B. von BD, Heidelberg, Deutschland)). Weiterhin bevorzugt erfolgt für die FACS-Analyse eine Doppelfärbung zur Identifizierung der Zellen, z. B. CD86/CD11c (verwendeter Antikörper z. B. mouse anti-human CD11c:FITC (z. B. von Serotec, Düsseldorf, Deutschland)) oder CD86/HLA-DR (verwendeter Antikörper z. B. FITC- conjugated mouse anti-human HLA-DR (z. B. von BD)) oder CD86/CD1c (z. B. human CD1c-FITC von Miltenyi, Bergisch Gladbach, Deutschland). Dabei sind Sensitivität und Reproduzierbarkeit der Detektion sensibilisierender Substanzen deutlich besser als bei bisherigen Assays.The proposed test kit can detect sensitizing substances by increasing the maturity markers of dendritic cells. Preferably, CD86 expression on the dendritic cells is measured by FACS analysis (used antibody eg R-phycoerythrin mouse anti-human CD86 (eg from BD, Heidelberg, Germany)). Further preferred for the FACS analysis is a double staining to identify the cells, eg. CD86 / CD11c (used antibody eg mouse anti-human CD11c: FITC (eg from Serotec, Dusseldorf, Germany)) or CD86 / HLA-DR (used antibody eg FITC-conjugated mouse anti-human HLA-DR (eg from BD)) or CD86 / CD1c (eg human CD1c-FITC from Miltenyi, Bergisch Gladbach, Germany). Sensitivity and reproducibility of the detection of sensitizing substances are significantly better than in previous assays.

Dosis-Wirkungs-Beziehungen bislang ungeprüfter Substanzen können bestimmt und mit bekannten Allergenen verglichen werden, deren Potential z. B. aus LLNA-Assays verifiziert ist. Damit lässt sich die Stärke des sensibilisierenden Potentials einer Testsubstanz in Katagorien bezüglich ihrer Wirkstärke einteilen. Es können effektive Konzentrationen angegeben werden.Dose-response relationships Unchecked substances can be determined and compared with known allergens, their potential z. B. is verified from LLNA assays. This can be the strength of the sensitizing potential of a test substance divide into categories according to their potency. Effective concentrations can be specified.

Eine messbare Reifung der Dendritischen Zellen durch Kontakt mit sensibilisierenden Substanzen erfolgt bereits weit unterhalb irritierender oder toxischer Konzentrationen. Die Viabilität der Dendritischen Zellen kann durch die Färbrate mit 7-AAD (z. B. von BD, Heidelberg, Deutschland) oder Propidiumiodid (z. B. von Sigma, Deisenhofen, Deutschland) bestimmt werden. Damit kann das Testkit sensibilisierend und irritierend wirkende Konzentrationen einer Substanz differenzieren.A measurable maturation of the dendritic cells by contact with sensitizing Substances are already far below irritating or toxic Concentrations. The viability of dendritic cells can be prepared by the staining with 7-AAD (eg from BD, Heidelberg, Germany) or propidium iodide (eg from Sigma, Deisenhofen, Germany). This can make the test kit sensitizing and differentiating irritant concentrations of a substance.

Die Zugabe des Cytokins TGF-β bedingt, dass die Dendritischen Zellen im Testkit nicht zu spontaner Reifung neigen.The Addition of the cytokine TGF-β causes the dendritic Cells in the test kit are not prone to spontaneous maturation.

Es besteht kein nennenswerter Einfluss der Zell-Spender auf die Testergebnisse. Es ist nicht notwendig, Spender zu poolen.It There is no appreciable influence of the cell donors on the test results. It is not necessary to pool donors.

Da keine Zellen des spezifischen Immunsystems ins Testkit integriert sind, müssen Keratinozyten und Dendritische Zellen, bzw. ihre Vorläufer nicht vom selben Spender stammen.There no cells of the specific immune system integrated into the test kit are keratinocytes and dendritic cells, or their forerunners did not come from the same donor.

Das Testkit lässt sich aus cryokonservierten Zellen herstellen.The Test kit can be made from cryopreserved cells.

Die Kokultur erfolgt serumfrei (z. B. in KGM (PromoCell, Heidelberg, Deutschland)).The Co-culture is serum-free (eg in KGM (PromoCell, Heidelberg, Germany)).

Zitierte LiteraturQuoted literature

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Das Verfahren zur Herstellung eines solchen Testkits wird angegeben. Zudem wird ein Anwendungsverfahren angegeben. Die folgenden Beispiele erläutern die Erfindung, ohne dass sie einschränkend zu verstehen sind.The A method of making such a test kit is given. In addition, an application method is specified. The following examples explain to understand the invention without being limiting are.

Beispiel für die Herstellung eines Testkits, bestehend aus einer lockeren Kokultur von humanen Keratinozyten und beweglichen humanen Dendritischen Zellen, die aus Vorläuferzellen durch die Cytokine IL-4, GM-CSF und TGF-β in Anwesenheit der Keratinozyten generiert werden.example for the production of a test kit, consisting of a loose coculture of human keratinocytes and human mobile Dendritic cells from the progenitor cells through the Cytokines IL-4, GM-CSF and TGF-β in the presence of keratinocytes to be generated.

1. Vorbereitende Arbeiten1. Preparatory work

1.1 Selektion und Cryokonservierung primärer humaner Keratinozyten1.1 Selection and Cryopreservation Primary human keratinocytes

Normale menschliche Hautproben sind mit der Erlaubnis lokaler Ethikkomitees als übrigbleibendes Material nach chirurgischen Eingriffen, wie einer Mammareduktion, erhältlich. Alle Arbeiten erfolgen unter sterilen Bedingungen. Die Haut wird in Streifen geschnitten und 18 Stunden lang bei 4°C in einem Medium inkubiert, das 3 RMB units Dispase I (z. B. von Roche, Mannheim, Deutschland) in PBS (z. B. von Gibco, Invitrogen Corporation, Karlsruhe, Deutschland) enthält. Danach lässt sich die Dermis abziehen. Die epidermalen Streifen werden 15 Minuten lang bei 37°C in einer Lösung inkubiert, die 0.25% Trypsin (z. B. von Biochrom, Berlin, Deutschland) und 0.01% DNase (z. B. von Roche, Mannheim, Deutschland) in PBS enthält. Dabei zerfällt die Epidermis in eine Einzelzellsuspension, die durch ein 40 μM Zellsieb (z. B. von BD, Heidelberg, Deutschland) passagiert und anschließend dreimal mit PBS gewaschen wird. Die Keratinozyten werden dann in einem serumfreien Zellkulturmedium wie KGM-2 mit Supplement Mix (PromoCell, Heidelberg, Deutschland) resuspendiert und auf Zellkulturflaschen ausgesät, die für adherierende Säugetierzellen geeignet sind (z. B. Costar Cell Culture Flask, von Corning Incorporated, Schiphol-Rijk, Niederlande). Die Dichte wird auf 2–5 × 105 Zellen/cm2 (Durchmesser > 8 μm) eingestellt, und die Zellkulturflaschen werden in einem Brutschrank bei 37°C, 5% CO2 und 95% Luftfeuchtigkeit inkubiert. Am Tag nach der Aussaat und danach an jedem zweiten Tag werden die Zellen mit PBS gewaschen und mit frischem Medium versorgt. Etwa 8 Tage nach der Aussaat erfolgt bei erfolgreicher Bildung von Keratinozyteninseln eine Selektion durch „Antrypsinieren". Die Zellen werden 3 Minuten lang bei Raumtemperatur 1 × Trypsin (Biochrom, Berlin, Deutschland) ausgesetzt, wodurch nur kräftig haftende, viable Zellen nicht abschwimmen. Nach Stoppen der Trypsinaktivität und Waschen mit PBS werden die selektierten Zellen bis zur Konfluenz weiterkultiviert. Die Ernte der Zellen erfolgt durch Ablösen von den Zellkuturflaschen durch Inkubation mit 1× Trypsin für 15 Minuten bei 37°C. Dann wird 5% hitzeinaktiviertes fötales Kälberserum (z. B. von Gibco, Invitrogen Corporation, Karlsruhe, Deutschland) in PBS zugegeben. Nach zweimaligem Waschen in PBS werden die Zellen in hitzeinaktiviertem fötalem Kälberserum mit 10% DMSO (z. B. Hybrimax von Sigma, Deisenhofen, Deutschland) in einer Dichte von etwa 6 × 106 Zellen > 8 μm/ml resuspendiert, 1 ml-weise portioniert, langsam in einer Cryobox (z. B. in einem Cryo 1°C Freezing Container von Nalgene (Batavia, IL, USA)) auf –80°C heruntergekühlt und dann in flüssigem Stickstoff cryokonserviert. Jede Charge wird einem Endotoxintest unterzogen, bevorzugt dem Limulus amoebocyte lysate Test (BioWhittaker, Walkersville, MD, USA).Normal human skin specimens are available with the approval of local ethics committees as residual material after surgical procedures such as mammary reduction. All work is done under sterile conditions. The skin is cut into strips and incubated for 18 hours at 4 ° C in a medium containing 3 RMB units Dispase I (eg from Roche, Mannheim, Germany) in PBS (eg from Gibco, Invitrogen Corporation, Karlsruhe, Germany). Then the dermis can be removed. The epidermal strips are incubated for 15 minutes at 37 ° C in a solution containing 0.25% trypsin (eg from Biochrom, Berlin, Germany) and 0.01% DNase (eg from Roche, Mannheim, Germany) in PBS contains. The epidermis breaks down into a single cell suspension, which is passaged through a 40 μM cell strainer (eg from BD, Heidelberg, Germany) and then washed three times with PBS. The keratinocytes are then resuspended in a serum-free cell culture medium such as KGM-2 supplemented (PromoCell, Heidelberg, Germany) and seeded on cell culture flasks suitable for adherent mammalian cells (e.g., Costar Cell Culture Flask, Corning Incorporated, Schiphol). Rijk, The Netherlands). The density is adjusted to 2-5 × 10 5 cells / cm 2 (diameter> 8 μm), and the cell culture bottles are incubated in an incubator at 37 ° C, 5% CO 2 and 95% humidity. On the day after sowing and every other day thereafter, the cells are washed with PBS and supplied with fresh medium. Approximately 8 days after sowing, keratinocyte islands are selected for selection by "trypsinization." The cells are exposed to 1 × trypsin (Biochrom, Berlin, Germany) at room temperature for 3 minutes, whereby only vigorously adhering, viable cells do not float Stopping the trypsin activity and washing with PBS, the selected cells are further cultivated to confluence the cell culture bottles by incubation with 1 × trypsin for 15 minutes at 37 ° C. Then 5% heat-inactivated fetal calf serum (eg, from Gibco, Invitrogen Corporation, Karlsruhe, Germany) in PBS is added. After washing twice in PBS, the cells are resuspended in heat-inactivated fetal calf serum with 10% DMSO (eg, Hybrimax from Sigma, Deisenhofen, Germany) at a density of about 6 x 10 6 cells> 8 μm / ml, 1 ml-wise portioned, slowly cooled to -80 ° C in a cryobox (eg, in a Cryo 1 ° C freezing container from Nalgene (Batavia, IL)) and then cryopreserved in liquid nitrogen. Each lot undergoes an endotoxin test, preferably the Limulus amoebocyte lysate test (BioWhittaker, Walkersville, MD, USA).

1.2 Cryokonservierung humaner Monozyten1.2 Cryoconservation of human monocytes

Humane Monozyten werden bevorzugt aus Leukozytenkonzentrat (buffy coat, z. B. von DRK-Blutspendediensten, Deutschland) isoliert, können aber auch aus Vollblut gereinigt werden. Das Konzentrat wird 1:2 mit PBS verdünnt, und es wird eine Ficoll-(z. B. von Biochrom, Berlin, Deutschland)Dichtegradientenzentrifugation durchgeführt. Die Leukozytenfraktion wird entnommen und solange mit PBS gewaschen, bis die Überstände klar sind. Anschließend werden aus den Leukozyten die Monozyten bevorzugt durch magnetisches Sortieren (MACS) weiter aufgereinigt. Bevorzugt werden hier das Oberflächenantigen CD14 nach Herstellerprotokoll (CD14 MicroBeads, human (Miltenyi, Bergisch Gladbach, Deutschland)) oder das human Monocyte Isolation Kit II (Miltenyi) benutzt. Die isolierten CD14+-Monozyten werden in fötalem Kälberserum (z. B. von Gibco) mit 10% DMSO (z. B. Hybrimax von Sigma, Deisenhofen, Deutschland) in einer Dichte von 2–3 × 10 Zellen > 8 μm/ml resuspendiert und cyryokonserviert wie oben für die Keratinozyten beschrieben. Jede Charge wird einem Endotoxintest unterzogen, bevorzugt dem Limulus amoebocyte lysate test (BioWhittaker, Walkersville, MD, USA).Human monocytes are preferably isolated from leukocyte concentrate (buffy coat, eg from DRK blood donation services, Germany), but can also be purified from whole blood. The concentrate is diluted 1: 2 with PBS and a Ficoll (eg from Biochrom, Berlin, Germany) density gradient centrifugation is performed. The leukocyte fraction is removed and washed with PBS until the supernatants are clear. Subsequently, the monocytes are further purified from the leukocytes, preferably by magnetic sorting (MACS). The surface antigen CD14 according to the manufacturer's protocol (CD14 MicroBeads, human (Miltenyi, Bergisch Gladbach, Germany)) or the human monocyte isolation kit II (Miltenyi) are preferably used here. The isolated CD14 + monocytes are in fetal calf serum (eg from Gibco) with 10% DMSO (eg Hybrimax from Sigma, Deisenhofen, Germany) at a density of 2-3 x 10 cells> 8 μm / ml resuspended and cryopreserved as described above for the keratinocytes. Each lot undergoes an endotoxin test, preferably the Limulus amoebocyte lysate test (BioWhittaker, Walkersville, MD, USA).

1.3. Cryokonservierung humaner Leukozyten1.3. Cryoconservation of human leukocytes

Alternativ werden Monozyten über ihre im Vergleich zu anderen Leukozyten stärkere Adhärenz an Zellkulturschalen und an Keratinozyten angereichert. Für diese spätere Anwendung werden nach der Dichtegradientenzentrifugation die gewaschenen Leukozyten (PBMC) ohne weitere Aufreinigung cryokonserviert wie oben beschrieben.alternative monocytes are over their versus other leukocytes stronger adherence to cell culture dishes and to Enriched keratinocytes. For this later After the density gradient centrifugation, the washed leucocytes are used (PBMC) cryopreserved without further purification as described above.

2. Aussaat des Testkits2. Sow the test kit

2.1. Aussaat der cryokonservierten Keratinozyten2.1. Sow the cryopreserved keratinocytes

Cryokonservierte Keratinocyten, beschrieben in 1.1, werden in einem 37°C-Wasserbad aufgetaut und mehrfach in PBS gewaschen. Danach werden die Keratinozyten in ein serumfreies Zellkulturmedium überführt. Vorzugsweise wird KGM-2 mit Supplement Mix (PromoCell, Heidelberg, Deutschland) benutzt. Die Zelldichte wird so gewählt, dass sich nach der Aussaat 2–6 × 104 Zellen > 8 μm/cm2 in der Testschale (z. B. Costar Cell Culture Cluster, von Corning Incorporated, Schiphol-Rijk, Niederlande) befinden. Die Zellkulturen werden am nächsten Tag mit PBS gewaschen und mit frischem Zellkulturmedium versorgt. Die Kultivierung erfolgt solange bis die heranwachsenden Keratinozyteninseln die Fläche der Testschale halb bedecken (halbkonfluent). Dies wird innerhalb von 2–4 Tagen erreicht.Cryopreserved keratinocytes, described in 1.1, are thawed in a 37 ° C water bath and washed several times in PBS. Thereafter, the keratinocytes are transferred to a serum-free cell culture medium. Preferably, KGM-2 is used with Supplement Mix (PromoCell, Heidelberg, Germany). The cell density is chosen so that after sowing 2-6 × 10 4 cells> 8 μm / cm 2 are in the test dish (eg Costar Cell Culture Cluster, from Corning Incorporated, Schiphol-Rijk, Netherlands). The cell cultures are washed the next day with PBS and supplied with fresh cell culture medium. The cultivation takes place until the growing keratinocyte islands cover the area of the test dish half-confluent. This is achieved within 2-4 days.

2.2 Aussaat der cryokonservierten Monozyten2.2 Sowing the cryopreserved monocytes

Cryokonservierte Monozyten, beschrieben in 1.2, werden in einem 37°C-Wasserbad aufgetaut und mehrfach in PBS gewaschen. Danach werden die Monozyten in ein serumfreies Zellkulturmedium überführt, das für sie und für Keratinozyten gleichzeitig geeignet ist. Vorzugsweise wird KGM-2 mit Supplement Mix (PromoCell, Heidelberg, Deutschland) benutzt. Die Testschale, die bereits halbkonfluent adherierende Keratinozyten, wie in 2.1 beschrieben, enthält, wird mit PBS gewaschen, und dann wird die Flüssigkeit abgesaugt. Auf die Keratinozyten werden die Monozyten ausgesät. Die Zelldichte der Monozyten wird so gewählt, dass sich nach der Aussaat 2–5 × 105 Zellen der Monozytenpräparation > 8 μm/cm2 zusätzlich zu den Keratinozyten in der Testschale befinden. Die Monozyten adherieren zunächst an das Plastikmaterial und an die Keratinozyten, lösen sich aber nach einigen Stunden größtenteils wieder ab. Es entsteht eine Kokultur aus adhärenten Kerationzyten, auf denen locker Monozyten aufliegen. Die Viabilität der Monozyten wird durch die Keratinozyten aufrecht erhalten.Cryopreserved monocytes, described in 1.2, are thawed in a 37 ° C water bath and washed several times in PBS. Thereafter, the monocytes are transferred to a serum-free cell culture medium which is suitable for them and for keratinocytes simultaneously. Preferably, KGM-2 is used with Supplement Mix (PromoCell, Heidelberg, Germany). The test dish, which already contains semi-confluent adherent keratinocytes as described in 2.1, is washed with PBS and then the liquid is aspirated. The monocytes are seeded on the keratinocytes. The cell density of the monocytes is selected such that after sowing 2-5 × 10 5 cells of the monocyte preparation are> 8 μm / cm 2 in addition to the keratinocytes in the test shell. The monocytes initially adhere to the plastic material and the keratinocytes, but dissolve mostly after a few hours. The result is a coculture of adherent Kenterzyten on which loose monocytes rest. The viability of the monocytes is maintained by the keratinocytes.

2.3 Aussaat cryokonservierter Leukozyten2.3 Sowing of cryopreserved leukocytes

Alternativ zu 2.2 können, nachdem in der Testschale die Keratinozyten gewaschen sind und die Flüssigkeit abgesaugt ist, cryokonservierte Leukozyten, wie in 1.3 beschrieben, nach Auftauen, Waschen und Aufnahme ins Zellkulturmedium (vorzugsweise wird KGM-2 mit Supplement Mix (PromoCell) benutzt) auf die Keratinozyten ausgesät werden. Hierbei werden Zelldichten der Leukozyten so gewählt, dass in der Zellkulturschale Leukozytendichten von 1–2 × 106 Zellen > 8 μm/cm2 vorliegen. Nach 2–3 Stunden werden Zellen, die nicht an das Plastikmaterial oder an die Keratinozyten haften, mit 37°C warmer Zellkulturmedium abgespült und abgesaugt. Anschließend wird frisches Zellkulturmedium zugegeben.As an alternative to 2.2, after the keratinocytes are washed in the test dish and the liquid is aspirated, cryopreserved leukocytes, as described in 1.3, after thawing, washing and uptake into the cell culture medium (preferably KGM-2 is used with Supplement Mix (PromoCell)) the keratinocytes are sown. In this case, cell densities of the leukocytes are selected such that leukocyte densities of 1-2 × 10 6 cells> 8 μm / cm 2 are present in the cell culture dish. After 2-3 hours, cells that do not adhere to the plastic material or the keratinocytes are rinsed with 37 ° C warm cell culture medium and aspirated. Subsequently, fresh cell culture medium is added.

3. Generierung Dendritischer Zellen3. Generation of dendritic cells

2–3 Stunden nach der Aussaat der Monozyten auf die Kerationozytenkulturen, wie in 2.2 beschrieben, bzw. nach dem Abspülen nicht-adhärenter Leukozyten, wie in 2.3 beschrieben, werden die rekombinanten humanen Cytokine IL-4 ad 100 ng/ml (z. B von Immuntools, Friesoythe, Deutschland), GM-CSF ad 100 ng/ml (z. B. von Immuntools) und TGF-β1 ad 10 ng/ml (z. B. von R&D, Wiesbaden-Nordenstadt, Deutschland) zugegeben. Am Tag 2 und am Tag 4 der Kokultur werden GM-CSF ad 100 ng/ml und TGF-β1 ad 10 ng/ml zugegeben. Am Tag 5–6 ist die Kokultur als Testkit benutzbar.2-3 Hours after seeding of the monocytes on the Kerationozytenkulturen, as described in 2.2, or after rinsing non-adherent leukocytes, as described in 2.3, the recombinant human cytokines IL-4 ad 100 ng / ml (ex: Immuntools, Friesoythe, Germany), GM-CSF ad 100 ng / ml (eg from Immuntools) and TGF-β1 ad 10 ng / ml (eg from R & D, Wiesbaden-Nordenstadt, Germany) was added. On day 2 and on the day 4 of the coculture are GM-CSF ad 100 ng / ml and TGF-β1 ad 10 ng / ml added. On days 5-6 the coculture is a test kit usable.

Anwendungsbeispielexample

Bestimmung der Konzentration, bei der eine Testsubstanz im Testkit eine halbmaximale Reifung der Dendritischen Zellen induziertDetermination of the concentration at which a test substance in the test kit a half-maximal maturation of the dendritic Cells induced

Ein Testkit wird in einer 12-Loch Testschale (Costar Cell Culture Cluster von Corning Inc.) mit Zellen wie im Herstellungsverfahren in 1.1 und 1.2 beschrieben nach Herstellungsverfahren 2.1 und 2.2 hergestellt. Die zu testende Substanz wird in einem geeigneten Lösungsmittel (wie Wasser, DMSO, Ethanol, Ölgemische) in maximal möglicher Konzentration gelöst. Den Vertiefungen der Testschale mit den Kokulturen wird zu jeweils 2 ml Zellkulturmedium KGM-2 mit Supplement Mix (PromoCell) die Testsubstanz in einer Verdünnungsreihe zugegeben. Dabei wird die Testsubstanz vor Zugabe zum Testkit so verdünnt, dass in den Kokulturen gleiche Endkonzentrationen des Lösungsmittels vorliegen. Es werden jeweils Doppelwerte angelegt. Das Testkit wird nach Zugabe der Testsubstanz 24 Stunden lang in einem Brutschrank inkubiert. Danach werden die nicht-adhärenten Zellen mit dem Zellkulturmedium abgezogen und in FACS-Röhrchen überführt (5 ml Polystyrene Round-Bottom Tube, BD Falcon, Heidelberg, Deutschland). Nach einer Zentrifugation (330 rpm, 4 min., 20°C) werden für mögliche spätere ELISA-Assays die Medien abgenommen, ihr exaktes Volumen bestimmt und bei –80°C gelagert. Die Zellen werden 2× in PBS gewaschen, und jede Probe wird in zwei Aliquote aufgeteilt. Je eins der Aliquote wird mit je 5 μl der Antikörperlösungen CD11c:FITC (von Serotec) und CD86-PE (von BD) für die FACS-Analyse gefärbt. Das jeweils zweite Aliquot wird mit 20 μl 7-AAD (Cell Viability Solution von BD) gefärbt. Es werden nur Proben mit mehr als 85% vitalen Zellen hinsichtlich ihrer CD86-Expression ausgewertet. Die mittleren Fluoreszenzintensitäten (MFI) für CD86 werden für CD11c-positive Zellen bestimmt. Die MFI-Werte der Testsubstanz-Proben werden durch den MFI-Mittelwert der Lösungsmittel-Proben dividiert. Die resultierenden Faktoren werden gegen die Konzentration der Testsubstanz aufgetragen, und aus der Kurve wird die Konzentration der Testsubstanz bestimmt, die zu halbmaximaler Erhöhung der MFI-Werte für CD86 führt. Nach Durchführung unabhängiger Tests unter Verwendung von Zellen, die von jeweils 3 verschiedenen Spender für die Keratinozyten und für die Monozyten stammen, ergibt sich die Konzentration, die eine mittlere halbmaximale CD86-MFI-Erhöhung bewirkt.One Test kit is placed in a 12-well test dish (Costar Cell Culture Cluster from Corning Inc.) with cells as in the preparation process in 1.1 and 1.2 described according to manufacturing methods 2.1 and 2.2. The substance to be tested is in a suitable solvent (such as water, DMSO, ethanol, oil mixtures) in maximum possible Concentration solved. The wells of the test dish with The cocultures are supplemented with 2 ml each of cell culture medium KGM-2 Mix (PromoCell) the test substance in a dilution series added. The test substance is added before addition to the test kit diluted, that in the cocultures same final concentrations of the solvent. There are double values each created. The test kit becomes 24 hours after addition of the test substance long incubated in an incubator. After that, the non-adherent Cells are removed with the cell culture medium and transferred to FACS tubes (5 ml Polystyrene Round-Bottom Tube, BD Falcon, Heidelberg, Germany). After centrifugation (330 rpm, 4 min., 20 ° C) for possible later ELISA assays the Media taken, their exact volume determined and at -80 ° C. stored. The cells are washed 2X in PBS, and each Sample is divided into two aliquots. One each of the aliquot becomes with 5 μl each of the antibody solutions CD11c: FITC (from Serotec) and CD86-PE (from BD) for the FACS analysis colored. The second aliquot is filled with 20 μl 7-AAD (Cell Viability Solution of BD) stained. It will only Samples with more than 85% vital cells for their CD86 expression evaluated. The mean fluorescence intensities (MFI) for CD86 are determined for CD11c-positive cells. The MFI values of the test substance samples are determined by the MFI mean divided the solvent samples. The resulting Factors are plotted against the concentration of the test substance and from the curve, the concentration of the test substance is determined to half maximum increase in MFI values for CD86 leads. After carrying out independent Tests using cells of 3 different each Donor for keratinocytes and monocytes result in the concentration, which is a mean half-maximal CD86 MFI increase effected.

ZITATE ENTHALTEN IN DER BESCHREIBUNGQUOTES INCLUDE IN THE DESCRIPTION

Diese Liste der vom Anmelder aufgeführten Dokumente wurde automatisiert erzeugt und ist ausschließlich zur besseren Information des Lesers aufgenommen. Die Liste ist nicht Bestandteil der deutschen Patent- bzw. Gebrauchsmusteranmeldung. Das DPMA übernimmt keinerlei Haftung für etwaige Fehler oder Auslassungen.This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

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Claims (18)

In-vitro Testkit zur tierversuchsfreien Bestimmung des sensibilisierenden Potentials einer Substanz, dadurch gekennzeichnet, dass es eine Kokultur humaner Keratinozyten und humaner Monozyten enthält.In vitro test kit for the animal-free determination of the sensitizing potential of a substance, characterized in that it contains a co-culture of human keratinocytes and human monocytes. Testkit nach Anspruch 1, dadurch gekennzeichnet, dass die Monozyten während der Kokultur mittels der Cytokine IL-4, GM-CSF und TGF-β zu Dendritischen Zellen generiert werden.Test kit according to claim 1, characterized that monocytes during coculture by means of cytokines IL-4, GM-CSF and TGF-β are generated to dendritic cells become. Testkit nach Anspruch 2, dadurch gekennzeichnet, dass die kokultivierten Dendritischen Zellen locker auf den adhärenten Keratinozyten aufliegen oder im Zellkulturmedium schwimmen.Test kit according to claim 2, characterized that the co-cultured dendritic cells loosen up on the adherent Lie keratinocytes or float in the cell culture medium. Testkit nach Anspruch 1, dadurch gekennzeichnet, dass die Keratinozyten während der Kokultur aktiviert werden.Test kit according to claim 1, characterized that the keratinocytes are activated during coculture. Testkit nach einem der Ansprüche 1–4, dadurch gekennzeichnet, dass außer einem für die Kultivierung von Säugerzellen geeignetem Plastikmaterial keine weiteren Täger, wie Kollagen/Fibroblastenschichten, eingesetzt werden.Test kit according to one of claims 1-4, characterized in that except one for the Cultivation of mammalian cells suitable plastic material no further carriers, such as collagen / fibroblast layers, be used. Testkit nach einem der Ansprüche 1–4, dadurch gekennzeichnet, dass das verwendete Zellkulturmedium eine Differenzierung der Keratinozyten nicht unterstützen muss.Test kit according to one of claims 1-4, characterized in that the cell culture medium used is a Differentiation of keratinocytes does not support. Testkit nach einem der Ansprüche 1–6, dadurch gekennzeichnet, dass sensibilisierende Substanzen anhand einer erhöhten Expression von Reifungsmarkern Dendritischer Zellen, bevorzugt von CD86, detektiert werden können.Test kit according to one of claims 1-6, characterized in that sensitizing substances based an increased expression of maturation markers dendritic Cells, preferably from CD86, can be detected. Verfahren zur Herstellung eines in-vitro Testkits zur tierversuchsfreien Bestimmung des sensibilisierenden Potentials einer Substanz, dadurch gekennzeichnet, dass in einem für die Kultur von Säugerzellen geeignetem Plastikmaterial humane Keratinozyten und humane Monozyten kokultiviert werden.Method of making an in vitro test kit for animal-free determination of the sensitizing potential a substance, characterized in that in a for the culture of mammalian cells suitable plastic material human keratinocytes and human monocytes are cocultivated. Verfahren nach Anspruch 8, dadurch gekennzeichnet, dass die Keratinozyten cryokonserviert gewesen sein können.Method according to claim 8, characterized in that that the keratinocytes may have been cryopreserved. Verfahren nach Anspruch 8, dadurch gekennzeichnet, dass bevorzugt Keratinozyten eingesetzt werden, die während ihrer ersten Kultivierung durch Antrypsinieren hinsichtlich starker Adhärenz an Plastikmaterial selektiert wurden.Method according to claim 8, characterized in that that keratinocytes are preferably used during the their first cultivation by Antrypsinieren with regard to strong Adherence to plastic material were selected. Verfahren nach Anspruch 8, dadurch gekennzeichnet, dass die Monozyten nach erfolgter Adhäsion der Keratinozyten zugefügt werden.Method according to claim 8, characterized in that that the monocytes after adhesion of the keratinocytes be added. Verfahren nach Anspruch 11, dadurch gekennzeichnet, dass die Monozyten bereits vor dem Einfügen in die Kokultur durch magnetisches Sortieren aus Vollblut oder Lymphozytenkonzentrat angereichert wurden oder alternativ, dass die Monozyten erst in der Kokultur angereichert wurden, indem aus einer ausgesäten Leukozytenfraktion Zellen selektiert wurden, die stärker an die Plastikoberfläche des Zellkulturmaterials oder an die bereits enthaltenen Keratinozyten haften.Method according to claim 11, characterized in that that the monocytes already in the coculture before insertion by magnetic sorting from whole blood or lymphocyte concentrate were enriched or alternatively, that the monocytes first in of coculture were enriched by sown from one Leukocyte fraction cells were selected that were stronger on the plastic surface of the cell culture material or on the already contained keratinocytes adhere. Verfahren nach Anspruch 12, dadurch gekennzeichnet, dass die magnetisch angereicherten Monozyten und die Leukozytenfraktion cryokonserviert gewesen sein können.Method according to claim 12, characterized in that that the magnetically enriched monocytes and the leukocyte fraction may have been cryopreserved. Verfahren nach einem der Ansprüche 8–13, dadurch gekennzeichnet, dass nach der Zugabe der Monozyten die Cytokine IL-4, GM-CSF und TGF–β zugegeben werden, so dass die Monozyten zu Dendritischen Zellen differenzieren und die Keratinozyten aktiviert werden.Method according to one of claims 8-13, characterized characterized in that after the addition of monocytes, the cytokines IL-4, GM-CSF and TGF-β are added so that differentiate the monocytes into dendritic cells and activate the keratinocytes become. Verfahren nach Anspruch 14, dadurch gekennzeichnet, dass die Kokultur nach erfolgter Differenzierung der Monozyten zu Dendritischen Zellen als Testkit benutzt werden kann.Method according to claim 14, characterized in that that coculture after differentiation of monocytes to Dendritic cells can be used as a test kit. Verfahren nach einem der Ansprüche 8–15, dadurch gekennzeichnet, dass serumfreie Zellkulturmedien benutzt werden können.Method according to one of claims 8-15, characterized characterized in that serum-free cell culture media are used can. Verfahren nach einem der Ansprüche 8–15, dadurch gekennzeichnet, dass Kerationozyten und Monozyten nicht vom selben Spender stammen müssen.Method according to one of claims 8-15, characterized characterized in that karyocytes and monocytes are not of the same Donors must come. Verfahren nach einem der Ansprüche 8–15, dadurch gekennzeichnet, dass die Zellen nicht von verschiedenen Spender gepoolt werden müssen.Method according to one of claims 8-15, characterized characterized in that the cells are not from different donors need to be pooled.
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