WO1987001385A1 - Long term culture, identification, characterisation and use of endothelial cells - Google Patents

Long term culture, identification, characterisation and use of endothelial cells Download PDF

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Publication number
WO1987001385A1
WO1987001385A1 PCT/DE1986/000355 DE8600355W WO8701385A1 WO 1987001385 A1 WO1987001385 A1 WO 1987001385A1 DE 8600355 W DE8600355 W DE 8600355W WO 8701385 A1 WO8701385 A1 WO 8701385A1
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medium
cells
microcarriers
endothelial cells
endothelial
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PCT/DE1986/000355
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German (de)
French (fr)
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Ulrich-Christoph Von Arnim
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Von Arnim Ulrich Christoph
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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
    • C12N5/069—Vascular Endothelial cells
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal 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
    • C12N2531/00—Microcarriers

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  • the invention relates to a method for long-term cultivation of any endothelial cells, a method for the unambiguous unambiguous identification and characterization of the endothelial cells, the use of such endothelial cells for the unambiguous and unequivocal diagnosis and clarification of the etiopathogenesis of disease states.
  • Contamination with sprouting cells is even more difficult to avoid because, in contrast to the smooth muscle cells, they show an almost ultrastructural and immunofluorescence-identical morphology (factor VTII, angiotensin converting enzyme, blood group antigens). In addition, these cells (similar to the pit cells of the liver) also show lipid and lipoprotein uptake. It can be seen from this that unambiguous identification is extremely problematic even when using the most modern laboratory methods.
  • High-quality growth factors endothelial cell growth supplement, multiple stimulating activity, heparin, insulin and fibroblast growth factor
  • endothelial cell growth supplement multiple stimulating activity
  • heparin heparin
  • insulin and fibroblast growth factor also have only a minimal influence on the growth behavior and potency of the endothelial cell in comparison to smooth muscle cells and sprouting cells, since unfortunately they too stimulate their growth behavior.
  • a high endothelial cell culture is first grown in vitro from reticulo-histocytic system tissue (RHS tissue). Cultivation takes place with optimal results by washing the RHS tissue several times in a buffered seed line or in culture medium in the presence of calcium or magnesium. gnesiumionen and in the presence 'of an antibiotic.
  • RHS tissue reticulo-histocytic system tissue
  • the washed tissue should be bathed for a short time (eg 10 minutes) in a cell-friendly disinfectant (eg a polyvidone such as Betaisodona, diluted in buffered saline in a ratio of 1:10, v: v), after washing again the tissue homogenized and the homogenate washed again several times.
  • a cell-friendly disinfectant eg a polyvidone such as Betaisodona, diluted in buffered saline in a ratio of 1:10, v: v
  • the homogenization is carried out, for example, by comminution with the aid of a knife in a mixer, and all other homogenization methods can also be used.
  • the homogenate is advantageously washed in the culture medium (RPMI 1640 + antibiotic + L-glutamine and serum; RPMI 1640 is commercially available).
  • the homogenate is then first freed from the medium by centrifugation and incubated in 0.1 to 0.2% by weight collagenase for a time up to 60 minutes at 37 ° C.
  • the incubated homogenate is advantageously kept in suspension by careful shaking.
  • the suspension is then filtered (filter size, for example 100 ⁇ m).
  • the high-endothelial cells cleaned and isolated in this way are then grown in a cell culture system, preferably in the cell culture system from Bühler, Tübingen.
  • the endothelial cells are grown on micro-carriers (for example Cyto-dex-III, Pharmacia Fine Chemicals).
  • the micro-carriers for growing the endothelial cells are prepared as follows: the denatured, pulverized Pig collagen is slurried in buffered saline for 12 hours, aliquoted and sterilized.
  • the pork collagen prepared in this way is mixed in a defined concentration (from 0.1 to 0.5 mg dry weight per ml medium) with the resuspended cells and then filled into the bio-reactors of the cell culture plant.
  • the cultivation in the cell culture plant takes place at 37 ° C with rotation.
  • Microcarriers loaded with endothelial cells are aspirated with medium over 4 to 10 days; the same volume of unloaded microcarriers and medium is always added; In this way, a continuous course is obtained, since the added, unloaded microcarriers load themselves in the bioreactor.
  • the removed loaded microcarriers are separated from the medium by centrifugation.
  • the so-called conditional medium is then frozen (e.g. at -70 ° C); the cells sitting on the microcarrier are detached enzymatically (e.g. with trypsin) and the enzyme is removed by repeated centrifugation and repeated washing.
  • the conditional medium obtained in this way is used to cultivate e.g. parenchymatous endothelia of a neighboring organ; the harvested high-endothelial cells can be grown, e.g. on chamber slides, and subsequent fixation can be used for the diagnosis and clarification of the etiopatogenesis of various disease states.
  • the high-endothelial cells can be stored for as long as desired at temperatures below -70 ° C.
  • the frozen, so-called Conditioned Medium enables the in vitro cultivation of any endothelial cell with a mixture ratio of one part of Conditioned Medium to one part of fresh culture medium (serum, ECGS, MSA, insulin and heparin).
  • the High Endothelial Cells should come from the RHS tissue that the Organ that contains the desired endothelia is adjacent.
  • any other tissue can also be used instead of the RHS tissue, for example liver, kidney, lung, central nervous system.
  • the fixed and labeled cells are embedded in a mixture of glycerol and p-phenylenediamine derivatives or 1,4-diazobicyclo (2,2) octane derivatives.
  • the evaluation should best be carried out by quantitative fluorescence microscopy. The method itself is not endothelial cell-specific, but rather the insulin and heparin receptor density per membrane area is specific.
  • This identification method has also made an alternative method for cell isolation possible.
  • An organ homogenate is labeled with fluorescence or rhodamine-coupled insulin and heparin receptor antibodies, the antibodies always having to be coupled in a corresponding manner;
  • the cells are then separated using fluorescence-activated cell sorters (FACS).
  • FACS fluorescence-activated cell sorters
  • the cell filtrate purified after the incubation described above can also be used.
  • the cells obtained via FACS can then be used for cloning.
  • cultured ⁇ fourth and fixed cells can be used for diagnostic purposes in the following way: High Endothelial Cells for the diagnosis and clarify the pathogenesis of de-myelinating diseases and disorders of the rheuma ⁇ disorders circle; Liver endothelia for hepatopathy, kidney endothelia for rhenopathy; CNS tissue capillaries for non-demyelizing diseases of the central nervous system.
  • High Endothelial Cells for the diagnosis and clarify the pathogenesis of de-myelinating diseases and disorders of the rheuma ⁇ disorders circle
  • Liver endothelia for hepatopathy kidney endothelia for rhenopathy
  • CNS tissue capillaries for non-demyelizing diseases of the central nervous system.
  • the isolation, cultivation and fixation of organ-specific endotheliums is always of crucial importance for the diagnosis and clarification of the etiopathogenesis of specific clinical pictures.
  • Healthy lymphocytes of defined properties and concentration are treated with patient serum over a defined period (eg 60 minutes) incubated at 37 ° C simultaneously with vital dyes such as acridine orange colors and gently washed with buffered saline.
  • vital dyes such as acridine orange colors and gently washed with buffered saline.
  • the endothelia that are most specific for a clinical picture are fixed on a slide. This fixed slide is incubated with the lymphocyte treated in the manner described above for a defined period of time (for example 30 minutes).
  • the adhesion and migration behavior of these lymphocytes can now be recorded by light microscopy or quantitative microscopy.
  • lymphocytes instead of lymphocytes, other leukocytes of a defined concentration and properties can also be used.
  • a youthful tonsil after tonsillectomy is in three times
  • beta-soda a virocide, fungicide and bacteria
  • the suspension is filtered through a 100 ⁇ m filter, then the collagenase is removed by washing three times with the same washing medium as described above and centrifuging for 5 minutes at 300 g. The centrifugation pellet is gently resuspended in the culture medium.
  • the culture medium consists of RPMI 1640 + 20 mM 1-glutamine + 2 to 5 ⁇ g gentamycin per 100 ml medium + 30 vol.% J -globulin-free inactivated fetal calf serum + 20 ⁇ g ECGS (Endothelial Cell Growth Supplement, Sigma) + 10 ⁇ g heparin (Sigma H 3125) per ml medium + 20 ⁇ g MSA (Multiple Stimulating Activity, Sigma) per ml medium + 15 ⁇ g human insulin per ml medium.
  • the ECGS, MSA and insulin are added shortly before the cells are resuspended.
  • the vitality is tested with acridine orange, diluted 1:20 " in PBS.
  • the cells are cultivated in Primaria culture bottles (from Falcon) or in the cell culture system from Bühler, Tübingen.
  • the Bottle cultures are washed again 3 times after 2 hours and provided with a culture medium. After 24 hours, the medium is changed again, afterwards every other day. From the 2nd to the 10th day, the media are collected and frozen (-70 ° C). After 8 to 10 days, the culture is confluent. With subcultivation, the doubling time increases from once a day to once every 8 days.
  • the microcarriers loaded with endothelial cells are suctioned off with medium over a period of 4 to 10 days.
  • the same volume of unloaded microcarriers and medium is always added again.
  • the removed loaded microcarriers are separated from the medium by centrifugation, the conditioned medium obtained in this way is frozen at -70.degree.
  • the cells on the microcarriers are detached enzymatically with Trpysin-EDTA and purified by repeated centrifugation and repeated washing of enzyme. The doubling time has been found at least once a day.
  • Muscle tissue is freed of fat, 3 times in phosphate-puffed
  • betaisodoma a vericide, fungicide and bacterial
  • Type I for 60 minutes at 37 ° C with gentle shaking.
  • the suspension is filtered through a 100 ⁇ m filter, then the collagenase is removed by washing three times with the same washing medium as described above and centrifuging for 5 minutes at 300 g.
  • the centrifugation pellet is gently resuspended in the culture medium.
  • the culture medium consists of RPMI 1640 + 20 M 1-glutamine + 2 to 5 ⁇ g gentamycin per 100 ml medium + 30% by volume ⁇ -globulin-free inactivated fetal calf serum + 20 ⁇ g ECGS (Endothelial Cell Growth Supplement, Sigma) + 10 ⁇ g heparin (Sigma H 3125) per ml medium + 20 ⁇ g MSA (Multiple Stimulating Activity, Sigma) per ml medium + 15 ⁇ g human insulin per ml medium.
  • the ECGS, MSA and insulin are added shortly before the cells are resuspended. Vitality is tested with acridine orange diluted 1:20 in PBS. Approximately 95% of the cells are vital.
  • the cells are cultivated in Primaria culture bottles (from Falcon) or in the cell culture system from Bühler, Tübingen. The
  • Bottle cultures are washed again 3 times after 2 hours and provided with a culture medium. After 24 hours, the medium is changed again, afterwards every other day. From the 2nd to the 10th day, the media are collected and frozen (-70 ° C). After 8 to 10 days, the culture is confluent. With subcultivation, the doubling time increases from once a day to once every 8 days.
  • the microcarriers loaded with endothelial cells are aspirated with medium over 4 to 10 days.
  • the same volume of unloaded microcarriers and medium is always added again.
  • the removed loaded microcarriers are centrifuged by the dium separated, the conditioned medium obtained in this way is frozen at -70 ° C.
  • the cells sitting on the microcarriers are detached enzymatically with Trpysin-EDTA and cleaned of enzyme by repeated centrifugation and repeated washing. The doubling time was found at least once a day.
  • the recovered cells according to Examples 1 and 2 are cultured on chamber slides and fixed in methanol (95 vol .-%, 5 vol .-% glacial acetic acid), the thus prepared Chamber slides are coated with monoclonal insulin receptor antibodies, • in the ratio Diluted 1 to 50 with PBS and incubated at room temperature for 30 minutes. Wash three times for 15 minutes each with PBS and overlay with fluorescent or rhodamine-coupled anti-human antibodies.
  • the diagnostic kite consists of 10 slides with fixed high endothelial cells and one tube each containing a lymphocyte suspension in RPMI 1640 (Gibco); the suspension is characterized by a certain concentration and by certain properties of the lymphocytes.
  • a control consisting of a physiological mixture of leukocytes belongs to the set, furthermore standard tables suitable for the respective lymphocytes and leukocytes.

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Abstract

A process for the long term culture of any endothelial cell is provided, as well as a clear, specific and reliable process for the identification of endothelial cells and a process for the clear and reliable clarification and diagnosis of the etiopathogenesis of pathological states.

Description

Langzeit-Züchtung, Identifizierung, Charakterisierung und Verwendung vonEndothelzellen.Long-term cultivation, identification, characterization and use of endothelial cells.
Die Erfindung betrifft eine Methode zur Langzeit-Züchtung von beliebigen Endothelzellen, eine Methode zur unzweifel¬ haften eindeutigen Identifizierung und Charakterisierung der Endothelzellen, die Verwendung solcher Endothelzellen für die eindeutige und unzweifelhafte Diagnose und Klärung der Ätiopathogenese von Krankheitszuständen.The invention relates to a method for long-term cultivation of any endothelial cells, a method for the unambiguous unambiguous identification and characterization of the endothelial cells, the use of such endothelial cells for the unambiguous and unequivocal diagnosis and clarification of the etiopathogenesis of disease states.
Die von E.A. Jaffee et al . (J. Clin. Invest. 52., 2745- 2756, 1973a) und M.A. Gimbrone et al . (J. Cell. Biol. 60. 673-684, 1974) entwickelte Methode zur Züchtung von Endo¬ thelzellen hat entscheidende Nachteile: ihre Durchführung durch die enzymatische Ablösung der Endothelzelle mit Hilfe von Collagenase aus den Nabelschnurgefäßen ist hoch¬ gradig diffizil und in der Ausbeute zu gering. Die Nabel¬ schnur enthält 3 Gefäße und zahlreiche Vasa-vasorum, wobei die letzteren den Hauptanteil der Endothelien enthalten. Durch diese Methode ist es nicht möglich, diese Endothe¬ lien zur Züchtung in vitro zu gewinnen; außerdem ist sie nur an mittleren und großen Gefäßen, zum Beispiel Aorta, Vena cava, anwendbar. Der entscheidenste Nachteil ist, selbst bei schonenster enzymatischer Zellisolierung läßt sich eine Kontamination mit Smooth-Muscle-Cells und voral¬ lem Sprouting-Zellen nict vermeiden. In neuerer Zeit (vgl. A. Ager et al . , Thrombosis Res. 3_5., 43-52, 1984) konnte zwar die unerwünschte Kontamination homologer Zellpopula- tionen mit Smooth-Muscle-Zellen durch Anwendung der Elek- tronenmikroskopie und anderer Laboratoriumsmethoden ver¬ bessert werden, doch ist auch heute noch in bis zu dreißig Prozent der Fälle ein Verwerfen der Kulturen wegen Kontamination mit Smooth uscle Cells notwendig. Die Kontamination mit Sprouting Cells ist noch schwerer zu ver¬ meiden, denn im Gegensatz zu den Smooth Muscle Cells zeigen sie eine nahezu ultrastrukturell und immunfluoreszenz-iden- tische Morphologie (Faktor VTII, Angiotensin Converting Enσym-Antikörper, Blutgruppenantigene) . Außerdem zeigen auch diese Zellen (ähnlich den Pit-Zellen der Leber) eine Lipid- und Lipoprotein-Aufnahme. Daraus ist zu ersehen, daß eine eindeutige Identifizierung selbst bei Anwendung modern¬ ster Laboratoriumsmethoden extrem problematisch ist.The EA Jaffee et al. (J. Clin. Invest. 52., 2745-2756, 1973a) and MA Gimbrone et al. (J. Cell. Biol. 60, 673-684, 1974) developed method for the cultivation of endothelial cells has decisive disadvantages: their implementation by enzymatic detachment of the endothelial cell with the help of collagenase from the umbilical cord vessels is extremely difficult and difficult Yield too low. The umbilical cord contains 3 vessels and numerous vasa vasorum, the latter containing the majority of the endothelia. This method does not make it possible to obtain these endotheliums for in vitro cultivation; moreover, it can only be used on medium-sized and large vessels, for example aorta, vena cava. The most decisive disadvantage is that even with the most gentle enzymatic cell isolation, contamination with smooth muscle cells and, in particular, sprouting cells cannot be avoided. More recently (cf. A. Ager et al., Thrombosis Res. 3_5., 43-52, 1984) the undesired contamination of homologous cell populations with smooth muscle cells has been able to be verified by using electron microscopy and other laboratory methods ¬ be improved, but even today, culture is rejected in up to thirty percent of cases necessary due to contamination with smooth uscle cells. Contamination with sprouting cells is even more difficult to avoid because, in contrast to the smooth muscle cells, they show an almost ultrastructural and immunofluorescence-identical morphology (factor VTII, angiotensin converting enzyme, blood group antigens). In addition, these cells (similar to the pit cells of the liver) also show lipid and lipoprotein uptake. It can be seen from this that unambiguous identification is extremely problematic even when using the most modern laboratory methods.
Ein kleiner Fortschritt wurde in diesem Zusammenhang mit der Etablierung der Elutriation erzielt (vgl. Maroin L. Meis- trich, Cell Separation, Academic Press, London, Vol 2^, S. 33 bis 61) . Der Vorteil der Elutriation ist die gleichzeitige Ausnutzung mehrerer physikalisch-chemischer Eigenschaften (Zentripetalkraft, Dichte, Fliehkraft, Gravitation, Ge¬ schwindigkeit und optische Eigenschaften) . Bedauerlicher¬ weise ist diese Methode extrem teuer und aufwendig. Bei An¬ wendung all dieser Methoden muß trotzdem eine der bedeutend¬ sten biologischen Eigenschaften der Endothelzelle beachtet werden: achstumspotenz und Wachstumsverhalten. Auch hoch¬ wertige Wachstumsfaktoren (Endothelial Cell Growth Supple¬ ment, Multiple Stimulating Activity, Heparin, Insulin und Fibroblasten-Wachstumsfaktor) beeinflussen Wachstumsverhal¬ ten und -potenz der Endothelzelle im Vergleich zu Smooth Muscle Cells und Sprouting Cells nur minimal, da sie leider auch deren Wachstumsverhalten stimulieren.A little progress has been made in this connection with the establishment of elutriation (cf. Maroin L. Meis- trich, Cell Separation, Academic Press, London, Vol 2 ^ , pp. 33 to 61). The advantage of elutriation is the simultaneous exploitation of several physico-chemical properties (centripetal force, density, centrifugal force, gravity, speed and optical properties). Unfortunately, this method is extremely expensive and complex. When all these methods are used, one of the most important biological properties of the endothelial cell must nevertheless be taken into account: growth potential and growth behavior. High-quality growth factors (endothelial cell growth supplement, multiple stimulating activity, heparin, insulin and fibroblast growth factor) also have only a minimal influence on the growth behavior and potency of the endothelial cell in comparison to smooth muscle cells and sprouting cells, since unfortunately they too stimulate their growth behavior.
Für die Langzeit-Züchtung von beliebigen Endothelzellen lassen sich alle diese Nachteile ausschließen, wenn erfin¬ dungsgemäß zunächst eine High Endothelial-Zellkultur aus Retikulo-Histocytärem-Systemgewebe (RHS-Gewebe) in vitro ge¬ züchtet wird. Die Züchtung erfolgt mit optimalem Ergebnis durch mehrfaches Waschen des RHS-Gewebes in gepufferter Sa¬ line oder in.Kulturmedium in Gegenwart von Calzium- oder Ma- gnesiumionen und in Gegenwart' eines Antibiotikums. Das ge¬ waschene Gewebe sollte für kurze Zeit (z.B. 10 Minuten) in einem zellfreundlichen Desinfizienz (z.B. einem Polyvidonjo- did wie Betaisodona, verdünnt in gepufferter Saline im Ver¬ hältnis 1:10, v:v) gebadet werden, nach erneutem Waschen wird das Gewebe homogenisiert und das Homogenisat erneut mehrmals gewaschen. Die Homogenisierung erfolgt beispiels¬ weise durch Zerkleinerung mit Hilfe eines Messers in einem Mixer, auch alle übrigen Homogenisationsmethoden sind an¬ wendbar. Die Waschung des Homogenisats erfolgt vorteilhaf¬ terweise im Kulturmedium (RPMI 1640 + Antibiotikum + L-Glu- tamin und Serum; RPMI 1640 ist im Handel erhältlich) . An¬ schließend wird das Homogenisat zunächst durch Zentrifugie- ren vom Medium befreit und in 0,1 bis 0,2 Gew.-%iger Colla¬ genase für eine Zeit bis zu 60 Minuten bei 37°C inkubiert. Vorteilhafterweise wird das inkubierte Homogenisat durch vorsichtiges Schütteln in Suspension gehalten. Die Suspen¬ sion wird anschließend filtriert (Filtergröße z.B. 100 μm) . Das Filtrat wird durch'mehrmalige kurzzeitige Zentrifugation (z.B. 5 Minuten) und durch mehrmaliges Waschen mit Kulturme¬ dium von der Collagenase befreit; der Zentrifugationspellet wird vorsichtig in Anzuchtmedium resuspendiert (Anzuchtme¬ dium RPMI 1640 + 20 mMol L-Glutamin + Antibiotikum + 20 bis 30 Vol. % gammaglobulinfreies, inaktiviertes Foetales-Käl- berserum + 20 μg/ml ECGS Medium (Endothelial-Cell-Growth Supplement) + 100 μg/ml Heparin (Sigma-H-3125) + 20 μg MSA (Multiple Stimulating Activity, Fa. Sigma) pro ml Medium + 15 μg Humaninsulin pro ml Medium) . Die so gereinigten und isolierten High-Endothelial Cells werden nun einer Zellkul¬ turanlage, am besten in der Zellkulturanlage der Fa. Bühler, Tübingen, angezüchtet.For the long-term cultivation of any endothelial cells, all of these disadvantages can be excluded if, according to the invention, a high endothelial cell culture is first grown in vitro from reticulo-histocytic system tissue (RHS tissue). Cultivation takes place with optimal results by washing the RHS tissue several times in a buffered seed line or in culture medium in the presence of calcium or magnesium. gnesiumionen and in the presence 'of an antibiotic. The washed tissue should be bathed for a short time (eg 10 minutes) in a cell-friendly disinfectant (eg a polyvidone such as Betaisodona, diluted in buffered saline in a ratio of 1:10, v: v), after washing again the tissue homogenized and the homogenate washed again several times. The homogenization is carried out, for example, by comminution with the aid of a knife in a mixer, and all other homogenization methods can also be used. The homogenate is advantageously washed in the culture medium (RPMI 1640 + antibiotic + L-glutamine and serum; RPMI 1640 is commercially available). The homogenate is then first freed from the medium by centrifugation and incubated in 0.1 to 0.2% by weight collagenase for a time up to 60 minutes at 37 ° C. The incubated homogenate is advantageously kept in suspension by careful shaking. The suspension is then filtered (filter size, for example 100 μm). The filtrate (eg 5 minutes) dium removed by 'repeated brief centrifugation and by washing several times with Kulturme¬ from the collagenase; the centrifugation pellet is carefully resuspended in culture medium (culture medium RPMI 1640 + 20 mmol L-glutamine + antibiotic + 20 to 30 vol.% gamma globulin-free, inactivated fetal calf serum + 20 μg / ml ECGS medium (endothelial cell growth supplement ) + 100 μg / ml heparin (Sigma-H-3125) + 20 μg MSA (Multiple Stimulating Activity, Sigma) per ml medium + 15 μg human insulin per ml medium). The high-endothelial cells cleaned and isolated in this way are then grown in a cell culture system, preferably in the cell culture system from Bühler, Tübingen.
Die Endothelzellen werden auf Micro-carriers (z.B. Cyto- dex-III, Fa. Pharmacia Fine Chemicals) angezüchtet. Die Prä¬ paration der Micro-carriers zur Anzucht der Endothelzellen erfolgt folgendermaßen: das denaturierte, pulverisierte Schwe'incollagen wird in gepufferter Saline für 12 Stunden aufgeschlämmt, aliquotiert und sterilisiert. Das so präpa¬ rierte Schweinecollagen wird in definierter Konzentration (von 0,1 bis 0,5 mg Trockengewicht pro ml Medium) mit den resuspendierten Zellen gemischt und anschließend in die Bio¬ reaktoren der Zellkulturanlage eingefüllt. Die Zucht in der Zellkulturanlage erfolgt bei 37°C unter Rotation. Über 4 bis 10 Tage werden mit Endothelzellen beladene Microcarriers mit Medium abgesaugt, es wird stets dasselbe Volumen an unbela- denen Microcarriers und Medium zugegeben; auf diese Weise erhält man einen kontinuierlichen Verlauf, da sich die zuge¬ gebenen, unbeladenen Microcarriers im Bioreaktor von selbst beladen.The endothelial cells are grown on micro-carriers (for example Cyto-dex-III, Pharmacia Fine Chemicals). The micro-carriers for growing the endothelial cells are prepared as follows: the denatured, pulverized Pig collagen is slurried in buffered saline for 12 hours, aliquoted and sterilized. The pork collagen prepared in this way is mixed in a defined concentration (from 0.1 to 0.5 mg dry weight per ml medium) with the resuspended cells and then filled into the bio-reactors of the cell culture plant. The cultivation in the cell culture plant takes place at 37 ° C with rotation. Microcarriers loaded with endothelial cells are aspirated with medium over 4 to 10 days; the same volume of unloaded microcarriers and medium is always added; In this way, a continuous course is obtained, since the added, unloaded microcarriers load themselves in the bioreactor.
Die entnommenen beladenen Microcarriers werden durch Zentri¬ fugation vom Medium getrennt. Das so gewonnene, sogenannte Conditional Medium wird anschließend eingefroren (z.B. bei -70°C) ; die auf dem Microcarrier sitzenden Zellen werden enzymatisch (z.B. mit Trypsin) abgelöst und durch mehrmalige Zentrifugation und mehrmaliges Waschen vom Enzym befreit. Das so gewonnene Conditional Medium dient der Anzucht von z.B. parenchymatösen Endothelien eines benachbarten Organs; die geernteten High-Endothelial-Cells können durch Anzucht, z.B. auf Chamber-Slides, und anschließende Fixierung für die Diagnostik und Klärung der Ätiopatogenese verschiedener Krankheitszustände genutzt werden. Die High-Endothelial Cells lassen sich bei Temperaturen unter -70°C beliebig lang aufbewahren.The removed loaded microcarriers are separated from the medium by centrifugation. The so-called conditional medium is then frozen (e.g. at -70 ° C); the cells sitting on the microcarrier are detached enzymatically (e.g. with trypsin) and the enzyme is removed by repeated centrifugation and repeated washing. The conditional medium obtained in this way is used to cultivate e.g. parenchymatous endothelia of a neighboring organ; the harvested high-endothelial cells can be grown, e.g. on chamber slides, and subsequent fixation can be used for the diagnosis and clarification of the etiopatogenesis of various disease states. The high-endothelial cells can be stored for as long as desired at temperatures below -70 ° C.
Das eingefrorene, sogenannte Conditioned Medium ermöglicht bei einem Mischungsverhältnis von einem Teil Conditioned Medium zu einem Teil frischem Kulturmedium (Serum, ECGS, MSA, Insulin und Heparin) die in-vitro-Kultivierung jeder beliebigen Endothelzelle. Dabei ist, will man besonders gute Ergebnisse erzielen, zu beachten, daß die High Endothelial Cells (HEC) aus dem RHS-Gewebe stammen sollten, welches dem Organ, das die gewünschten Endothelien enthält, benachbart ist. Zur Zucht in der oben beschriebenen Weise kann auch an¬ stelle des RHS-Gewebes ein beliebiges anderes Gewebe benutzt werden, z.B. Leber, Niere, Lunge, Zentrales Nervensystem.The frozen, so-called Conditioned Medium enables the in vitro cultivation of any endothelial cell with a mixture ratio of one part of Conditioned Medium to one part of fresh culture medium (serum, ECGS, MSA, insulin and heparin). If you want to achieve particularly good results, it should be noted that the High Endothelial Cells (HEC) should come from the RHS tissue that the Organ that contains the desired endothelia is adjacent. For breeding in the manner described above, any other tissue can also be used instead of the RHS tissue, for example liver, kidney, lung, central nervous system.
Die oben genannten Zusatzfaktoren im Kultur- bzw. Anzuchtme¬ dium (Serum, ECGS, MSA, Insulin und Heparin) müssen nicht zugesetzt werden, sie optimieren jedoch auf die Länge der Zeit gesehen das Ergebnis und erhöhen vor allen Dingen die Widerstandskraft. Nun ist es endlich möglich geworden belie¬ bige Endothelzellen unter physiologischen Bedingungen für kommerzielle Zwecke zu nutzen.The above-mentioned additional factors in the culture or culture medium (serum, ECGS, MSA, insulin and heparin) do not have to be added, but they optimize the result over time and, above all, increase the resistance. Now it is finally possible to use any endothelial cells under physiological conditions for commercial purposes.
Wissenschaftlich war es notwendig, eine Methode zu finden, die eine eindeutige spezifische und unzweifelhafte Identifi¬ zierung der Endothelzellen erlaubt; dies läßt sich erfin¬ dungsgemäß dadurch erreichen, daß man an den zu identifizie¬ renden Zellen eine quantitative Immunofluorenszenz mit Hilfe von monoklonalen Insulin- und Heparinrezeptorantikörpern durchführt. Dies läßt sich beispielsweise dadurch erreichen, daß die zu identifizierenden Zellen auf einem Chamber-Slide kultiviert und fixiert werden: die Zellen werden an¬ schließend mit einem monoklonalen Insulinrezeptorantikörper überschichtet (für ca. 30 Minuten) , gründlich mit gepuffer¬ ter Saline gewaschen (3 x 15 Minuten) und mit einem Fluor¬ eszenz- oder Rhodamin-gekoppelten Antihumanantikörper über¬ schichtet und erneut gewaschen. Anschließend erfolgt eine Überschichtung mit einem Heparinrezeptorantikörper (für ca. 30 Minuten) , gefolgt von einer gründlichen Waschung mit ge¬ pufferter Saline, danach Überschichtung mit dem kontra¬ korrespondierenden Fluoreszenz- oder Rhodamin-gekoppelten Antihumanantikörper. Diese Überschichtungen können auch in umgekehrter Reihenfolge erfolgen. Um zu verhindern, daß das Präparat austrocknet oder die Fluoreszenz verschwindet, bettet man die fixierten und markierten Zellen in ein Ge¬ misch aus Glycerin und p-Phenylendiaminderivaten oder l,4-Diazobicyclo-(2,2) -octanderivaten. Die Auswertung sollte am besten durch quantitative Fluoreszenzmikroskopie erfol¬ gen. Die Methode an sich ist nicht endothelzell-spezifisch, sondern spezifisch ist die Insulin- und Heparin-Rezeptor- dichte pro Membranfläche.Scientifically, it was necessary to find a method that would allow the endothelial cells to be identified clearly and unambiguously; This can be achieved according to the invention by carrying out quantitative immunofluorescence on the cells to be identified with the aid of monoclonal insulin and heparin receptor antibodies. This can be achieved, for example, by cultivating and fixing the cells to be identified on a chamber slide: the cells are then covered with a monoclonal insulin receptor antibody (for approx. 30 minutes), washed thoroughly with buffered saline (3 x 15 minutes) and covered with a fluorescence or rhodamine-coupled anti-human antibody and washed again. This is followed by an overlay with a heparin receptor antibody (for about 30 minutes), followed by a thorough washing with buffered saline, followed by an overlay with the corresponding fluorescent or rhodamine-coupled anti-human antibody. These overlays can also be done in reverse order. In order to prevent the preparation from drying out or the fluorescence disappearing, the fixed and labeled cells are embedded in a mixture of glycerol and p-phenylenediamine derivatives or 1,4-diazobicyclo (2,2) octane derivatives. The evaluation should best be carried out by quantitative fluorescence microscopy. The method itself is not endothelial cell-specific, but rather the insulin and heparin receptor density per membrane area is specific.
Durch diese Identifikationsmethode ist auch eine alternative Methode zur Zellisolierung möglich geworden. Ein Organhomo- genisat wird mit Fluoreszenz- oder Rhodamin-gekoppelten In¬ sulin- und Heparin-Rezeptorantikörpern markiert, wobei die Antikörper stets kontrakorrespondierend gekoppelt sein müssen; die Trennung der Zellen erfolgt dann über Fluor- eszenz-Activated Cell-Sorter (FACS) . Anstelle des Organhomo- genisats kann auch das nach der oben beschriebenen Inkuba¬ tion gereinigte Zellfiltrat eingesetzt werden. Die über FACS gewonnenen Zellen können anschließend zur Klonierung genutzt werden.This identification method has also made an alternative method for cell isolation possible. An organ homogenate is labeled with fluorescence or rhodamine-coupled insulin and heparin receptor antibodies, the antibodies always having to be coupled in a corresponding manner; The cells are then separated using fluorescence-activated cell sorters (FACS). Instead of the organ homogenate, the cell filtrate purified after the incubation described above can also be used. The cells obtained via FACS can then be used for cloning.
Die auf geeigneten Unterlagen, z.B. Chamber-Slides, kulti¬ vierten und fixierten Zellen lassen sich auf folgende Weise zu diagnostischen Zwecken einsetzen: High Endothelial Cells für die Diagnose und Klärung der Ätiopathogenese von de- myelinisierenden Erkrankungen und Erkrankungen des rheuma¬ tischen Formenkreises; Leberendothelien für Hepatopathien, Nierenendothelien für Rhenopathien; ZNS-Gewebekapillaren für nicht demyelisierende Erkrankungen des zentralen Nerven¬ systems. Von entscheidender Bedeutung ist immer die Isolie¬ rung, Kultivierung und Fixierung organspezifischer Endo- thelien für die Diagnose und Klärung der Ätiopathogenese spezifischer Krankheitsbilder. Um zu einer Diagnose zu kommen bzw. um die Ätiopathogenese eines spezifischen Krank¬ heitsbildes zu klären, muß man sich an folgende Versuchsan¬ ordnung halten: Gesunde Ly phozyten definierter Eigenschaf¬ ten und Konzentration werden über einen definierten Zeitraum (z.B. 60 Minuten) mit Patientenserum bei 37°C inkubiert gleichzeitig mit Vitalfarbstoffen, wie Acridinorange, ange- färbt und mit gepufferter Saline schonend gewaschen. Auf einem Objektträger werden die für ein Krankheitsbild am ehesten spezifischen Endothelien fixiert. Dieser fixierte Objektträger wird mit dem auf die oben beschriebene Weise behandelten Lymphozyten über einen definierten Zeitraum in¬ kubiert (z.B. 30 Minuten) . Man kann nun durch Lichtmikrosko¬ pie oder quantitative Mikroskopie das Adhäsions- und Migra¬ tionsverhalten dieser Lymphozyten messend festhalten. Mit Hilfe erstellter Standardkurven kann Diagnose, Klärung der Ätiopathogenese, Verlauf und Stadieneinteilung der Krank¬ heitsbilder eindeutig und unzweifelhaft erfolgen. Anstelle von Lymphozyten können auch andere Leukozyten definierter Konzentration und Eigenschaften benutzt werden.Including by appropriate documents, eg Chamber slides, cultured ¬ fourth and fixed cells can be used for diagnostic purposes in the following way: High Endothelial Cells for the diagnosis and clarify the pathogenesis of de-myelinating diseases and disorders of the rheuma¬ disorders circle; Liver endothelia for hepatopathy, kidney endothelia for rhenopathy; CNS tissue capillaries for non-demyelizing diseases of the central nervous system. The isolation, cultivation and fixation of organ-specific endotheliums is always of crucial importance for the diagnosis and clarification of the etiopathogenesis of specific clinical pictures. In order to arrive at a diagnosis or to clarify the etiopathogenesis of a specific clinical picture, one must adhere to the following experimental arrangement: Healthy lymphocytes of defined properties and concentration are treated with patient serum over a defined period (eg 60 minutes) incubated at 37 ° C simultaneously with vital dyes such as acridine orange colors and gently washed with buffered saline. The endothelia that are most specific for a clinical picture are fixed on a slide. This fixed slide is incubated with the lymphocyte treated in the manner described above for a defined period of time (for example 30 minutes). The adhesion and migration behavior of these lymphocytes can now be recorded by light microscopy or quantitative microscopy. With the help of standard curves, diagnosis, clarification of the etiopathogenesis, course and stage classification of the clinical pictures can be carried out clearly and unquestionably. Instead of lymphocytes, other leukocytes of a defined concentration and properties can also be used.
Es ist unbedingt notwendig, von nicht durch pathologische Stimuli veränderten Leukozyten auszugehen, da im Rahmen der Homöostase ein biologisches Prinzip stets auf einen Gleich¬ gewichtszustand zustrebt, d.h. kommt es zu einer Entgleisung des Gleichgewichts infolge eines aktiven Krankheitsschubs, wie z.B. bei der Multiplen Sklerose, so vermehrt sich auto¬ matisch die Zahl der Suppressorzellen bzw. ihre Stoff¬ wechselaktivität, es werden Hemmsubstanzen gebildet, welche wieder zur Einstellung des Gleichgewichtes im Sinne einer Remission führen. Es findet dabei keine Heilung statt, es stellt sich lediglich ein Status quo ein.It is absolutely necessary to start from leukocytes that have not been altered by pathological stimuli, since in the context of homeostasis a biological principle always strives for a state of equilibrium, i.e. there is a derailment of the equilibrium due to an active relapse, e.g. In the case of multiple sclerosis, the number of suppressor cells or their metabolic activity automatically increases, inhibitory substances are formed which again lead to the establishment of the equilibrium in the sense of remission. There is no healing, only a status quo arises.
Die folgenden Beispiele sollen das Wesen der Erfindung näher erläutern: Beispiel 1The following examples are intended to illustrate the essence of the invention: example 1
High Endothelial Cell-KulturHigh endothelial cell culture
Eine jugendliche Mandel nach Tonsillektomie wird dreimal inA youthful tonsil after tonsillectomy is in three times
2+ 2+ phosphatgepufferter Salme (PBS + Ca , Mg ; Seromed) mit Calzium- und Magnesiumionen und 20-50 μg Gentamycin pro2+ 2+ phosphate buffered salts (PBS + Ca, Mg; Seromed) with calcium and magnesium ions and 20-50 μg gentamycin per
100 ml PBS gewaschen und zerkleinert. Nach der Zerkleinerung100 ml PBS washed and crushed. After crushing
2+ 2+ wird nochmals 3 mal in PBS und Gentamycin, Ca + Mg2+ 2+ is repeated 3 times in PBS and Gentamycin, Ca + Mg
2+ 2+ (=PBSG + Ca + Mg ) gewaschen. Es erfolgt ein lOminü- tiges Baden in Betaisodoma (einem Virozid, Fungizid und Bak-2+ 2+ (= PBSG + Ca + Mg) washed. There is a 10 minute bath in beta-soda (a virocide, fungicide and bacteria).
2+ 2+ terizid) , 1:10 verdünnt mit PBSG + Ca + Mg . Danach wird durch Zentrifugation und 3-maliges Waschen das Beta¬ isodona entfernt. Anschließend wird homogenisiert und erneut 3 mal gewaschen mit dem Waschmedium, bestehend aus RPMI 1640 + 2 bis 5 μg Gentamycin/1 ml Medium + 20 mM L-Glutamin und 10 Vol.-% gammaglobulienfreies, inaktiviertes Foetales-Käl- berserum. Danach erfolgt die Inkubation in 0,1 % Collagenase Typ I für 60 Minuten bei 37°C unter schonendem Schütteln. -2+ 2+ tericide), 1:10 diluted with PBSG + Ca + Mg. The beta-isodona is then removed by centrifugation and washing 3 times. The mixture is then homogenized and washed again 3 times with the washing medium, consisting of RPMI 1640 + 2 to 5 μg gentamycin / 1 ml medium + 20 mM L-glutamine and 10% by volume, inactivated fetal calf serum free of gamma globules. Then incubate in 0.1% collagenase type I for 60 minutes at 37 ° C with gentle shaking. -
Die Filtrierung der Suspension erfolgt über ein 100 μm-Fil- ter, danach wird durch 3-maliges -Waschen mit dem gleichen Waschmedium, wie oben beschrieben, und Zentrifugation für jeweils 5 Minuten bei 300 g die Collagenase entfernt. Der Zentrifugationspellet wird schonend im Kulturmedium resus¬ pendiert. Das Kulturmedium besteht aus RPMI 1640 + 20 mM 1-Glutamin + 2 bis 5 μg Gentamycin pro 100 ml Medium + 30 Vol.-% J -globulinfreies inaktiviertes Foetales-Kälberserum + 20 μg ECGS (Endothelial Cell Growth Supplement, Fa. Sigma) + 10 μg Heparin (Sigma H 3125) pro ml Medium + 20 μg MSA (Multiple Stimulating Activity, Fa. Sigma) pro ml Medium + 15 μg Humaninsulin pro ml Medium. Das ECGS, MSA und Insulin werden erst kurz vor der Resuspension der Zellen zugesetzt. Die Vitalität wird mit Acridinorange, verdünnt 1:20" in PBS, getestet. Ca. 95 % der Zellen sind vital. Kultiviert werden die Zellen in Primaria-Kulturflaschen (der Fa. Falcon) oder in der Zellkulturanlage der Fa. Bühler, Tübingen. Die Flaschenkulturen werden nach 2 Stunden erneut je 3 mal ge¬ waschen und mit einem Kulturmedium versehen. Nach 24 Stunden erfolgt erneut Mediumwechsel, hernach jeden 2. Tag. Ab dem 2. bis zum 10. Tag werden die Medien gesammelt und eingefro¬ ren (-70°C). Nach 8 bis 10 Tagen ist die Kultur konfluent. Bei Subkultivierung steigt die Verdoppelungszeit von 1 mal pro Tag auf 1 mal alle 8 Tage.The suspension is filtered through a 100 μm filter, then the collagenase is removed by washing three times with the same washing medium as described above and centrifuging for 5 minutes at 300 g. The centrifugation pellet is gently resuspended in the culture medium. The culture medium consists of RPMI 1640 + 20 mM 1-glutamine + 2 to 5 μg gentamycin per 100 ml medium + 30 vol.% J -globulin-free inactivated fetal calf serum + 20 μg ECGS (Endothelial Cell Growth Supplement, Sigma) + 10 μg heparin (Sigma H 3125) per ml medium + 20 μg MSA (Multiple Stimulating Activity, Sigma) per ml medium + 15 μg human insulin per ml medium. The ECGS, MSA and insulin are added shortly before the cells are resuspended. The vitality is tested with acridine orange, diluted 1:20 " in PBS. About 95% of the cells are vital. The cells are cultivated in Primaria culture bottles (from Falcon) or in the cell culture system from Bühler, Tübingen. The Bottle cultures are washed again 3 times after 2 hours and provided with a culture medium. After 24 hours, the medium is changed again, afterwards every other day. From the 2nd to the 10th day, the media are collected and frozen (-70 ° C). After 8 to 10 days, the culture is confluent. With subcultivation, the doubling time increases from once a day to once every 8 days.
Verwendet man anstelle der Flaschenkulturen die Zellkultur¬ anlage der Fa. Bühler, Tübingen, so werden über 4 bis 10 Tage die mit Endothelzellen beladenen Microcarriers mit Me¬ dium abgesaugt. Es wird stets dasselbe Volumen an unbelade- nen Microcarriers und Medium wieder zugegeben. Die entnom¬ menen beladenen Microcarriers werden durch Zentrifuation vom Medium getrennt, das so gewonnene Conditioned Medium wird bei -70°C eingefroren. Die auf den Microcarriers sitzenden Zellen werden enzymtisch mit Trpysin-EDTA abgelöst und durch mehrmalige Zentrifugation und mehrmaliges Waschen von Enzym gereinigt. Die Verdoppelungszeit ist zu mindestens 1 mal pro Tag gefunden worden.If, instead of the bottle cultures, the cell culture system from Buhler, Tübingen, is used, the microcarriers loaded with endothelial cells are suctioned off with medium over a period of 4 to 10 days. The same volume of unloaded microcarriers and medium is always added again. The removed loaded microcarriers are separated from the medium by centrifugation, the conditioned medium obtained in this way is frozen at -70.degree. The cells on the microcarriers are detached enzymatically with Trpysin-EDTA and purified by repeated centrifugation and repeated washing of enzyme. The doubling time has been found at least once a day.
Beispiel 2Example 2
KapillarendothelienCapillary endothelia
Muskelgewebe wird vom Fett befreit, 3 mal in phosphatgepuf-Muscle tissue is freed of fat, 3 times in phosphate-puffed
2+ 2+ ferter Saline (PBS + Ca , Mg ; Seromed) mit Cal- zium- und Magnesiumionen und 20-50 μg Gentamycin pro 100 ml2+ 2+ ferrous saline (PBS + Ca, Mg; Seromed) with calcium and magnesium ions and 20-50 μg gentamycin per 100 ml
PBS gewaschen und zerkleinert. Nach der Zerkleinerung wirdPBS washed and crushed. After crushing
2+ 2+ nochmals 3 mal in PBS und Gentamycin, Ca + Mg (=PBSG2+ 2+ again 3 times in PBS and gentamycin, Ca + Mg (= PBSG
2+ 2+ + Ca + Mg ) gewaschen. Es erfolgt ein lOminütiges2+ 2+ + Ca + Mg) washed. There is a 10 minute walk
Baden in Betaisodoma (einem Verizid, Fungizid und Bakteri-Bathing in betaisodoma (a vericide, fungicide and bacterial
2+ 2+ zid) , 1:10 verdünnt mit PBSG + Ca + Mg . Danach wird durch Zentrifugation und 3-maliges Waschen das Betaisodona entfernt. Anschließend wird homogenisiert und erneut 3 mal gewaschen mit dem Waschmedium, bestehend aus RPMI 1640 + 2 bis 5 μg Gentamycin/1 ml Medium + 20 mM L-Glutamin und 10 Vol.-% gam aglobulienfreies, inaktiviertes Foetales-Käl- berserum. Danach erfolgt die Inkubation in 0,1 % Collagenase2+ 2+ zid), 1:10 diluted with PBSG + Ca + Mg. The betaisodona is then removed by centrifugation and 3 washes. Then it is homogenized and again 3 times washed with the washing medium, consisting of RPMI 1640 + 2 to 5 μg gentamycin / 1 ml medium + 20 mM L-glutamine and 10 vol.% gam agglobule-free, inactivated Foetales calf serum. The incubation then takes place in 0.1% collagenase
Typ I für 60 Minuten bei 37°C unter schonendem Schütteln. Die Filtrierung der Suspension erfolgt über ein 100 μm-Fil- ter, danach wird durch 3-maliges Waschen mit dem gleichen Waschmedium, wie oben beschrieben, und Zentrifugation für jeweils 5 Minuten bei 300 g die Collagenase entfernt. Der Zentrifugationspellet wird schonend im Kulturmedium resus¬ pendiert. Das Kulturmedium besteht aus RPMI 1640 + 20 M 1-Glutamin + 2 bis 5 μg Gentamycin pro 100 ml Medium + 30 Vol.-% ^-globulinfreies inaktiviertes Foetales-Kälberserum + 20 μg ECGS (Endothelial Cell Growth Supplement, Fa. Sigma) + 10 μg Heparin (Sigma H 3125) pro ml Medium + 20 μg MSA (Multiple Stimulating Activity, Fa. Sigma) pro ml Medium + 15 μg Humaninsulin pro ml Medium. Das ECGS, MSA und Insulin werden erst kurz vor der Resuspension der Zellen zugesetzt. Die Vitalität wird mit Acridinorange, verdünnt 1:20 in PBS, getestet. Ca. 95 % der Zellen sind vital. Kultiviert werden die Zellen in Primaria-Kulturflaschen (der Fa. Falcon) oder in der Zellkulturanlage der Fa. Bühler, Tübingen. DieType I for 60 minutes at 37 ° C with gentle shaking. The suspension is filtered through a 100 μm filter, then the collagenase is removed by washing three times with the same washing medium as described above and centrifuging for 5 minutes at 300 g. The centrifugation pellet is gently resuspended in the culture medium. The culture medium consists of RPMI 1640 + 20 M 1-glutamine + 2 to 5 μg gentamycin per 100 ml medium + 30% by volume ^ -globulin-free inactivated fetal calf serum + 20 μg ECGS (Endothelial Cell Growth Supplement, Sigma) + 10 μg heparin (Sigma H 3125) per ml medium + 20 μg MSA (Multiple Stimulating Activity, Sigma) per ml medium + 15 μg human insulin per ml medium. The ECGS, MSA and insulin are added shortly before the cells are resuspended. Vitality is tested with acridine orange diluted 1:20 in PBS. Approximately 95% of the cells are vital. The cells are cultivated in Primaria culture bottles (from Falcon) or in the cell culture system from Bühler, Tübingen. The
Flaschenkulturen werden nach 2 Stunden erneut je 3 mal ge¬ waschen und mit einem Kulturmedium versehen. Nach 24 Stunden erfolgt erneut Mediumwechsel, hernach jeden 2. Tag. Ab dem 2. bis zum 10. Tag werden die Medien gesammelt und eingefro¬ ren (-70°C). Nach 8 bis 10 Tagen ist die Kultur konfluent. Bei Subkultivierung steigt die Verdoppelungszeit von 1 mal pro Tag auf 1 mal alle 8 Tage.Bottle cultures are washed again 3 times after 2 hours and provided with a culture medium. After 24 hours, the medium is changed again, afterwards every other day. From the 2nd to the 10th day, the media are collected and frozen (-70 ° C). After 8 to 10 days, the culture is confluent. With subcultivation, the doubling time increases from once a day to once every 8 days.
Verwendet man anstelle der Flaschenkulturen die Zellkultur¬ anlage der Fa. Bühler, Tübingen, werden über 4 bis 10 Tage die mit Endothelzellen beladenen Microcarriers mit Medium abgesaugt. Es wird stets dasselbe Volumen an unbeladenen Microcarriers und Medium wieder zugegeben. Die entnommenen beladenen Microcarriers werden durch Zentrifuation vom Me- dium getrennt, das so gewonnene Conditioned Medium wird bei -70°C eingefroren. Die auf den Microcarriers sitzenden Zel¬ len werden enzymtisch mit Trpysin-EDTA abgelöst und durch mehrmalige Zentrifugation und mehrmaliges Waschen von Enzym gereinigt. Die Verdoppelungszeit ist zu mindestes 1 mal pro Tag gefunden worden.If, instead of the bottle cultures, the cell culture system from Bühler, Tübingen, is used, the microcarriers loaded with endothelial cells are aspirated with medium over 4 to 10 days. The same volume of unloaded microcarriers and medium is always added again. The removed loaded microcarriers are centrifuged by the dium separated, the conditioned medium obtained in this way is frozen at -70 ° C. The cells sitting on the microcarriers are detached enzymatically with Trpysin-EDTA and cleaned of enzyme by repeated centrifugation and repeated washing. The doubling time was found at least once a day.
Beispiel 3Example 3
EndothelzellidentifizierungEndothelial cell identification
Die nach den Beispielen 1 und 2 gewonnenen Zellen werden auf Chamber-Slides kultiviert und fixiert (Methanol 95 Vol.-%, 5 Vol.-% Eisessig), die so präparierten Chamber-Slides werden überschichtet mit monoklonalen Insulin-Rezeptorantikörpern, • im Verhältnis 1 zu 50 mit PBS verdünnt und bei Raumtempera¬ tur für 30 Minuten inkubiert. Man wäscht 3 mal für jeweils 15 Minuten mit PBS und überschichtet mit Fluoreszenz- oder Rhodamin-gekoppelten Antihumanantikörpern. Danach wird noch¬ mals 3 mal für 15 Minuten gewaschen; man überschichtet das Präparat mit einem monoklonalen Heparin-Rezeptorantikörper, es wird wiederum 3 mal mit der obengenannten Waschflüssig¬ keit gewaschen, mit einem kontrakorrespondierenden Fluor¬ eszenz- oder Rhodamin-gekoppelte Antihumanantikörper über¬ schichtet, erneut 3 mal gewaschen; sodann wird, um zu ver¬ hindern, daß das Präparat austrocknet und die Fluoreszenz verschwindet, dasselbe in ein Gemisch aus Glycerin und p-Phenylendiamin-derivaten oder 1,4-Diazobicyclo(2,2)oktan- derivaten gebettet. Die Auswertung erfolgt über quantitative Fluoreszenzmikroskopie. Beispiel 4The recovered cells according to Examples 1 and 2 are cultured on chamber slides and fixed in methanol (95 vol .-%, 5 vol .-% glacial acetic acid), the thus prepared Chamber slides are coated with monoclonal insulin receptor antibodies, • in the ratio Diluted 1 to 50 with PBS and incubated at room temperature for 30 minutes. Wash three times for 15 minutes each with PBS and overlay with fluorescent or rhodamine-coupled anti-human antibodies. Then it is washed 3 more times for 15 minutes; one overlays the preparation with a monoclonal heparin receptor antibody, it is again washed 3 times with the above-mentioned washing liquid, covered with a counter-corresponding fluorescence or rhodamine-coupled anti-human antibody, washed again 3 times; then, in order to prevent the preparation from drying out and the fluorescence disappearing, the latter is embedded in a mixture of glycerol and p-phenylenediamine derivatives or 1,4-diazobicyclo (2,2) octane derivatives. The evaluation is carried out using quantitative fluorescence microscopy. Example 4
Diagnose-Kit für demyelinisierende ErkrankungenDiagnostic kit for demyelinating diseases
Der Diagnose-Kite besteht aus 10 Objektträgern mit fixierten High Endothelial Cells und je einem Gefäß enthaltend eine Lymphozytensuspension in RPMI 1640 (Fa. Gibco) ; die Suspen¬ sion zeichnet sich durch eine bestimmte Konzentration und durch bestimmte Eigenschaften der Lymphozyten aus. Eine Kon¬ trolle bestehend aus einem physiologischen Leukozytengemisch gehört mit zu dem Set, desweiteren Standardtabellen passend zu den jeweiligen Lymphozyten und Leukozyten. The diagnostic kite consists of 10 slides with fixed high endothelial cells and one tube each containing a lymphocyte suspension in RPMI 1640 (Gibco); the suspension is characterized by a certain concentration and by certain properties of the lymphocytes. A control consisting of a physiological mixture of leukocytes belongs to the set, furthermore standard tables suitable for the respective lymphocytes and leukocytes.

Claims

Patentansprüche Claims
1. Verfahren zur Langzeit-Züchtung beliebiger Endothelzel¬ len, dadurch gekennzeichnet, daß eine High-Endothelial-Zell- kultur aus Retikulo-Histocytärem-Systemgewebe in-vitro ge¬ züchtet wird, die isolierten und gereinigten High-Endothe- lial-Zellen in einer Zellkulturanlage auf Microcarriers an¬ gezüchtet werden und das dabei gewonnene Medium, gegebenen¬ falls angereichert mit frischem Kulturmedium, zur in-vitro- Kultivierung beliebiger Endothelzellen verwendet wird und die geernteten isolierten und gereinigten High-Endothial- Zellen zur Diagnose und Klärung der Ätiopathogenese ver¬ schiedener Krankheitszustände verwendet werden.1. A method for long-term cultivation of any endothelial cells, characterized in that a high-endothelial cell culture from reticulo-histocytic system tissue is grown in vitro, the isolated and purified high-end cell cells in a cell culture plant are grown on microcarriers and the medium obtained, optionally enriched with fresh culture medium, is used for the in vitro cultivation of any endothelial cells and the harvested isolated and purified high-endothial cells are used for diagnosis and clarification of the etiopathogenesis ¬ different disease states can be used.
2. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, daß zur Züchtung von High-Endothelial-Zellkulturen aus Reti- kulo-Histocytärem-Systemgewebe zuerst dieses Gewebe mit ge¬ pufferter Saline oder Kulturmedium in Gegenwart von Kal¬ zium- und Magnesiumionen und eines Antibiotikums gewaschen wird, das Gewebe anschließend homogenisiert, das Homogenisat mit einem Kulturmedium aus RPMI 1640 + Antibiotikum + L-Glu- tamin und Serum gewaschen, das Homogenisat durch Zentrifu- gieren vom Medium befreit, in 0,1 bis 0,2 gew.-%iger Colla¬ genase bei 37°C inkubiert, die Suspension filtriert, das Filtrat durch Zentrifugation und Waschen mit dem Kulturmedi¬ um von der Collagenase befreit und der Zentrifugationspellet in einem Anzuchtmedium aus RPMI 1640 + 20 mMol L-Glutamin + Antibiotikum + 20 bis 30 Vol.-% gammaglobulinfreiem, inakti¬ vierten Foetales-Kälberserum + 20 μg Medium Endothelial- Cell-Growth-Supplement + 100 μg/ml Heparin + 20 μg Multiple Stimulating Activity Factor pro 1 ml Medium + 15 μg Humanin¬ sulin pro 1 ml Medium resuspendiert wird und die so isolier¬ ten und gereinigten High-Endothelial-Zellen auf Micro¬ carriers, bestehend aus denaturierten, pulverisierten Schweincollagen, aufgebracht, die so beladenen Microcarriers bei 37°C bebrütet werden, wobei über 4 bis 10 Tage die mit Endothelzellen beladenen Microcarriers mit dem Medium abge¬ saugt und durch dasselbe Volumen an unbeladenen Micro¬ carriers und Medium ersetzt werden, die entnommenen belade¬ nen Microcarriers anschließend durch Zentrifugation vom Me¬ dium getrennt, das Medium zur Anzüchtung anderer Endothel¬ zellen bereitgestellt und die auf den Microcarriers sitzen¬ den Zellen enzymatisch abgelöst und gereinigt werden.2. The method according to claim 1, characterized in that for the cultivation of high-endothelial cell cultures from reticulo-histocytic system tissue, this tissue is first washed with buffered saline or culture medium in the presence of calcium and magnesium ions and an antibiotic If the tissue is then homogenized, the homogenate is washed with a culture medium composed of RPMI 1640 + antibiotic + L-glutamine and serum, and the homogenate is freed from the medium by centrifugation in 0.1 to 0.2% by weight Collagenase incubated at 37 ° C., the suspension filtered, the filtrate freed of the collagenase by centrifugation and washing with the culture medium and the centrifugation pellet in a culture medium made from RPMI 1640 + 20 mmol L-glutamine + antibiotic + 20 to 30 vol % gamma globulin-free, inactivated fetal calf serum + 20 μg medium endothelial cell growth supplement + 100 μg / ml heparin + 20 μg multiple stimulating activity factor per 1 ml medium + 15 μg human insulin per 1 ml medium is resuspended and the high-endothelial cells isolated and purified in this way are pulverized on microcarriers consisting of denatured Pig collagen, applied, the microcarriers loaded in this way are incubated at 37 ° C., the microcarriers loaded with endothelial cells being suctioned off with the medium over 4 to 10 days and replaced by the same volume of unloaded microcarriers and medium, the loaded loads Microcarriers are then separated from the medium by centrifugation, the medium is provided for culturing other endothelial cells, and the cells sitting on the microcarriers are detached and purified enzymatically.
3. Verfahren zur eindeutigen, spezifischen und unzweifelhaf¬ ten Identifizierung von Endothelzellen, dadurch gekennzeich¬ net, daß an den zu identifiziernden Zellen eine quantitative Immunofluoreszenz mit Hilfe von monoklonalen Insulin- und Heparinrezeptorantikörpern durchgeführt wird.3. Method for the clear, specific and unquestionable identification of endothelial cells, characterized in that quantitative immunofluorescence is carried out on the cells to be identified with the aid of monoclonal insulin and heparin receptor antibodies.
4. Verfahren nach Anspruch 3, dadurch gekennzeichnet, daß die zu identifizierenden Zellen auf einem Chamber-Slide kul¬ tiviert und fixiert werden, die Zellen anschließend mit einem Insulinrezeptorantikörper überschichtet, mit gepuffer¬ ter Saline gewaschen, mit einem Fluoreszenz- oder Rhodamin- gekoppeltem Antihumanantikörper überschichtet, erneut ge¬ waschen, mit einem Heparinrezeptorantikörper überschichtet, erneut mit gepufferter Saline gewaschen, dann mit dem kon¬ trakorrespondierenden Fluoreszenz- oder Rhodamin-gekoppelten Antihumanantikörper überschichtet und, zur Verhinderung der Austrocknung, mit einem Gemisch aus Glycerin und p-Phenylen- diaminderivaten oder 1,4-Diazobicyclo(2,2)octanderivaten überschichtet werden, wobei die Auswertung der Insulin- und Heprain-Rezeptordichte pro Membranfläche durch ein quantita¬ tives Fluoreszenzmikroskop erfolgen kann.4. The method according to claim 3, characterized in that the cells to be identified are cultivated and fixed on a chamber slide, the cells are then covered with an insulin receptor antibody, washed with buffered saline, coupled with a fluorescence or rhodamine Anti-human antibodies overlaid, washed again, overlaid with a heparin receptor antibody, washed again with buffered saline, then overlaid with the counter-corresponding fluorescence or rhodamine-coupled anti-human antibody and, to prevent dehydration, with a mixture of glycerol and p-phenylene Diamond derivatives or 1,4-diazobicyclo (2,2) octane derivatives are overlaid, the insulin and heprain receptor density per membrane area being able to be evaluated by means of a quantitative fluorescence microscope.
5. Verfahren zur Zellisolierung, dadurch gekennzeichnet, daß ein Organhomogenisat mit Fluoreszenz- oder Rhodamin-gekop¬ pelten Insulin- und Heparin-Rezeptorantikörpern markiert. wobei die Antikörper stets kontrakorrespondierend gekoppelt sein müssen, die markierten Zellen mit Hilfe eines Fluor- eszenz-activated Cell Sorters getrennt und die entsprechen¬ den Zellen anschließend kloniert werden.5. A method for cell isolation, characterized in that an organ homogenate with fluorescence or rhodamine-gekop¬ marked insulin and heparin receptor antibodies marked. the antibodies must always be coupled in a corresponding manner, the labeled cells separated with the aid of a fluorescence-activated cell sorter and the corresponding cells then cloned.
6. Verwendung der nach dem Verfahren gemäß Anspruch 1 gewon¬ nenen Endothelzellen für die eindeutige und unzweifelhafte Diagnose und Klärung der Ätiopathogenese von Krankheitszu- ständen. 6. Use of the endothelial cells obtained by the method according to claim 1 for the clear and unequivocal diagnosis and clarification of the etiopathogenesis of disease states.
PCT/DE1986/000355 1985-09-07 1986-09-04 Long term culture, identification, characterisation and use of endothelial cells WO1987001385A1 (en)

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DEP3531966.6 1985-09-07
DE19853531966 DE3531966A1 (en) 1985-09-07 1985-09-07 METHOD FOR LONG-TERM BREEDING OF ANY ENDOTHEL CELLS, METHOD FOR IDENTIFYING AND CHARACTERIZING THEM AND THEIR USE FOR DIAGNOSIS AND CLEARING OF THE AETIOPATOGENESIS OF DISEASES

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Publication number Priority date Publication date Assignee Title
EP0415695A1 (en) * 1989-08-30 1991-03-06 Eli Lilly And Company Selective cloning for high monoclonal antibody secreting hybridoma

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EP0233925A1 (en) 1987-09-02
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DE3531966A1 (en) 1987-03-12

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