EP1523550A2 - Cellules epitheliales du colon immortalisees et leur utilisation - Google Patents

Cellules epitheliales du colon immortalisees et leur utilisation

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Publication number
EP1523550A2
EP1523550A2 EP03771082A EP03771082A EP1523550A2 EP 1523550 A2 EP1523550 A2 EP 1523550A2 EP 03771082 A EP03771082 A EP 03771082A EP 03771082 A EP03771082 A EP 03771082A EP 1523550 A2 EP1523550 A2 EP 1523550A2
Authority
EP
European Patent Office
Prior art keywords
colon
epithelial cells
colon epithelial
cells
crypts
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP03771082A
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German (de)
English (en)
Inventor
Pablo Steinberg
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitaet Postdam
Original Assignee
Universitaet Postdam
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Filing date
Publication date
Application filed by Universitaet Postdam filed Critical Universitaet Postdam
Publication of EP1523550A2 publication Critical patent/EP1523550A2/fr
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Classifications

    • 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
    • C12N5/0679—Cells of the gastro-intestinal tract
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells

Definitions

  • the invention relates to immortalized colon epithelial cells and cell lines and the use of the colon epithelial cell lines for the study of colon-relevant physiological processes and the development of colon cancer.
  • Numerous possibilities are known for examining physiological processes or processes of cancer development on cells.
  • One possibility is, for example, an examination of a living object.
  • An alternative is the investigation on cell cultures.
  • Cell cultures which are primary cultures and can be maintained in vitro for approximately one week at a 24 hour division. By definition, primary cultures are all in vitro cultivations of cells, tissues and organs that have been taken directly from the organism. A complete dissociation of the tissue or the organ is particularly necessary to obtain a cell culture.
  • Primary cultures or cell cultures of numerous cells are known in the prior art, for example cardiac muscle cells from chickens or from neonatal rat hearts, from fresh skin samples of human origin and from solid human tumors and the like. a.
  • cell cultures are known which are not primarily obtained and cultivated from the organism, but represent permanent cultures. There will be secondary cultures and Cell lines distinguished. Secondary cultures are cell strains that reach different generations from a primary culture. Cell lines are permanent viable cultures. Numerous cell lines have been established by various tumors. Cell lines are mostly uniform in their morphology and in their functional properties. Certain cell lines are characterized by selective growth properties that certain cell culture media with defined growth factors require; these cell lines are therefore valuable objects for the study of growth and differentiation processes, including the processes of malignant transformation. With the help of cell cultures, it is also possible to carry out special tests, such as tests for in vitro or in vivo toxicity, for testing acute cell toxicity, testing for cell viability or tests for measuring viability and normal and degenerate Growth.
  • special tests such as tests for in vitro or in vivo toxicity, for testing acute cell toxicity, testing for cell viability or tests for measuring viability and normal and degenerate Growth.
  • Cell cultures are also used to detect mutagenic substances. It is also possible to use cultivated cell lines for transfection, for example for transfection by means of electroporation or for cloning and for testing the proliferation rate.
  • cultivated cell lines for transfection, for example for transfection by means of electroporation or for cloning and for testing the proliferation rate.
  • examinations such as the proliferation control or the mutagenicity test in vitro in such a way that they allow as many conclusions as possible to be drawn about the in vivo behavior of the cells, it is necessary for certain types of tumor, such as liver cancer or colon cancer, healthy, untreated liver tissue or To establish colon cells or cell lines.
  • tumor such as liver cancer or colon cancer
  • colon cells or cell lines are available in the prior art for examining and monitoring the development of important tumors.
  • Colon cancer is one of the most common cancers in western industrialized countries. In the Federal Republic of Germany, an estimated 24,000 men and 28,000 women develop colon
  • the human colon tumor cells are already malignant transformed cells, so that they cannot be used to deal with the question of colon cancer;
  • the small intestine epithelial cells like the IEC-6 and IEC-18 cells, behave completely differently from colon colon epithelial cells as a cell culture, and (iii) the colon colon primary cell cultures have only a limited lifespan, usually 7 to 10 days, so that they not suitable for studying processes that take a long time, such as testing mutagenic substances or the development of cancer.
  • Lacy and Colony (1985) were able to show that the expression of carbonic anhydrase can only be detected in the colon cells at the time an organism is born. Whereas saccharase activity is present in fetal colon cells and is practically no longer measurable at birth. Furthermore, Guer and Verity (1990) found that NA + / K + -ATPase activity in fetal colon cells is very low and only increases sharply after birth. Takahashi et al. (1998) further disclosed that the IIß-hydroxysteroid dehydrogenase type II activity is hardly detectable in fetal colon cells up to the 40th week of pregnancy and only increases sharply after birth.
  • fetal human large bowel epithelial cells are much less sensitive to apoptosis-inducing agents, such as, compared to adult human large bowel epithelial cells.
  • the present invention solves this technical problem by providing non-tumorigenic adult human or rat colon epithelial cells, which are obtained by removing crypts containing colon epithelial cells from the colon tissue of untreated humans or rats, diluting and plating the crypts and the in- Contacting the crypts with a retrovirus comprising the gene for the -SV40 large T antigen, thereby infecting the colon epithelial cells with the retrovirus and recovering the non-tumorigenic adult colon epithelial cells.
  • the colon is removed from an untreated rat or a piece of the human colon.
  • the large intestine can then be cut into small pieces or broken up differently and incubated in suitable solutions known to those skilled in the art.
  • the solutions can be selected such that they complex calcium, for example, which leads in particular to the fact that the crypts of the large intestine sit very loosely in the tissue.
  • Crypts are folds on the epithelial surface of the colon that serve to enlarge the surface.
  • the crypts are jointly responsible for secretion and cell renewal. Since there are no villi in the large intestine - as in the small intestine - the crypts have an important function.
  • Retroviruses containing the sequence for the SV 40 large T antigen are then brought into contact with the crypts in such a way that the colon epithelial cells located in the crypts are infected with the retroviruses.
  • Retroviruses are particularly well suited for transfection because they have a high integration guarantee.
  • the integration can take place anywhere in the genome, but in particular only one copy is integrated without it being advantageous. ' there are major changes at the integration site. If the integration in each cell takes place at a different location : several cells can each emerge from a different genome. However, it can also be provided that the integration takes place at the same or similar location in the genome.
  • nucleic acid sequences are known to the person skilled in the art, for example microinjection, DNA transfer using the gene gun, either using microprojectiles or using macroprojectiles, or using cosmids or plasmids. These methods can be used in combination or in addition to the retroviral infection.
  • microinjection DNA transfer using the gene gun, either using microprojectiles or using macroprojectiles, or using cosmids or plasmids.
  • cosmids or plasmids can be used in combination or in addition to the retroviral infection.
  • SV 40 large T antigen in the genome of the. Colon epithelial cells
  • these cells are immortalized in the crypts.
  • the fact that the cells are located in particular in the crypts advantageously keeps their degree of differentiation. Immortalization enables an unlimited number of cell culture passages, ie the cells can divide almost indefinitely and are therefore available for long-term experiments of up to half a year or longer.
  • the Cre recombinase of a selected bacteriophage catalyzes a site-specific recombination between the so-called Loci-LoxP. Since a ligation step in the classic sense is missing, this method is advantageously very quick and error-free.
  • the protein Cre is a recombinant enzyme, a recombinase that recognizes and binds to a 34 base long DNA segment called LoxP sites. The Cre recombinase advantageously mediates a recombination between two LoxP sites.
  • the recombination event advantageously advantageously specifically removes the sequence between the LoxP sites.
  • the nucleic acid fragments to be examined cannot replicate themselves, they must be coupled with a suitable replicon.
  • Such replicons are particularly vectors or cloning vehicles.
  • Small plasmids and bacteriophages are suitable as vectors and can be modified in many ways in order to improve the introduction of recombinant DNA or RNA molecules into cells, preferably colon epithelial cells, and to facilitate the selection of successfully transformed cells.
  • Cre is an enzyme isolated from viruses that cuts the chromosomes into specific sections, which advantageously stimulates an exchange of these sections between the chromosomes.
  • the vector mentioned advantageously enables transient expression of the SV 40 large T antigen. It is also possible that the vector includes genes for herpes simplex virus thymidine kinase (TK). These genes can be flanked by LoxP sites, the substrate of the bacteriophage recombinase Cre. However, the retrovirus can also include genes for the Cre recombinase and selection markers. With the markers used, it is advantageously possible to use the cell line for more than one To grow year in culture.
  • TK herpes simplex virus thymidine kinase
  • the crypts and the colon epithelial cells located in them are brought into contact with the retroviruses after removal of the intestine or after removal from the intestine without further intermediate steps, in particular cultivation steps. It is therefore preferred to use fresh colon epithelial cells, di ⁇
  • the invention also relates to the use of the cells according to the invention for establishing a colon epithelial cell line.
  • the primary culture of the individual colon epithelial cells according to the invention becomes a cell line which consists of numerous sublines of the cells from which the primary culture was originally composed.
  • the proliferation and growth of cells 1 in a cell line in vitro including cultures of single cells . is accordingly according to the invention as. Called cell culture.
  • a cell line consists of numerous sublines of the cells from which the primary culture originally consisted. In cell cultures, the cells no longer organize themselves in the tissue.
  • a continuously growing culture that has a large number of population doubles behind it is referred to in the sense of the invention as an established cell culture.
  • the colon epithelial cells and / or the colon epithelial cell lines can be used for the screening of drugs.
  • the cells and cell lines according to the invention in addition to screening - for other combinatorial tests.
  • the cells can be a probe that integrates with solid phase assays or other target structures, or it is possible that the cell line is used as a target in a combinatorial test, using selected probes - e.g. B. phages or molecules - interacts.
  • the colon epithelial cells or the colon epithelial cell lines are used to examine the development of colon cancer, physiological, pathophysiological and / or toxic processes. With the colon cells or colon epithelial cell lines according to the invention, it is advantageously possible to investigate the development of colon cancer or other physiological processes over a long period of time using homogeneous cultures. In particular, it is possible
  • the colon epithelial cells or cell cells according to the invention are lines used for long-term cultures, preferably for culture periods of 6 to 12 months.
  • Physiological processes or processes of colon cancer usually take periods of several weeks, months or more.
  • Known cell cultures can be established for a few weeks with stable quality. Test series that require a longer time frame cannot be examined with these cell cultures.
  • the cells and cell lines of the colon epithelium according to the invention are able to carry out examinations on one and the same cell line which take longer than 1 year;
  • the cells of the invention and / or cell lines can • be preferably continued for the following applications used: (a) for the identification of metabolites formed after incubation of the substance with the cells and are located in the cell culture supernatant and in the cells'
  • Substances in the sense of the invention can be all biological, chemical but also physical material effects, such as. B. lohen, complex compounds, drugs, carbohydrates, lipids, proteins, mixtures of these, environmental substances, food components, radioactive radiation, X-rays and others. That is to say, a substance in the sense of the invention is any agent whose effect on the cells or on substances which in turn act on the cells is to be tested.
  • the colon epithelial cells and cell lines according to the invention have several advantages over the known cell lines. Numerous cell lines that are used to examine the processes in the colon area come from colon tumor cells. Such cells are already malignant and can no longer be used for attempts to develop and develop tumors.
  • the known colon primary cell cultures have a limited lifespan of approximately 14 days, so that they are not suitable for studying processes which take a long time. With the cells and cell lines according to the invention, a system is made available for the first time, which makes it possible to carry out experiments on non-tumorigenic adult cells from rats or humans which take a physiologically long period of time.
  • Another advantage of the cells and cell lines according to the invention is that the tissues or the organisms - namely Humans and rats - from which they are taken, do not need to be pretreated. This also largely maintains the degree of differentiation of the cells in the fresh tissue.
  • the cells or cell lines according to the invention can advantageously be kept in culture media which are easy to prepare. This cultivation of the cells in simple culture media enables them to be used to investigate transport processes and to metabolize foreign substances in test systems to test the mutagenicity or cytotoxicity 1 of basic chemicals, food ingredients, pollutants and medicinal products and to investigate the molecular basis of colon cancer use.
  • the invention also relates to a method for producing the colon epithelial cells and cell lines according to the invention, this method comprising the following steps:
  • the invention also relates to a kit which comprises at least one colon epithelial cell according to the invention.
  • This kit can be used, for example, as a diagnostic kit to carry out a proliferation control or a mutagenicity test. With this kit, colon-relevant physiological processes as well as tumor development in vitro can be researched.
  • Colon cell includes.
  • the Ijit according to the invention can of course comprise further constituents which are necessary, for example, to ensure the survival of the cell or to cultivate it.
  • the kit according to the invention can advantageously comprise substances which are suitable for researching colon-relevant physiological processes or the development of tumors in vitro.
  • the cells of the invention in the kit - essentially separated from each other - be stored, so it that for example, by a professional. Examination of tumor development under the conditions desired by them can be merged.
  • the kit according to the invention can further comprise biological, chemical and / or physical agents which serve the research of physiological and patnophysiological processes.
  • Such a physical means can be, for example, an optical device with which metabolic processes or the growth of the cells can be observed and determined.
  • Biological or chemical agents are e.g. B. nutrient media, other cells or cell cultures, toxins, buffer solutions, drugs to be examined, food components and the like.
  • the colon is removed from an untreated rat.
  • the data content is rinsed out with 0.15 M NaCl solution.
  • a 4 cm long middle section of the intestine is then removed and cut open lengthways.
  • the middle part of the intestine is dissolved in 30 ml of a solution A (109 mM NaCl, 2.4 mM KC1, 1.5 mM KH 2 PO 4 , 4.3 mM Na 2 HPO 4 , 1.5 mM EDTA, 10 mM glucose, 5 mM Glutamine; pH 7.4) at 37 ° C, 50 min and 100 revolutions / min.
  • the center piece is then concentrated in solution B (Dulbecco's modified Eagle Medium lOx, 500 ml fetal bovine serum, 5%; insulin, 0.25 units / ml; NaHC0 3 , 7.5%; glutamine, 2.99%; gentamycin, 25 ⁇ g / ml; penicillin / streptomycin, each 100 ⁇ g / ml).
  • the crypts which are located in the central part of the large intestine, are diluted in solution B and plated out in 12 or 24 well plates. Through the use of EDTA (ethylenediaminotetraacetic acid disodium salt), the calcium complexes in the tissue and leads to the crypts only sitting loosely in the tissue. By slightly swiveling the middle part of the intestine in solution B, the crypts detach from the tissue and pass into solution B.
  • EDTA ethylenediaminotetraacetic acid disodium salt
  • the sequence for da's SV 40 large T antigen (in the form of the vector LoxP-HyTK-large T) is introduced into the amphotropic packaging cell line PA317 and GP + envAml2.
  • This method is Liang-Ping Li et al. , Human Gene Therapy 8, pp. 1695-1700 (September 20, 1997) and thus included in the disclosure of this invention.
  • the packaging cell lines release retroviruses, which contain the sequence for the SV 40 large T antigen, into the culture supernatant. To immortalize the colon epithelial cells, portions of the supernatant are made of these Packing cell lines placed on the colon epithelial cells.
  • the retroviruses infect the colon epithelial cells and in the nucleus of the host cells, ie in this case the colon epithelial cells, the viral DNA is incorporated into the host genome of the colon epithelial cells.
  • the colon epithelial cells thus produce SV 40 i large T antigen after the viral infection, which leads to immortalization of the colon epithelial cells by complexing the cellular p53 and Rb protein.
  • the colon cells obtained in this way can be used for test systems to test the mutagenicity or cytotoxicity of chemicals and other molecular tests.
  • the immortalized human colon epithelial cells are in a serum-free cell culture medium, such as. B. described in US Pat. No. 6,395,542 B1, sown thinly.
  • a substance e.g. 0.05 or 0.1 ⁇ g

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des cellules épithéliales du côlon non tumorigènes, adultes, de rats ou d'êtres humains, pouvant être obtenues par : prélèvement de cryptes, contenant des cellules épithéliales du côlon, dans le tissu du côlon d'êtres humains ou de rats non traités ; dilution et mise en culture des cryptes sur boîte de Pétri ; et mise en contact des cryptes avec un rétrovirus contenant le gène de l'antigène grand T du SV 40, pour infecter ainsi les cellules épithéliales du côlon avec le rétrovirus et obtenir les cellules épithéliales du côlon adultes non tumorigènes.
EP03771082A 2002-07-24 2003-07-24 Cellules epitheliales du colon immortalisees et leur utilisation Withdrawn EP1523550A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE2002134508 DE10234508A1 (de) 2002-07-24 2002-07-24 Immortalisierte Dickdarmepithelzellen und ihre Verwendung
DE10234508 2002-07-24
PCT/EP2003/008110 WO2004011629A2 (fr) 2002-07-24 2003-07-24 Cellules epitheliales du colon immortalisees et leur utilisation

Publications (1)

Publication Number Publication Date
EP1523550A2 true EP1523550A2 (fr) 2005-04-20

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EP03771082A Withdrawn EP1523550A2 (fr) 2002-07-24 2003-07-24 Cellules epitheliales du colon immortalisees et leur utilisation

Country Status (4)

Country Link
EP (1) EP1523550A2 (fr)
AU (1) AU2003254584A1 (fr)
DE (1) DE10234508A1 (fr)
WO (1) WO2004011629A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111172114B (zh) * 2019-12-10 2021-11-30 上海中医药大学附属岳阳中西医结合医院 一种人源化肠癌前病变永生化上皮细胞系、构建方法及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
None *
See also references of WO2004011629A3 *

Also Published As

Publication number Publication date
WO2004011629A3 (fr) 2004-04-15
AU2003254584A8 (en) 2004-02-16
DE10234508A1 (de) 2004-02-12
WO2004011629A2 (fr) 2004-02-05
AU2003254584A1 (en) 2004-02-16

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