EP0966522A1 - Lignee cellulaire humaine du foie - Google Patents
Lignee cellulaire humaine du foieInfo
- Publication number
- EP0966522A1 EP0966522A1 EP98916819A EP98916819A EP0966522A1 EP 0966522 A1 EP0966522 A1 EP 0966522A1 EP 98916819 A EP98916819 A EP 98916819A EP 98916819 A EP98916819 A EP 98916819A EP 0966522 A1 EP0966522 A1 EP 0966522A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- hepatocytes
- hepz
- cell line
- expression
- 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.)
- Withdrawn
Links
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
- C12N5/067—Hepatocytes
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/12—Hepatocyte growth factor [HGF]
-
- 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
Definitions
- the invention relates to a new human liver cell line that can be used for toxicological, physiological and particularly gene therapy studies. Areas of application are molecular biology, medicine and the pharmaceutical industry.
- Gene therapy for diseases of the liver is an important goal of molecular medicine, since a large number of genetic diseases, but also infectious and tumor diseases, originate in this organ.
- the complex route of ex vivo gene therapy has been opened up internationally, in which part of the liver is removed and the cells isolated therefrom are treated in the cell culture with the therapeutic gene and then transplanted back into the liver.
- Gene transfers in cells in culture can be carried out relatively easily using various physical or chemical means.
- the highest effectiveness is achieved with vectors that are derived from viruses and have been genetically modified in a targeted manner. Retroviruses and adenoviruses have been used particularly successfully.
- Dividing differentiated hepatocytes are necessary for such tests. Attempts have long been made to establish lines of differentiated hepatocytes in rodents and humans.
- human hepatocytes were immortalized with the SV40 T-Ag using retroviral infection.
- the lines THLE-2 and -3 obtained were not tumorigenic in nude mice and did not express AFP.
- the cells from previous passages were able to produce albumin and cytokeratin 8 (CKN8, characteristic of hepatocytes) and to metabolize a number of chemical carcinogens.
- the expression of cytokeratin (CKN19, characteristic of bile duct epithelium) and a reduction in albumin expression were observed in cells of later passages (Pfeifer et al., 1993).
- Two lines of differentiated, immortalized hepatocytes were obtained from transgenic mice that express the gene for the hepatocyte growth factor TGF- ⁇ .
- the cells of the two lines AML-12 and AML-14 were not tumorigenic in nude mice and expressed mRNA for albumin, a-1-antitrypsin (AAT) and transferrin. However, the expression of the hepatocyte-specific genes decreased with increasing number of passages (Wu et al., 1994).
- the aim of the invention is to establish a new human liver cell line which can be used for toxicological, physiological and, in particular, gene therapy studies.
- the object of the invention is to achieve this goal by genetically influencing the cell cycle regulation of human hepatocytes.
- the cell line starting from primary human hepatocytes, the following steps were carried out according to the invention (which are also generally applicable to liver cells)
- the expression of the gene p53 was additionally blocked with the aim of avoiding the apoptotic death of the genetically modified primary hepatocytes.
- This blockade was carried out as follows: The hepatocytes obtained from the liver segment were sown with a cell density of 5 ⁇ 10 5 per 6 cm dish and cultivated for 2 hours with WM E with growth factors and 10% FCS. 2 hours after plating out, the medium was changed with serum-free WM E. HGF (10ng / ml) was added to the medium. HGF levels in serum are known to increase in the first hours after partial liver resection. The peak of DNA synthesis does not take place until 24-48 hours (in different species) after the operation; that is, about 20-40 hours after the start of the HGF effect. It was expected that the peak of DNA synthesis triggered by plating and HGF influence in primary human hepatocytes should also take place in the same period.
- hepatocytes were transfected using lipofectamine using the following plasmids (in a ratio of 1: 1: 1: 1):
- hepatocytes were transfected in 5 dishes (total cell number 2.5 ⁇ 10 6 ). Furthermore, 5 hours after the transfection when changing the medium, 10% FCS was also added to the medium (as well as growth factors). 18 days after the transfection, the majority of the transfected cells had degenerated (while the hepatocytes in the control dishes retained their viability for up to 6 weeks). During this period, only 4 colonies were observed in one dish. One of the colonies was transferred to a 24-well plate. Fresh WM E and conditioned supernatant from colonies formed (in a ratio of 1: 1) were added to these cells. The cells grew and after 2 days the number of cells doubled.
- the morphology of the cells in the center of the colony was very similar to that of primary hepatocytes, while the cells at the edge of the colony were very extended.
- the cells of the colony had rounded and then peeled off the surface of the dishes, after which cell death was observed.
- the cells If they are sown as a single cell suspension in low cell density, then they have an extended morphology. After the formation of line-to-line contacts, the cells become polygonal Hepatocyte-like morphology. After confluence, the cells do not form a "three-dimensional foci", but detach from the surface of the shell. This indicates that the cells had not been transformed.
- HepZ The established hepatocytes were called HepZ.
- the established cells were analyzed for hepatocyte-specific parameters using immunofluorescence.
- Primary human hepatocytes (fixed 24 hours after isolation - HuPrimHep) served as controls, and the cells of the human hepatoma lines HepG2 and HuH7. The results are shown in Table 1.
- Hepatocyte-specific markers of established human hepatocytes (HepZ) in immunofluorescence
- HepZ (Fig. 3a) as well as HuH7 (Fig. 3b), HepG2 cells (Fig. 3c) and primary hepatocytes (Fig. 3d) are strongly positive for albumin.
- AAT Alpha-1-Antitrypsin
- the HepZ were still positive after 3 passages.
- HepZ are negative for alpha-fetoprotein (AFP) (Fig. 3f). This indicates that the established cells come from differentiated hepatocytes. All cells (including primary human hepatocytes) were positive for both CKN8 and CKN19.
- the HepZ line was also tested for expression of p53 and pRb.
- the hepatoma lines mentioned above were used as controls (Table 2).
- HepG2 and HuH7 cells served as controls.
- Hepatocyte line HepZ established from primary human hepatocytes using lipofectamine-mediated transfection with a plasmid combination: pAlbasp53 + pAlbas Rb + pCMVE2F + pSV2neoDl (after 15 passages) (enlargement 1: 100)
- HuH7 and HepG2 hepatoma lines served as controls. a. strong expression of the mutant p53 in HuH7 cells, b.
- HepZ cells The expression of pRb in d. hepG2, e. HepZ.
- Lane 1 HepG2 cells
- Lane 2 HepZ cells
- Lane 3 HuH7 cells
- b. p53 expression Lane 1: HuH7 cells
- Lane 2 HepZ cells.
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (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)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
L'invention concerne une nouvelle lignée cellulaire humaine du foie, pouvant être utilisée pour des recherches toxicologiques, physiologiques et en particulier pour la thérapie génique. La biologie moléculaire, la médecine et l'industrie pharmaceutique constituent des domaines d'application de l'invention. Cette nouvelle lignée cellulaire humaine du foie est caractérisée par les paramètres suivants: albumine: positif, alpha-1-antitrypsine (AAT): positif, et alpha-foetoprotéine (AFP): négatif. Elle est déposée auprès de la Deutsche Sammlung von Mikroorganismen (DSMACC2302).
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19711266A DE19711266C2 (de) | 1997-03-05 | 1997-03-05 | Neue humane Leber-Zellinie |
DE19711266 | 1997-03-05 | ||
PCT/DE1998/000604 WO1998039417A2 (fr) | 1997-03-05 | 1998-03-03 | Lignee cellulaire humaine du foie |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0966522A1 true EP0966522A1 (fr) | 1999-12-29 |
Family
ID=7823784
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP98916819A Withdrawn EP0966522A1 (fr) | 1997-03-05 | 1998-03-03 | Lignee cellulaire humaine du foie |
Country Status (7)
Country | Link |
---|---|
US (1) | US6046050A (fr) |
EP (1) | EP0966522A1 (fr) |
JP (1) | JP2001523953A (fr) |
AU (1) | AU7029198A (fr) |
CA (1) | CA2285677A1 (fr) |
DE (1) | DE19711266C2 (fr) |
WO (1) | WO1998039417A2 (fr) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2550452A1 (fr) * | 2003-10-10 | 2006-04-20 | Multicell Technologies, Inc. | Hepatocytes immortalises |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5665589A (en) * | 1988-12-14 | 1997-09-09 | The United States Of America As Represented By The Department Of Health And Human Services | Human liver epithelial cell lines |
ES2181677T3 (es) * | 1991-08-23 | 2003-03-01 | Univ Nebraska | Metodo y composiciones utiles en la reprogramacion celular. |
-
1997
- 1997-03-05 DE DE19711266A patent/DE19711266C2/de not_active Expired - Fee Related
-
1998
- 1998-03-03 AU AU70291/98A patent/AU7029198A/en not_active Abandoned
- 1998-03-03 JP JP53805498A patent/JP2001523953A/ja active Pending
- 1998-03-03 WO PCT/DE1998/000604 patent/WO1998039417A2/fr not_active Application Discontinuation
- 1998-03-03 EP EP98916819A patent/EP0966522A1/fr not_active Withdrawn
- 1998-03-03 CA CA002285677A patent/CA2285677A1/fr not_active Abandoned
- 1998-03-05 US US09/035,396 patent/US6046050A/en not_active Expired - Fee Related
Non-Patent Citations (1)
Title |
---|
See references of WO9839417A2 * |
Also Published As
Publication number | Publication date |
---|---|
US6046050A (en) | 2000-04-04 |
WO1998039417A3 (fr) | 1998-12-17 |
WO1998039417A2 (fr) | 1998-09-11 |
AU7029198A (en) | 1998-09-22 |
DE19711266C2 (de) | 1999-06-17 |
CA2285677A1 (fr) | 1998-09-11 |
JP2001523953A (ja) | 2001-11-27 |
DE19711266A1 (de) | 1998-09-17 |
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Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 19990921 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DK ES FI FR GB IT LI NL SE |
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RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: KIRILLOWA, IRA Inventor name: STRAUSS, MICHAEL |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: DEVELOGEN AKTIENGESELLSCHAFT FUER ENTWICKLUNGSBIOL |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20021001 |