EP1869167A1 - Procede de production de cellules tumorales de culture primaire, a partir de tissus tumoraux, notamment de carcinomes mammaires, cellules tumorales de culture primaire correspondantes et leur utilisation - Google Patents

Procede de production de cellules tumorales de culture primaire, a partir de tissus tumoraux, notamment de carcinomes mammaires, cellules tumorales de culture primaire correspondantes et leur utilisation

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Publication number
EP1869167A1
EP1869167A1 EP06722757A EP06722757A EP1869167A1 EP 1869167 A1 EP1869167 A1 EP 1869167A1 EP 06722757 A EP06722757 A EP 06722757A EP 06722757 A EP06722757 A EP 06722757A EP 1869167 A1 EP1869167 A1 EP 1869167A1
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EP
European Patent Office
Prior art keywords
tumor
cells
primary culture
culture
tumor cells
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EP06722757A
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German (de)
English (en)
Inventor
Ralf Hass
Jutta Rogoll
Hartmut Kueppers
Axel Dehn
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Medizinische Hochschule Hannover
Mylan Healthcare GmbH
Original Assignee
Medizinische Hochschule Hannover
Solvay Arzneimittel GmbH
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Application filed by Medizinische Hochschule Hannover, Solvay Arzneimittel GmbH filed Critical Medizinische Hochschule Hannover
Publication of EP1869167A1 publication Critical patent/EP1869167A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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

Definitions

  • the present invention relates to methods for obtaining primary culture tumor cells from tumor tissue. More particularly, the present invention relates to a method for obtaining primary culture tumor cells from tumor tissue, in particular breast cancer, wherein in one process step the tumor tissue is comminuted into tumor tissue of a particular size and then these tissue pieces are cultured under certain conditions. The invention further relates to the primary culture tumor cells obtainable by this method from the tumor tissue, in particular primary culture tumor cells from mammary carcinoma. Finally, the invention relates to the use of these primary culture tumor cells u. a. for the determination of an individual tumor therapy or for the testing and screening of new tumor therapeutics.
  • Tumors and related cancers are among the most common causes of death in humans.
  • Breast cancer as a form of malignant breast disease, is by far the most common cancer in women. Due to the very limited treatment options, surgery, chemo- / hormone therapy, radiotherapy - with diversified tumor progression, breast cancer is also one of the most common causes of death in women. But other cancers, such as bladder cancer, lung cancer, skin cancer or other cancers of epithelial origin as well as other types of glandular cancer, such as pancreatic cancer are a common cause of death in humans.
  • adjuvant drug therapy with cytotoxic drugs is often followed by surgery and the associated removal of the primary tumor, breast cancer.
  • this adjuvant medicinal therapy becomes effective performed with cytostatics.
  • Other therapeutic methods include radiotherapy or hormone therapy or combinations of these forms of therapy.
  • the hormone receptor status of the tumor tissue has been taken to perform stratification of the patient, ie to determine the strategy for the therapy of the patient. The right choice of therapy is very important.
  • Hormone therapies can only be used for treatment if hormone receptor positive cancer cells, eg Estrad iol-positive breast cancer cells, are detected in the tumor. Furthermore, it is known that a certain percentage of these hormone receptor-positive tumors do not respond to hormone therapy. The same applies to other types of therapy or combined therapies such as simultaneous chemo- and hormone therapy or a combined chemo- and radiotherapy.
  • the right choice of therapy, the so-called individualized therapy of the diseased person is therefore essential in the treatment of tumor diseases. In particular, since the time after diagnosis of the cancer also plays an essential role in the success of the therapy.
  • the classic prognostic factors in patients with primary breast cancer are tumor size, Differentiation type, hormone receptor status and lymph node involvement.
  • tumors which according to these established prognostic factors should actually be associated with a good prognosis, can take an aggressive course of the disease.
  • Approximately 30% of patients with nodal-negative breast cancer experience recurrence within 10 years of diagnosis. Therefore, the stratification of the individual patient and thus the determination of the individual tumor therapy is an essential point in a successful treatment of tumor patients.
  • immortalized mammalian carcinoma cell lines are known. By immortalization they represent in contrast in vivo conditions a more artificial cell culture. Amongst other things, these immortalized cell lines are distinguished by the fact that their response to active substances, for example for testing drugs, is different than the reaction of cells in vivo, ie in patients. Hass et al. (Signal Transduction, 3, pages 9-17, 2003) for differences in the responses of normal mammary epithelial cells (HMEC-1) to breast tumor cell lines MDA-MB-231 and MCF-7, respectively, to increased estrogen concentration. Attempts to obtain primary culture cells from tumor tissue, eg from breast cancer, were performed.
  • HMEC-1 normal mammary epithelial cells
  • the object of the present invention is therefore to provide a method which allows the preservation of primary culture tumor cells from the tumor tissue and thereby overcomes the above-mentioned disadvantages, i. which allows prolonged cultivation of the tumor cells without substantial modification of them.
  • These primary culture tumor cells thus obtained permit stratification of patients for therapy but also use of these obtained cells in other areas, e.g. in the field of screening and testing of new potential tumor therapeutics as well as in the development and testing of appropriate application regimens of new and old tumor therapeutics.
  • tumor tissue samples of tumors in particular epithelial tumors, such as mammary carcinomas
  • This direct expansion of ex vivo tumor primary cultures offers a range of individual diagnostic and therapeutic applications.
  • the present invention is therefore also directed to the use of these tumor primary cultures in screening assays and test systems for stratifying patients, ie for determining the individual tumor therapy of the patient.
  • the tumor primary cultures allow the provision of systems for testing and screening new potential tumor therapeutics and for determining the application regimens of new and old tumor therapeutics.
  • the method according to the invention comprises the processing of the tumor tissue without an enzymatic digestion, in particular without a .Proteaseverdau, before culturing the tumor tissue.
  • the tumor tissue is crushed into tumor tissue of a size with a diameter of about 0.5 mm to about 3 mm for cultivation.
  • the tumor tissue pieces are cultivated in medium suitable for tumor cells.
  • This medium is preferably supplemented with bovine pituitary extract, hydrocortisone, insulin and human epidermal growth factor. In other preferred embodiments, this medium contains no serum and / or antibiotics.
  • the tumor tissue is preferably primary breast cancer.
  • these tumor primary cultures form multilayer cell aggregates on cultivation.
  • FIG. 1 is an electron micrograph of breast cancer primary culture cells.
  • FIGS. 2 A to D are fluorescence images of breast cancer primary culture cells labeled with different markers.
  • FIG. 2A panCytokeratin
  • FIG. 2B vimentin
  • FIG. 2C DAPI.
  • Figure 2 D shows a superposition of the Einzelfluoreszenzingn, as shown in Figures 2 A to C are shown.
  • Figure 3 is a graph of cell cycle analysis of various passages of the culture of breast cancer primate tumor cells.
  • P2 to P7 mean the corresponding passages, passage 2 to passage 7.
  • FIG. 4 is a western blot which in turn shows the expression of panCytokeratin, vimentin and actin at the protein level in various passages during the cultivation of breast cancer tumor cells.
  • the invention provides a method for obtaining primary culture tumor cells from tumor tissue, in particular tumor primary cultures from mammary carcinomas, as well as primary culture tumor cells obtainable by this method, in particular tumor primary cells of mammary carcinoma.
  • the primary culture tumor cells according to the invention which in a preferred embodiment are derived from epithelial cell carcinomas, in particular mammary carcinomas, can be obtained from tumor tissue samples by the method according to the invention and propagated in culture, so that e.g. be available as a test system for various applications. These preserved primary culture tumor cells can be stored in the meantime conserved.
  • primary tumor tumor cells from tumor tissue is understood to mean those cells which essentially express the same markers and exhibit essentially the same properties as tumor cells in vivo in the tumor tissue from which these primary culture tumor cells originate "and" tumor primary cultures "synonymous used.
  • the primary culture tumor cells are not artificially (eg by viral transformation) immortalized cell lines.
  • tumor covers all types of tumors, in particular solid tumors, in particular solid tumors of epithelial origin, such as
  • tumor includes not only primary tumors but also metastasis tumors, in particular organ metastases and bone marrow metastases as well as cells from recurrent breast cancer tumors
  • Glandular tissue is detected as a mammary gland, e.g. of pancreatic carcinoma.
  • tumor therapeutics refers to drugs that have a negative impact on tumor growth. These tumor therapeutics include compounds such as chemotherapeutics and hormones as well as other agents for the treatment of tumors such as radiation or non-conservative forms of therapy.
  • system or "test system”, as used here side by side, includes, inter alia, so-called kits or other units containing the primary culture tumor cells according to the invention, as well as a guide to perform, inter alia, the inventive method. If appropriate, these systems or test systems may further comprise further components necessary for carrying out the method according to the invention, such as reagents, vessels, etc.
  • the individual components of the kit according to the invention may be packed in separate containers or together in a container.
  • the system according to the invention can e.g. a screening assay as is well known to those skilled in the art.
  • the method according to the invention for obtaining tumor primary cultures from tumor tissue is based on tumor tissue, which is obtainable, for example, during a breast cancer operation or when taking biopsies.
  • This tissue which is obtained as sterile as possible, is optionally sterilized washed with a washing solution, such as PBS or other physiological solutions, and then minced into tumor tissue under sterile conditions to subsequently cultivate the tumor tissue in media suitable for tumor cells.
  • a washing solution such as PBS or other physiological solutions
  • tumor cell-suitable medium means a medium that promotes the growth of the tumor cells in the tumor tissue.
  • the processing of the tumor tissue obtained for cultivating the tumor tissue pieces does not involve treatment with protease or, in a preferred embodiment, any enzymatic treatment.
  • no trypsin treatment of the tumor tissue is carried out prior to culturing the tumor tissue.
  • the medium to be used in the method according to the invention for cultivating the tumor cells in the tumor tissue is supplemented with bovine pituitary extract, glucocorticoids, such as hydrocortisone, insulin and recombinant human growth factors, such as the human epidermal growth factor.
  • bovine pituitary extract glucocorticoids, such as hydrocortisone
  • insulin recombinant human growth factors, such as the human epidermal growth factor.
  • these substances in amounts of 500 to 5 ⁇ g / ml for the bovine pituitary extract, 0.05 to 5 ⁇ g / ml for the glucocorticoids, such as hydrocortisone, 0.5 to 50 ⁇ g / ml insulin and 1 to 100 ng / ml of human epidermal growth factor in the medium.
  • a particularly suitable medium is, for example, the mammary epithelial cell growth medium from Promocell GmbH, Heidelberg, Germany, hereinafter also referred to as MaCa medium.
  • this mammary epithelial cell growth medium is 2 ml bovine pituitary extract (13 mg / ml), 0.5 ml hydrocortisone (0.5 mg / ml), 0.5 ml human recombinant epidermal growth factor (10 ⁇ g / ml) and 0.5 ml insulin (5 mg / ml) were added to 500 ml culture medium.
  • this medium suitable for culturing tumor cells contains no serum, neither human serum nor fetal calf serum, as is customarily used for cell cultivation. In another embodiment, this medium is not mixed with antibiotics.
  • the tumor tissue pieces of the particular size are cultured in a specific density per square centimeter of culture vessel area, such as a culture flask or culture dish, or per culture volume in medium, preferably in one of the aforementioned media.
  • the cultivation takes place at 37 ° C. in an atmosphere with 5% CO 2 in the culture vessels in suitable devices, such as an incubator.
  • the crushing of tumor tissue according to the invention is carried out so that the tumor tissue pieces edge lengths in
  • the tumor tissue obtained by an operation or from a biopsy is sufficiently prepared that primary culture tumor cells from tumor tissue, in particular mammary carcinoma-derived tumor tissue, are formed during cultivation.
  • the comminution is preferably carried out by cutting, for example with a scalpel or a pair of scissors, to a size of the tissue pieces in which these lengths have a cutting edge of at most 3 mm, preferably 2 mm, such as 1, 5 mm, more preferably 1 mm, preferably two substantially mutually perpendicular side edges have this length, more preferably three substantially mutually perpendicular cut edges this preferred length exhibit.
  • the particular size to which the tumor tissue is comminuted is a shape in which the length of at least one cut edge is 0.5 to 2 mm, and the maximum thickness is in the range of 0.5 to 3 mm, preferably 2 to 1 mm.
  • cubes or cuboids having an edge length in the range of 0.5 to 3 mm, preferably 1 to 2 mm, most preferably about 1 mm can be cited.
  • the tissue pieces of the given dimensions are seeded in a number of 5 to 20 pieces, more preferably 10 to 15 pieces per 100 cm 2 surface of the culture vessel, with sufficient medium to adequately cover the tumor tissue, preferably by more than 1 to 2 mm to cover.
  • the tumor tissue pieces comminuted to a certain size are prepared by filtering out too small tissue pieces and single cells, such as foreign cells, e.g. Blood cells, or fragments of these single cells, e.g. with a filter with an exclusion limit of 20 ⁇ m, more preferably with an exclusion limit of at least 50 ⁇ m, e.g. at least 80 microns, in particular of at least 100 microns.
  • single cells such as foreign cells, e.g. Blood cells, or fragments of these single cells
  • the content of fibroblasts may be selectively removed in the preparation before or after tissue disintegration or after initial culture by known methods such as MACS anti-fibroblast kit or a similar method.
  • the present invention furthermore relates to the isolated primary culture tumor cells obtainable by the methods according to the invention.
  • these are primary culture tumor cells from a breast carcinoma.
  • tumor cells are characterized by having many properties of tumor cells in vivo in the tumor tissue. They continue to express many the marker molecules similar to those of the tumor cells in vivo, eg cytokeratins. More particularly, in a preferred embodiment, the primary culture tumor cells co-express both cytokeratin and vimentin.
  • the primary culture tumor cells according to the invention also differ from e.g. the normal human mammary gland epithelial cells, which do not form a multi-layered, three-dimensional structure but only grow in a single layer to confluence in the cell culture. Furthermore, it has been shown that these normal cells lose their proliferative abilities during cultivation.
  • these tissue pieces are removed from the culture vessel.
  • the preservation of primary culture tumor cells obtained according to the invention can be stored, for example by cryopreserving, ie slow freezing of trypsinized cells in cryopreservation medium (eg 10% DMSO, 10% fetal calf serum in supplemented medium suitable for culturing tumor cells, as described above) in liquid nitrogen Following the cryopreservation, primary culture tumor cells according to the invention can be brought back into culture and are ready for tests or further cultivation.
  • cryopreserving ie slow freezing of trypsinized cells in cryopreservation medium (eg 10% DMSO, 10% fetal calf serum in supplemented medium suitable for culturing tumor cells, as described above) in liquid nitrogen Following the cryopreservation, primary culture tumor cells according to the invention can be brought back into culture and are ready for tests or further cultivation.
  • cryopreservation medium eg 10% DMSO, 10% fetal calf serum in supplemented medium suitable for culturing tumor cells, as described above
  • the culture-enhanced primary culture tumor cells i. the cells grown out of the tumor tissue and further propagated adhering to the surface of the culture vessel, used in tests in the confluent or subconfluent state, or further propagated by trypsinization and usual culture passengers.
  • the cells and the cell tissue can grow continuously in culture for more than one year without any protease treatment, ie without trypsinization for passage.
  • the properties of the resulting primary culture tumor cells remain essentially unchanged over a maximum of 7 passages, preferably up to 5, more preferably up to 3 passages, i.e. preferably show no or only slightly altered properties compared to the cells that are present in the tumor tissue in vivo.
  • the properties of the cultured primary culture tumor cells in the example of tumor cells from a breast carcinoma, change and degenerate so much that the culture begins to die.
  • this dying also proves that the primaryculture tumor cells according to the invention are not artificially immortalized cell lines and that no immortalization of the tumor cells takes place with the method according to the invention.
  • the primary culture tumor cells thus obtained can be used in test systems for diagnosis, for stratifying the therapy or for testing new and known tumor therapeutics.
  • the cells of the invention find application in the testing and screening of new tumor therapeutics.
  • a test system called a bank
  • this library comprises at least ten different, such as at least twenty, different cultures obtainable by the method of the invention
  • Primary tumor cells which come from different tumor tissues.
  • the tumor tissue of a tumor such as breast cancer, or come from different types of tumors.
  • tumor cell material Due to the expansion of the individual cultures, sufficient tumor cell material is available to compare, for example, effects of singular and persistent substance deliveries and to optimize therapeutic application times, ie to optimize the application schedules of the selected tumor therapy. Due to the above-mentioned properties of the obtained primary culture tumor cells, these are also suitable for use in standardized test and screening systems, such as in high-throughput screening methods for the development and application of new and known tumor therapeutics and their application schemes in therapeutic measures.
  • An advantage of the method according to the invention is in particular that it is capable of producing primary tumor cultures, for example of breast cancer, within a few days and thus makes it possible to quickly and accurately create an individualized therapy plan for the patient and thus also to forego potentially ineffective standard therapies ,
  • Tumor tissue susceptible to breast cancer surgery was washed under sterile conditions three times in phosphate buffered saline (PBS) at room temperature and in MaCA medium, (Mammary Epithelial Cell Growth Medium, Promocell, Heidelberg, Germany), containing 2 ml per 500 ml mL bovine pituitary extract (13 mg / mL), 0.5 mL_ hydrocortisone (0.5 mg / mL), 0.5 mL recombinant human epidermal growth factor (EGF, 10 ⁇ g / mL), 0.5 mL insulin (5 mg / mL) and 0.5 mL gentamycin (50 mg / mL) mixed with amphotericin B (50 ⁇ g / mL) was cut with a scalpel into pieces of tissue having a maximum edge length of 1 to 1.5 mm, which is a cuboidal shape exhibited.
  • PBS phosphate buffered saline
  • MaCA medium MaCA medium
  • tissue pieces were up to 10 to 15 units in 50 mL of the above medium supplemented MACA-cultured at 37 0 C in a water-saturated 5% CO 2 atmosphere.
  • cell aggregates grown out of the tumor pieces and adhering to the surface of the culture dish can be observed, forming multi-layered cell aggregates.
  • trypsin / EDTA solution the cell clusters grown on the surface of the culture dish can be detached and subcultured, optionally after washing and gentle centrifugation.
  • the mixture of adult tumor cells ie the growing cells on the surface of the culture dish in mixture with the original tumor tissue twice with PBS at 37 0 C and then after treatment with 0.25 percent trypsin / EDTA solution (1:10 , 0.25% trypsin / EDTA, Invitrogen GmbH, Düsseldorf, Germany) for about 10 to 15 min at 37 0 C replaced.
  • the cell suspension is passed through 100 ⁇ m filters (DaKoCytomation, Hamburg, Germany) to remove the original tumor tissue.
  • the cells contained in the filtrate are called passage 1 cells, centrifuged (400 xg / 5 min), resuspended in fresh supplemented MaCA medium and cultured in cell culture bottles to a density of about 5 x 10 4 cells / mL.
  • the breast cancer primary cells of this first passage can be detached in the subconfluent state by a renewed trypsin / EDTA treatment and are then available as passage 2 cells in sufficient numbers as individual breast cancer primal tumor cells for functional analysis.
  • Example 2 Additional reduction of fibroblasts in preparations of tumor tissue
  • Example 1 primary cell cultures are generated from tumor tissue, and additionally the cells of the first passage can be treated with a MACS anti-fibroblast kit (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany).
  • Mouse monoclonal anti-fibroblast antibodies immobilized on colloidally dispersed superparamagnetic beads selectively remove any fibroblasts present from the primary cell culture of the first passage. Subsequently, the tumor cells are cultured in supplemented MaCA medium according to Example 1. In the preparation of this invention, however, it has been shown that additional removal of fibroblasts from primary cell cultures of the first passage is largely dispensable, since the culture already shows no significant contamination with fibroblasts due to the method according to the invention.
  • Housekrebsprimärzellulturen prepared according to Example 1 are trypsinized in the usual way after the first, second or third passage in supplemented MaCA medium and supplemented to about 5 ⁇ 10 5 cells / mL in a tube with MaCA medium, according to Example 1, in addition to 10 % (VoI ./Vol.) Of fetal calf serum (Invitrogen) and 10% (v / v) DMSO (Sigma) frozen over a period of about 12 h at -80 0 C and then in liquid nitrogen (-196 0 C ) shock-frozen and stored.
  • Frozen shock primary culture cells can be stored for a short time at -80 0 C., but preferably in liquid nitrogen.
  • the cryopreserved tumor primary culture cells are rapidly heated to 37 0 C and cultured after removal of fetal calf serum and DMSO in supplemented MaCA medium at 37 0 C, 5% CO 2 .
  • Example 1 an initial cell / tissue culture was cultivated from tumor tissue pieces of a breast carcinoma with a maximum edge length in the range of 1 to 2 mm on a coverslip and then in a solution with 3% glutaraldehyde, 2% formaldehyde in 0.1 M sodium cacodylate at pH 7.4 for at least 24 hours at 4 0 C fixed. Subsequently, the samples were postfixed in 1% OsO 4 , then dehydrated in an ethanol gradient. The samples dried at the critical point were coated with gold-palladium (SEM coating system E5400, Polaron, Watford, England) and then analyzed in a scanning electron microscope (JEOL SSM-35CF) at 15 kV.
  • SEM coating system E5400 Polaron, Watford, England
  • FIG. 1 An overview image is shown in Figure 1 at 200-fold magnification, in which the cultivation of the individual pieces of tumor on the glass surface and the growth of the tumor primary cells is visible.
  • the cell morphology of bulky cell bodies with strange structures is atypical for contaminating foreign cells, such as fibroblasts, since the latter have very elongated and spindle-shaped leaking cell structures.
  • Figure 1A also partially illustrates the stacking of cells within this three-dimensionally growing tumor culture, with individual cells forming both long and short cell spurs.
  • the tumor cells form a multiplicity of cytoplasmic extensions to their neighboring cells. It is believed that these intercellular compounds serve transcellular transport and other intracellular intracellular communication processes. These intercellular compounds are also typical of tumor tissue in vivo, so it is clear here that the primary cell cultures according to the invention have a structure which closely resembles the structure of the tumor in vivo.
  • breast tumor primary culture cells according to the invention form a relatively homogeneous population with three-dimensional structure similar to the cells in the connective tissue of a tumor in vivo.
  • cytokeratin was analyzed as a marker in primary tumor cultures in MaCA medium.
  • vimentin intermediate filaments can also be expressed in breast tumor tissue. Vimentin expression is also typical of fibroblasts, but no cytokeratins are found here.
  • these two intermediary filaments are useful as markers for detecting any contamination by other cell types, such as fibroblasts.
  • Fibroblasts usually present a problem in the cultivation of primary cells, as fibroblasts have a higher cell division rate than other cell populations and thus overgrow the primary cells desired in the culture.
  • cells of a tumor primary culture according to Example 1 were prepared, passaged three times and washed on a microscope slide three times with PBS / Tween-20 for 15 min, then dried for 60 min. Thereafter, the samples were fixed in ice-cold acetone for 10 minutes and rehydrated with PBS for 5 minutes. Non-specific binding sites were blocked by incubation in 5% (w / v) BSA in PBS, then incubated for 30 min with a mouse anti-vimentin antibody (clone V9 (1: 100), DakoCytomation).
  • a mouse anti-vimentin antibody clone V9 (1: 100
  • FIG. 2 A shows the uptake of the green fluorescence of the cytokeratin filaments in the primary tumor culture
  • FIG. 2 B the vimentin filaments in red fluorescence
  • FIG. 2 C the cell nuclei of the respective tumor primary culture cells in blue DAPI staining.
  • FIG. 2D The superimposition of the individual images of FIGS. 2A-C produced with the aid of the image processing program bündle analySIS'B (Olympus) is shown in FIG. 2D.
  • FIG. 2 D makes it clear that tumor primary culture cells express either only cytokeratin or exhibit more or less strong co-expression of the vimentin and cytokeratin filaments, whose intracellular localization differs, however.
  • Example 6 Use of breast tumor primary culture cells to analyze the efficacy of drugs against in vivo tumor tissue
  • tumor primary culture cells For efficacy tests of substances that could be used as medicaments, preference is given to using tumor primary culture cells according to the invention of the second to fourth passage. Because it has been shown that the primary cell cultures in later passages contain a higher proportion of cell cycle arrested and apoptotic cells, suggesting that the cell characteristics differ from the original ones.
  • the method according to the invention is capable of producing tumor primary cultures, for example of breast cancer, within a few days, it is suitable for providing a test system for individual patients, with which the effect of medicaments, for example chemotherapeutics, as well as the possible success of radiation therapy can be tested.
  • medicaments for example chemotherapeutics
  • the method according to the invention is capable of producing tumor primary cultures, for example of breast cancer, within a few days, it is suitable for providing a test system for individual patients, with which the effect of medicaments, for example chemotherapeutics, as well as the possible success of radiation therapy can be tested.
  • chemotherapeutics for example chemotherapeutics
  • the therapeutic approach against the original tumor can be determined individually, so that effective forms of therapy can be selected, while ineffective standard therapies are dispensable.
  • Example 7 Characterization of tumor primary culture cells by analysis of cell cycles in successive culture runs
  • the decrease in cell number that is in the S-phase of the cell cycle shows the decrease in the ability to divide in the course of successive passages. From the 5th to the 7th passage, the G2 / M phase is no longer detectable, while at the same time a continuous increase in the population in the sub-G1 phase is detectable, which indicates predominantly apoptotic cells.
  • Example 8 Characterization of the tumor primary culture cells in the course of the passage through Westemblot
  • Example 6 For analysis, cells obtained from the individual passages of Example 6 were washed in PBS, spun down, homogenized in SDS loading buffer with a cannula, and then electrophoresed in a denaturing polyacrylamide gel. The proteins were transferred to ImmobilonP membranes and detected after blocking with BSA with specific antibodies to panCytokeratin, and vimentin, as well as to -ctin as charge control. The result is shown in Figure 4 for panCytokeratin, vimentin and ⁇ -actin, respectively. It equal amounts of protein (10 ⁇ g) were applied; the fragment sizes given were determined using parallel separated marker proteins.
  • panCytokeratin shows that cytokeratin is expressed at a significantly higher level only with an increasing amount of culture passages, ie when the primary culture has already reached a growth-arrested state.
  • panCytokeratin shows that cytokeratin is positive
  • Cytokeratins in tumor tissue does not appear to be a marker to identify malignant metastatic tumor cells or tumor recurrences.

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Abstract

L'invention concerne des procédés permettant de produire des cellules tumorales de culture primaire à partir de tissus tumoraux. L'invention concerne plus précisément un procédé permettant de produire des cellules tumorales de culture primaire à partir de tissus tumoraux, notamment de carcinome mammaire. Dans une première étape du procédé, le tissu tumoral est fractionné en fragments de tissu tumoral de dimension déterminée et ces fragments de tissu sont ensuite mis en culture, dans des conditions déterminées. L'invention concerne en outre les cellules tumorales de culture primaire pouvant être obtenues à l'aide dudit procédé, à partir de tissus tumoraux, notamment des cellules tumorales de culture primaire issues du carcinome mammaire. L'invention concerne pour finir l'utilisation de ces cellules tumorales de culture primaire, entre autres pour déterminer une thérapie tumorale individuelle ou pour tester et sélectionner de nouveaux agents thérapeutiques tumoraux.
EP06722757A 2005-04-07 2006-04-05 Procede de production de cellules tumorales de culture primaire, a partir de tissus tumoraux, notamment de carcinomes mammaires, cellules tumorales de culture primaire correspondantes et leur utilisation Withdrawn EP1869167A1 (fr)

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DE102005015953A DE102005015953A1 (de) 2005-04-07 2005-04-07 Verfahren zur Anreicherung und ex vivo Kultivierung von Brustprimärzellen
PCT/DE2006/000608 WO2006105777A1 (fr) 2005-04-07 2006-04-05 Procede de production de cellules tumorales de culture primaire, a partir de tissus tumoraux, notamment de carcinomes mammaires, cellules tumorales de culture primaire correspondantes et leur utilisation

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JP5774496B2 (ja) * 2010-01-19 2015-09-09 株式会社ルネッサンス・エナジー・インベストメント 癌組織由来細胞塊または癌細胞凝集塊の培養方法、評価方法および保存方法
JP5891172B2 (ja) * 2010-07-07 2016-03-22 エーザイ・アール・アンド・ディー・マネジメント株式会社 正常乳腺上皮細胞からの腫瘍細胞の製造方法
KR101832133B1 (ko) 2011-10-20 2018-02-27 주식회사 차바이오텍 폐암세포의 분리 및 증식 방법
JP2019068790A (ja) * 2017-10-11 2019-05-09 地方独立行政法人 大阪府立病院機構 薬剤および／または放射線に対する癌細胞の感受性および／または抵抗性評価方法
WO2022039219A1 (fr) * 2020-08-21 2022-02-24 凸版印刷株式会社 Procédé de production d'un milieu conditionné pour la culture de cellules cancéreuses issues d'un patient

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US20040023375A1 (en) * 2002-07-30 2004-02-05 Precision Therapeutics, Inc. Method for preparing cell cultures from biological specimens for chemotherapeutic and other assays
US6074874A (en) * 1997-08-29 2000-06-13 University Of Pittsburgh Epithelial cell cultures for in vitro testing
DE19912798C1 (de) * 1999-03-10 2000-02-17 Andreas Jordan Verfahren zur Kultivierung von Krebszellen aus Humangewebe und Vorrichtung zur Aufbereitung von Gewebeproben
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WO2006105777A1 (fr) 2006-10-12
CA2603133A1 (fr) 2006-10-12

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