EP1869167A1

EP1869167A1 - Method for obtaining primary-culture tumour cells from tumour tissue, in particular breast carcinoma, primary-culture tumour cells and their use

Info

Publication number: EP1869167A1
Application number: EP06722757A
Authority: EP
Inventors: Ralf Hass; Jutta Rogoll; Hartmut Kueppers; Axel Dehn
Original assignee: Medizinische Hochschule Hannover; Solvay Arzneimittel GmbH
Current assignee: Medizinische Hochschule Hannover; Mylan Healthcare GmbH
Priority date: 2005-04-07
Filing date: 2006-04-05
Publication date: 2007-12-26
Also published as: CA2603133A1; US20080176267A1; JP2008534022A; WO2006105777A1; DE102005015953A1; DE112006001500A5

Abstract

The present invention relates to methods for obtaining primary-culture tumour cells from tumour tissue. More precisely, the present invention relates to a method for obtaining primary-culture tumour cells from tumour tissue, in particular breast carcinoma, where, in one method step, the tumour tissue is divided into tumour-tissue sections of a certain size, and these tissue sections are then cultured under defined conditions. The invention also concerns the primary-culture tumour cells that can be obtained by this method from the tumour tissue, in particular primary-culture tumour cells from breast carcinoma. Finally, the invention relates to the use of these primary-culture tumour cells for, among other things, determining an individual course of tumour treatment, or for testing and screening of new therapeutic agents against tumours.

Description

Method for obtaining primary culture tumor cells from tumor tissue, in particular breast cancer, primary culture tumor cells and their use

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.

State of the art

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.

In breast cancer patients, adjuvant drug therapy with cytotoxic drugs is often followed by surgery and the associated removal of the primary tumor, breast cancer. Particularly in the case of metastasis of the primary tumor that has already occurred, ie in the case of simultaneous tumor infiltration of the axillary lymph nodes, for example, this adjuvant medicinal therapy becomes effective performed with cytostatics. Other therapeutic methods include radiotherapy or hormone therapy or combinations of these forms of therapy. In fact, hitherto, among other things, 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, eg with tamoxifen, 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 necessary for a sufficient form of therapy screening independent of the surgical procedure, ie the testing of hormone, chemo and / or radiotherapeutic measures, has been limited to immortalized breast cancer cell lines (eg MCF-7, etc.) in vitro and / or on animal experiments in vivo , However, immortalized cell lines represent an artificial test system and do not correspond to the actual in vivo situation. The same applies to animal models, since these are usually induced tumor animal models. It is difficult to transfer the results obtained in the animal model or with immortalized cell lines to the in vivo situation. The stratification of the patient to be treated therefore continues to be a major problem. In particular, multidrug-resistant tumors (MDR tumors) are a major problem in the therapy of a tumor patient.

As already mentioned above, for example in the area of breast cancer, the classic prognostic factors in patients with primary breast cancer are tumor size, Differentiation type, hormone receptor status and lymph node involvement. In everyday clinical practice, it is always noticeable that 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.

As already stated above, the in vitro screening methods and the animal experimental methods have only a generalized and with regard to the diversification of the individual case completely unspecific value. The consequence of this is that coarse-meshed therapy regimens have been developed for routine use, which in some patients only achieve inadequate effects or even completely fail. In the worst case, these regimens may even induce secondary tumors in the healthy proliferating tissue even by the DNA damaging and mutagenic effect. For this reason, e.g. In the case of breast cancer, the individual diagnostic tools extremely limited and beyond there are in principle no possibilities for individualized therapeutic approaches.

As mentioned above, 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. For example, Bartsch et al, LifeSciences 67, pages 2953-2960, 2000 describes the study of tumors from patients with breast and ovarian carcinoma. The tumor material was then divided into small fragments and these fragments were digested to isolate the cells with enzyme reagents, usually containing proteases, such as trypsin or chemotrypsin, and DNases and RNases. Subsequently, these cells thus obtained, isolated by protease digestion, were cultured.

However, the prior art does not disclose a method of simply obtaining primary culture tumor cells from the tumor tissue, which cells have not been altered by immortalization, but nevertheless can be maintained in culture for a prolonged period of time without substantial change in their properties existing methods for the isolation and testing of breast cancer cells have their limitation also in the cultivation, eg Stampfer.MR, J. Tissue Culture Methods, 9: 107-115 (1985).

Summary of the invention

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.

With the help of the new method, it has been possible to enrich ex vivo from tumor tissue samples of tumors, in particular epithelial tumors, such as mammary carcinomas, and multiply them over a long period of time. 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. Furthermore, 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.

In preferred embodiments, 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. According to the invention, 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. Subsequently, 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. In embodiments according to the invention, these tumor primary cultures form multilayer cell aggregates on cultivation.

Brief description of the illustrations

FIG. 1: FIG. 1 is an electron micrograph of breast cancer primary culture cells.

FIG. 2: 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 Einzelfluoreszenzaufnahmen, as shown in Figures 2 A to C are shown.

Figure 3: 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: 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.

Detailed illustration of the invention

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.

In the following, the term "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.

The term "tumor" covers all types of tumors, in particular solid tumors, in particular solid tumors of epithelial origin, such as

Breast carcinoma, bladder carcinoma or lung carcinoma and skin cancer and their

Metastases. That The term "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.

The term "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.

The term "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, such as mammary carcinomas, 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.

The term "tumor cell-suitable medium" means a medium that promotes the growth of the tumor cells in the tumor tissue.

According to the invention, 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. In particular, no trypsin treatment of the tumor tissue is carried out prior to culturing the tumor tissue.

In a preferred embodiment, 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. Particularly preferred are these substances in amounts of 500 to 5 μg / ml for the bovine pituitary extract, 0.05 to 5 μg / ml for the glucocorticoids, such as hydrocortisone, 0.5 to 50 μg / ml insulin and 1 to 100 ng / ml of human epidermal growth factor in the medium. Particularly preferred is a medium containing 20 to 80 μg / ml, such as 52 μg / ml bovine pituitary extract, 0.1 to 2.5 μg / ml, such as 0.5 μg / ml hydrocortisone, 1 to 25 μg / ml 5 μg / ml insulin and 2 to 50 ng / ml, such as 10 ng / ml human epidermal growth factor.

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. In a preferred embodiment, 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 μg / ml) and 0.5 ml insulin (5 mg / ml) were added to 500 ml culture medium.

In a further preferred embodiment, 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.

Of course, other media are also suitable, such as the MCDB170 medium mentioned in Hammond et al., PNAS, 1984, 81: 5435-5449.

In a further preferred embodiment, 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.

By crushing the tumor tissue to the specific size, it is now possible to obtain primary culture tumor cells. The crushing of tumor tissue according to the invention is carried out so that the tumor tissue pieces edge lengths in

Range of 0.5 mm to 3mm obtained. In these tumor tissue pieces, 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. As a result, 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. As an example of forms taking this particular size of tumor tissue, 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.

Preferably, 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.

In a further preferred embodiment, 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 μm, more preferably with an exclusion limit of at least 50 μm, e.g. at least 80 microns, in particular of at least 100 microns.

Alternatively, 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. Preferably, these are primary culture tumor cells from a breast carcinoma.

These cells obtainable in this way 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.

Detailed morphological studies, for example scanning electron micrographs, show that the above-described methods according to the invention produce primary cell cultures of mammary carcinoma cells which are substantially free of other cell types, in particular free of fibroblasts.

During cultivation, under the conditions mentioned above, primary cultures from the tumor tissue pieces grow out as adherent cells, where they form a multi-layered, three-dimensional cell culture on further proliferation. The three-dimensional structure already forms in the subconfluent growth of the primary culture tumor cells. Upon reaching the confluent state in the cell culture, no contact inhibition of proliferation takes place, but the cells are not limited in their proliferation. In this way, 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.

Preferably, after outgrowth of the primary culture tumor cells from the tumor tissue pieces, 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.

Depending on the individual growth of the tumor cells, 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.

In connection with the long-term cultivation and a partially necessary passaging of the primary culture tumor cells according to the invention, it has been found that 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.

By passaging by trypsinating the cells and then further culturing, it has been found that 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.

As can be seen from one of the following examples, it has been shown that after approximately 7 passages, 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. However, 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. This means that uses of these cells according to the invention can be found, inter alia, in drug discovery, since the cells according to the invention better represent the in vivo situation and thus also the reaction in vivo in the tumor tissue compared to the known cell lines. All types of previous and future therapies, such as hormone therapy, cytostatics or radiotherapy, can be tested alone or in combination and, for example, an individualized therapy plan for an individual can be developed. By simulating different forms of therapy, various conservative or innovative therapeutic approaches and their possible combination can be tested and individually adapted before use in the patient.

Furthermore, the cells of the invention find application in the testing and screening of new tumor therapeutics. Thus, e.g. With the help of a test system, called a bank, which has at least two different cultures of

Primary tumor cells from various tumor tissues, e.g. on one

Array, the efficacy and also the side effects of potential new drugs or other physical or biological methods, the pharmacokinetic efficiencies are tested on a homogeneous ex vivo material. Preferably, 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.

In this case, the tumor tissue of a tumor, such as breast cancer, or come from different types of tumors.

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 ,

Example 1: Generation of a Primary Cell Culture from Breast Cancer

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 μg / mL), 0.5 mL insulin (5 mg / mL) and 0.5 mL gentamycin (50 mg / mL) mixed with amphotericin B (50 μg / 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.

The 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.

After a culture period of several days, 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. With conventional 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. For this purpose, 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, Karlsruhe, Germany) for about 10 to 15 min at 37 ⁰ C replaced. The cell suspension is passed through 100 μm 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

According to 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.

Example 3: Cryopreservation of Breast Cancer Primary Cell Cultures

Brustkrebsprimärzellulturen 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. For renewed cultivation or use, 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 4 Characterization of Inventive Breast Cancer Primary Cells by Scanning Electron Microscopic Analysis

According to 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.

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 bizarre 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.

Moreover, it becomes clear that 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.

Overall, the electron micrograph shows that 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.

Example 5: Functional Characterization of Tumor Primary Cultures

Breast tumor tissue is so far known and among other things in addition to the morphology characterized by the formation of certain intermediary filaments, in particular the cytokeratins. For functional characterization according to the invention Therefore, cytokeratin was analyzed as a marker in primary tumor cultures in MaCA medium.

In addition to cytokeratins, vimentin intermediate filaments can also be expressed in breast tumor tissue. Vimentin expression is also typical of fibroblasts, but no cytokeratins are found here.

Thus, 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.

Specifically, 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). After washing three times with PBS / Tween-20 for 5 min each, incubation is carried out with a secondary anti-mouse antibody labeled TRITC (1:40, DakoCytomation). After washing three times with PBS / Tween-20 for 5 min each, the samples were treated with mouse serum (1:40, DakoCytomation) for 5 min and then incubated for 90 min with a FITC-coupled monoclonal anti-panCytokeratin antibody (clone MNF 116, 1: 20, DakoCytomation). After washing three times with PBS / Tween-20 for 5 min each, the samples were preserved in DAPI-containing mounting medium (DakoCytomation) until immunofluorescence microscopy. As negative controls, parallel cultures were treated identically, but instead of the mouse anti-vimentin antibodies a corresponding mouse serum of the respective IgG subclass was used, which all showed no immunofluorescence. Fluorescence images were each taken with an Olympus SIS F-view II CCD camera in an Olympus IX-50 fluorescence microscope. The analysis of the fluorescence images as well as the superimposition of the fluorescence images were generated with the aid of the image processing program SIS bundle analySIS'B (Olympus).

The analysis showed that the expression of cytokeratins and vimentin is not distributed to two different cell types, but that vimentin detected is always expressed in a single cell together with cytokeratins, so that in any case the combination of these two intermediary filament types Exclusion criterion for fibroblasts represents. For this purpose, a triple fluorescence dyeing method was used, wherein the respective Einzelfluoeszenzaufnahmen, shown in Figures 2 A-C, and the associated superposition, shown in Figure 2 D, were used for the analysis.

FIG. 2 A shows the uptake of the green fluorescence of the cytokeratin filaments in the primary tumor culture, in FIG. 2 B the vimentin filaments in red fluorescence and in FIG. 2 C the cell nuclei of the respective tumor primary culture cells in blue DAPI staining. 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. These images are clear evidence that regardless of the morphology of the imaged cells no fibroblasts are detectable in the culture of the invention, since all cells express at least cytokeratins.

Example 6: Use of breast tumor primary culture cells to analyze the efficacy of drugs against in vivo tumor tissue

Brustkrebsprimärkulturzellen according to Example 1 or cryopreserved Krebsprimärzellkulturen according to Example 3 were just like the original Tumor tissue by further markers, such as Ki-67, characterized in detail and it was found that tumor primary culture cells according to the invention have a very similar expression pattern of other markers, such as isolated from the patient tumor tissue, The cryopreservation of cell cultures did not change this property.

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.

Individual tumor primary culture cells were exposed to different chemotherapeutic agents in different concentrations and by the change of their

Morphology or apoptosis, the effectiveness of each compound could be estimated. Due to the great similarity of the tumor primary culture cells with the properties of the tumor tissue in vivo, the result of these ex vivo tests can be directly included in the expected therapeutic success in the patient.

Since 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. In this way, from a tumor biopsy tumor primary culture cells can be generated, by means of which a variety of drugs or other therapeutic methods can be tested for their effectiveness. Based on the reaction of the primary cell cultures ex vivo, 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

To characterize the passaged tumor primary culture cells, their cycle was determined by determining their DNA content in FACS measurements.

In Figure 3, the results of the measurements are shown graphically. The generation of the tumor primary culture cells was carried out according to Example 1, the passage was carried out by trypsinization, washing three times in PBS and sowing at 10 ⁵ cells / ml in supplemented MaCA medium.

The data shown in FIG. 3 make it clear that the cell properties degrade after the fifth phase. Thus, 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

Cell samples derived from passages of Example 6 were analyzed in the Western blot on pan Cytokeratin, Vimentin and Actin.

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 β-actin, respectively. It equal amounts of protein (10 μg) were applied; the fragment sizes given were determined using parallel separated marker proteins.

The detection of 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.

This expression pattern of panCytokeratin shows that cytokeratin is positive

Cells have primarily non-malignant properties, so that the expression of

Cytokeratins in tumor tissue does not appear to be a marker to identify malignant metastatic tumor cells or tumor recurrences.

The detection of β-actin as a control shows that approximately equal amounts of protein were applied.

Expression of the vimentin confirms the results from immunofluorescence (FIG. 2) that in addition to cytokeratins partial co-expression of vimentin in the breast cancer primary cultures can be detected, but whose expression pattern does not change significantly in the course of the culture passages.

Furthermore, it has been shown that treatment of the primary culture tumor cells obtained according to the invention with known tumor therapeutics has induced cell death. The primary culture tumor cells thus showed the expected response, which is another indication that they are primary cultures of tumor cells.

Claims

claims

A method for obtaining substantially primary culture tumor cells from tumor tissue, comprising the steps of a) crushing the tumor tissue into tumor tissue of a size of about 0.5 mm to about 3 mm in diameter, and b) cultivating the tumor tissue pieces in a manner suitable for tumor cells

Medium, characterized in that until the cultivation of the tissue pieces, the tumor tissue or the tumor tissue pieces with no treatment

Proteases is subjected.

2. The method according to claim 1, characterized in that no trypsin treatment is carried out before culturing the tumor tissue in suitable for tumor cells medium.

3. The method according to claim 1 or 2, characterized in that no enzymatic treatment of the tumor tissue or the tumor tissue pieces is carried out prior to culturing the tumor tissue pieces in suitable for tumor cells medium.

4. The method according to any one of the preceding claims, characterized in that before the crushing of the tumor tissue, a sterile washing of the tumor tissue takes place.

5. The method according to any one of the preceding claims, characterized in that the tumor tissue pieces are cultured in a number of 5 to 20 per 100 cm ² area of a culture vessel.

6. The method according to any one of the preceding claims, characterized in that before the step of cultivating the Tumor tissue smaller fragments or single cells by filtration with a pore size of 20 to 100 microns are separated.

7. The method according to any one of the preceding claims, characterized in that the medium for cultivating the

Tumor tissue is not supplemented with serum.

8. The method according to any one of the preceding claims, characterized in that the medium for cultivating the tumor tissue pieces is not supplemented with antibiotics.

9. The method according to any one of the preceding claims, characterized in that the medium is supplemented with at least bovine pituitary extract, hydrocortisone, insulin and / or human epidermal growth factor.

10. The method according to any one of the preceding claims, characterized in that the tumor tissue is an epithelial cell carcinoma.

11. The method according to any one of the preceding claims, characterized in that the tumor tissue is a breast carcinoma, lung carcinoma or bladder carcinoma.

12. The method according to any one of the preceding claims, wherein the culture medium is a suitable medium for mammary carcinomas.

13. The method according to any one of the preceding claims, characterized in that after a growth of primary culture tumor cells from the tissue pieces, the tumor tissue pieces are removed from the culture vessel.

14. The method according to any one of the preceding claims, characterized in that the primary culture tumor cells form in cultivation multilayer cell aggregates.

15. Isolated primary culture tumor cells obtainable by a method according to any one of claims 1 to 14.

16. Primary culture tumor cells according to claim 15, which are primary culture tumor cells from a breast carcinoma.

17. Primary culture tumor cells according to one of claims 15 or 16, characterized in that the cells co-express either only cytokeratins or vimentin and cytokeratin.

18. Primary culture tumor cells according to one of claims 15 to 17, characterized in that they form a multi-layered three-dimensional structure in the subconfluent state.

19. Use of primary culture tumor cells obtainable according to one of claims 1 to 14 for testing compounds as tumor therapeutics against tumor types from which these primary culture tumor cells originate.

20. Use of primary culture tumor cells of an individual obtainable according to any one of claims 1 to 14 for the determination of the individual tumor therapy of this individual.

21. Use of the primary culture tumor cells obtainable according to any one of claims 1 to 14 in a method for determining the Metastasierungspotenzials of these cells.

22. Use of the primary culture tumor cells obtainable according to any one of

Claims 1 to 14 for the development and testing of application regimens of tumor therapeutics.

23. Use of at least two types of primary culture tumor cells of at least two different tumors obtainable according to one of claims 1 to 14 in a test system for testing potential tumor therapeutics.

24. Test system for testing and / or screening of new potential

Tumor therapeutics comprising primary culture tumor cells obtainable according to one of claims 1 to 14.

A system for determining individual tumor therapy of an individual comprising primary culture tumor cells of that individual obtainable according to any one of claims 1 to 14.

26. System according to claim 24, characterized in that said system comprises primary culture tumor cells from at least two different tumors.

27. System according to one of the preceding claims 24 to 26 further comprising cell culture medium for culturing the primary culture tumor cells.