METHOD FOR CULTURING A SUBPOPULATION OF CIRCULATING EPITHELIAL TUMOUR CELLS FROM A BODY FLUID

Abstract

The invention relates to a method for culturing a subpopulation of circulating epithelial tumour cells from a body fluid of a human or animal suffering from an epithelial tumour, wherein cells contained in the body fluid each containing at last one cell nucleus are separated from the body fluid and cultured over at least 24 hours in suspension, with formation of spheroids.

Claims

1. A method for culturing circulating epithelial tumor cells from a body fluid from a human or animal affected by an epithelial tumor, comprising: (i) separating cells present in the body fluid and containing at least one nucleus in each case from the body fluid without selection of certain of these cells; (ii) transferring said separated cells to a cell culture medium; and (iii) culturing said transferred separated cells under cell culture conditions in said cell culture medium containing at least an animal serum, a growth stimulator and one growth factor, wherein the growth stimulator is insulin and hydrocortisone and wherein the growth factor is Epidermal growth factor, and wherein the cells are cultured for at least 24 hours in suspension at least until a subpopulation of tumor cells, which does not require adherence to a surface for proliferation, has formed spheroids by proliferation, and wherein tumor cells which have formed the spheroids during culturing are separated from the cultured tumor cells by separating the spheroids formed during culture.

2. The method as claimed in claim 1, wherein the body fluid is lymph or blood.

3. The method as claimed in claim 1, wherein the body fluid contains intact erythrocytes and the cells containing at least one nucleus in each case are separated from the intact erythrocytes.

4. The method as claimed in claim 1, wherein the cell culture conditions comprise a temperature within the range of 35.5° C. to 37.5° C., and an atmosphere containing 4.5% to 5.5% CO.sub.2.

5. The method as claimed in claim 1, wherein fresh cell culture medium is added to the cell culture medium repeatedly or regularly, or wherein the cells are transferred to fresh cell culture medium repeatedly or regularly.

6. Spheroid-forming tumor cells which have been cultured according to a method as claimed in claim 1 for use in the immunological treatment of a tumor disease in a human or animal, wherein the tumor cells present in the spheroids are treated such that they can no longer proliferate and are subsequently administered, suspended in an adjuvant, to the human or animal.

7. The method as claimed in claim 2, wherein the body fluid is peripheral blood.

8. The method as claimed in claim 3, wherein the cells containing at least one nucleus in each case are separated from the intact erythrocytes by lysing the intact erythrocytes.

9. The method as claimed in claim 4, wherein the temperature is within the range of 36.8° C. to 37.1° C.

10. The method as claimed in claim 4, wherein the temperature is 37° C. and the atmosphere contains 5% CO.sub.2.

11. A method for examining a self-renewal capacity of tumor cells comprising: (i) providing the spheroids obtained by the method according to claim 1; and (ii) examining the self-renewal capacity of tumor cells present in the spheroids and/or of progenitor cells generated in the spheroids.

Description

[0025] The invention will now be described in more detail on the basis of an exemplary embodiment and the figures. What are shown are:

[0026] FIG. 1a, b, c spheroids of tumor cells from peripheral blood from a patient suffering from colon carcinoma after culturing for 7 days (FIG. 1a), 14 days (FIG. 1b) and 21 days (FIG. 1c) and

[0027] FIG. 2 a fluorescence microscopy image of a tumor spheroid fluorescently stained with antibodies and propidium iodide.

[0028] The methods described below were carried out using the following chemicals, reagents, buffers, solutions, antibodies, instruments and consumables and also the indicated medium:

TABLE-US-00001 Chemicals and reagents Chemicals and reagents Company Ammonium chloride Sigma-Aldrich, USA (NH.sub.4Cl) (155 mM) Potassium bicarbonate Sigma-Aldrich, USA (KHCO.sub.3) (10 mM) Ethylenediaminetetraacetate Sigma-Aldrich, USA (EDTA) (0.5 M) Dulbecco's Phosphate Buffered GIBCO, USA Saline (D-PBS) Flow-Check 770 Fluorospheres Beckman Coulter, Ireland Propidium iodide solution (1 mg) Sigma-Aldrich, USA RPMI 1640 (1X) 500 ml Invitrogen GmbH, Germany Hepes buffer solution (1 M) Invitrogen GmbH, Germany Penicillin/streptomycin Invitrogen GmbH, Germany (10000 U/ 10000 μg) L-Glutamine (200 mM) Biochrom AG, Germany Fetal bovine serum (FBS) Invitrogen GmbH, Germany Insulin (100 U/mL) Sanofi-Aventis, USA Hydrocortisone 100 mg Pfizer, USA EGF Sigma-Aldrich, USA

TABLE-US-00002 Buffers and solutions Buffers and solutions Constituents Amount D-PBS CaCl.sub.2 0.901 mM MgCl.sub.2—6H.sub.2O 0.493 mM KCl 2.67 mM KH.sub.2PO.sub.4 1.47 mM NaCl 137.93 mM Na.sub.2HPO.sub.4—7H.sub.2O 8.06 mM D-PBS-EDTA D-PBS 500 ml EDTA 2 ml Erythrocyte lysis buffer NH.sub.4Cl 8.3 g (dissolve in 1 L of KHCO.sub.3 1 g distilled water) EDTA 2 ml Propidium iodide solution PI 3.5 μl (in 1 ml of distilled water)

TABLE-US-00003 Antibodies Article Antibodies Company number CD 326 (EpCAM) Miltenyi Biotec 130-080-301 conjugated with GmbH, Germany fluorescein isothiocyanate (FITC)

TABLE-US-00004 Instruments Instruments Company Centrifuge 5810 R Eppendorf, Germany Laser scanning CompuCyte Corporation, microscope iCys ™ USA Vortexer Bender und Hobein GmbH, Germany

TABLE-US-00005 Consumables Consumables Company 15 ml Falcon tubes Labor Schubert, Germany 1.5 ml Eppendorf tubes Labor Schubert, Germany Micropipettes Eppendorf, Germany Disposable Pasteur pipettes, ROTH, Germany graduated (3.2 ml) Microtiter plates (MTP) Greiner bio-one, Germany with glass base Culture flasks (25 cm.sup.2, 65 ml) neoLab, Germany

TABLE-US-00006 Composition of the medium Chemicals Final concentration A culture medium, preferably RPMI 1640 A serum additive, 5% preferably fetal calf serum L-Glutamine   4 mM Hepes   15 mM Growth stimulator,   5 μg/ml preferably insulin Hydrocortisone  0.5 μg/ml Antibiotics, preferably  100 U/ml; 100 μg/ml penicillin/streptomycin Growth factors,   40 ng/ml preferably EGF

[0029] Blood samples were collected from the peripheral vein in 2-7 ml tubes containing EDTA as anticoagulant. The viability of the circulating epithelial tumor cells was on average 95%.

[0030] In a sample tube, 1 ml of the particular blood sample was topped up to a total volume of 15 ml using the erythrocyte lysis buffer and incubated in the refrigerator for 15 min at a temperature of 4° C. Next, the sample was centrifuged at 2000 rpm for 7 min and at a temperature of 18° C. The supernatant was then decanted. Thereafter, the pellet was resuspended with 2 ml of medium and transferred to a culture flask into an initially charged 3 ml of medium (depending on the leukocyte count: 5 ml of medium per 10 000 leukocytes). This was incubated for 21 days at 5% CO.sub.2 and 37° C. Every 5 days, 2 ml of fresh medium were added. Every 7 days, i.e., on the 7th, 14th, 21st and 28th day, the tumor spheroids were examined under the microscope. For the analysis, the tumor spheroids were collected by means of a gentle centrifugation and then the pellet was resuspended with 500 μl of D-PBS-EDTA. 50 μl of this mixture were then transferred to a 1.5 ml Eppendorf tube. Thereafter, 5 μl of a (FITC)-monoclonal antibody against the human epithelial antigen (EpCAM) were pipetted thereinto. This was followed by another 15-minute cooling at 4° C. Lastly, 430 μl of D-PBS-EDTA were added and the samples, now completed, were stored overnight in the refrigerator at 4° C.

[0031] On the following day, 100 μl of cell suspension of the sample to be measured and 5 μl of propidium iodide (PI) were pipetted in each case into a well of an ELISA plate. The plate was then covered and allowed to rest for about 20 min so that the cells could sediment on the base. Subsequently, the cells were measured using the laser scanning cytometer and the measurement results were evaluated.

[0032] What were obtained were nonadherent, three-dimensional spheroids, which are typical of the presence of tumor stem cells. FIG. 1a shows the spheroids after 7 days of culturing, FIG. 1b shows them after 14 days of culturing, and FIG. 1c shows them after 21 days of culturing. FIG. 2 shows a red-stained necrotic tumor cell in the middle of a tumor spheroid cell aggregate. The death of this tumor cell may possibly be due to a lack of diffusion of growth factors through the tight cell packing surrounding said cell.

[0033] It was possible to culture a subpopulation of the circulating epithelial tumor cells which forms spheroids and has a high capacity for proliferation.