NO801388L - PROCEDURE FOR PREPARING A SUSPENSION OF WEAKED CARCINOMA CELLS - Google Patents

Description

Treatment of carcinoma with cytostatic agents has been

known for a long time. Almost all cytostatics have the disadvantage that they are highly toxic and generally also have a detrimental effect on the growth of normal cells. It has also been found that the known cytostatics are not equally effective against all types of carcinoma. Even different patients who have the same carcinoma will often show different responses to one and the same cytotoxic drug. In order to choose the optimal cytostatic agent in such cases and perform a selective treatment with this, Limburg and Krahe have proposed testing tissue cultures taken from the particular carcinoma for sensitivity to different cytostatic agents to provide an onco-biogram. See Deutsche Medizinische Wochenschrift,

Vol. 89 (1964), page 1938.

Although the above method entails the advantage that it

(the) affected organ(s) in the patient are not exposed to the toxic effect of cytostatic agents that are not effective against the type of tumor in question, the patient is still directly affected by a cytostatic agent. This effect has been shown to be harmful, particularly when large doses are administered over a longer period of time.

It is an object of the present invention to produce

a preparation to be used for the treatment of carcinoma, and which is suitable for use either alone or together with a cytostatic drug which is particularly effective against the type of tumor in question.

The preparation must be satisfactorily acceptable to the patient.

This and other objects of the invention have been achieved as such

will appear from the following description, the examples and the requirements.

Carcinoma cells are used as starting material in the method according to the present invention, and can be obtained by conventional methods. For example, cancer cells can be released from surgically explanted tumor tissue by treatment with trypsin; see Limburg. et al, Deutsche Medizinische Wochenschrift,

Vol. 89 (1964), page 1938, which is hereby described by reference. A preferred method for producing carcinoma cells is as explained in the following: To begin with, the desired malignant tumor tissue is excised under sterile conditions by a surgical procedure. It is then treated with a trypsin solution and then cultured on mica flakes in Leighton tubes. The removed tissue is macerated and split

mechanically under sterile conditions to form a mass of tissue. It must be ensured that only solid cancer tissue that is structurally intact and free of significant components of blood and dead cells is used. A solution of 0.25% trypsin in phosphate-buffered saline (PBS), containing 100 units/ml penicillin and 0.1 mg/ml streptomycin for sterilization, is added to this mass which is then stirred at 37°C. For certain types of firm tissue with a harder consistency, the concentration of the trypsin solution can be increased to 2%. After approx. After 20 minutes, the freed individual cells, without parts of tissue as far as possible, are poured into another sterile flask.

To interrupt further action of trypsin on the cell suspension poured into the second sterile flask, cool the suspension to +4°C using an ice bath. This method of stirring and decanting is repeated until there is no more solid tissue in the flask that was used for the trypsin treatment. When the trypsin treatment is complete, the cooled cell suspension is coarsely filtered, preferably through a gas layer that is sterile but has not been treated with any chemicals. The filtrate is centrifuged at approx. 800 to 1000 rpm, and the separated cell sediment is isolated from the filtrate.

The trypsin treatment can be omitted in the case of carcinoma cells that are already available in the form of individual cells, such as ascites carcinoma cells and leukemia cells.

The cancer cells are then cultured to form a cell culture, or a cell peat according to known methods, e.g. as described in the above article by Limburg et al in the Deutsche Medizinische Wochenschrift. The cells are cultured as illustrated below.

The cell sediment is diluted with a liquid nutrient medium to give a concentration of from 2 to 4 million viable cells per ml, and distribute equally among 30 tubes after precise pipetting., A drop is placed on a slide so that the degree of dilution can be ascertained. The dilution is satisfactory when cal 20 cells can be observed in the field of view at a magnification of approx. 125. For the test tube test, 1 ml of liquid is filled into each Leighton tube. The nutrient medium used preferably has the following composition:

100 units of penicillin per ml and 0.1 mg streptomycin per ml is added to the nutrient medium to ensure that it is sterile.

The cell suspension is incubated in the incubator at 37°C. Celie metabolism is indicated by a change in the color of the nutrient medium from red to yellow. The nutrient medium is constantly renewed when metabolism has taken place. The growth of the culture is examined and monitored in a chemist's microscope with reflected light. Depending on the type of starting material, the cultures have different growth characteristics. For example, cells from endometrial or ovarian carcinoma can grow and form an excellent cell monolayer within 48 hours. Cells from other malignant growths, such as cells from squamous cell carcinoma, need up to 120 hours, depending on the degree of development.

The sensitivity test of the cell cultures to different cytostatic agents is carried out, e.g. also according to methods as described by Limburg et al, Deutsche Medizinische Wochenskrift, indicated above. An illustrative method is as follows: Only well-developed cell cultures are tested. Depending on the amount of cultured material available, the culture can be sensitivity-tested against any cytostatic agent. A single culture can be tested for sensitivity against 10 to 20 chemotherapeutic agents at the same time, and these are added to the respective tubes in a sufficient weight concentration. Each agent used for sensitivity testing is administered in the same concentration as it is administered to humans in for continuous or intermittent treatment) the values in the following table are given as a guide. The different cytostatics are prepared in a pharmaceutical form and added to the different test tubes.

Continuous monitoring with a chemist's microscope will

reveal visible morphological damage in most cases after 24 hours, and when antimetabolites are used, after 48 hours.

For in vitro experiments, the following concentrations are used which are suitable for treatment. These doses are calculated according to the concentrations for treating humans recommended by the manufacturer, in relation to an average body weight of 60 kg or to square meters of body surface and approx. 5000 ml blood volume, as the case may be.

When using this method, the dilution can

of any other cytostatic agent is easily calculated.

In vitro sensitivity testing

The sensitivity or resistance of a culture of human cancer tissue is determined by a semi-quantitative method. A culture that is completely resistant will correspond exactly to that in the control tube. Partial activity is indicated by partial dissolution of approx. 50% of the cells in the visual field. High sensitivity is revealed by the fact that the field of view is free of viable cells and only includes a few shrunken cells that have been killed. Only such a result is considered a sign of complete sensitivity. On average, at least two cultures treated with different cytostatic agents are found to be sensitive.

Only these cultures should be used in the cell treatment, as will be explained below. Such investigations have been carried out on more than 3000 individual cases, and have given the unequivocal result that cells from each of many tumors from similar locations and/or with similar histology, but from different patients, can give very different responses to the same cytostatic agent, and further that cells from metastases can react differently to cells from the primary tumour.

Sensitivity testing of carcinoma cell cultures to different cytostatic agents can be performed in vivo in the abdomen of rats.

In vivo sensitivity testing

The sensitivity test to cytostatics is performed in vivo

in diffusion chambers placed in the abdomen of rats when less than 20 g of tumor material is available. The cell suspension is prepared in the same way as with the in vitro method. The diffusion chamber consists of two filters placed in two Teflon rings which are connected by screws. The filters act as a semi-permeable membrane. The pore size is chosen so that liquid, electrolyte and proteins can penetrate the membrane, while cells such as leukocytes, macrophages and the like are held back. A suitable pore size for the filters is approx. 0.25 ym. This chamber is filled with 0.3 ml of the prepared cell suspension.

Immediately after they are repaired, they are implanted

chambers in the abdominal cavity of rats of the inbred strain BD2. Two chambers are used for each animal. Once the chambers are implanted,

the peritoneum and abdominal wall are closed. Using this method, 100% of the tumor cells retained on the membrane filters grow.

On the fifth day after implantation of the diffusion chambers

in the abdomen of the rats, various cytostatic agents are injected into the abdominal cavity of the respective rats. In the same way as with the in vitro method, the concentration used is the same as that used in the treatment of humans. 48 hours after the last injection, the rats are euthanized and the diffusion chambers are removed. The membrane filters are isolated and fixed, and the cells are labeled according to the Papanicolaou method. The cytotoxic effect is determined according to the morphological criteria described above in the in vitro test.

Cell-weakening treatment

When the cells have been tested for sensitivity against various cytostatic agents as described above, it has been found that untreated carcinoma cells which have the same origin as the previously tested cells are weakened by the treatment and the method according to the invention.

In order to carry out the method according to the invention, it is essential that when the optimal cytostatic agent for a given cell culture has been determined, the cells are treated with the selected agent in such a way that they are not completely killed, but only partially weakened. To achieve this, care must be taken that the incubation time in the presence of the cytostatic agent is not too long. As might be expected, the incubation time for each culture will vary with the different cell cultures, and with the activity of the cytostatic agent. The increasing weakening can be observed under a microscope with a magnification of approx. 125. This can be carried out by a skilled professional without difficulty. The incubation is preferably allowed to continue until a microscopic examination shows that only approx. 30 to approx. 70%, preferably only approx. 40 to approx. 60% of the cells appear to be intact. Incubation time from approx. 4 to approx. 15 hours, preferably from approx. 6 to approx. 12 hours, has generally been found to be suitable. The incubation is preferably carried out at a temperature of approx. 37°C.

For the debilitating treatment, each cytotoxic agent is used in the same concentration as in the test described above.

The cell weakening treatment is preferably carried out as follows. Cells obtained by trypsinization, as described above, and which have not been treated with a cytostatic agent, are suspended in a nutrient medium, placed in Roux flasks and heated in an incubator to 37°C. The particular cytostatic that has given optimal results in the above test is introduced into the bottles in the concentration indicated in table 1.

When the cells have been suitably attenuated to the desired extent, the culture is washed thoroughly to remove the cytostatic agent. This step serves to reduce the effect of the cytostatic agent on the cells and mainly to prevent a continued reaction between the cytostatic agent and the cells which could possibly lead to the cells being killed. A phosphate-buffered saline solution is preferred as the washing liquid, although other suitable physiologically compatible solutions can also be used.

The cells, which have been weakened in this way to the desired extent, are centrifuged at approx. 800 to approx. 1000 rpm for 10 minutes and preferably suspended in a physiologically acceptable liquid,

such as a physiological saline solution or calf serum. The concentration of the thus treated cells in the resulting pharmaceutical preparation which is ready for administration after dilution is preferably approx. 10 cells per ml.

The pharmaceutical preparations to be prepared are preferably prepared from tumor cells taken from the particular patient to be treated. This will ensure a particularly high tolerance and optimal effect. It is clear that there will be cases where, for various reasons, it is not possible to prepare preparations from cells taken from the patient to be treated, e.g. because there is insufficient time available, or because professionals who can prepare and test carcinoma cell cultures and the necessary equipment for this work are not available. In such cases, preparations made from carcinoma cells from other patients can be used. It is of course most preferred to treat a patient with a preparation made from carcinoma cells which corresponds to the carcinoma to be treated. E.g. should patients suffering from ovarian carcinoma be treated with preparations made from cells taken from ovarian carcinoma in other patients. It is also possible to prepare mixed preparations containing cells from different types of carcinoma, such as weakened cells from breast and ovarian carcinomas.

The pharmaceutical preparations are stable in suspension i

about. 4 weeks at a storage temperature of approx. +4°C. To increase the shelf life of the preparations, the pharmaceutical preparations can be quickly deep-frozen to temperatures as low as -80°. Deep-frozen, the preparations are stable for approx. 1 year. For this purpose, they are preferably sealed and packed with a small excess of nutrient medium solution. The pharmaceutical preparations can also be stored for a longer time in a lyophilized state.

The pharmaceutical compositions may be administered by any suitable and effective means, such as subcutaneously or intramuscularly, or by intravenous infusion. Administration by subcutaneous or intramuscular injection is preferred.

With a preferred treatment method, approx. 1 ml of the cell suspension prepared according to the invention, diluted to 10 times the volume and containing approx. 10 cells, with each individual treatment. The treatment is suitably repeated 5 or 6 times at 48 hour intervals. It has been found that treatment with the preparations produced according to the invention will result in greatly improved tolerance in the patient to the corresponding cytostatic agent. For this reason, it is highly desirable to treat a patient with the cytostatic agent alone in the usual way<1> according to known methods for approx. 8 days after the treatment sequence with the preparations prepared according to the invention. Further details in this respect will be seen from the following clinical examinations which serve to illustrate and explain the invention.

Example

The following active ingredients and solutions were

used:

Washing solution - this was prepared by conducting

80 ml. of solution A, 100 ml of solution B, 100 ml of

solution C and 5 ml of an antibiotic solution into a container together with double distilled water to make 1000 ml of solution.

Solution A - was prepared by dissolving the following ingredients in 400 ml of double-distilled water:

Solution B - was prepared by dissolving in 500 ml of double-distilled water: CaCl20.5 g

Solution C - was prepared by dissolving in 500 ml double-distilled water of: MgCl2-6H20 0.5 g

Solutions A, B and C were sterilized in an autoclave

and stored at 4°C.

Antibiotic solution

was dissolved in 300 ml double-distilled water and stored at -20°C.

Trypsin solution - was prepared in a water bath at

37°C.

The solution was filtered under sterile conditions and then filled into small bottles and stored at -20°C. For the trypsin treatment, this solution was adjusted to the desired concentration with washing solution.

Behring IV nutrient medium

The mixture was filtered under sterile conditions and was poured into bottles and stored at -20°C. Before use, the nutrient medium was adjusted to a pH value in the range 6.8 to 7.2 with sodium bicarbonate solution (22 g NaHCO 3 and 478 ml double-distilled water).

H ank's stock solution A - the following ingredients were dissolved in 250 ml of double-distilled water and stored at 4°C:

Hank's Stock Solution B - the following ingredients

was dissolved in 240 ral of double-distilled water and stored at 4°C:

Phenol red solution

Phenol red was dissolved cold in the sodium hydroxide solution

during approx. 30 minutes, and the solution was made up to a volume of 50 ml with double-distilled water. The solution was stored at 4°C.

TCM solution

Double distilled water was added to the two stock solutions for

to give 500 ml. The powder was dissolved in the resulting solution.

G glutamine solution

Double distilled water was added to the stock solutions in a volume of 100 ml. The glutamine powder was dissolved in the resulting solution.

Vitamin solution

Double-distilled water was added to the stock solutions to a volume of 900 ml and the vitamins were added. The resulting solution was then filtered under sterile conditions and

filled into small bottles of 100 ml and stored at -20°C.

Calf serum

Fetal calf serum (Boehringer, Mannheim) was stored at

-20°C and thawed in a water bath at 37°C before use.

Tissue (50 g) was excised from an ovarian carcinoma in

a 46-year-old patient at stage T4N5Mlc and was manually minced together with 700 ml of a trypsin solution diluted to a concentration of 0.5% and filled into a 1000 ml flask. The mixture was stirred for 10 minutes with a magnetic stirrer. The liquid on top was then poured into an ice-cooled flask of the same size. This step of stirring the tissue-trypsin mixture and decanting the supernatant was repeated 4 times. The fourth repetition resulted in a clear overlying liquid. The pooled decanted fluids and the remaining tissue were then filtered through sterile gas. The filtrate was centrifuged at 800 to 1000 rpm. for 10 minutes. Washing solution (100 ml.) was poured over the precipitate. The resulting mixture was centrifuged again and precipitated.

The resulting sediment was accurately pipetted with

5 ml nutrient medium and was then filled up with nutrient medium to a total volume of approx. 400 ml. After this dilution, examination with a Zeiss inverted microscope with a magnification of 125 showed approx. 20 to 25 cells in the visual field. The diluted cell suspension was distributed among a series of test tubes, 1 ml per tube, and in Roux bottles, 100 ml per bottle. The growth and metabolism of the cells were observed and recorded every day for 6 days. As soon as the nutrient medium solution showed a color change from red to yellow, the nutrient medium was renewed.

Good cell growth was recorded after the cell suspension had been incubated in an incubator at o7°C for 6 days.

The cell cultures in the tubes were subjected to the in vitro test explained above with the cytostatic agents indicated in Table 1

in the concentrations indicated there. After 24 hours, the morphological damage was determined under a microscope. The cell suspension proved to be sensitive to the following cytostatics: Adriblastin, Vincristine and Proresid. All the other indicated cytostatic agents, 6 in this case, were ineffective.

Four Roux flasks containing the cell suspension obtained according to the above method were then reconstituted. Adriblastin, Vincristine and Proresid were used to treat the contents of the respective bottles in the concentrations indicated in Table 1. The cell suspension in the remaining bottle was not treated and served as a control sample. After the cytostatic agent had been allowed to act on the cells for 12 hours, the samples were examined under a microscope, and only 60% of the cells were found to be intact. The cells were then separated by centrifugation at 800 to 1000 rpm. for 10 minutes^10 ml of fresh nutrient medium was poured onto the sediment. The thus obtained weakened cells were suspended in fresh nutrient medium at a concentration of 10 g cells per ml. The suspension was stored at 4°C.

In vitro test

1 ml of an untreated cell suspension containing

10 c cells were placed in each of several test tubes. These cells had been obtained by the above treatment of carcinoma tissue with trypsin, but had not been treated with a cytostatic agent. For 6 days, 0.3 ml of one of the above preparations consisting of cells that had been weakened with Adriblastin, Vincristine or Proresid, was added daily to each tube. During this investigation, the tubes were kept in an incubator at a temperature of 37°C. After 7-9 days approx. 80 to 90% of the untreated cells in each tube destroyed by the preparation prepared according to the invention. Since the cytostatic agent was thoroughly washed out of the preparations, it is assumed that this was the result of an intercellular reaction.

Clinical treatment

Based on the results of the above surveys

as well as further experiments of a similar nature and after corresponding experiments on cell types from animal tumors, the preparations produced according to the present invention were used for clinical therapy. With one exception, all patients suffered from cancer at an incurable stage and, despite operations, radiotherapy and/or cytostatic treatment, were at stage T 4 N3-5 Mlc according to the classification introduced by the Union International contre le Cancer. The remaining life expectancy of such patients: averages less than 60 days.

The preparations produced according to the invention were used mainly as supportive immunotherapy in an attempt to

strengthen the immune system before a cytostatic treatment with the corresponding cytostatics. A total of 22 patients were treated,

all of whom were fully informed about their condition and the planned method of treatment. 14 of the patients suffered from malignant ovarian tumor (one of them from mesosigmoid tumor as recurrent growth),

four from breast carcinoma, one from pancreatic carcinoma, one from a retothelial sarcoma, one from coecum carcinoma and one from uterine carcinoma.

Tissue that had been attacked by cancer was taken from the patients during surgery and was treated according to the method described above for the preparation of the new preparations. Every other day, individually prepared cell suspensions totaling 6 to 8 10 ml portions were administered intragluteally to each patient. Each 10 ml portion contained 10 cells,

i.e. 1 ml of the preparation in nutrient solution and 9 ml of physiological salt solution or calf serum. In some cases, 1 ml of Lidocaine solution (Xylocaine) in a concentration of 0.5% was administered with each 10 ml portion. In this first sequence, the number of injections depended on the number of cells available, and in most cases this resulted in 5 to 8 injections at 48 hour intervals. After a waiting period of 8-20 days (in the case of patients in extremely poor condition the waiting period should be closer to the upper limit) the cytostatic treatment was carried out according to the individual test results, in most cases according to the following scheme: 1 mg Vincristine injected intravenously, 6 hours later Adriblastin injected intravenously in an amount of 40 mg per m body surface; 48 hours later cyclophosphamide or Proresid in a quantity of 25 mg per kg body weight, dissolved in the same concentration in 500 ml physiological saline solution. Due to the toxicity of Adriblastin, a 3-week delay was allowed before the next cytostatic treatment sequence, until a total of 550 mg of that agent had been administered. If resistance occurred after test results, it was switched to other cytostatic agents, such as 5-FU, methotrexate Leukoverin and others.

at weekly intervals.

This treatment resulted in a subjective and objective improvement in all patients. There were no local or general side effects. Although the cell suspension must be considered toxic, the injection caused no local or general reddening or swelling. Pain or a corresponding reaction to the preparation was not registered. The improvement was so lasting

that in all cases the normally dangerous cytostatic treatment could begin after a maximum period of 20 days.

Four of the above 22 patients were not given

subsequent cytostatic treatment because they refused, or because the tumors were clearly too advanced. Still, these patients survived exceptionally long. E.g. a 60-year-old woman who had a breast tumor larger than a fist and bone metastases and brain metastases was also detected, was subjected to a simple removal of the breast and given subsequent treatment with the preparation produced according to the invention. Without further treatment, she lived for 270 days.

Five patients were also treated with heterologous carcinoma cell suspensions and showed very good tolerance.

The average survival time for all patients is so far 350 days, which is almost 6 times the previous average. Five patients were alive after 245 to 665 days. Of these, three have not had recurrent tumors and two

have had recurrent tumors that have responded to treatment. All the treated patients are in good general health.

The result of the treatment with the prepared preparations

according to the invention, on two of the above-mentioned 22 patients and on a further patient, will be described in the following.

1) A 46-year-old patient was admitted to the hospital because she

suffered from breast tumor on both sides which had existed for 2 years,

and large abdominal tumors. The general condition of the patient was very poor. After a general preliminary treatment, both breasts, which were heavily attacked by cancer, as well as the associated lymph nodes in the armpits, were removed in two operations in June 1978. 14 days later, the uterus and the two tumors on the ovaries were removed. The remaining cancerous tissue in the omentum and on the peritoneum, in the lymph nodes and in other places could not be removed by surgery. The patient did not receive radiotherapy, but received 12 injections of the preparation prepared according to the invention, from pre-treated carcinoma cells from her

own body (prepared as indicated in the example above) and was then subjected to the combined chemotherapy. The patient recovered completely during the course of the immunotherapy, and then showed a high tolerance to the cytostatic agents. After 12 months, the patient said she felt in excellent shape.

She was fully able to carry out her work, and had no complaints. The clinical examination showed no recurrent tumour, either in the abdomen or in the chest. At the start of treatment, she was at tumor stage T4 N5 Mlc.

2) A 32-year-old patient had a large tumor in her abdomen when she

was admitted to the hospital. The uterus and the two adnexa had been removed at another hospital a year earlier for treatment of quite large ovarian cancer. She had also received cytostatic treatment for the previous case. After the relapse, a mesosigma tumor about the size of a child's head was removed. This had developed as a recurrent tumor after the previous ovarian carcinoma. It was impossible to completely remove the mesosigma tumor in fresh tissue. She also had metastases in the lymph nodes on the pelvic wall. The patient was given post-operative supportive immune therapy which consisted of 12 injections of preparations made by processing cells from her own tumours. Because the patient subsequently moved, she could not receive subsequent cytostatic treatment. The patient returned several times for examination. 11 months after the immunotherapy was started, she was in excellent shape, with no clinical indication of recurrent tumors. The entire abdomen is free of tumor.

3) A 41-year-old patient, who is not included in it

the group mentioned above because she was at the Tl stage, was admitted to the hospital because she had a small, possibly cancerous nodule in her left mammary gland. The results of mammography, thermography and puncture cytology were positive for the left side and doubtful for another small nodule on the right side (Papanicolaou 3). Histology of tumor tissue removed from the left breast revealed a solid mammary carcinoma (Tl Nx Mo) approx. 2 cm in diameter. This patient's case is described separately, because she was the only early case. The patient agreed to simple mastectomy of the left breast, but refused further measures, particularly on the right breast. She also refused cyto-

static or other subsequent treatment, but accepted cellular therapy. She was given heterologous cell therapy with breast carcinoma cells obtained from another patient,

and treated according to the method described according to the invention. The cell treatment was carried out according to the routine described above, and included 6 intramuscular injections of 10 ml cell suspensions containing 10 cells each, every other day. 12 months after treatment, the patient had no indications of recurrent tumor growth. The suspected tumor on the right breast had completely disappeared.

It will be clear from the above that it was not possible

to administer sufficiently large doses in most cases due to lack of material. It would be desirable to give every patient suffering from carcinoma, even at an early stage, a treatment consisting of a sequence of 5 or 6 injections every 48 hours for a month, then every three months, then in corresponding months for four consecutive months six months and then in corresponding months for several consecutive years. Such treatment can only be carried out with heterologous cancer cells, but these should, if possible, be taken from corresponding organs, in cases where no autologous cells are available. The heterologous cancer cells can be mixed in a 1:1 ratio with lyophilized cancer cells,

which does not need to be pre-treated.

Without being bound by any particular theory or mode of action, it seems that the preparations produced according to the invention are effective in the treatment of carcinoma on the following basis: This method of cancer treatment was developed based on the assumption that malignant tissue growth begins on basis of self-directed, ever-expanding and excessive multiplication of body-specific cells. The principle of the method seems to largely consist in an improvement of the immunological condition of the patient before the cytostatic treatment by repeated injections of ultrafiltrates of body-specific, cytostatically pretreated carcinoma cells, as described above.

Claims (10)

1. Procedure for the isolation, cultivation and cultivation of selected carcinoma cells, characterized by the following steps: (1) carcinoma cells are provided; (2) the cells are cultured and incubated in a nutrient growth medium under cell culture conditions to produce a cell culture; (3) multiple predetermined portions of the cell culture according to step (2) are exposed to various cytostatic agents, and the visible morphological damage to each portion is observed; (4) the cytostatic agent which causes visible morphological damage to these susceptible cells is selected; (5) the remaining part of the cell culture from step (2) is treated with the cytostatic agent from step (4), and incubated to such an extent that from approx. 30 to 70% of these cells are intact under microscopic examination, and that these cells are not completely killed, but only partially weakened; (6) the weakened cells from step (5) are washed with a physiologically acceptable solution to remove substantially all cytostatic agent from said weakened cells, and ■ (7) the thus treated cells are suspended in a physiologically acceptable liquid for the preparation of a pharmaceutically acceptable preparation. 2. Method as stated in claim 1, characterized in that the incubation according to step (5) is carried out over a period of approx. 4-15 hours, preferably approx. 6 to 12 hours. 3. Method as stated in claim 1, characterized in that the incubation according to step (5) is carried out at a temperature of approx. 37 C. 4. Method as stated in claim 1, characterized in that the incubation according to step (5) is carried out so that from approx. 40 to approx. 60% of the cells remain intact. 5. Method for producing a suspension of weakened selected carcinoma cells which are susceptible to the cytostatic effect of a selected cytostatic agent, characterized in that a cell culture of carcinoma cells is treated with a cytostatic agent to which the cells are sensitive, and incubated over such a long time that microscopic examination shows that only approx. 30 to approx. 70% of the thus treated cells are intact, and then the thus weakened cells are washed with a physiologically acceptable solution to remove the cytostatic agent. 6. Method as stated in claim 1, characterized in that according to step (1) ascites carcinoma cells or leukemia cells are provided. 7. Method as stated in claim 5, characterized in that the resulting solution of weakened cells is deep-frozen. 8. Method as stated in claim 5, characterized in that the resulting solution of weakened cells is lyophilized. 9. Method as stated in claim 5, characterized in that a portion of the resulting solution of weakened cells is deep-frozen and a portion of the resulting solution of weakened cells is lyophilized, the deep-frozen and the lyophilized portion are kept under storage conditions, and then the frozen portion is thawed and mixed with an equal amount of lyophilized cells. 10. Method as stated in claim 1, characterized in that the weakened cells from step (5) are separated by centrifugation at a speed of approx. 800 to 1000 rpm, and then washed according to step (6).