Medium and method for measuring the efficacy of a tumour therapy

The invention relates to a medium for measuring the efficacy of tumor therapy on individual cell suspensions, as well as to a method for measuring the effectiveness of tumor therapy using this medium.

In order to test the effectiveness of tumor therapies, it would be desirable to be able to determine the influence of tumor therapy on the viability of this cell on an isolated tumor cell. Such a method would enable a patient to determine in advance whether a particular therapy is effective in its particular tumor or not. For this purpose, tumor parts were to be removed, converted into a single-cell suspension by enzymatic digestion, and then the life activity of the individual cells thus obtained was determined under the influence of the therapy, typically the chemotherapeutic agent. A comparison of the metabolic activity of untreated tumor cells with the metabolic activity of therapeutically treated tumor cells can then directly determine the effectiveness of the therapy by means of the reduced metabolic activity of the treated cell compared to the untreated tumor cell. In this way, it would be possible to determine within a very short time which of a particular tumor is suitable or less suitable for the treatment of the patient.

A crucial problem for such a method, however, is to adjust the conditions in the cell suspension in such a way that the isolated tumor cells can develop a maximum metabolic activity. Only then can a reduction in metabolic activity be measured with sufficient accuracy. However, individual cell suspensions of the tumor cells produced by a compact tumor tissue by enzymatic digestion have very low metabolic activities in the known media so that it is very difficult or even impossible to determine a further reduction in the metabolic activity with sufficient accuracy.

The invention is based on the object of providing a cell culture medium, which allows the individual metabolic suspensions of tumors produced in this way to have a maximum metabolic activity and a lifetime, and to provide a method for assessing tumor therapy with this medium.

According to the invention, this object is achieved by a medium for measuring the efficacy of tumor therapy on individual cell suspensions containing the essential amino acids, vitamins, salts and carbon donors, characterized in that the medium contains 0.1 to 1 mM buffer pH 7.0 to 7.4, To 10 g / l glucose and 2 to 5 mM glutamine as carbon sources and 5 to 20% fetal calf serum, the medium containing no other carbon sources.

<patcit id="pcit0001" dnum="US6221873B"><text>US 6,221,873</text></patcit> Describes the use of a medium based on DMEM (Dulbecco's modified Eagle's Medium) containing more than 44 mM of buffer as well as pyruvate. <nplcit id="ncit0001" npl-type="s"><text>Metzger et al. (Toxicology 166 (2001), 97-108</text></nplcit>) Describes the use of a medium based on RPMI (ImM phosphate buffer) containing no fetal calf serum.

The medium according to the invention is distinguished from known media by a very low buffering capacity, a higher content of glucose, glutamine and fetal calf serum. Carbon sources other than glucose and glutamine and other buffers do not contain the medium.

The composition of the medium according to the invention depends to a certain extent on the metabolic properties of the cells contained in the tumor being examined. The adjustment of the variables in the medium according to the invention aims at setting the conditions for glycolysis for the tumor cells since a high pH change is to be expected. At the same time, the normal cells which are unavoidably also present in the suspension are, as far as possible, kept in aerobic metabolism and thus have a negligibly small effect on the pH.

Since tumor cells consume more glucose than many non-tumor cells and, on the other hand, in tumor cells, the achievement of the highest possible glycolysis is achieved in which the proton formation and thus the acidification of the medium per produced ATP mofeculin is highest 2 g / l, particularly preferably 4 to 6 g / l of glucose. Other carbon sources, with the exception of the glutamine present as essential component of the medium, should not be present. Typical examples of such undesired carbon sources are pyruvate, lipids and similar known carbon sources in nutrient media. Lipids, in particular, have proved to be harmful in combination with glutamine, since the initial activity of all tumor cells is greatly reduced.

The best results in terms of increasing the activity of the tumor cells while at the same time minimizing the possible influence of the non-tumor cells on the change in pH resulted therefore in a content of 5 g / l of glucose, 2 mM of glutamine and 10% by volume of fetal calf serum ± 10% can fluctuate.

The medium according to the invention can be produced by construction from the components. For example, conventional vitamin mixtures and protein hydrolysates can be constructed as the source of the essential amino acids with the above-mentioned essential amounts of glucose, glutamine, buffer and fetal calf serum. Alternatively, it is also possible to proceed from known media and its composition can be supplemented by the addition of the important amounts of essential components as explained above. In particular, the RUN medium is suitable as the starting point. Further, the medium may contain antibiotics.

The invention further provides a method for measuring the efficacy of tumor therapy on individual cell suspensions of tumor cells by comparing the formation of acid in a medium in the case of treated and non-treated tumor cells, which is characterized in that the measurement is carried out in a medium as described above. The method is well suited for use with commercially available devices such as the cytosensor^®Microgysiometer from Molecular Devices Corp., Sunnyvale, CA., USA, but is not limited to this.

The latter device and its use for the analysis of cell membrane bound receptors is shown in FIG <nplcit id="ncit0002" npl-type="s"><text>Biosensors &amp; Bioelectronics 15 (2000) 149-158</text></nplcit> , The pH changes caused by the physiological effects of such receptors being measured as a measure of the metabolic effects.

The method according to the invention for measuring the efficacy of a tumor therapy is described subsequently, for example, using the cytosensor^®Microphysiometer.

For carrying out the method according to the invention using the medium according to the invention, a sample of the tumor to be examined is removed and converted into a single-cell suspension by enzymatic digestion in a known manner. The cells are separated from the digestion medium and their pH curve is then taken up in the medium according to the invention. A typical medium according to the invention has, for example, the composition to be taken from the following Table 1.<tables id="tabl0001" num="0001"><table frame="none"><title>Table 1</title><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="25mm" /><colspec colnum="2" colname="col2" colwidth="59mm" /><thead><row><entry valign="top">concentration</entry><entry align="center" valign="top">substance</entry></row></thead><tbody><row><entry>Mg / l</entry></row><row><entry>4.45</entry><entry>Alanine, L-</entry></row><row><entry>147.5</entry><entry>Arginine L-, HCL</entry></row><row><entry>7.5</entry><entry>Asparagine, L-, H2O</entry></row><row><entry>6.65</entry><entry>Aspartic acid, L-</entry></row><row><entry>24</entry><entry>Cystine, L-</entry></row><row><entry>17.56</entry><entry>Cysteine, L-, HCL, H2O</entry></row><row><entry>7,35</entry><entry>Glutamic acid, L-</entry></row><row><entry>18.75</entry><entry>glycine</entry></row><row><entry>31.48</entry><entry>Histidine, L-, HCl H2O</entry></row><row><entry>20</entry><entry>Hydroxyproline, L-4-</entry></row><row><entry>54.45</entry><entry>Isoleucine, L-</entry></row><row><entry>59.05</entry><entry>Leucine, L-</entry></row><row><entry>91.25</entry><entry>Lysine, L-HCl</entry></row><row><entry>17.24</entry><entry>Methionine, L-</entry></row><row><entry>35.48</entry><entry>Phenylalanine, L-</entry></row><row><entry>17.25</entry><entry>Proline, L-</entry></row><row><entry>26.25</entry><entry>Serine, L-</entry></row><row><entry>53.45</entry><entry>Threonine, L-</entry></row><row><entry>9.02</entry><entry>Tryptophan, L-</entry></row><row><entry>38.7</entry><entry>Tyrosine, L-</entry></row><row><entry>52.85</entry><entry>Valine, L-</entry></row><row><entry>2000</entry><entry>D + glucose</entry></row><row><entry>12.6</entry><entry>Inositol, myo-, inositol, meso-</entry></row><row><entry>0.00365</entry><entry>Biotin, D-</entry></row><row><entry>2.24</entry><entry>Calcium D-pantothenate</entry></row><row><entry>8.98</entry><entry>choline chloride</entry></row><row><entry>1</entry><entry>Glutathione, L-</entry></row><row><entry>2.02</entry><entry>nicotinic acid amide</entry></row><row><entry>2.031</entry><entry>Pyridoxine, HCL</entry></row><row><entry>2.17</entry><entry>Thiamine chloride, thiamine HCl</entry></row><row><entry>0.01</entry><entry>Tocopherol succinate DL-alfa</entry></row><row><entry>0.1</entry><entry>Vitamin A acetate</entry></row><row><entry>0.68</entry><entry>Vitamin B 12 (cyanocobalamin)</entry></row><row><entry>2</entry><entry>Vitamin C (ascorbic acid)</entry></row><row><entry>0.1</entry><entry>vitamin D2</entry></row><row><entry>8.1</entry><entry>phenol red</entry></row><row><entry>154</entry><entry>Calcium chloride 2 H2O</entry></row><row><entry>0.05</entry><entry>Iron (III) nitrate, 9H2O</entry></row><row><entry>0.417</entry><entry>Iron (II) sulfate, 7H2O for analysis</entry></row><row><entry>311.8</entry><entry>potassium chloride</entry></row><row><entry>0.00125</entry><entry>Copper (II) sulfate, 5H2O</entry></row><row><entry>100</entry><entry>Magnesium sulfate, 7 H2O</entry></row><row><entry>61</entry><entry>Magnesium chloride, 6 H2O</entry></row><row><entry>6999.5</entry><entry>sodium chloride</entry></row><row><entry>14.2</entry><entry>Di-sodium hydrogen phosphate (anhydro)</entry></row><row><entry>0.43</entry><entry>Zinc sulfate, 7 H2O</entry></row><row><entry>2 mM</entry><entry>glutamine</entry></row><row><entry>10 vol%</entry><entry>fetal calf serum</entry></row><row><entry>100 units / ml</entry><entry>penicillin</entry></row><row><entry>100 μg / ml</entry><entry>streptomycin</entry></row><row><entry>100 μg / ml</entry><entry>kanamycin</entry></row><row><entry>2.5 μg / ml</entry><entry>Fungizone</entry></row></tbody></tgroup></table></tables>

For this purpose, the individual cells thus obtained are then preferably immobilized on a pH electrode. The immobilization can be carried out by suitable substances as are customary in cell culture. Agarose, which can immobilize the obtained individual cells on the electrode surface, but also other immobilization agents used in the cell culture, are also suitable.

The device used has eight channels in which the pH value can be simultaneously determined in flow cells. A pH electrode, on which the immobilized cell suspension is located, is arranged in each channel. The medium according to the invention is then pumped by means of this immobilized cell suspension for a suitable period by means of a pump, and at the same time the pH measurement is started. In a typical case, a measurement cycle lasts 120 seconds. 90 seconds, only medium is pumped in and a pH value is determined every second. The pH value change is then measured for 30 seconds. This process is continued over a longer period of time, generally 14 to 24 hours, resulting in a pH curve which is directly equivalent to the metabolic activity of the immobilized cells.

In the above apparatus, there are eight parallel channels as mentioned above. To determine the efficacy of a tumor therapy, only medium according to the invention is expediently pumped through in a channel for the determination of a basic curve; in the other channels, various antitumor agents (cytostatics) can be simultaneously determined. Since the cytostatics, when acting against the tumor cells used herein, have an effect of reducing the metabolic activity, a pH curve is obtained over time, which has a lower pH value due to the reduced metabolic activity due to the cytostatic action Change. The activity of the respective cytostatically active compounds can be determined directly from the comparison of the curve inclinations, which are then obtained in the curves measured in the presence of cytotoxic substances in comparison to the cytostatic-free measurement curve. In the same way, it is also possible not to examine different cytostatically or cytotoxically active compounds in the different channels, but to examine different concentrations with respect to their efficacy for a particular cytostatic agent. The latter embodiment has the advantage that possible influences of the examined cytostatics on the pH can be compensated for by adjusting the medium within the limits of the invention, in particular with respect to the buffer capacity. The buffer capacity as such is kept as low as possible, but must not be less than 0.1 mM. The buffer quantity is preferably set as close as possible to this lower limit value. In the case of cytostatics, which themselves can influence the pH value, however, the buffer quantity can then be increased up to a maximum of 1 mM.

According to the invention, it is possible to measure the effectiveness of a tumor therapy in a very extracorporeal manner within a very short time, and to adjust the treatment accordingly on the basis of the measurement results. This not only makes a decisive gain in time during the treatment of the patient but also the risk of undesirable side effects of the therapy without corresponding effectiveness against the tumor itself is reduced or completely avoided. This leads to a considerable advance in tumor therapy.