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Biocrush^®

The test of the anti-tumoural effectivness of the preparation Biocrush® in vitro

The prove of the cytotoxicity of Biocrush^® on cancer cells was done by the MTT test.
This a colorimetric test, which bases on the reduction of the yellow tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide (MTT) to purple formazan crystals through cellular dehydrogenase. (fig. 1). Because dehydrogenase is only active in living cells, the transformation of MTT is a measure for the degree of viability and the metabolic activity of the cells (Mosmann et al.1983). The absorption of the formed formazan is determined photometrically at the wave length – 550 nm and reference filter - 690 nm.

Fig. 1: Reduction of the yellow MTT to the purple formazan.

In vitro tests  

For the tests were used four different tumour cell lines :

Tab.1: overview of the tested cell lines

The four cell lines were procured at the German collection for microorganisms and cell cultures (DSMZ, Braunschweig, Germany).

All tumour cells grew adherent as mono-layer and were cultivated in T 25 cell culture flasks at 37°C and 5% CO2 in an incubator.

EFO-27 and EFM-192A were cultivated in RPMI 1640 Medium, HELA-S3 in Hams F12 and EN in DMEM-media. To all media were added respectively 10-20% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin and 100 µg/ml L-glutamine.

Half of the respective media (table1) were exchanged every 2-3 days by fresh media. The cell passaging took place shortly before the confluence (80%-90%) and then the cells were splitted weekly according their growth rate 2-3 x in the ratio 1:5 to 1:10. Therefore the cells were washed twice with PBS and then enzymatically flaked of with 2.5 ml trypsin/EDTA from the growth surface. The flaking was controlled by microscopy. The trypsination was inactivated by addition of 10% FCS-containing media. The cell suspension was poured into sterile 15ml-centrifuge tubes and centrifuged for 4 minutes at 220 x g. The excess liquid was carefully sucked out and the cell pellet was resuspensed in 5 ml of fresh media. After this the number of cells per ml were counted with a Neubauer counting chamber. The cells were desseminated to the requested cell number in new cell culture flasks.

Toxicity of Biocrush® on tumour cells

To test the toxicity of Biocrush®, 5000 cells per well were disseminated in a 96 well plate in a volume of 200 µl. After 24 h the media was take away and replaced by 200 µl fresh media with 1 % FCS, which additionally contained the testing substance. After 72h incubation were added to the cells with the test substance per Well 20 µl MTT-solution (5 mg/ml) and the cells were incubated for 4 h at 37°C and at 5%-CO2 in a incubator. The cells then were desintergrated with 100 µl MTT-lysis buffer per well und the formed formazan crystals were given time to disolve over night. Then the extinctions of each well at 550-690 nm was determined with a titer-plate-photometer.

As 0-control were used the untreated cells and defined as non-toxic level. SDS (sodium dodecyl sulphate) as known cytotoxic reference substance was used for comparison in the concentrations 6.3, 12.5, 25, 50 and 100 µg/ml. Biocrush® was used respectively in 5 different concentrations:

A =10.000 µg/ml, B = 1.000 µg/ml, C = 100 µg/ml, D = 10 µg/ml and E = 1 µg/ml.
The extinction of the untreated cells was set to 100 %, the other values are related percentually. Showed are the mean value from n = 8 parallels with the standard deviation. The level of significance relates to the comparison of the control.

Results

Determination of the cytotoxic effect of the preparation  Biocrush^®  on tumour cells in vitro

Tab.2: HELA-S3

Tab. 3: EN

Tab. 4: EFO-27

Tab. 5: EFM-192A

Tab. 6: NHDF-c adult

Fig. 2: Cytotoxic effect of Biocrush® on the four tumour cell lines

Normal human dermal fibroblasts

Fig.3: testing of the Biocrush®‘s metabolic activity on human fibroblast

The test showed that the preparation „Biocrush® “ has a cytotoxic effect on tumour cells.
The effect depends on the concentration and increases with an enlarging concentration.
In the concentration level of 1 to 1000 mg/ml the cytotoxic effect on various tumour cells varies.

For the Cervix cancer cell HELA-S3 the cytotoxic effect of Biocrush® in a concentration level of 1 to 10 mg/ml was negligible small. At a concentration level of 100 to 10000mg/ml a maximum inhibition of the cancer cells of app. 15% was determined.

For the breast cancer cells EFM-192A Biocrush® in the concentration level of 1, 10 und 100 mg/ml also only a small effect was observed, i.e. the inhibition was at app. 3, 5 and 8%.
At a concentration of 1000mg/ml a clear inhibition of the cancer cells at app. 34% was observed.

A good inhibition effect was observed for the endometrial cancer cells EN and the  ovary adeno cancer cells  EFO-27. In the tested concentrations of 1, 10, 100 and 1000mg/ml were observed for EN cells inhibition rates of app. 10, 51, 60 and 65 % and for the EFO-27 cells of app. 33, 34, 44 and 66%.

At the max. tested concentration of 10000 mg Biocrush®/ml the inhibition effect for all cancer cells was > 95%.

In comparison to the influence of Biocrush® on cancer cells furthermore human dermal fibroblasts NHDF-c adult were tested. As shown in figure 3, the influence of Biocrush® is negligible in a concentration level of 1 to 1000 mg/ml, i.e. no toxic effects on human fibroblasts were observed.