Characterization of bioactive substances MHGF-68 on tumour cell lines with LiveFlow In Vitro Technology
Article Category: Original Paper
Publié en ligne: 18 juin 2021
Pages: 24 - 29
Reçu: 11 nov. 2020
Accepté: 22 mars 2021
DOI: https://doi.org/10.2478/afpuc-2020-0021
Mots clés
© 2021 Hodoši R. et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Comparison of IVTech, in vitro and in vivo testing.
| Lack of human complexity | Ethically controversial | Human organ environment simulation |
| Lack of side effects tests | Time ineffective | Multi-organ models |
| Lack of geometrical complexity | Expensive (2–30 times more than in vitro) | 3D and dynamic cell cultures |
| Cells cultivated in static conditions | No high-throughput monitoring | Real time monitoring |
In our experiments, we have confirmed that MHGF-68 has a two-component character and its Mr is < 1000. We used MALDI technique, which is also suitable for checking the samples during the purification procedure for checking the mass of the protein/peptide content after fractionation after RP-HPLC, FPLC respectively (Olejníková
MHGF-68 compounds were obtained by the cultivation of BHK-21 cells (baby hamster kidney) infected with murine herpesvirus-68 (MHV-68). Monolayers of BHK-21 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) enriched with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine and 0.1% gentamicin. Subsequently, cells were infected with MHV-68 with multiplicity of infection (MOI) 0.01 and incubated for 24 hours in 41°C. Afterwards, cells were transferred to 37°C and cultivated for another 24 hours. After cultivations, media from these cells were pooled, lyophilized and stored in +4°C until other experiments were performed (Šupolíková et al., 2015). The fraction of MHGF-68 used in experiments was acquired by FPLC separation on Sephadex G15 column, eluted by redistilled water, so the transformation suppressing activity remained. Experimental conditions: 5 ml of medium obtained from infected cells MHV-68 was loaded onto an HPLC analyser (Shimadzu, Kyoto, Japan) with column of dimensions 950 × 20 mm containing sorbent Sephadex G15 Fine (Pharmacia, Lund, Sweden). Deionized was used as the mobile phase water or saline (pH 7.2). Flow mobile phase was 0.8 ml/min. Fractions were collected based on the signal from the UV detector at the wave detectors lengths 220 and 254 nm (Vojs Staňová et al., 2015).
Experiments were performed on adherent fibroblast cell lines Hepa1c1c7 and NIH3T3. Cell line Hepa1c1c7 (ATCC® CRL-2026™) is a stabilized tumour cell line isolated from murine hepatocellular carcinoma, whereas cell line NIH3T3 is a normal cell line derived from murine embryonal cells (ATCC® CRL1658™). Cells were cultivated in DMEM enriched with 7% FBS, 1% PSA (antibiotics: 100 U/ml penicillin/streptomycin, 100 μg/ml amphotericin) and 1% L-glutamine, and they were incubated in 37°C with 5% CO2.
Sterile circular glass cover slides (20 mm in diameter) were placed in 6-well plates (1 slide per well). 1 × 105 cells were seeded onto glass slides in volume of 0.25 ml per slide. After a 20-minute-long incubation, 2 ml of DMEM was added to each well. After a 24-hour-long cultivation, glass slides were placed into LiveFlow system, as shown in Figure 1, and the cultivation medium with MHGF-68 (diluted 1:100) was added. In every experiment, control untreated cells (without MHGF-68) were used. Volume of circulating medium was 7.5 ml per LiveBox (1 slide). Cells were cultivated 24, 48 and 72 hours with continuous flow of medium at a speed of 180 μl per minute (Figure 2). Incubation was performed at 37°C, 5% CO2. For information: LiveFlow system is commercially manufactured by In Vitro Technologies (in short IVTech) and distributed by Scintilla spol. s r.o.
Figure 1
LiveFlow system scheme. Blue and green – LiveBox with samples, parallel circuits of medium, Red – controls, series circuit of medium.
Figure 2
Scheme of dynamic cultivation using LiveFlow (IVTech). LiveBox with adherent cell line on the glass slide inside, reservoir with cultivation medium and LiveFlow ensuring continual flow of the medium.
Sterile circular glass cover slides (20 mm in diameter) were placed in 6-well plates (1 slide per well). 1 × 105 cells were seeded onto glass slides in volume of 0.25 ml per slide. After a 20-minute-long incubation, 2 ml of DMEM was added to each well. After a 24-hour-long cultivation, the medium was exchanged with a medium with MHGF-68 (diluted 1:100). In every experiment, control untreated cells (without MHGF-68) were used. Cells were cultivated for 24, 48 and 72 hours at 37°C, 5% CO2.
After incubation, the cells were fixed with 4% paraformaldehyde solution and permeabilized with 0.01% Triton X-100 (PBS solution). Alexa Fluor 555 conjugated with Phalloidin was used to visualize actin filaments. To visualize cell nuclei, DAPI stain was used. Prepared slides were observed using Leica TCS SP8 AOBS (Leica Microsystems, Germany) confocal microscope with HC PL APO CS2 63x/1.40 OIL lens.
The cytoskeleton plays an important role in the regulation of various cellular processes associated with transformation, such as proliferation, contact inhibition, and cell growth in thin agar or apoptosis. In general, loss of actin filaments is considered a marker of oncogenicity (Pawlak and Helfman, 2001; Etienne-Manneville, 2004; Shutova and Alexandrova, 2010). In this study, morphological changes in cellular actin cytoskeleton after treatment with bioactive substances MHGF-68 were observed. These changes were significantly more pronounced on Hepa1c1c7 cells, especially after dynamic cultivation in LiveFlow system. After dynamic cultivation in LiveFlow, the shape of untreated (control) cells was rhomboidal (Figure 3A, B, C). Actin cytoskeleton of untreated cells was diffusive, probably depolymerized, with small aggregations of actin molecules with filamentous actin near cell margin (Figure 3C). Cells treated with MHGF-68 after 24 hours of cultivation had enlarged nuclei and changed shape (Figure 3E). After 72 hours, cells gained spindle-like shape (Figure 3F). On the contrary, highly organized actin filaments and high granulation of cytoplasm were found in whole-cell volume of cells incubated with MHGF-68 (Figure 3D). Any significant changes in NIH 3T3 cells were not detected. In comparison with dynamic cultivation, cytoskeleton structure of Hepa1c1c7 cells after static cultivation was different. Highly organized actin filaments were not present in the whole volume of cells, but were largely arranged along the edges of the cells (Figure 4E, F). After 72 hours of static cultivation, signs of spindle-shaped cell formation were observed (Figure 4F). On the normal NIH 3T3 cell line cultured with MHGF-68, we did not observe any changes in the cell cytoskeleton during the dynamic culture (data not shown). Comparison of 72 hours static and dynamic cultivation of Hepa1c1c7 with MHGF-68 showed that during stationary cultivation, changes were less visible (Figure 5). In the case of untreated cells, the formation of highly organized actin filaments and the enlargement of nuclei were observed only after dynamic cultivation in LiveFlow (Figure 5D).
Figure 3
Distribution of actin filaments in Hepa1c1c7 cells incubated with MHGF-68 after dynamic cultivation.
Hepa1c1c7 cells were incubated with MHGF-68. After 0, 24 and 72 h, the cells were fixed and labeled with Alexa Fluor 555 phalloidin. Nuclei were stained with DAPI. The intracelular distribution of actin filamets (green) and nuclei (blue) were imaged by confocal laser scanning fluorescence microscopy (Leica TCS SP8 AOBS) with HC PL APO CS2 63×/1.40 OIL lens.
Figure 4
Distribution of actin filaments in Hepa1c1c7 cells incubated with MHGF-68 after static cultivation.
Hepa1c1c7 cells were incubated with MHGF-68. After 0, 24 and 72 h, the cells were fixed and labeled with Alexa Fluor 555 phalloidin.
Nuclei were stained with DAPI. The intracelular distribution of actin filamets (green) and nuclei (blue) were imaged by confocal laser scanning fluorescence microscopy (Leica TCS SP8 AOBS) with HC PL APO CS2 63×/1.40 OIL lens.
Figure 5
Distribution of actin filaments in Hepa1c1c7 cells incubated 72 h with MHGF-68 after static and dynamic cultivation.
Hepa1c1c7 cells were incubated with MHGF-68. After 72 h, the cells were fixed and labeled with Alexa Fluor 555 phalloidin. Nuclei were stained with DAPI. The intracelular distribution of actin filamets (green) and nuclei (blue) were imaged by confocal laser scanning fluorescence microscopy (Leica TCS SP8 AOBS) with HC PL APO CS2 63×/1.40 OIL lens.
Our results from monitoring the effect of MHGF-68 on cell proliferation, metabolic activity and viability of the cells were confirmed by xCELLigence analyser too (Šupolíková et al., unpublished data). We found that the effect of the MHGF-68 fractions was relatively short-lived and decreased with longer time of cultivation. Since the chemical structure of MHGF-68 fractions is not yet known, the described biological activities require further studies.
Changes of cell size and shape and distribution of actin filaments were observed during both types of cultivation in normal and tumour cell line. Comparison of stationary and dynamic cultivation showed that the effect of MHGF-68 on cytoskeletal structures is more significant during dynamic cultivation. Regarding quantitative determination of the activity of bioactive compounds MHGF-68, it is more suitable to use stationary cultivation. We confirmed by MTT assay that the effect of MHGF-68 in a static cultivation showed a significant inhibitory effect on the proliferation and viability of NIH 3T3 and Hepa1c1c7 cells and that the effect in the dynamic cultivation did not have a significant effect on the proliferation and viability of Hepa1c1c7 cells (data not shown). However, dynamic cultivation in LiveFlow system is suitable for the optimization of experiments before