Exploring the toxicity and carcinogenic potential of 300 KDA “Mito Organelles”™ cellular extracts through MTT and BALB/C-3T3 cell transformation assays: a vital component of peptide and protein biomedical research and safety study
Authors: Klokol Dmytro , Chan Mike KS , Wong Michelle BF , Nishkumai Olha , Mostbauer Halyna , Dietrich Heike , Skutella Thomas 
Published: October 2024

The MTT assay, an essential component of our research, evaluates cellular metabolic activity to indicate cell viability, proliferation, and the cytotoxic effects of therapeutic products utilized as cellular therapy agents. The objectives of this study were: to investigate and assess the potential cytotoxicity and carcinogenesis of the selected range of the medicinal biological products - 300 kDa cellular extracts “Mito Organelles” of the following types: heart, brain, kidney, cartilage, thymus, placenta, lungs, connective tissue, and a combo LPPSIMKE (liver, pancreas, placenta, kidney, intestines, retina); evaluate carcinogenic potential; compare toxicity; provide recommendations for biomedical research and application. Materials and methods: The MCF-7 human breast cancer cell line was used for the MTT assay. Cells were cultured in standard MCF-7 medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) under controlled conditions at 37°C and 5% CO₂ in a humidified incubator. Different protein solutions (designated as MOs) were tested for their effects on MCF-7 cells. The BALB-CTA offers essential information to assess cell viability and carcinogenic potential following treatment with MOs 300 kDA cellular peptide/protein extracts. Material processing was carried out using MS Excel and Statistica EZR version 1.62-2023 statistical programs. Dunn's test, Kruskal-Wallis rank univariate analysis, Scheffe, Student's parametric t-test were used to assess differences between groups. The difference was considered statistically significant at p <0.05. Results: According to MTT assays, the 300 kDA peptide/protein cellular extracts “MitoOrganelles” (MOs) tested did not affect the viability of MCF-7 cells. In addition, cells that were stressed with low doses of H2O2 were able to improve their vitality through the addition of MOs 300 kDa. The BALB/c-3T3 two-stage in vitro transformation assay (CTAs), a model for studying carcinogenesis, is another important tool in our research. It showed chemical transformation with morphologically aberrant foci after treatment with MCA and TPA. In contrast, various 300 kDA peptide/protein cellular extracts were tested, and no carcinogenic activity was observed, reinforcing the safety profile of these cellular extracts. The confidence in our research methods, particularly the MTT and BALB/c-3T3 assays, is crucial in understanding the safety profile of these cellular extracts. Conclusion: The comparative study conducted on the cytotoxicity and potential adverse effects of these extracts on cell viability and metabolic activity revealed that the selected range of medicinal biological products-specifically, the cellular extracts known as "Mito Organelles" from heart, brain, kidney, cartilage, thymus, placenta, lungs, connective tissue, and the combo LPPSIMKE (liver, pancreas, placenta, kidney, intestines, retina)-showed no cytotoxic effects on human cells. Additionally, no potential for malignant transformation or morphological changes were observed in the treated cell lines, and there were no negative impacts on cell viability or transformation rates. The results of these assays support recommendations for the safe use of Mito Organelles cellular extracts in biomedical research and therapeutic applications within regenerative medicine.