DMYTRO VAVRYSH

• Project title: Yeast extracellular vesicles and their application as a new-generation drug delivery platform
• The main project parts: 1) Isolation of extracellular vesicles (EVs) from cultures of two probiotic yeasts species Saccharomyces boulardii and Kluyveromyces marxianus 2) Development of a method of introducing selected biologically active substance (Doxorubicin hydrochloride) inside isolated yeast extracellular vesicles 3) Contemplation of releasement kinetics of doxorubicin substance out of extracellular vesicles under application of different physicochemical conditions (various periods of incubation, pH, temperature) 4) Study of substance transfer property by extracellular vesicles, prefilled with doxorubicin, to human colorectal adenocarcinoma cell lines (HT-29 and HCT 116).
• Acquired skills: gained an extensive experience in both upstream and downstream processing of biotechnological products. Particularly, I got experience in working with yeast culture - cultivation (in small-scale bioreactors), primary separation of biomass; working in aseptic conditions (including Flow Biological Safety Cabinet BSL-2), preparation of media (exp. Sabouraud Dextrose Agar, and Yeast Extract Peptone Dextrose), DLS analysis; ultracentrifugation, centrifugation with cut-off filters; measurements of optical density; fluorescence spectroscopy analysis;electroporation-for induction cellular uptake of macromolecules, investigation using fluorescent and inverted microscopy, SDS-PAGE, working with human cell culture (cancer cell line), and staining nuclei with fluorescent dyes.

• I have trained and acquired a solid knowledge background and laboratory experience in the following courses: protein engineering, proteomics, structural biochemistry, molecular biology, drug delivery and formulation, experimental pharmacology, molecular immunology and diagnostic microbiology, bioinformatics, and computational biology laboratories
• Acquired technical skills and strategies by courses:

1) Protein engineering training course: primers design, site-directed mutagenesis - PCR cloning: the transformation of competent cells, DNA purification, analysis of purified DNA: sequencing, agarose gel electrophoresis, analysis of protein expression by SDS - PAGE, analysis of protein content by BCA enzymatic assay, etc.;
2) Advanced molecular biology training course: design the strategy for cloning α-SG fused to the tag protein GFP into the expression vector pCDNA3; transfection of the plasmid into the HEK293 cell line; analysis of different cellular compartments and the cyto-localization of α-SG by an immunofluorescence assay;
3) Structural biochemistry training course: Protein fluorescent spectroscopy, Circular dichroism (CD) spectroscopy;
4) Experimental pharmacology training course: cell cycle analysis, cell seeding, cell treatment, cytotoxicity assay (MTT assay);
5) Diagnostic microbiology training course: evaluation of dividing cells and generation of colonies on agar medium following the Miles-Misra Method and Surface-Spread Method, antimicrobial susceptibility test: broth dilution method, microscopic observation, and staining, analysis of cell morphology;

Examples of laboratory techniques: Gel - filtration chromatography; UV-spectrophotometry; Mass spectrometry; Preparation of liposome and hydrogel formulations; Mutagenic overlapping PCR cloning, Agarose gel electrophoresis; trained in use various diagnostic microbiology tests: Spread Plate method, Pour-Plate Method, Miles-Misra Method, Immunofluorescence (IF) assay, MTT assay, Protein fluorescent spectroscopy, Circular dichroism (CD) spectroscopy, SDS - PAGE.

Research Area:
- Research of the effectiveness of dermal equivalents intended for the treatment of serious burn wounds (3rd and 4th degree stage) – in vivo, used mouse models
Research Activities:
The aim of our project was to estimate the efficiency of a new dermal equivalent in vivo. In our institute, we have obtained the new human cell line 4BL from lymphocytic fraction of peripheral blood of adult healthy donors (cells line 4BL is stable immortalized line).

We have tried to use formulations containing defrosted live cell cultures and cell-free preparations based on a complex of active compounds secreted by growing cell culture. Gels containing 4BL and MSU-1.1 cell suspensions were shown to accelerate the epithelization process beginning.

Acquired knowledge and skills: Learned the crucial principles of regulatory agency review processes learned how to manage projects and prioritize tasks got broad experience across the field of RegulatoryAffairs got business skills and understanding of the pharmaceutical industry have built a knowledge of the drug development process, as well as regulatory agency guidelines and expectations