Tissue Culture Unit

Responsible person: Tomasz Witold Radaszkiewicz, Ph.D., M.Sc. 

Tissue cultures (TC) have become an essential tool in biomedical research in recent decades. Methods of in vitro cell cultivation are constantly evolving, and tissue culture methods and procedures across their entire spectrum, including ensuring the necessary standardization, will be the main focus of this PC unit. The TC unit will ensure the creation of new models such as genetically modified cell and organoid lines – either for use in the laboratories of external clients or in other PC units. It will also develop customized cell assays for both the local academic sector and the pharmaceutical industry (pharmacodynamic assays, cytotoxicity assays, etc.). The unit's operating mode will also enable the validation of the biocompatibility of new biomaterials, the validation of the origin and identity of cell lines, and the performance of functional whole-genome screens (e.g., Crispr/Cas9).

The TC and genetic modification unit will function as a place where live cell material of animal (from other PC units) or human (from cooperating clinical workplaces) origin will be processed for applications in other PC units. The structure of the unit will also allow the use of mammalian cells for the expression and preparation of proteins for biochemical applications. Last but not least, this unit will provide laboratory facilities for molecular biological analyses.

What services/products will the unit provide?

 Consultation

• one-on-one planning of experiments (overexpression, knock-in, knock-out)
• advice on genome editing (CRISPR/Cas9) and classical transfection/transduction
• recommendations for tags, reporters, and selection markers

Cloning & Plasmids

• subcloning into viral or standard mammalian expression plasmids
• preparation of inducible/conditional systems (Tet-On)
• RNA/DNA/pDNA preparations
• pilot tests to optimize DNA/RNA amounts, reagents, and cell density

Cellular Model Preparation

• antibiotic selection (G418, puromycin, hygromycin, blasticidin…)
• fluorescence-based selection (FACS of reporter-positive cells)
• generation of inducible or reporter cell lines (g., fluorescent, luciferase)
• western blot or other validation of derived cell lines
• antibody specificity testing
• tissue lysate prep and analysis

Genome Editing

• CRISPR/Cas9 knock-out
• endogenous tagging

Mechanism of Action & Pathway Assays (also with collaborators)

• reporter gene assays
• signal transduction analysis
• omics (with others)

Imaging & Functional Assays

• immunofluorescence and confocal microscopy
• migration assays (fixed or live-cell imaging)
• functional assays (drug-response curves etc.)

(please discuss specific details in advance with Dr. Radaszkiewicz; email: radaszkiewicztomasz@gmail.com)

 - Subunit name: Extracellular vesicles

Responsible person: RNDr. Vendula Hlaváčková Pospíchalová, Ph.D.

Isolation and characterization of extracellular vesicles (EVs)

 We offer expert isolation/enrichment and basic characterization of extracellular vesicles (EVs), including exosomes, from cell cultures (human cell lines) and biological fluids, particularly blood plasma, serum, ascites, cerebrospinal fluid, and other relevant samples.

 The service includes an initial expert consultation focused on designing the optimal isolation and analytical strategy regarding the type of input material, project objectives, and planned follow-up analyses.

We provide isolation and/or enrichment of:

• total populations (EVs)
• specific EV subpopulations, g., brain cells-derived EVs circulating in peripheral blood 

Basic characterization of EVs

performed as quality control and serves to verify:

• presence and integrity of EVs and potential contaminants (e.g., lipoproteins)
• basic physical and molecular parameters
• suitability of the isolate for further experimental use

All procedures are performed in accordance with current international recommendations in the field, in particular MISEV 2023 guidelines.

(please consult specific parameters relating to the isolation and characterization of EVs in advance with Dr. Hlaváčková Pospíchalová; email: pospich@sci.muni.cz)