possibilities for replacing living animals Eszter Tuboly Assistant Professor Institute of Surgical Research

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1 Animal experiments- In vitro possibilities for replacing living animals Eszter Tuboly Assistant Professor Institute of Surgical Research

2 Alternatives Replacement is needed Social assessment, growing demand Associations prohibition Legislation Advantages only focuse on the investigated mechanism Easier to manage, less risk and responsibility Requirement for publishing Disadvantages Not definitely more cost-effective Does not reflect real life enough Cell cultures: infections (mycoplasma)

3 Possibilities for replacing animals Only chemicals in the system Tissue homogenates, isolated organelles Ex-vivo experiments Cell-and tissue cultures Artificially growth organs Treatment instead of invasive intervention

4 Biochemical tests Immunochemical reactions (for the identification of bacterial toxins) Microorganisms Higher plants A few metazoa Computer-simulation modells Nanotechnology (cancer therapies)

5 Cell-Tissue culturing Used from 1907 Widespread from the 50 Breakthroughes: antibiotics, media, tripsin Societies, Cell-and Tissue Banks Isolated cell lines are maintained up to now (HeLa) Fast developement of the equipements Basis of the gene-and biotech industry (cloning) Stem-cell and gene therapy (ethic questions) Synthetic biology (artificial organs, programmed cells) Virology (vaccinating)

6 Appliances Description of physiological mechanisms within a cell of interest Cell-cell interactions, communication (neuronal wires) Cell responses for a treatment (drug developement) Protein products originated from a cell (biotech industry) tissue engineering Cells are from Tissue explants (explant culture) Cell suspensions (suspensional culture)

7 Cell culture types Primer cultures We have to create From embryotic or adult tissue Limitated sustaining ( mitosis) limitated lifespan (few weeks or months) Cell lines Often abnormal, transformed cells Homogenous cell population Immortal cells, unlimited proliferation For cancer research purpose Easier to maintain, a lot knowledge Pros: No other effects on the cells but dissociation methods (chemical or mechanical) No transformed or modified cells for sure Contras: Limitated lifespan and growth Each isolations are a little different from

8 What we definitely need Laminar box with HEPA filter for ensuring sterile air-flow Horizontal Air flows towards the researcher It is inappropriate for work with dangerous material Vertical Airflow from up to down Proper for work with dangerous material CO 2 Incubators (5-10 %, 100% humidity) Phase-contrast inverse microscope

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10 Sterilized room without windows Steril clothing UV-protection Special breeding vessels Own tools water-bath, refrigerator 70% alcohol Desinficient special medium Ionic homeostasis Vitamines, co-factors, metals Proteins, lipids Energy Serum Bakteriocid-fungicid cocktail

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13 For the treatement of the breeding vessel s surface (ECM) Collagen Fibronectines Laminin Poly L-lysine Poly-L-Ornithin Supplements for supporting the cells Foetal calf or bovine serum Growth factors Insulin

14 Proper cell types Usually any kind of cells, neurons and myocytes are harder to work with Blood cells: are uncapable of proliferating after going to the circulation Fibroblast: growing and developement are fast, short generation period Epithel: growing is fast, easy to deal with Embryotic cells: fast growing, proliferative, long generation period, delicate Cell lines: Easier to maintain, a lot knowledge, immortal cells, cancer research (HeLa, HEK, CHO)

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16 How to create a cell culture? Isolation: Chose the desired organ in virtue of cycle, organellum, connection or motion, perhaps financial or material limitation In vitro proofs added to in vivo results Neonatal or adult cells, embryotic cells, hybridomas, transformed cells Starter cell number, lifespan, growing are different Adult cells are capable of growing only in adherent way: laminin, or collagen plate, coated-plate (own recipe)

17 Gaining identical cells from tissues: chemically (enzymes, heat, time) or mechanically (shear force minimalization) Washing, screening Addiction into media, treatment Breeding in incubator Counting time after time (epetri) It is worth to titrate in case of any types (fibroblast) Passage Viability-assay Proteomics, freezing (DMSO!)

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19 Our nightmare Infection Chemically (out of date media components) Biologically: bacteria, funghi (mycoplasma tests, mask, alcohol) Indicator in the media: phenol red: ph change indicates metabolic activity increase In case of suspicious culture: throw away and microbiological investigation Freshly sterilization The role of autoclave, water-exchange in the incubator Special cleaning-up in every half-year

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21 Tissue-culturing Cells growing into tissues on a special scaffold Aim: to repair or supply damaged tissues, organs which are unable to functioning anymore Regenerative medicine-stem cell researchsynthetic biology Important to minimalise the response of the immune system (graft vs. host), the best is to apply autologous cells Sometimes allogenous (immunsupression, MHC compatibility) Xenogenous cells (pigs, anti-imflammatory genes-way of the futue?)

22 Scaffold Netty polymer, is made from different materials (protein, polysacharide, polypeptide) Let the cells grow on, permeabile for nutritions, appropriate for ECM Have to keep the original 3D structure Have to ensure the proper microenvironement Allowed the cells to migrate

23 An ideal scaffold 3D Contains cross-bindings Porous Biodegradible Appropriate chemical circumstances on the surface Endures mechanical charge Biocompatible Facilitates the natural healing processes Easy to available Large-scale producible

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26 Most used types Polymers Collagen Laminin Fibrin Decellularized matrix (heart) Ceramicals: Hydroxyapatite Calcium phosphate Bioglass

27 Cartillage replacement Cartillage cells Collagen scaffold Well-vascularisation is not a requirement

28 Artificial bones Stem cells differentiate into bone tissue Up to the added growth factor The scaffold should not be too large, otherwise oxygene supplement wont be enough 3D Calcium- scaffold

29 Artificial skin Collagene-chitozan, or hyaluronic acid instead One cell layer in one culture, 3 layer It has already been succesful among burned patients

30 ß-cells replacement for diabetic patient In vivo Islet of Langerhans in pancreas

31 Artificial vessels Often applied during by-pass operations h/sci/tech/9805tumdom.html

32 Regeneration of the injured heart Cardyomyocytes, vessels Special scaffold (decellularized matrix) Many different cell types

33 Bioprinting Inventor: Gábor Forgách (University of Missouri) Hydro-gele scaffold 3D inkjet printers 2 printer head, one is for cells, the other for gellike material is rich in nutrients Calibration with laser, software control Appliance: vessels for by-pass operations Further plans: printing whole organs, skin regeneration as a rutin, ambulant treatment

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35 Thank You so much!!!