AnimalAnimal experimentsexperiments-- InIn vitrovitro

possibilitiespossibilities forfor replacingreplacing

livingliving animalsanimals

Eszter Tuboly

Assistant Professor

Institute of Surgical Research

AlternativesAlternatives

• 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)

PossibilitiesPossibilities forfor replacingreplacing animalsanimals

• Only chemicals in thesystem

• Tissue homogenates, isolated organelles

• Ex-vivo experiments

• Cell-and tissue cultures

• Artificially growth organs

• Treatment instead of invasive intervention

• Biochemical tests

• Immunochemical reactions (for theidentification of bacterial toxins)

• Microorganisms

• Higher plants

• A few metazoa

• Computer-simulation modells

• Nanotechnology (cancer therapies)

CellCell--TissueTissue culturingculturing

• Used from 1907• Widespread from the 50’

– Breakthroughes: antibiotics, media, tripsin

• Societies, Cell-and Tissue Banks

• Isolated cell lines are maintained upto now (HeLa)

• Fast developement of theequipements

• Basis of the gene-and biotechindustry (cloning)

• Stem-cell and gene therapy (ethicquestions)

• Synthetic biology (artificial organs, programmed cells)

• Virology (vaccinating)

AppliancesAppliances

• Description of physiological mechanisms withina cell of interest

• Cell-cell interactions, communication (neuronalwires)

• Cell responses for a treatment (drugdevelopement)

• Protein products originated from a cell (biotechindustry)

• tissue engineering

• Cells are from…

• Tissue explants (explant culture)

• Cell suspensions (suspensional culture)

Cell Cell cultureculture typestypesPrimer cultures

• We have to create• From embryotic or adult tissue• Limitated sustaining (50-100

mitosis)• limitated lifespan (few weeks or

months)

• Pros:

– No other effects on the cells butdissociation methods (chemicalor mechanical)

– No transformed or modifiedcells for sure

• Contras:

– Limitated lifespan and growth– Each isolations are a little

different from

Cell lines

• Often abnormal, transformed cells

• Homogenous cell population• Immortal cells, unlimited

proliferation

• For cancer research purpose• Easier to maintain, a lot knowledge

WhatWhat wewe definitelydefinitely needneed……

• 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

• CO2 Incubators (5-10 %, 100% humidity)

• Phase-contrast inverse microscope

• special medium

– Ionic homeostasis

– Vitamines, co-factors,

metals

– Proteins, lipids

– Energy

– Serum

– Bakteriocid-fungicidcocktail

• Sterilized room

without windows

• Steril clothing

• UV-protection

• Special breeding

vessels

• Own tools

• water-bath,

refrigerator

• 70% alcohol

• Desinficient

• 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

ProperProper cellcell typestypes

• 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)

HowHow toto createcreate a a cellcell cultureculture??

• Isolation: Chose the desired organ in virtue of cycle, organellum, connection or motion, perhaps financial ormaterial 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)

• Gaining identical cells from tissues: chemically(enzymes, heat, time) or mechanically (shearforce 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!)

OurOur nightmarenightmare……

Infection

• Chemically (out of date media components)

• Biologically: bacteria, funghi (mycoplasma tests, mask, alcohol)

• Indicator in the media: phenol red: pH change indicatesmetabolic 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

TissueTissue--culturingculturing

• 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 research-

synthetic 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?)

ScaffoldScaffold

• Netty polymer, is made from differentmaterials (protein, polysacharide, polypeptide)

• Let the cells grow on, permeabile fornutritions, appropriate for ECM

• Have to keep the original 3D structure

• Have to ensure the proper micro-environement

• Allowed the cells to migrate

An An idealideal scaffoldscaffold……

• 3D

• Contains cross-bindings

• Porous

• Biodegradible

• Appropriate chemical circumstances on thesurface

• Endures mechanical charge

• Biocompatible

• Facilitates the natural healing processes

• Easy to available

• Large-scale producible

Most Most usedused typestypes

• Polymers

– Collagen

– Laminin

– Fibrin

– Decellularized matrix (heart)

• Ceramicals:

– Hydroxyapatite

– Calcium phosphate

– Bioglass

CartillageCartillage replacementreplacement

– Cartillage cells

– Collagen scaffold

– Well-vascularisation is not a requirement

ArtificialArtificial bonesbones

• 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

ArtificialArtificial skinskin

• Collagene-chitozan,

or hyaluronic acid

instead

• One cell layer in one

culture, 3 layer

• It has already been

succesful among

burned patients

ßß--cellscells replacementreplacement forfor diabeticdiabetic

patientpatientIn vivo Islet of Langerhans in pancreas

ArtificialArtificial vesselsvessels

• Often applied during

by-pass operations http://popularmechanics.com/popmec

h/sci/tech/9805TUMDOM.html

RegenerationRegeneration of of thethe injuredinjured heartheart

• Cardyomyocytes,

vessels

• Special scaffold

(decellularized matrix)

• Many different cell

types

BioprintingBioprinting

• Inventor: Gábor Forgách (University of Missouri)

• Hydro-gele scaffold

• 3D inkjet printers

• 2 printer head, one is for cells, the other for gel-like material is rich in nutrients

• Calibration with laser, software control

• Appliance: vessels for by-pass operations

• Further plans: printing whole organs, skinregeneration as a rutin, ambulant treatment

ThankThank YouYou soso muchmuch!!!!!!