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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
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