341ZOO Tissue Culture Presentation

7/31/2019 341ZOO Tissue Culture Presentation

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Cell culture technique and

its implication

341 Zoo

Dr. Gamal Badr

Associate professor


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History of tissue culture

1885: Roux maintained embryonic chick cells in saline

1965:Ham introduced serum free culture medium that supportliving cells

1965: Harris & Watkins successfully fused human and mousecells by virus

1975: Kohler & Milstein produced the first Hybridomas capable

of secreting monoclonal antibodies. 1978: Sato developed a serum free medium with a cocktail of

hormone and growth factors

!982: human insulin is produced

1985: human growth factors were produced

1986: lymphoblastoid gamaIFNlicences

1987:tissue type plasminogen activator(tPA) becamecommercially available

1989:Recombinant erythropoitin in trial

1990:Recombinent products in trial (factorVI.HBsAg, Il2, EGF,mAbs)


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What is cell culture?

Cells, previously growing in a human or animalmodified to grow in plastic or glass In the body = in vivo

On plastic or glass = in vitro

Kept in an incubator to stay at body temperature

We use special media with nutrients so the cellscan grow and divide


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What can we do with cells?

Test pharmaceutical drugs

Watch disease mechanisms

Design potential treatments

Observe the regenerative process

How do cells and tissues repair themselves afterdamage from illness or injury?

Observe the developmental process


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The Dos and Donts of cell culture

The Dos

Use personal protective equipment (PPE)

Wear dedicated PPE for tissue culture facility and keep separate from PPE in the

general lab environment.

Keep all work surfaces free of clutter.

Correctly label reagents including flasks, medium.

Only handle one cell line at a time.

Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol).

Wherever possible maintain separate bottles of media for each cell line. Examine cultures and media daily for evidence of gross bacterial or fungal

contamination.

Quality control all media and reagents prior to use.

Ensure that incubators, cabinet, centrifuges and microsocpes are cleaned.

Test cells for mycoplasma on a regular basis.

Cell Culture Protocols


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The Donts

Do not continuously use antibiotics in culture medium.

Dont allow waste to accumulate particularly within the incubators

and culture area.

Dont have too many ppl in the lab at any one time.

Dont handle cells from unauthenticated sources in the main cellculture suite.

Avoid keeping cell line continually in culture without returning to

frozen stock.

Avoid cell culture becoming fully confluent. Always sub-culture 70-80% confluency or as advised on ECACCs cell culture data sheet.

Dont allow media to go out of date.

Avoid water baths dirty.

Dont allow essential equipment to become out of ccalibration.


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Technique and instrument

Laminar flow


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Carbon dioxide incubator


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Microscope


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Tissue culture Ware


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Culture Media Sterilization


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Cell counting


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Changing Medium


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Passaging cells (subculturing cells)

Process of diluting cell number in

order to keep cells actively growing

For adherent cells, when they cover the

tissue culture dish, they need to be

passaged

Otherwise, the cells will become

unhealthy and stop growing


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Other miscellaneous Equipment

Fridge Freezer for storing medium

Liquid nitrogen Container for cryopreservation

of cells

Incubator for warming up of the medium

Bench centrifuges to separate out cell pellet.


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Sterile technique

Procedures by which cultures are manipulated

without infecting the worker or contaminatingthe cultures or the laboratory environment

Important for the cell culture

You want to be sure you are growing only the cellsyou want to grow a single unwanted cell can ruin an

experiment or a multimillion $ production run

Important for the lab worker Some cultured cells can pose health threats to

workers if they are inhaled, ingested, or absorbed--

sterile technique prevents exposure of the worker to

cultured cells


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Sterile technique: Bacteria

Media is sterilized in an autoclave

Very high pressure and temperature kills

Minimizing contamination through awareness

Inoculation loop, pipets, pipet tips, etc. should never touch

contaminating surfaces

Containers holding media and other cell additives should be kept

closed until needed, then opened briefly

Tubes and flasks are flamed whenever they are opened

The purpose of flaming is not to sterilize, but to warm the tubeand create warm air convection currents up and away from the

opening. This "umbrella" of warm, rising air will help to prevent

the entrance of dust particles carrying contaminating microbes


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Sterile technique: Tissue culture Media is usually sterilized by filtration

Standard biological filters are 0.22 mm - 0.45 mm; these remove

most microbes by trapping them in the filter This does not remove all microbes (such as mycoplasm), and will not

remove viruses

Unlike bacterial media, animal cell media cannot be autoclaved,because this would destroy many of the growth factors and other

molecules needed for cell growth Working with cells in a laminar flow hood

HEPA filter

Disinfect

70% Ethanol is sprayed in hood, onto bottles entering hood Minimizing contamination through awareness

Inoculation loop, pipets, pipet tips, etc. should never touchcontaminating surfaces

Containers holding media and other cell additives should be keptclosed until needed, then opened briefly


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Sterilization methods Autoclave

Applies heat under high pressure; this increases the boiling point

of water to 121C (normal boiling point of water is 100C) 15-20 min. is sufficient to kill most microbes

Filtration

Large volumes: suction filter

Small volumes: syringe filter

UV radiation

Causes mutations to form in the DNA of microbes, causing

genetic damage and eventual death

Used to sterilize surfaces (such as the surface of laminar flow

hoods)


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What is in the media?

Dulbecco Modified Eagles Media (DMEM) Contains glucose, some proteins, and essential salts

Contains a pH indicator (phenol red) Media looks

pink/red at pH 7.2

Acidic -yellow or orange (cell growth,

bacterial growth)

Basic -purple (no cell growth, not enough

CO2)


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Media preparation

Process of combining and sterilizing ingredients of a

particular medium Proper media prep is essential for cell culture systems!

If the media is lacking any components, the cells would

either die or be unhealthy

If the media is not properly sterilized, cells will be

contaminated with microorganisms

If glassware, pipettes, etc. used to prepare media are not

properly cleaned, cell cultures can be contaminated with

chemical residues

As a result, the cells would not produce the desired product

effectively


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More media components

Antibiotics (penicillin and streptomycin) Prevent bacterial contamination

Salts and buffers

To simulate in vivo environment Serum

Portion of blood after the cells and fibers have clotted

From cow, horse, sheep

added to media as a nutrient source for growing cells Lipids, proteins


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Phosphate Buffered Saline

Used to wash/remove excess serum thatinhibits the function of TRED.

Must be warmed in the water bath before

use so cells are not shocked by cold liquid.

Trypsin EDTA An enzyme used to detach the cells from a culture

dish.

EDTA binds calcium ions in the media that would

normally inhibit trypsin.


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Bleach

Used to destroy any remaining cells in

dishes and tubes before they are tossed in

the trash can.

Add enough to change media to clear,

wait 5 minutes,

rinse solution down sink

throw away the dish/flask/plate in the trash can.


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Potential sources of contamination in

cell culture

Equipment

Glassware, instruments, incubators.

Solutions

Media or reagents added into media

Room air

Work surfaces Operators

Hands, hair, clothing, breath, etc.

Incoming cells


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Types of contamination in animal cell

culture systems

Biological

Bacteria

Fungi

Cross-contamination by other cell cultures Chemical

Residues left from detergents or disinfectants on

glassware, pipettes, instruments, etc.

Metal ions, other impurities in water

Endotoxin: highly bioreactive part of the cell wall of

some types of bacteria (endotoxin molecules are shed

from bacteria and are left behind even after bacteria die)


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Characteristics of microbial

contamination in cell cultures

pH Sudden change in pH is often a strong

indicator of contamination

Turbidity

Media looks cloudy

Microscopic evaluation Can see individual microorganisms, often

because their motion can be seen easily under

the microscope

F th d t ti f t i ti i ll


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Further detection of contamination in cell

cultures Mycoplasma

Smallest free-living prokaryotes Not killed easily by many antibiotics

Contamination cant be seen by microscopicevaluation

Mycoplasma testing should be doneroutinely (several tests are available)

Long-term effects of mycoplasma contamination includereduced growth rate, changes in cell shape and

metabolism, and chromosome abnormalities Endotoxin

LAL test: an extract from the blood ofhorseshoe crabs is used to test for endotoxin (horseshoecrab blood contains a protein that binds endotoxin & can be

detected)


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Minimizing contamination

Contamination is a fact of life when dealing

with cell cultures

Very difficult to prevent entirely, but good lab

practices can keep contamination incidents to a

minimum

Proper cleaning and sterilization of glassware,

pipettes, and other lab instruments. Practicing sterile technique when working with

cell cultures


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Types of cell culture

Primary Cell culture

Continuous Cell lines

Adherent s S spension cells for tiss e


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Adherent vs Suspension cells for tissue

culture

Adherent cells: cells grow in asingle layer (called a monolayer)attached to the tissue culture dish Cell growth is limited by available

surface area on which cells can grow

To passage adherent cells, the cellsmust be released from the dish (doneeither enzymatically, chemically, ormechanically)

Suspension cells : cells aresuspended in liquid as single cells oras free-floating clumps of a few cells To passage suspension cell cultures, a

proportion of the cells in culture are

diluted into a larger volume of medium


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Secondary or continuous

Cell LinesAttached Cell Lines

Name Species and tissue of origin Morphology

MRC-5 (Prod. No. 84101801) Human lung Fibroblast

HELA (Prod. No. 93021013) Human cervix EpithelialVERO (Prod. No. 84113001) African Green Monkey Kidney Epithelial

NIH 3T3 (Prod. No. 93061524) Mouse embryo Fibroblast

L929 (Prod. No. 85011425) Mouse connective tissue Fibroblast

CHO (Prod. No. 85050302) Chinese Hamster Ovary Fibroblast

BHK-21 (Prod. No. 85011433) Syrian Hamster Kidney Fibroblast

HEK 293 (Prod. No. 85120602) Human Kidney Epithelial

HEPG2 (Prod. No. 85011430) Human Liver Epithelial

BAE-1 (Prod. No. 88031149) Bovine aorta Endothelial


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SUSPENSION Cell lines

Suspension Cell Lines

Name Species and tissue of origin Morphology

NSO (Prod. No. 85110503) Mouse myeloma Lymphoblastoid-like

U937 (Prod. No. 85011440)Human Hystiocytic

Lymphoma Lymphoblastoid

Namalwa (Prod. No. 87060801) Human Lymphoma Lymphoblastoid

HL60 (Prod. No. 98070106) Human Leukaemia Lymphoblastoid-like

WEHI 231 (Prod. No. 85022107) Mouse B-cell Lymphoma Lymphoblastoid

YAC 1 (Prod. No. 86022801) Mouse Lymphoma Lymphoblastoid

U 266B1 (Prod. No. 85051003) Human Myeloma Lymphoblastoid

SH-SY5Y (Prod. No. 94030304) Human neuroblastoma Neuroblast


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Storing and maintaining Continuous

Cell Lines


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Advantages of Cell Culture

Cell types kept Constant and homogenous.

There is direct access to the cells so effect oftoxicity of drug and chemicals studied without

being lost to other tissues and excreted. Replace/ reduce the number of animals. Legal,

moral, ethical issues?(animal rights Group).

Control of the environment (pH, osmoticpressure, temperature, oxygen and Carbondioxide tension.)


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Disadvantages of Cell Culture

Contamination can be Chemical (culturemedium) Or Biological (adding antibiotics).

Finding a Happy Environment.

De-differentiation

Origin of Cells

Major differences from in-vivo


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Implication for Cell Culture

Model system (many specialized functions are restored) Toxicity Test (viability of cells)

Cancer research

Virology

Cell Based manufacturing

Genetic councelling

Genetic engineering

Drug screening & development Gene Therapy


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Production of monoclonal antibodies


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Tissue Engineering

Carbon nanotube

scaffolding

Name of

scaffoldig

Made up of

Nanofiber Like carbon

Textile Polyglycolide

Gas Foam Foam like

structure due

to CO2 gas
http://en.wikipedia.org/wiki/Image:Kohlenstoffnanoroehre_Animation.gif


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Growing cells in 3D Forming

Tissue in Bioreactor
http://science.nasa.gov/NEWHOME/headlines/images/heart/construct.jpeghttp://en.wikipedia.org/wiki/Image:Pg166_bioreactor.jpg


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Application of Monoclonnal

Antibodies

IMMUNOLOCALIZATION

IMMUNOBlotting

Cancer Treatment


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Immunolocalization


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Immunoblotting


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Cancer Treatment
http://en.wikipedia.org/wiki/Image:MonoclonalAb.jpg


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Stem cell research

Embryonic Cell Lines

Adult Cell Line

Homeiopoitic Stem Cell


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Different types of stem cells

Early embryonic stem cells

Totipotent: can become any kind of cell in the body

Blastocyst embryonic stem cells

Pluripotent: can become virtually any kind of cell inthe body

Umbilical cord stem cells:

Multipotent: can differentiate into only a limited

number of cell types

Adult stem cells:

Multipotent: can differentiate into only a limited

number of cell types

F t f t ll di t d th


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Future of stem cell-mediated therapy{stem cells to replace damaged organs}

Stem cells are induced todifferentiate into a cell type that is

damaged or missing in the patient

Cells are grown on tissue-promoting matrix or scaffolding

Healthy tissue is transplanted into

patient--unlike other organ

transplants, tissue is an immune

match to patient, so no

immunosuppression drugs would

be necessary

Medical applications of embryonic stem


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Medical applications of embryonic stem

cell research

Diabetes Caused by bodys

destruction of insulin-producing cells in thepancreas.

Researchers in Spain(2001) used mouseembryonic stem cells thatthey differentiated intoglucose-responsive,insulin-producing cells; thecells reversed diabetessymptoms when injectedinto the spleens of mice.

A i l l i


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Animal cloning

Remove nucleusfrom egg cell Add somatic cellfrom adult donor Grow in culture to produce anearly embryo (blastocyst)

Implant blastocyst insurrogate mother

Remove embryonic stemcells from blastocyst andgrow in culture

Induce stem cells toform specialized cells(therapeutic cloning)

Clone of donor is born(reproductive cloning)

Donorcell

Nucleus fromdonor cell

Reproductive cloning

The successful "cloning" of mammals has resulted in a flurry ofresearch, Dolly July 96

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341ZOO Tissue Culture Presentation