Industrial Cell Cultuer

8/2/2019 Industrial Cell Cultuer

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In the name ofGod


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Summer SchoolInfluenza Unit,

Pasteur Institute of Iran

summer 2011


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Industrial Cell Culture


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Introduction Cell culture is the process by which

prokaryotic, eukaryotic or plant cells are

grown under controlled conditions. But in

practice it refers to the culturing of cells

derived from animal cells.

Cell culture was first successfully undertaken

by Ross Harrison in 1907


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Historical events in the development of cell culture

1878: Claude Bernard proposed that physiological systems of anorganism can be maintained in a living system after the death .

1880: Roux maintained embryonic chick cells in a saline culture.

1911: Lewis made the first liquid media consisted of sea water,serum, embryo extract, salts and peptones. They observed limitedmonolayer growth.


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Contd..

1916: Rous and Jones introduced proteolytic enzyme trypsin forthe subculture of adherent cells.

1923: Carrel and Baker developed 'Carrel' or T-flask as the firstspecifically designed cell culture vessel.

1940s: The use of the antibiotics penicillin and streptomycin inculture medium decreased the problem of contamination in cellculture.

1952: Gey established a continuous cell line from a human cervicalcarcinoma known as HeLa (Helen Lane) cells. Dulbecco developedplaque assay for animal viruses using confluent monolayers ofcultured cells.


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History

Aseptic techniques

Carrel Flask


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Contd..

1955: Eagle studied the nutrient requirements of selected cells inculture and established the first widely used chemically definedmedium.

1965: Ham introduced the first serum-free medium which was ableto support the growth of some cells.

1978: Sato established the basis for the development of serum-free media from cocktails of hormones and growth factors.


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Major developments in cell culture technology

First development was the use of antibiotics which inhibits the

growth of contaminants.

Second was the use of trypsin to remove adherent cells tosubculture further from the culture vessel.

Third was the use of chemically defined culture medium.


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Why is cell culture used for?

Areas where cell culture technology is currently playing a majorrole.

Model systems for Studying basic cell biology, interactions between disease causingagents and cells, effects of drugs on cells, process and triggering of aging & nutritional

studies.

Toxicity testing , Study the effects of new drugs.

Cancer research, Study the function of various chemicals, virus & radiation to convertnormal cultured cells to cancerous cells.


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Contd.

VirologyCultivation of virus for vaccine production, also used to study there

infectious cycle.

Genetic Engineering

Production of commercial proteins, large scale production of viruses

for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B &

measles

Gene therapy

Cells having a functional gene can be replaced to cells which are

having non-functional gene


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Terminology

Primary Cell Culture Derived from an explant, directly from the animal Usually only survive for a finite period of time Involves enzymatic and/or mechanical disruption of the

tissue and some selection steps to isolate the cells of

interest from a heterogeneous populationClone A population derived from a single cellSub-culture Transplantation of cells from one vessel to anotherEstablished or Continuous Cell Lines A primary culture that has become immortal due to

some transformation Most commonly tumour derived, or transformed with a

virus such as Epstein-Barr


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Primary culture

Cells when surgically or enzymatically removed from an organism andplaced in suitable culture environment will attach and grow are calledas primary culture

Primary cells have a finite life span

Primary culture contains a very heterogeneous population of cells

Sub culturing of primary cells leads to the generation of cell lines Cell lines have limited life span, they passage several times before they

become senescent

Cells such as macrophages and neurons do not divide in vitro so can beused as primary cultures

Lineage of cells originating from the primary culture is called a cellstrain


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Continous cell lines

Most cell lines grow for a limited number of generations after which

they ceases

Cell lines which either occur spontaneously or induced virally or

chemically transformed into Continous cell lines

Characteristics of continous cell lines

-smaller, more rounded, less adherent with a higher nucleus/cytoplasm ratio

-Fast growth and have aneuploid chromosome number

-reduced serum and anchorage dependence and grow more in

suspension conditions

-ability to grow upto higher cell density-different in phenotypes from donar tissue


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Types of cells

On the basis of morphology (shape & appearance) or on

their functional characteristics. They are divided into

three.

Epithelial like-attached to a substrate and appears

flattened and polygonal in shape

Lymphoblast like- cells do not attach remain in

suspension with a spherical shape

Fibroblast like- cells attached to an substrate appears

elongated and bipolar


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Common cell lines Human cell lines

-MCF-7 breast cancer

HL 60 Leukemia

HEK-293 Human embryonic kidney

HeLa Henrietta lacks Primate cell lines

Vero African green monkey kidney epithelial cells

Cos-7 African green monkey kidney cells

And others such as CHO from hamster, sf9 & sf21 from insect

cells


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Monkey Kidney cells

Polio vaccine first product primary monkey kidneycells human diploid lung fibroblast


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Canine KidneyEpithelial

Common cell linesBHK

CHO

PER-C6MDCK

Vero

3T3

Baby hamster kidneyFibroblast

Chinese Hamster OvaryEpithelial

Mouse fibroblastFibroblast

Monkey KidneyFibroblast

Human KeratonocytEpithelial


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HeLa Cells

Classic example of an

immortalized cell line.

These are human epithelial cells from afatal cervical carcinoma transformed by human

papillomavirus 18 (HPV18).


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HeLa Cells

Adherent cells which maintain contact inhibition in vitro:

As they spread out across the culture flask, when two

adjacent cells touch, this signals them to stop growing.

Loss of contact inhibition is a classic sign of oncogenic cells:

Cells which form tumors in experimental animals.

Such cells not only form a monolayer in culture but also pile up on

top of one another.

HeLa cells are not oncogenic in animals, but they may become so if

further transformed by a virus oncogene.


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Number of Cell Divisions

Growing cells in culture. Place cells in a culture dish.

Give them nutrients, growth

factors, keep them free from

bacterial.

Cells will grow to cover the

surface of the dish.

Can take cells out of this culture

and start a new culture.

Splitting cells from one dish to

another is a passage.


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Number of Cell Divisions

This ability to split cells and

have them continue to divide is not

without limits however.

Normal cells have a limit to

the number of times which

they can be passed in culture.

This number does vary from cell

type to cell type, but commonly the

limit is between 50 and 100

passages.


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Contact Inhibition

The phenomenon observed in normal animal cells that causes themto stop dividing when they come into contact with one another.

Cells in a culture flask with the appropriate nutrients and the cellsgrow and divide.

Continues until the cells are covering the entire surface.

At that point they stop dividing.

These cells can be triggered to begin dividing again by givingthem more room.

The cells now being in an environment where they are not in

contact with one another begin to divide again.


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Contact Inhibition

Cancer cells do not display contact inhibition.

Put them in a culture dish, they will grow to create a

single layer of cells

Then they will continue to grow multiple layers and

create piles of cells.


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GROWTH CYCLE IN ATTACHEMENT

CULTURE

Eukaryotic cells in attachment culture have a characteristic growth cyclesimilar to bacteria.

The growth cycle is typically divided into three phases.

Lag Phase

Log Phase

Plateau Phase


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Lag Phase

This is the time following subculture and reseeding during which

there is little evidence of an increase in cell number.

It is a period of adaptation during which the cell replaceselements of the glycocalyx lost during trypsinization, attaches to

the substrate, and spreads out.

During spreading the cytoskeleton reappears and its reappearanceis probably an integral part of the spreading process.


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Log Phase

This is the period of exponential increase in cell number followingthe lag period and terminating one or two doublings afterconfluence is reached.

The length of the log phase depends on the seeding density, thegrowth rate of the cells, and the density at which cell proliferationis inhibited by density.

In the log phase the growth fraction is high (usually 90%-100%) andthe culture is in its most reproducible form.

It is the optimal time for sampling since the population is at its mostuniform and viability is high.

The cells are, however, randomly distributed in the cell cycle and, for

some purposes, may need to be synchronized.


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Plateau Phase

Toward the end of the log phase, the culture becomes confluent

All the available growth surface is occupied and all the cells are

in contact with surrounding cells.

Following confluence the growth rate of the culture is reduced,and in some cases, cell proliferation ceases almost completelyafter one or two further population

doublings.

At this stage, the culture enters the plateau (or stationary)

phase, and the growth fraction falls to between 0 and10%.

There may be a relative increase in the synthesis of

specialized versus structural proteins and the constitution

and charge of the cell surface may be changed.


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Culture media

Choice of media depends on the type of cell being cultured

Commonly used Medium are GMEM, EMEM,DMEM etc.

Media is supplemented with antibiotics viz. penicillin, streptomycin

etc.

Prepared media is filtered and incubated at 4 C


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Culture Media Ions

Na, K, Ca, Mg, Cl, P, Bicarbonate

Trace elements

iron, zinc, selenium

Sugars

glucose is the most common

Amino acids

13 essential Vitamins

Serum

Contains a large number of growth promoting activities such as

buffering toxic nutrients by binding them, neutralizes trypsin and other

proteases Contains peptide hormones or hormone-like growth factors that

promote healthy growth.

Antibiotics - although not required for cell growth, antibiotics are

often used to control the growth of bacterial and fungal

contaminants.


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Serum

Serum/protein free media

Growth factors

Lipid concentrate Extracts Yeast extract

Insulin

Bovine Serum Albumin ( transport/detoxification)


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Basic equipments used in cell culture

Laminar cabinet-Vertical are preferable

Incubation facilities- Temperature of 25-30 C for insect & 37 C formammalian cells, co2 2-5% & 95% air at 99% relative humidity. Toprevent cell death incubators set to cut out at approx. 38.5 C

Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) &media components (e.g. glutamine & serum) at -20 C

Microscope- An inverted microscope with 10x to 100x magnification

Tissue culture ware- Culture plastic ware treated by polystyrene


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Equipment

Class II CabinetsThese cabinets are designed togive operator protection as wellas a sterile environmentThe air is directed downwards

from the top of the cabinet to thebase, when working in thesecabinets it is important not topas non-sterile objects over

sterile ones


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Equipment

Centrifuges

There are centrifuges in each cell culture area which arerefrigerated

100 x g is hard enough to sediment cells, higher g forcesmay damage cells

Incubators The incubators run at 37C and 5% Carbon Dioxide to

keep the medium at the correct pH

They all have meters on them to register temperature and

gas level There are alarms to indicate when these deviate from set

parameters

Keep the door open for as short a time as possible


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Why sub culturing? Once the available substrate surface is

covered by cells (a confluent culture) growthslows & ceases.

Cells to be kept in healthy & in growing state

have to be sub-cultured or passaged Its the passage of cells when they reach to

80-90% confluency in flask/dishes/plates

Enzyme such as trypsin, dipase, collagenase incombination with EDTA breaks the cellularglue that attached the cells to the surface


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Culturing of cells Cells are cultured as anchorage dependent or

independent

Cell lines derived from normal tissues are considered

as anchorage-dependent grows only on a suitable

substrate e.g. tissue cells Suspension cells are anchorage-independent e.g. blood

cells

Transformed cell lines either grows as monolayer or as

suspension


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Adherent cells

Cells which are anchorage dependent

Cells are washed with PBS (free of ca & mg ) solution.

Add enough trypsin /EDTA to cover the monolayer

Incubate the plate at 37 C for 1-2 min.

Tap the vessel from the sides to dislodge the cells

Add complete medium to dissociate and dislodge the

cells

with the help of pipette which are remained to be

adherent

Add complete medium depends on the subculture

requirement either to 75 cm or 175 cm flask


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Suspension cells Easier to passage as no need to detach them

As the suspension cells reach to confluency

Asceptically remove 1/3rd of medium

Replaced with the same amount of pre-warmed

medium


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Culturing Animal Tissue- the Steps

Animal tissue is obtainedeither from a particularspecimen, or from a tissue

bank ofcryo-preserved (cryo= frozen at very low

temperatures in a specialmedium)

Establishment of the tissue isaccomplished in the requiredmedium under aseptic

conditions

Culture vessels and medium

for animal cell culture


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Working with cryopreserved cells

Vial from liquid nitrogen is placed into 37 C water bath, agitate vial

continuously until medium is thawed

Centrifuge the vial for 10 mts at 1000 rpm at RT, wipe top of vial with

70% ethanol and discard the supernatant

Resuspend the cell pellet in 1 ml of complete medium with 20% FBS

and transfer to properly labeled culture plate containing the

appropriate amount of medium

Check the cultures after 24 hrs to ensure that they are attached to the

plate

Change medium as the colour changes, use 20% FBS until the cellsare established


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Freezing cells for storage Remove the growth medium, wash the cells by PBS and remove the

PBS by aspiration

Dislodge the cells by trypsin-versene

Dilute the cells with growth medium

Transfer the cell suspension to a 15 ml conical tube, centrifuge at

200g for 5 min at RT and remove the growth medium by aspiration

Resuspend the cells in 1-2ml of freezing medium

Transfer the cells to cryovials, incubate the cryovials at -80 C overnight

Next day transfer the cryovials to Liquid nitrogen


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Cell viability Cell viability is determined by staining the cells with trypan

blue

As trypan blue dye is permeable to non-viable cells or

death cells whereas it is impermeable to this dye

Stain the cells with trypan dye and load tohaemocytometer and calculate % of viable cells

- % of viable cells= Nu. of unstained cells x 100

total nu. of cells


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Cell toxicity

Cytotoxicity causes inhibition of cell growth

Observed effect on the morphological alteration in the cell layer or

cell shape

Characteristics of abnormal morphology is the giant cells,

multinucleated cells, a granular bumpy appearance, vacuoles in the

cytoplasm or nucleus

Cytotoxicity is determined by substituting materials such as medium,

serum, supplements flasks etc.


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Contaminants of cell culture

Cell culture contaminants of two types

Chemical-difficult to detect caused by endotoxins,

plasticizers, metal ions or traces of disinfectants that

are invisible

Biological-cause visible effects on the culture they aremycoplasma, yeast, bacteria or fungus or also from

cross-contamination of cells from other cell lines


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Cell Culture Enemies

Micro-organisms grow ~10-50 times faster thanmammalian cells, which take ~8-16 hours todivide. They are more tolerant to variations intemperature, pH and nutrient supply than cells.

Cells are most vulnerable to contamination whenour aseptic technique is bad and the culturebecomes infected with bugs.

This can lead to the development of antibiotic

resistant micro-organisms.


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Cells are more susceptible to infection at certain times When they have been stressed after recovery from

liquid nitrogen

Primary cells are often generated by enzymaticdisruption and selection procedures

Cultures prepared from live animals will often beaccompanied by micro-organisms

Splitting cells at too high a dilution can allow micro-organisms to dominate the culture

Cells release Autocrine growth factors whichcondition the medium and favour cell growth


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IF YOU ARE IN DOUBT ABOUT THE CONDITION OF YOUR CELLS,ASK FOR ADVICE

NEVER USE CONTAMINATED CELLS. THEY MAY NOT REACT IN

THE SAME WAY AS UNCONTAMINATED CELLS

POOR ASEPTIC TECHNIQUE IS THE MAJOR CAUSE OFINFECTIONS


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Effects of Biological Contaminations

They competes for nutrients with host cells

Secreted acidic or alkaline by-products ceses the growth

of the host cells

Degraded arginine & purine inhibits the synthesis ofhistone and nucleic acid

They also produces H2O2 which is directly toxic to cells


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Detection of contaminants

In general indicators of contamination are turbid culture

media, change in growth rates, abnormally high pH, poor

attachment, multi-nucleated cells, graining cellular

appearance, vacuolization, inclusion bodies and cell lysis

Yeast, bacteria & fungi usually shows visible effect on the

culture (changes in medium turbidity or pH)

Mycoplasma detected by direct DNA staining with

intercalating fluorescent substances e.g. Mycoplasma also

detected by enzyme immunoassay by specific antisera or

monoclonal abs or by PCR amplification of mycoplasmalRNA


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Basic aseptic conditions If working on the bench use a Bunsen flame to heat the

air surrounding the Bunsen

Swab all bottle tops & necks with 70% ethanol

Flame all bottle necks & pipette by passing very quicklythrough the hottest part of the flame

Avoiding placing caps & pipettes down on the bench;practice holding bottle tops with the little finger

Work either left to right or vice versa, so that allmaterial goes to one side, once finished

Clean up spills immediately & always leave the work

place neat & tidy


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Safety aspect in cell culture Possibly keep cultures free of antibiotics in order to be able to recognize the

contamination

Never use the same media bottle for different cell lines. If caps are dropped or

bottles touched unconditionally touched, replace them with new ones

Necks of glass bottles prefer heat at least for 60 secs at a temperature of 200 C Switch on the laminar flow cabinet 20 min prior to start working

Cell cultures which are frequently used should be subcultered & stored as

duplicate strains

Safety


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Safety

Use of Cell Culture areas

The cell culture area, as any otherlaboratory is a working area

Do not bring your friends in withyou

Do not eat, drink or smoke in theseareas

Do wear a lab coat at all timeswhether in a cell culture area or a

laboratory Do wear disposable gloves, but

make sure that you dispose ofthem in the correct way before you

leave the area


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Other key facts.? Use actively growing cells that are in their log phase of

growth, which are 80-90% viable

Keep exposure to trypsin at a minimum

Handle the cells gently. Do not centrifuge cells at high

speed or roughly re-suspend the cells Feeding & sub culturing the cells at more frequent

intervals then used with serum containing conditions

may be necessary

A lower concentration of 10 4cells/ml to initiate

subculture of rapidly growing cells & a higherconcentration of 105cells/mlfor slowing growing cells


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Industrial cell culture Aims

1) Vaccine Production

2) Production of commercial proteins


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Industrial cell culture cell production in large scale

1) Suspension cell line

2) Hyper Flask

3) Rolling Bottle

4) Microcarrier


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Industrial cell culture Suspension cell lines

BHK-21


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Industrial cell cultureSuspension cell lines:

Adaptation of cells to serum free medium

Culture in suspension condition


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Industrial cell culture Hyper Flask: Each of the 10 layers of all HYPERFlask cell culture vessels

contain a gas permeable surface, and an air gap exists between each

layer.


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Industrial cell culture Rolling Bottle


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Industrial cell culture Rolling Bottle incubator


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Industrial cell culture Attachment of cells


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Industrial cell culture Microcarrier

In microcarrier cell culture technology, anchorage-dependent animal

cells are grown on the surface of small (~150 diameter) spheres that

are maintained in stirred suspension cultures.


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Industrial cell culture Microcarrier


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Industrial cell culture Microcarrier or suspension cell scale up


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Industrial Cell Cultuer

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