Cell Culture and handling of the cell lines

By Shakira Ghazanfar

PhD ScholarPIASA

20-04-2011

Contents

• Cell Culture• Cell Lines• Continues Cell Lines• Subculture• Handling of cell lines• Cryopreservation of cell lines

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 R. Harrison in 1907

Cell Culture

Definition:

In vitro cultivation of cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is known as Cell Culture.

Cell Culture

• Tissues Individual cells trypsin & mechanical shaking

• Cells are washed, counted & suspended in a growth medium.

• Growth medium – Minimum Essential Medium (MEM): essential aminoacids, vitamins, salts, glucose & bicarbonate in 5% CO2 with 5% fetal calf or calf serum, antibiotics & phenol red indicator

MAIN TYPES OF CELL CULTURE according to the cultivation period (and survival in culture)

• Routinely used for growing viruses• Classified into 3 types:

– Primary cell culture – normal cells taken from body & cultured, limited growth1. Chick embryo fibroblast2. Human amnion cell culture

– Diploid cell strains – cells of single type (fibroblast cells) that can be sub cultivated for limited number of times, mostly 501. WI-38: human embryonic lung cell

– Continuous cell lines – indefinite subcultivtion1. HeLa: Human Ca of cervix cell line2. HEP-2: Human epithelioma of larynx3. Vero: Vervet monkey kidney

Classification 1. Primary Cultures

– Derived directly from tissue and cultured either as • Outgrowth of tissue in culture • Cut into single cells (by enzymatic digestion or

mechanical dispersion)– Advantages:

• usually retain many different characteristics of the cell in vivo

– Disadvantages:• initially heterogeneous but later become dominated by

fibroblasts. • the preparation of primary cultures is labor intensive• can be maintained in vitro only for a limited period of

time.

Classification2. Continuous Cultures

– derived from subculture (or passage, or transfer) of primary culture • Subculture = the process of dispersion and re-culture

the cells after they have increased to occupy all of the available substrate in the culture

– can be serially propagated in culture for several passages

Types:– There are two types of continuous cultures

• Cell lines• Continuous cell lines

Types of continuous cultures

1) Cell lines • short time life, die after approximately thirty cycles of

division• usually diploid and maintain some degree of

differentiation.

Types of continuous cultures

• 2. Continuous cell lines :Most cell lines grow for a limited number of generations after which they ceases

• Cell lines which either occur spontaneously or induced or chemically transformed into Continous cell lines

• Characteristics of continous cell linesSmaller, More Rounded, Less Adherent With a Higher

Nucleus /Cytoplasm RatioFast growthReduced serum and anchorage dependence and grow

more in suspension conditions

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

American Type Culture Collection .

Practical

Design and Equipment for the Cell Culture Laboratory

• 1. Laboratory design- in a safe and efficient manner

• 2. safety cabinets

Cell Culture Room

Close Small AC/Heater

ROOM FOR ANIMAL CELL CULTURE

sterile conditions (disinfection of the work surfaces, microbiological safety cabinets)

Hood

Basic equipments used in cell culture• Laminar cabinet- • Incubation- Temperature is 37 C for mammalian cells, Co2 2-

5% & 95% air at 99% relative humidity.• Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin)

& media components (e.g. glutamine & serum) at -20 C• Microscope- An microscope with 10x to 100x magnification• Cell culture tubes-• Autoclave-

Laminar- flow hood

Laminar- flow hood• The working environment is protected from dust

and contamination by a • constant, stable flow of filtered air• Two types: Horizontal, airflow blow from the side facing

you, parallel to the work surface, and is not circulating;

Vertical, air blows down from the top of the cabinet onto the work surface and is drawn through the work surface and recalculated

Laminar- flow hood

Laminar- flow hood

• Routine maintenance checks of the primary filters are required (every 3-6 months).

• They might be removed and discarded or washed in soap and water.

• Every 6 months the main high efficiency particulate air (HEPA) filter above the work surface should be checked for airflow and hole

Precaution Measure Inside The Hood

Incubator Gloves arealways worn

The pipettes are disposable

Lab coat

Cell Culture Incubator

Cell Culture Incubator

• It requires a controlled atmosphere with high humidity and super controlled of CO2.

• The incubator should be large enough, like 50-200 l have forced air circulation

• Temperature should be + 0.5oC • It should be stainless steel, and easily cleaned

Autoclave A simple autoclave may be sufficient.

Refrigerators and Freezers

• 4C•-20C•-80C• Liquid N2 tank

MicroscopeLarge stage so plates and flasks can be used.Magnification; 5X, 10X, 20X, 40X

Culture vessels and mediumfor animal cell culture

Culture vessel

Culture Mediaremoves a tray of stem cell cultures from an incubator

Anaerobic Jar

Multiwell plates

Cell Culture Bottles / Tubes

Heating of media on heater

Centrifuge

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• Culture Medium

– Basic Composition of Media• Inorganic salts• Trace elements• Buffering systems

– pH range should be 7,2 – 7,4• Carbohydrates• Aminoacids• Vitamins• Proteins and peptides• Fatty acids and lipids• Serum – fetal bovine serum• Antibiotics

Before use

• Ultraviolet lights are used to sterilize the air and exposed work surfaces in laminar flow cabinets between use.

• Detergent • 70% alcohol

Culture Morphology

• Suspension (as single cells or small free-floating clumps)

• or as a monolayer that is attached to the cell culture flask.

• The form taken by a cell line reflects the tissue from which it was derived ...

• From blood tend to grow in suspension • From solid tissue (lungs, kidney) tend to grow as

monolayer's. • Attached cell lines can be classified as endothelial,

epithelial, or fibroblasts and their morphology tell the area within the tissue of origin

Anchorage dependent or independent

• Cell lines derived from normal tissues are considered as anchorage-dependent grows only on a suitable substrate.

• Suspension cells are anchorage-independent e.g. blood cells

Adherent cells• Cells which are anchorage dependent • Add enough trypsin/EDTA to cover the monolayer• Incubate the plate at 37 C for 5 mts• 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

Suspension cells

• Easier to passage as no need to detach them• As the suspension cells reach to confluency• Aseptically remove 1/3rd of medium• Replaced with the same amount of pre-

warmed medium

confluency

• Once the available substrate surface is covered by cells (a confluent culture) growth slows & ceases.

hep 3B - 70% confluency

Gaps

100% confluency

After 24 h

confluency

• Cells to be kept in healthy & in growing state have to be sub-cultured or passaged , It’s the passage of cells when they reach to 80-90% confluency in flask/dishes/plates

• Enzyme such as trypsin, collagenase in combination with EDTA breaks the cellular glue that attached the cells to the surface

Cell culture contaminants

• 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 are mycoplasma, yeast, bacteria or fungus or also from cross-contamination of cells from other cell lines

Safety

Why sub culturing.?

• Once the available substrate surface is covered by cells growth slows & ceases.

• Cells to be kept in healthy & in growing state have to be sub-cultured or passaged

• It’s the passage of cells when they reach to 80-90% confluency in flask/dishes/plates

• Enzyme such as trypsin, collagenase in combination with EDTA breaks the cellular glue that attached the cells to the surface

Rules for working with cell culture

Never use contaminated material within a sterile area Use the correct sequence when working with more than one

cell lines.• Diploid cells (Primary cultures, lines for the production of

vaccines etc.)• Diploid cells (Laboratory lines)• Continous, slow growing line• Continous, rapidly growing lines• Lines which may be contaminated• Virus producing lines

Cell Culture Used Areas where cell culture technology is currently playing a

major role.– Model systems for

Studying basic cell biology, interactions between disease causing agents 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 convert normal cultured cells to cancerous cells

• Virology Cultivation 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

Cell Culture Used

AdvantageViral Vaccines:

Continuous Cell Lines

Cell Substrate Live Vaccines Inactivated Vaccines

African green monkey kidney:Vero Poliovirus Poliovirus (Europe) (U.S.)

Disadvantages

• Cell characteristics can change• Cell can adapt to different nutrients• If mixed cells cultivated some types will

disappear. • Activity of enzymes may altered by

environment.

Cryopreservation

DefinitionCryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as, −196 °C (the boiling point of liquid nitrogen).

Liquid Nitrogen

Principles of cryopreservation

• Water in cell: Around 90% of water is free (water) while the remaining 10 % bounds to other molecular components of the cell (proteins, lipids, nucleic acids and other solutes). This water does not freeze and called hydrated water– Removal of water is necessary during freezing to avoid ice

crystal formation, dehydration is limited to the free water– Removal of hydrated water could have adverse effect on the

cell viability and the molecular function (freezing injuries)

Cryopreservation of Cell Lines • The aim of cryopreservation is to enable stocks of cells to be

stored to prevent the need to have all cell lines in culture at all times. It is invaluable when dealing with cells of limited life span. The other main advantages of cryopreservation are:

• Reduced risk of microbial contamination • Reduced risk of cross contamination with other cell lines • Reduced risk of genetic drift and morphological changes • Work conducted using cells at a consistent passage number

(refer to cell banking section below) • Reduced costs (consumables and staff time)

Successful Cryopreservation of cell lines

• There has been a large amount of developmental work undertaken for successful cryopreservation of a wide variety of cell lines of different cell types.

• The basic principle of successful cryopreservation is a • Slow freeze • Quick thaw. • Cell lines should be cooled at a rate of –1ºC to –3ºC

per minute and thawed quickly by incubation in a 37ºC water bath for 3-5 minutes..

• A high concentration of serum/protein (>20%) should be used. In many cases serum is used at 90%.

• Use a cryoprotectant such as dimethyl sulphoxide (DMSO) or glycerol to help protect the cells from rupture by the formation of ice crystals.

• The most commonly used cryoprotectant is DMSO at a final concentration of 10%.

Successful Cryopreservation of cell lines

Vocabulary Cell-Culture-In vitro cultivation

of cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is known as Cell Culture.

Culture – growth of microorganisms in a special medium; the process of growing microorganisms in the laboratory.

Anchorage-dependent- Cell lines derived from normal tissues are considered as anchorage-dependent grows only on a suitable substrate

Primary Culture :Cells when surgically or enzymatically removed from an organism and placed in suitable culture environment will attach and grow are called as primary culture

Cell Lines: Sub culturing of primary cells leads to the generation of cell lines

Cell Strain :Lineage of cells originating from the primary culture is called a cell strain

Anchorage-independent- Suspension cells are anchorage-independent e.g. blood cells

Next practical

• Transformation

• See you next week!

Thanks