Applications of cell lines

Presented by:

Arshvir kaur

M.pharm 1st year

Cell culture

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

Roux in 1885 for the first time maintained embryonic chick cells in a cell culture.

Why is cell culture used for?

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

Conti…..

Virology

Cultivation of virus for vaccine production, also used to study there infectious cycle. 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.

Tissue culture

•In vitro cultivation of organs, tissues & cells at defined temperature using an incubator

& supplemented with a medium containing cell nutrients & growth factors is collectively

known as tissue culture.

•Different types of cell grown in culture includes connective tissue elements such as

fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, kidney) and many

different types of tumor cells.

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.

•Primary cells have a finite life span.

•Primary culture contains a very heterogeneous population of cells.

•Treated by proteolytic enzyme (Trypsin).

Cell lines

A cell line is a product of immortal cells that are used for biological research.

Cells used for cell lines are immortal, that happens if a cell is cancerous.

The cells can perpetuate division indefinitely which is unlike regular cells which can only

divide approximately 50 times.

WRL-68HeLa Jurkat

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.

Cell line

Species of origin

Tissue of origin

Cell morphology

Growth in suspension?

3T3 Mouse Connective Fibroblast No

CHO ChineseHamster

Ovary Epithelial Yes

BHK21 SyrianHamster

Kidney Fibroblast Yes

HeLa Human CervicalCarcinoma

Epithelial Yes

Continuous cell lines

Cell lines which either occur spontaneously or induced virally or chemically

transformed into Continuous cell lines

Characteristics of continuous cell lines

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

ratio

• -Fast growth and have aneuploid chromosome number

Introduction of Cell Culture Lab(Equipment)

CO2-thermostats

Airflow

Solutions

Dishes

Freezers

Liquid nitrogen

Centrifuges

Vacuum ovens

Cryotubes

Microscopes

ELISA-readers

Autoclave

Co2 incubator Dishes

CentrifugesAutoclaves

Vacuum Ovens

ELISA readers

What is in the media?

Dulbecco’ Modified Eagle’s 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)

Antibiotics (penicillin and streptomycin) Prevent bacterial contamination

Salts and buffers To simulate in vivo environment

Conti……

SerumPortion 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

Use of tissue cultures in toxicity testing

Mammalian cell cultures can be a suitable alternative for the use of whole animal tests

to establish the potential toxicity of compounds.

This due to many reasons:

•They can overcome the disadvantages of the whole animal tests including:

High cost

Variability of results

•Cell culture tests are ripid,allow more efficient screening of novel compounds and sometimes can

allow identification of metabolic target of inhibition.

Conti…..

Cell culture tests can be designed to evaluate various effect:

Reduced growth rate

Breakdown of membrane permeability

Tissue specificity response

Ability to metabolize toxic compound

Genetic effect

Use of tissue culture for biological products

Production of vaccines:

• Two factors stimulated the use of tissue cultures for vaccine production:

▫ The ability to grow viruses in cell culture

Current egg vaccine production require long time(9 month) that hinder the response to

unanticipated demands.

In (1949),Enders discovered that the poliomyelitis virus could be grown from primary

monkey cells in culture.

The polio vaccine, produced in 1954, was the first human vaccine to be produced using

large-scale cell culture techniques.

Conti…

Animal cell technology is considerably developed for the production of a range of human and veterinary viral vaccines against a variety of diseases.

(b) Production of antibodies:

Also, the in vitro methods for production of mABs are the methods of

choice because of:

The ease of culture for production.

Less economic consideration compared with the use of animals.

Conti…..

Practical uses of the in vitro produced mABs:

Diagnostic tests for the identification of small quantities of specific antigens.

mABs also are used therapeutically: OKT3 recognizes a surface antigen (CD3) on T cell

and is one of the most effective agents in preventing immunological rejection of

transplanted kidneys.

Conti…..

Various mAbs designed to destruct tumor cells by targeting a membrane bound

protein antigens specifically expressed by these cells.

The conjugation of radiactive or toxic compounds to the antibody can result in a

localized high concentration resulting in cytotoxicity to the target cells.

Conti….

(c)Recombinant proteins:

Proteins extracted from biological sources have been important for the substitution

therapy.

since the 1920s when Best and Banting used insulin to treat diabetes.

Conti….

Some examples for these biological products:

Interferone:

Discovered when Isaacs and Lindenmann (1957) found that culture medium taken from cells that

had supported viral growth could protect non-infected cells from a subsequent viral infection.

Tissue plasminogen activator (t-PA ):

t-PA was produced in large scale by Genenteck from transfected CHO-K1 cells. It is used to prevent

undesirable formation fibrin clots in the bloodstream.

Blood clotting factors:

For example, factor VIII is produced in large scale by Bayer through transfection of the mammalian

kidney cell line (BHK) with an appropriate gene.

Cell therapy

Literally, cell therapy means treatment with cells, i.e replacing diseased or

dysfunctional cells with healthy functioning ones.

For example:

•When hematopoietic cells are vulnerable to destruction by any cytotoxic drugs used in

chemotherapy to eradicate residual tumor cells.

Drug screening & development

• Cell based assay have become increasingly important for the pharmaceutical industry,

not just for cytotoxicity testing but also for high throughput screening of compounds

that may have potential use of drugs. Originally these cell culture tests were done in 96

well plates, but increasing use is now being made of 384 & 1536 well plate.

Corning micro plate

Respiratory cell line

Alveolar cells

A549 cells

Bronchial epithelial cells

BEAS 2B, DMS53, SHP-77,NCI-H23, 292, 524, 727,11655,1299HBE4-E6/E7 etc….. (>140)

Macrophage

Differentiated U937/MonoMac6 cellMurine RAW264.7

• Respiratory cell lines are useful for evaluating of molecular mechanisms of inflammation and other physiological /pathological events.

Pancreatic beta cell lines and their applications in diabetes mellitus research

During the past 30 years great effort has been put into establishing an insulin-

secreting beta cell line that retains normal regulation of insulin secretion, but only few

of these attempts have been successful.

To overcome the limited availability of primary beta cells into the field of diabetes

mellitus research, numerous investigators used X-rays or viruses to induce insulinomas,

in vitro transformation, derivation of cells from transgenic mice or even non-islet cells to

produce immortalized beta cell lines.

The most widely used insulin-secreting cell lines are RIN, HIT, MIN, INS-1 and TC

cells.

These cells produce insulin and small amounts of glucagon and somatostatin.

Despite problems associated with beta cell cultures, these cell lines have provided

some valuable information about physiological processes.

However, an urgent need to establish a "normal" beta cell line of human or pig

origin remains.

Vivalis, a Nantes, France-based biotechnology company, has developed EB66®, a

novel cell line derived from duck embryonic stem cells for the cell culture

production of viral vaccines.

As a result, EB66 cells have become a superior industry alternative for the safe,

cost effective manufacturing of viral vaccines.

Cell line in vaccinesCell lines in vaccines

Vivalis offers research and commercial licenses for this cell line to pharmaceutical

and biotechnology companies for the production of prophylactic and therapeutic

vaccines.