Welcome to our

presentation

Syed Shahariar Bappy(BG-13034)

Rasedul Islam(BG-13031)
Sorna Sultana(BG-13030)
Atokia Bilkis(BG-13033)
Khairul Azam Bokul(BG-13013)
Ramkrishno Sorkar(BG-13039)
Jorina jabin mili(BG-13012)
Mabia Zaman Mukta(BG-13014)

Mass culture

Mass cultureTechnique
Large number of primary cells are obtained from the animal.

They are added to a culture dish where they attach to the bottom.

The ones which survive,they proliferate on the culture dish.

After a number of generations of proliferations,they form a monolayer of

cells.

These cells cover the entire bottom portion of the culture dish.

Here different type of cells are obtained in culture,hence different type of

cells lines.

Hybridoma Technology
A method for producing large numbers of identical antibodies
(also called monoclonal antibodies) or a Hybrid cell.

These cells are produced by fusing B-lymphocyte with tumour

cell.

These hybrid cells have got the ability to produce antibodies due
to the B-lymphocyte genetic material and also capacity to divide
indefinitely in the culture due to the presence of tumour cell
involved in the production of hybrid cells.

Procedure of hybridoma technology

B-lymphocytes are excreted from the spleen of an animal,which has been immunized
with the required antigen against which monoclonal antibodies are produced.

The specific antibody producing B-lymphocytes are then mixed with the selected
myeloma cells and are induced to fuse to form hybrid cells.

Then cultured in selective media known as HAT medium along with the drug
aminoprotein.

Mutant cells deficient or laking Tk and HGPRT cannot grow in the HAT medium
because aminopterin bloks endogenous(de novo) synthesis of purin and pyrimidin

Only the hybridoma cells(HGPRT+ and TK+) have got the ability to divide and
proliferate on the HAT medium by utilizing exogenous hypoxanthine and thymidine
via salvage pathway of nucleotide synthesis.

Fig:procedure of hybridoma technology

Selection process through HAT

medium

Identification and isolation of the hybridoma cells

The specific antibodies present in the each microwell.
Microwell

are

identified

using

one

of

the

method

such

as

agglutination method.
Most commonly used and most sensitive and rapid method is ELISA.
Wells which contain the antibodies specific to the antigens are
identified and hebridoma cells are isolated from these wells and
cultured(cloned).
This ensure that these hybridoma cells have the capacity to produce
same single type of antibodies.
After these hybridoma cells are muitiplied using in vitro or in vivo
method.

Uses
Hybridoma technology is used for
producing monoclonal antibodies.

Cell line
A population of animal or plant cells that can be grown in a
suitable nutrient culture medium in the laboratory .
This cell culture developed from a single cell and therefore
consisting of cells with a uniform genetic make-up.

Types of cell line

On the basis of the life span of culture, the cell lines are
categorized into two typesFinite cell Lines- The cell lines which have a limited
life span and go through a limited number of cell
generations (usually 20-80 population doublings) are
known as Finite cell lines.
Continuous Cell Lines- Cell lines transformed under
in vitro culture conditions give rise to continuous
cell lines.

Properties of Finite Cell

Lines
Euploid, diploid
Normal transformation
Anchorage dependence
Monolayer
Low Cloning efficiency
Low Yield
Cyclic

Properties of Continuous Cell Lines

Aneuploid, heteroploid
Immortal,growth controlled altered
,tumerigenic .
No anchorage dependence
No contact inhibition
High cloning efficiency
High yeild

Significance of Cell Morphology

Granularity around the nucleus,
Cytoplasmic vacuolation,
Rounding up of the cells with detachment from
the substrate.
Also know about-

Medium change
Indicate a more serious problem,such as
inadequate or toxic medium or serum, microbial
contamination.

CHOOSING A CELL LINE

Finite or continuous
Normal or transformed
Species

Growth characteristics
Population-doubling time
Saturation densityyield per flask
Plating efficiency
Growth fraction
Ability to grow in suspension

Availability
Validation.
Phenotypic expression
Control cell line
Stability

Replacement of Medium
Four factorsindicate the need for the replacement
of culture medium-

A drop in pH
Cell concentration.
Cell type.
Morphological deterioration.

Subculture
A subculture is a microbiological culture made by
transferring some or all cells from a previous culture
to fresh growth medium.This action is called
subculturing or passaging the cell.

Criteria for Subculture

Density of culture.
Exhaustion of medium.
Time since last subculture.
Requirements for other procedures.

Subculture of a Monolayer Cell

Monolayer and Suspension Cultures Comparison

Monolayer
Culture requirements
Cyclic maintenance
Trypsin passage
Limited by surface area
Growth properties
Contact inhibition
Cell interaction
Diffusion boundary
Useful for
Cytology
Mitotic shake-off
Continuous product harvesting
Applicable to
Most cell types, including
Primaries

Suspension
Steady state
Dilution
Volume (gas exchange)
Homogeneous suspension

Bulk production
Batch harvesting

Only transformed cells

Fibroblast

A fibroblast is a type of
cell that is responsible for
making the extracellular
matrix and collagen.

Together, this
extracellular matrix and
collagen form the structural
framework of tissues in
animals and plays an
important role in tissue
repair. Fibroblasts are the
main connective tissue.

Origin of fibroblast

Fibroblasts are derived from primitive mesenchyme,

like all connective tissue cells.

Their ability to express filament protein vimentin

alludes to their mesodermal origin.

Function of fibroblast
Maintain the structural integrity of the connective tissue by continuously
secreting extracellular matrix proteins like
collagens,
glycosaminoglycans, and
glycoproteins.
Injury of tissue displays a proliferative stimulus for fibroblasts and
induces them to produce wound healing proteins.
Fibroblasts sometimes undergo MET to produce epithelia.
When tissue damage has occurred, the fibrocytes are stimulated to
undergo mitosis.

Isolation Dermal Fibroblast

Place the foreskin directly after circumcision into CnT-PR-F medium.

Place the foreskin into a Petri dish. Cut it into 4 equally sized pieces
using a scalpel.

Place the pieces of skin in a 15 mL centrifuge tube containing 10 mL

of 1x dispase solution and 2 x antibiotics / antimycotics.

Incubate the skin over night (~ 16 h) at 4C. Next day transfer the skin
together with the dispase solution into a Petri dish.

Transfer each tissue piece to a new Petri dish containing PBS to wash
away excess dispase.

While holding the skin submerged in PBS, gently separate the dermis
from the epidermis with two pairs of curved forceps.

If the separation does not occur easily, put the tissue back into the Petri
dish with dispase solution for another 30 to 60 min at room temperature.

Cut the dermis into very small pieces using a new scalpel blade.

Transfer the pieces into a 50 mL centrifugation tube containing 5 mL of

collagenase A , plus 0.5x CnT-GAB10 antibiotic/antimycotic.

Incubate at 37C for 4-8 hours with occasional mixing.

Add 15 mL CnT-PR-F medium to dilute the collagenase, mix by pipetting up

and down, and then pass through 70 um cell strainer to obtain a single-cell
suspension.

Centrifuge the single cell suspension for 5 minutes. Discard the supernatant
and re-suspend the pellet in 1 mL CnT-PR-F.

Perform a cell count, and seed at approximately 4000 cells cm2 in CnT-PR-F.