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Isolation of Primary Cells: Tissue Culture & Animal Biotechnology

Primary cell cultures are obtained by directly culturing cells from tissue. These cells retain characteristics of their original tissue such as diploid karyotype and differentiated functions. There are two main methods to initiate primary cell cultures - mechanical and enzymatic disaggregation. Mechanical disaggregation involves finely mincing tissue while enzymatic uses enzymes like collagenase and trypsin to further break down tissue into single cells. Primary cultures provide a way to study cell properties and functions as they exist in vivo.

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Sama Al Hayek
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263 views30 pages

Isolation of Primary Cells: Tissue Culture & Animal Biotechnology

Primary cell cultures are obtained by directly culturing cells from tissue. These cells retain characteristics of their original tissue such as diploid karyotype and differentiated functions. There are two main methods to initiate primary cell cultures - mechanical and enzymatic disaggregation. Mechanical disaggregation involves finely mincing tissue while enzymatic uses enzymes like collagenase and trypsin to further break down tissue into single cells. Primary cultures provide a way to study cell properties and functions as they exist in vivo.

Uploaded by

Sama Al Hayek
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Isolation of Primary Cells

Tissue Culture & Animal


Biotechnology
Primary Cultures
• Primary culture refers to the cells that are placed in culture
directly from the tissue of origin. These are called primary
cultures until the first subculture. After the first subculture, they
may be called secondary cultures, and thereafter, if continued
passage is possible, a cell line.

• These cells retain their diploid karyotype, i.e., they have the
chromosome number and morphology of their tissues of origin.
They also keep some of the differentiated characteristics that
they possessed in vivo

• There will always be a need to perform primary culture in order


to study the properties of cells that are only recently removed
from the in vivo situation in order to learn more about their
functions in vivo
Primary Cultures
• The cultures consist of a wide variety of cell types, most of which
are capable of very limited growth in vitro, usually <10 divisions.

• Attempts may be made to mechanically or enzymatically purify


the cell type of interest during the tissue dissociation or to
"clean" the cultures by culturing the cells in conditions that
specifically inhibit or kill some major contaminant cell type(s).

• Primary cultures can be maintained in conditions chosen to


positively select for the survival of only one cell type.

• In general, the method chosen must take into consideration the


desired balance between obtaining a high cell yield and
minimizing damage to the cells to be cultured and between
maximal yield and maximal purity
Initiation of Primary Cell Culture
1. Sample acquisition
2. Isolation of tissue
3. Dissection and disaggregation
4. Culture after seeding into vessel
• After isolation, a primary cell culture is obtained by
allowing cells to migrate out from the fragment of tissue
adhering to a suitable substrate
• Or by disaggregating the tissue mechanically and
enzymatically
Isolation of the Tissue
• Ensure that you are working within the ethical legislations of
experimentation with animals

• Sterilize the site of dissection with 70% ethanol

• Remove tissue aseptically and transfer to a tissue vessel


containing PBS as soon as possible

• Dissection of animals should be carried outside the tissue


culture facility as they may carry microbial contamination

• If a rapid transfer of tissue is not possible then it should be


kept at 4°C for 72 hrs.
Mouse Embryos
• Excellent source of undifferentiated mesenchymal cell cultures
which are referred to as “embryo mouse fibroblasts” and can be
used as feeder cells.

• Full term in mice is about 19-21 days depending on the strain

• The optimal age to isolate cells from whole embryos is 8-11


days

• Most of the organs are completely formed by the 18 th day


Chick Embryo
• Easier to dissect because they are bigger than mouse
embryos at the same stage of development.

• Used to provide mesenchymal cell primary cultures for


- cell proliferation analysis
- Feeder layers
- substrate for viral propagation

• Dissect individual organs to generate specific cell types ex.


Hepatocytes, cardiac muscle and lung epithelium
Options for the primary culture
• Several techniques have been devised for the
disaggregation of tissue isolated for the
primary culture.

• Purely mechanical techniques, involving


dissection with or without some form of
maceration.

• Techniques utilizing enzymatic disaggregation


Enzymatic disaggregation

And

Mechanical disaggregation
Primary Explant
• Suitable for small amount of tissues, for example skin
biopsy. Attachment on the substrate by using plasma
clots, or fibrinogen and thrombin. Disaggregation by
mechanical and enzymatic.
Mouse, mammals,
Embryo
Eggs
(best: for TC : embryo, young)
because stage of differentiation)

Finely cut

Finely cut
tissue or explant
explant Grow in media
•Explants
•Explants with outgrowth
organ
*

Primary Explant Culture


Types of Disaggregation
Techniques used for the disaggregation of tissue isolated from
primary cells:
1. Fine dissection involves chopping tissue with scalpels. It
involves special filters with pores large enough for cells to go
through but not clumps of tissue.
2. Enzymatic disaggregation using an enzyme to
disaggregate the tissue further.

• Mechanical disaggregation works well with soft tissues and


some firmer tissues.

• Enzymatic disaggregation gives better yields when more


tissues are available.
Tissue Culture & Hybridoma Technology
(1450361) / Ban Al-Joubori
•Mouse, mammals,
Cell culture
•Embryo
•Embryonated Eggs
(best: for TC : embryo, young)
because stage of differentiation)
Finely cut

Grow in media
-monolayer
Finely cut -suspension cells
tissue

explant

organ
Enzymic digestion
Enzymes used for Disaggregation

Collagenase
• most commonly derived from Clostridium histolyticum
• degrades the helical regions in native collagen
• most commonly used for tissue dissociation

Trypsin
• pancreatic serine protease with specificity for peptide bonds
involving the carboxyl group of the basic amino acids,
arginine and lysine
Warm Trypsinization
• This method is widely used for disaggregation of cells. The
chopped tissue is washed with dissection basal salt solution
(DBSS), and then transferred to a flask containing warm trypsin
(37° C). The contents are stirred, and at an interval of every thirty
minutes, the supernatant containing the dissociated cells can be
collected. After removal of trypsin, the cells are dispersed in a
suitable medium and preserved (by keeping the vial on ice).
• The process of addition of fresh trypsin (to the tissue pieces),
incubation and collection of dissociated cells (at 30 minutes
intervals) is carried out for about 4 hours. The disaggregated
cells are pooled, counted, appropriately diluted and then
incubated.
Cold Trypsinization
• An alternative method for disaggregation by placing the tissue in
trypsin at 4°C for 6-18 hrs to allow penetration of the enzyme with little
tryptic activity.
• The tissue is then incubated at 37°C for 20-30 min. for disaggregation
• This method gives higher yield of proliferating cells with improved
survival rate and preserve more different cell types
• Culture from mouse embryos contain more epithelial cells in this
method
• Erythroid cultures from 13-day fetal mouse liver respond to
erythropoietin with this method but not with warm trypsinization or
mechanical disaggregation
Elastase
• produced in the pancreas as an inactive zymogen, proelastase,
and activated in the duodenum by trypsin.
• used with other enzymes like trypsin or collagenase to
dissociate tissues which contain extensive intercellular fiber
networks.
• Unique in its ability to hydrolyze native elastin, a substrate not
attacked by trypsin, chymotrypsin or pepsin.

Hyaluronidase
A polysaccharidase, is often used in combination with a crude
protease such as collagenase, for the dissociation of connective
tissues.

Tissue Culture & Hybridoma Technology


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Papain
• is a sulfhydryl protease from Carica papaya
• has wide specificity and degrades most protein substrates more
extensively than the pancreatic proteases
• also exhibits esterase activity
• With some tissues has proved less damaging
• more effectively used for obtaining high yields of viable,
morphologically intact cortical neurons.

Chymotrypsin
• preferentially catalyzes the hydrolysis of peptide bonds involving
the aromatic amino acids tyrosine, phenylalanine, and
tryptophan

• Chymotrypsin is used to a limited extent in tissue dissociation,


usually in combination with trypsin and elastase.
Neutral Protease (Dispase)
• is a bacterial enzyme produced by Bacillus polymyxa
• hydrolyses N-terminal peptide bonds of non-polar amino acid
residues
• is classified as an amino-endopeptidase
• it possesses a mild proteolytic dissociation activity while
preserving the integrity of the cell membrane.
• used as a secondary enzyme in conjunction with collagenase
and/or other proteases in many primary cell isolation and tissue
dissociation applications.
• dissociates fibroblast-like cells more efficiently than epithelial-
like cells.
• Thus it has been used for differential isolation and culture
applications.

Tissue Culture & Hybridoma Technology


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Features of Disaggregation
1. Fat and necrotic tissues should be removed during dissection

2. Tissue chopped finely with scalpels

3. Enzymes used for disaggregation should be removed by


centrifugation

4. The conc. of cells in primary culture should be higher than


regular sub culturing density.

5. Rich medium such as Ham’s F12 is preferable, use FBS


instead of HS, some cell lines require serum free media.

6. Embryonic tissue disaggregates more rapidly, yields more


viable cells and proliferate faster than adult tissue
Mechanical Disaggregation
• Outgrowth of cells from primary explants is a slow
process and can be highly selective
• Enzymatic Disaggregation is more labor intensive but
gives a more representative culture of the cells in the
tissue.
Problem: Risk of proteolytic damage to cells during
enzymatic digestion, many prefer mechanical
disaggregation
Mechanical Disaggregation
Spillage: collecting the cells that spill out when the tissue is
sliced and the slices are scraped
Sieving: pressing the dissected tissue through a number of
sieves for which the mesh is reduced in size
Syringing: Forcing tissue through a syringe (with or without a
wide-gauge needle)
• The procedures gives a quicker cell suspension than
enzymatic digestion but may cause mechanical damage
• Only soft tissues, such as spleen, embryonic liver, embryonic
and adult brain and some tumors respond well to this
technique
Tissue Culture & Hybridoma Technology
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Viable and Nonviable Cells
• Only a portion of the cells will be able to survive and generate a
primary culture.
• Some cells are viable but not capable of attachment, others are
nonviable (necrotic or apoptotic).
• Nonviable cells are removed at the first change of medium.
• Primary suspension cultures, nonviable cells are diluted out when
cell proliferation starts
• Nonviable cells may be removed by centrifuging the cells on a
mixture of ficoll (a polysaccharide) and sodium metrizoate. Viable
cells collect at the interface between the medium and the
Ficoll/metrizoate and the dead cells form a pellet.
Tissue Culture & Hybridoma Technology
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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 after damage

from illness or injury?

• Observe the developmental process

• Production of vaccines
Tissue Culture & Hybridoma Technology
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Primary Cell Culture
• Cell culture refers to cultures derived from dissociated cells
taken from the original tissue ('primary cell culture').
• Tissue is dissociated by collagenase/Dispase into a cell
suspension which may then be cultured as a monolayer on a
solid substrate, or as a suspension in the culture medium.
• They can be propagated and hence expanded and divided to
give rise to replicate cultures.
• Cell cultures can be characterized and a defined population
can be preserved by freezing.
Cell Line Propagation
• Once a cell line is established, it needs to be propagated in order to
produce sufficient cells for characterization and storage, as well as
for particular experiments.

• If the cell line is likely to be viable for only a few sub-culturings or


generations each generation should be noted, by writing the
passage #.

• Once a culture is confluent (i.e., the cells cover 60-70% of the


growth surface available) it can be transferred into a holding or
maintenance medium which provides just enough nutrients to keep
the cells alive and healthy but reduces the replication rate so that
the cells do not overgrow.

• If cells are required for experiments or storage the contents of the


flask can be split or divided to seed 2-4 new flasks depending on the
vigor of the cell growth.
END

Tissue Culture & Hybridoma Technology


(1450361) / Ban Al-Joubori

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