Learning outcomes

Describe the methods to establish primary cell

culture
Discuss the applications of primary cell culture
in biomedical sciences.

 Cultures

established from a specific

organ site with specific processing

Sources

Adult-

human biopsy
materials, animal tumour
Embryo-mouse, chicks,
organ rudiments
Egg-embryonated

Primary cell cultures typically will have a finite life

span in culture (maximum passage 20-40X)

Continuous cell lines are, by definition, abnormal and

are often transformed cell lines (can be passaged
indefinitely).

Advantages

"mimic" in vivo conditions more

closely than cell lines.

able to accurately model functions of

an organ in various states and
conditions by the use of the actual in
vitro organ itself.

exclude the influence of other organs

and of the circulatory and immune
system, thus providing the possibility
to study direct effects on a cell
population.

Types

Cell culture

Explant culture.

Organ culture

Cell culture- from tissue that is

disaggregated by enzymatic,
chemical, or mechanical methods

Explant culture- from the outgrowth

of migrating cells from a piece of
tissue or from

Organ culture- whole organ or part of

the organ is maintained in culture

Fat and necrotic tissues are best removed during

dissection.

The tissue should be chopped finely with sharp

instruments to cause minimum damage.

Enzymes used for disaggregation should be

removed subsequently by gently centrifugation.

The concentration of cells in the primary culture

should be much higher than that normally used for
subculture, since the proportion of cells from the
tissue that survives in primary culture may be quite
low.

A rich medium is preferable to a simple medium

Embryonic tissue is preferable as it disaggregates

more readily, yields more viable cells and
proliferates more rapidly in primary culture than
does adult tissues.

Requirements

Methods

Primary
explant/outgrowth

Enzymatic
disaggregation

Mechanical
disaggregation

Explant/
Outgrowth
culture

Tissue is finely dissected and

placed into culture as "primary
explants
Cells migrate from tissue onto
culture substrate /vessel
Cell will begin to divide and grow
Cells can be harvested

Explant/
Outgrowth
culture

Harvest the cells

migrating out from the
explant

Enzymatic
disaggregation

Types of cells
-Fibroblasts
-Epithelial
-Muscle
-Bone
-Nerve

Tissue is finely dissected

Need to disrupt cell-cell adhesion
proteins
Enzymes such as collagenase or
trypsin are used to digest tissue
Cells can be damaged to point of
lysis
Centrifuged to harvest and wash
cells from enzymes

Enzymatic
disaggregation
Types of enzymes
Trypsin and pronase give the most complete disaggregation
but may damage the cells.
Collagenase and dispase give incomplete disaggregation but
are less harmful.
Hyaluronidase + collagenase digest the intracellular matrix.
DNase is employed to digest DNA released from lysed cells.

Enzymatic
disaggregation

Tissue
Disaggregation
by Collagenase

Types of Collagenases

Mechanical
disaggregation
Sieving
Syringing
Vigorous
pipetting

Tissue is finely dissected

Cells separated using syringing and

vigorous pipetting

Pressing tissue into a mesh

Wash cells through sieve

Faster than enzymes but less yield

May cause mechanical damage to cells

Mechanical
disaggregation
Types of cells

-Only soft tissues: spleen, thymus, embryonic liver,

embryonic and adult brain, and some human and animal
soft tumors, respond well to this technique.
- Brain - complete disaggregation can be obtained easily,
the viability of the resulting suspension is lower than that
achieved with enzymatic digestion.

Mechanical
disaggregation

Forcing tissue
through sieve with
syringe piston

Drawing tissue into

syringe

Pipetting tissue tissue

fragments up and
down

Isolation of organ

Dissection
Fine
dissection/
explant

Mechanical
disaggregation

Enzymatic disaggregation
Cold trypsin

Primary Explant
Overnight
storage, long
incubation
Explant

Outgrowth

Dispersed
primary
culture

Transfer
Subculture
Secondary
explant Culture

CELL LINE

Warm
trypsin

Short incubation,
repeated sampling

Centrifuge

Resuspend
and seed

Collagenase
Long
incubation,
complete
medium

Media- A rich medium, such as Hams F12, DMEM

are preferable to a simple medium, such as Eagles
MEM,
Serum - fetal bovine (FBS) often gives better
survival than does calf or horse.

Viability of cells- an inverted phase contrast

microscope.

Live cells - phase bright; suspension cells are typically

rounded and somewhat symmetrical; adherent cells will
form projections when they attach to the growth
surface.

Assessed using the vital dye, trypan blue, which is

excluded by live cells but accumulates in dead cells.

Cell numbers are determined using a hemocytometer.

Adherent primary culture - nonviable cells are removed at the first

change of medium (floating cells).

Cultures maintained in suspension- nonviable cells are gradually

diluted out when cell proliferation starts.
Nonviable cells may be removed from the primary disaggregate by
centrifuging the cells on a mixture of Ficoll and sodium metrizoate
(e.g., Hypaque or Triosil) [Vries et al., 1973].
The viable cells collect at the interface between the medium and the
Ficoll/metrizoate, and the dead cells form a pellet at the bottom of the
tube.

Cell Type

Tissue Source

Reference

Fibroblast

Skin, embryonic

Han et al., 1993; Lam et al., 1988.

Brain Cells

Brain, embryonic
Brain, post hatch

Nicholas et al., 1986; Shafren and Tannock,

1991. Adams, 1965.

Pituitary cell

Pituitary gland, post hatch

Preze et al., 1987.

Rigmented retinal cell

Retina, post hatch

Rosenberg et al., 1989.

Liver cell

Liver, post hatch

Schultz and Mistry, 1981 (surgical);

Legrand and Lemarchal, 1992 (nonsurgical).

Intestinal epithelial cell

Intestine

Ali and Rernolds, 1996.

Pancreatic cells

Pancreas, post hatch

Kodoma et al., 1995.

Kidney cell

Kidney, embryonic

Chomiak et al., 1960; El Zein et al.m 1971.

Thymic cell

Thymus, post hatch

Lam et al., 1988;

Bursal cell

Bursa, post hatch

Lam et al., 1988; Nunoya et al. 1991.

Spleen cell

Spleen, post hatch

Hurk, 1990;

Leukocyte

Peripheral blood, post hatch

Bone marrow, post hatch

Hurk, 1990;
Hurk, 1990;

Macrophage

Peritoneal exudate

Qureshi and Hagler, 1992.

Adipose cell

Abdominal fat, post hatch

Cryer et al., 1987.

Cartilage cell

Sternal cartilage, embryonic

Growth plates, post hatch

Coon and Cahn, 1966.

Rosselot, et al., 1992.

Tendon cell

Toes, embryonic

Riederer-Henderson et al., 1983.

Bone Cells
_Osteocyte
_Osteoblast
_Osteoclast

Tibiotarsi, embryonic
Calvaria, post hatch
Medulary bone, past hatch

van der Plas and Nijweid, 1992.

Teti et al., 1991a.
Teti et al., 1991b.

Muscle cell

Pectoralis major, post hatch

Breast muscle, embryonic
Thigh, embryonic

McFarland et al., 1988.

Armstrong et al., 1990.
Paterson and Strohman, 1972.

Adrenocartical cell

Adrenogland, past hatch

Rosenberg et al., 1989.

Endothelial cell

Fat pads

Twal and Leach, 1996.

Testicular cell

Testis, embryonic

Rombauts et al., 1995.

Granulosa

Follicles, laying hen

Yoshimura and Tamura, 1988.

Oviduct cell

Oviduct, laying quail

Kato et al., 1975.

Fundamental
Diagnostics-isolation
and identification of
agents, diseases,
mechanism

Applied Research

Bioreactor-production of
biological reagents
(monoclonal antibodies)

Stem cell research-embryonic

and adult (somatic) stem
cells, reproductive
biotechnology, gene therapy

Tissue engineering
(regenerative
medicine)

Drug discovery- screen for

effects of compounds like
hormones and drugs

Diagnostics
Clinical detection and
isolation of viruses, how
they grow and infect
organisms

Use of Cells as
replacement tissues
and organs
Artificial skinstreating burns
and ulcers.
Researchartificial organs
such as
pancreas,
kidney and liver

Cell-Based
Manufacturing
Large scale
production of
viruses for use
in vaccine
productionpolio, rabies,
chicken pox,
hepatitis B and
measles.

Cells (genetically
enginereed) to
produce proteins
(monoclonal
antibodies,
insulin,
hormones)

Gene therapy

Embryonic and adult

stem cells
Supply of
replacement
cells and tissues

Genetically engineer
cells-cells removed
from patient lacking
functional gene-.
Cells grown and
missing or damaged
gene replaced

Tissue
Engineering

Primary rodent
kidney cell
culture

-Propagation of many types

of viruses i.e. adenoviruses
and rabies.
-Studies related to kidney
and diabetic diseases.
-Study of the toxicity of
drugs

2.3. Primary cultures of cortical neurons

Effects of PSC833 on cerebral
glucose metabolism in rat
primary cultures of cortical
neurons and
Astrocytes (Cruz and Wolf,
2001)
(Biochemical Pharmacology 62
(2001) 129139)

Neuronal cultures were prepared from the cerebral cortex

of 17-day-old rat embryos as described by Gomeza et al.
[23]. Tissue was cleaned of meninges and dissociated
with papain (7 min, 37, 0.4 mg/mL in a 10-mM
phosphate buffer solution, pH 7.5, with 6 mM glucose)
and subsequently disaggregated by passing through
a Pasteur pipette. After centrifugation (800 g, 5 min),
pelleted cells were resuspended in DMEM containing 10%
FBS, penicillin (50 IU/mL), and streptomycin (0.5 mg/mL)
and plated on poly- L-lysine (10 mg/mL)-coated plates at
a density of 106 cells/ mL. Medium was replaced after 3 hr
by a serum-free medium prepared with DMEM
supplemented as described by Brewer et al. [24]. This
medium was changed every two days up to the sixth day
after seeding, when the experiment was performed. Cells
were maintained at 37, 95% air and 5% CO2. Neuronal
cultures were characterized immunohistochemically using
a monoclonal antibody antineurofilament 160 kD (antiNF160) [25]. The monoclonal antibodies antiglial fibrillary
acidic protein (specific for astrocytes) [26], antivimentin
(specific for astrocytes and fibroblast) [27], and
antifibronectin (specific for fibroblast) [28] were used to
verify the purity of the cultures. Neuron enrichment was
higher than 90%. Experiments were performed after cells
were maintained for 6 days in culture.

Development of a primary mouse intestinal

epithelial cell monolayer culture system to
evaluate factors that modulate IgA transcytosis
Original Research Article
Mucosal Immunology 7, 818-828 (July 2014) |
doi:10.1038/mi.2013.98

These monolayers
contained differentiated
epithelial cells that
displayed robust
transepithelial electrical
resistance.

Primary cell cultures of bovine colon epithelium: isolation

and cell culture of colonocytes Original Research Article
Toxicology in Vitro, Volume 14, Issue 5, October 2000, Pages
435-445

Susceptibility of primary cultures of proximal tubular and

distal tubular cells from rat kidney to chemically induced
toxicity Original Research Article
Toxicology, Volume 103, Issue 2, 30 November 1995, Pages 85103

WHY NEED
PRIMARY
CULTURE ??

2
3
4

Acquisition of tumor

Tumor dissociation techniques

Culture and Maintenance

Culture and Maintenance

Researchers developed a bone

marrow-on-a-chip that reportedly
can reproduce the structure,
functions, and cellular makeup
of bone marrow

An Organ-on-a-Chip, is a cell culture device that contains hollow channels

lined by living cells and tissues that mimic organ-level physiology.
The device is capable of producing levels of tissue and organ functionality not
possible with conventional culture systems and also allows real-time analysis
of biochemical, genetic, and metabolic activities within individual cells.

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