Cultures
Sources
Adult-
human biopsy
materials, animal tumour
Embryo-mouse, chicks,
organ rudiments
Egg-embryonated
Advantages
Types
Cell culture
Explant culture.
Organ culture
Requirements
Methods
Primary
explant/outgrowth
Enzymatic
disaggregation
Mechanical
disaggregation
Explant/
Outgrowth
culture
Explant/
Outgrowth
culture
Enzymatic
disaggregation
Types of cells
-Fibroblasts
-Epithelial
-Muscle
-Bone
-Nerve
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
Mechanical
disaggregation
Types of cells
Mechanical
disaggregation
Forcing tissue
through sieve with
syringe piston
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
Cell Type
Tissue Source
Reference
Fibroblast
Skin, embryonic
Brain Cells
Brain, embryonic
Brain, post hatch
Pituitary cell
Liver cell
Intestine
Pancreatic cells
Kidney cell
Kidney, embryonic
Thymic cell
Bursal cell
Spleen cell
Hurk, 1990;
Leukocyte
Hurk, 1990;
Hurk, 1990;
Macrophage
Peritoneal exudate
Adipose cell
Cartilage cell
Tendon cell
Toes, embryonic
Bone Cells
_Osteocyte
_Osteoblast
_Osteoclast
Tibiotarsi, embryonic
Calvaria, post hatch
Medulary bone, past hatch
Muscle cell
Adrenocartical cell
Endothelial cell
Fat pads
Testicular cell
Testis, embryonic
Granulosa
Oviduct cell
Fundamental
Diagnostics-isolation
and identification of
agents, diseases,
mechanism
Applied Research
Bioreactor-production of
biological reagents
(monoclonal antibodies)
Tissue engineering
(regenerative
medicine)
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
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
These monolayers
contained differentiated
epithelial cells that
displayed robust
transepithelial electrical
resistance.
WHY NEED
PRIMARY
CULTURE ??
2
3
4
Acquisition of tumor