CELL CULTURE

Tissue culture

In vitro
cultivation grow tissues
of organs, tissues and cells
outside the body
in an artificial environment
 Types of tissue culture
Organ culture
Entire embryos/organs
Explant culture
Fragments of excised tissue
Physiological functions maintained
Cells remain fully differentiated
Slow growth
Difficult to scale up
Requires fresh explant for every
experiment
Some physiological functions maintained
Possible for scale-up
Original organisation of
tissue is lost
 Types of tissue culture

Cell culture Give rise to cell lines

Dispersed cells from a tissue Possible for scale-up
explant
Cultured as monolayer or
suspension culture
Cells may lose some
differentiated characteristics
Why cell culture?

Controlled experimental environment

Homogenous samples
Less compound/drugs needed for experiments

Difficult to maintain
Only grow a small amount of tissue at high cost
Can dedifferentiate (depend on growth and
maintenance media, population density, etc)
Can undergo senescence
 Purpose of cell culture

Research
Disease modelling
Toxicity studies
Overcome problems in cellular behaviour
Confounding effects (e.g. ec. surrounding tissues)
Reduce animal use

Production of biological materials

Vaccine, monoclonal antibodies, hormones, etc
Gene therapy
General characteristics of animal cell culture

Nutritionally demanding
Sensitive to shear stress and extremes
Doubling time 12-48 hours
Cell density
 Animal cell culture

Primary culture
From excised tissue:
Outgrowth
Dissociated cells (by enzymatic digestion/mechanical
dispersion)
Retain differentiated phenotype
Needs extra work for preparation
Finite life span in vitro (ec. senescence)
Animal cell culture

Secondary culture
From primary culture
More homogenous population
Because isolated by selection
Finite life span in vitro (ec. senescence)
Animal cell culture

Continuous culture
From subculture (or passaging) of primary culture
Sub-culture : process of dispersion and re-culture after cells
having occupied available substrate in the culture
Comprised of a single cell type
Can be propagated several times

Divided into:
Cell lines
Continuous cell lines
Continuous culture

Cell lines
Finite life undergo senescence
Maintain some degree of differentiation
Requires a bank system to maintain lines for
long periods of time
Master bank and working banks
Continuous culture

Continuous cell lines

Immortal
Due to spontaneous mutation into tumor
Due to transformation by viral oncogenes, chemical
treatments
Retain very little of the original in vivo cell characteristics
Cell culture morphology

Suspension culture
Dont need to attach to cell culture vessel
E.g. cells from blood, spleen, bone marrow etc
(+) large numbers easy to harvest

Adherent culture
Monolayer attached to surface
Exhibit contact inhibition
From ectodermal/endodermal embryonic cells (e.g. fibroblasts,
epithelial cells)
Good for microscopy and other functional assays
Basics for cell culture

Laminar flow hood

Incubator
Fridge (-80oC, -196oC (liquid nitrogen))
Inverted microscope
Cell culture flasks/dish/plates
Media
Stem Cells
Stem cells: Unique characteristics

Can differentiate
into multiple cell
types (pluri-/multi-
potent)

Self renewal
while maintaining its
pluri-/multi-potency
Stem cells: why so valuable?

Cell therapy
E.g. Bone marrow transplant
Research
New drugs

Hope for cure / improving

treatments for many diseases!
Stem cells: How does it work?
Stem cells transplanted into the body

Arrive at the injured site

Stem cells come in contact with

growth factors in the body

These chemicals program stem cells to differentiate into

surrounding tissue
 Pluripotent stem cells

Can differentiate into all cell types of the three

germ lineages (i.e. ectoderm, mesoderm and
endoderm).
Safety concerns:
Tumorigenicity, immune rejection
 Pluripotent stem cells:
Embryonic stem cells (ESCs)

Derived from blastocysts

Pluripotent
Can become all cell types EXCEPT placenta
Undergo clonal expansion
Safety concerns:
Tumorigenicity, immune rejection, ethical controversy (from embryo)

vs Totipotent:
after the first few divisions of
fertilized egg
can become all cell types AND
placenta
Toti-potent Pluri-potent ESCs
 Pluripotent stem cells:
induced pluripotent stem cells (iPSCs)

Introduced in 2006 by Takahashi and Yamanaka

From reprogrammed somatic cells by retroviral
infection of mouse skin fibroblasts with four
transcription factors (Oct3/4, Sox2, c-Myc and Klf-4)
Source can be autologous can avoid immune
rejection
Safety concerns:
Tumorigenicity because one of the pluripotency gene is a
cancer gene
Cells may still have genetic defects
Viruses may insert genes in places we dont want them
causing mutation
Adult stem cells

Multipotent
Can become cells that are closely related (limited
number of cell types)
Can self-renew over a life time (until senescence)
Found in various organs and tissues such as:
Umbilical cord, amniotic fluid, bone marrow, adipose
tissue, skeletal muscle and heart tissue
Already used to treat patients (hematological
malignancy, immune system desease)
Usually difficult to isolate, identify and purify
 Adult stem cells:
Umbilical Cord and Amniotic Fluid-derived Stem Cells

Contain a heterogenous population of multipotent stem

cells
E.g. red blood cells, white cells, platelets

Safety concerns:
ethical dilemma as amniotic fluid extraction may endanger
the fetus and mother
immune rejection with allogeneic amniotic fluid stem cells
low yield of umbilical cord stem cells
Embryonic vs Adult stem cells
 Adult stem cells:
Adipose-derived stem cells
Human adipose tissue is readily available in large quantities
Harvesting methods such as liposuction is minimally
invasive and enable a repetitive access to ASCs
 Adult stem cells:
Bone marrow-derived mesenchymal and
hematopoietic stem cells

Most widely exploited stem cell source

Low occurrence in the bone marrow (0.001-0.01%)
Limited proliferative potential
 Adult stem cells:
Cardiac Resident Stem Cells (CRSCs)

Regenerative capability of the heart

9 identified sub-types:
c-Kit+
Isl+
Sca1+,
Cardiac mesoangioblasts
Side population
Epicardium-derived progenitor cells
Cardiac colony forming units-fibroblasts (CFU-F),
Cardiosphere-derived cells (CDC)
W8B2+ CRSCs
Stem cells: applications

Treat baldness
Stem cells from hair follicles (keratinocytes)
Grown into skin for burn patients (autologous skin graft)

Diabetes
hESCs grown in culture stimulate to form insulin-
producing cells transplan back into patient
Stem cells: applications

Treat baldness
Stem cells from hair follicles (keratinocytes)
Grown into skin for burn patients (autologous skin graft)

Diabetes
hESCs grown in culture stimulate to form insulin-
producing cells transplan back into patient
Stem cells: applications

Corneal disease

Parkinson
From bone marrow stem cells grown in culture
inject into brain

Brain damage, kidney, hearing loss, orthopedics,

myocardial infarction

Gene therapy