Topic:

Introduction to Biomedical Reaearch

Content of lecture:
The importance of animal model and cell culture in
Basic Medical Research

Prof. Dr. Supargiyono DTM&H, PhD, SpParK.

Department of Parasitology Faculty of Medicine,
Gadjah Mada University
Learning objective:

• Student have a broader view on the important

of animal model and cell culture in conducting
Basic Medical Research
 Biomedical Research
• Biomedical research (= experimental medicine= Medical Research,
• The Scope of Medical Research:
– basic research,
– applied research, and
– translational research
• Basic research (to study basic mechanism of certain activity)
– The strength of the binding of “C” and some protein molecules
– Changes of FC-R expression on M@ by LPS
• Translational (to explain feedbackloop from basic to clinic)
– The effect of different C concentration on the movement protein accross
cell membrane
– Changes of phagocytosis activity of mouse M@ injected with MTB extract
• Applied (study of aplication of certain teory on real life/clinic/com
– The effect of different dose of norit on diarhe patient
– The effect of curcumin consumption on the frequency of illnes
Medical research can also be divided into two general
categories:
– Clinical research: directly contribut to clinical situation
Evaluation the efficacy and safety of new
treatments/procedure
– Pre-clinical research:
mostly basic research , that contributes to the development
of new treatments or new clinical procedure
– A new paradigm: translational research,
• focuses on iterative feedback loops between the basic
and clinical research domains
• to accelerate knowledge translation from the bench to
bedside , and back again.
• The place where medical research be done:
– Hospital/clinical setting :
• Tx of infectious diseases (TB/HIV/thyphoid/Dengue, etc;
• non-infectious , : application of new technique in surgery, diabetes,
cardiovascular, etc.
– Community (public health):
• prevention of malaria outbreack  effect of LLIN distribution  applied
• Bionomic of Anopheles mosquito (breeding places, resting habit, feeding habit,
 translation
– Laboratory: biochemistry, microbiology, physiology, Parasitology  basic
Phagocytosis activity ; cytokine production; cell proliferation  basic

• The success of
• vaccines development for measles and polio,
Contribution of
• Tx of Diabetes with insulin,
• discovery of some antibiotics,
• Tx for high blood pressure, and for
basic to applied research cancer,
 Animal models for human diseases.
Animal Models are essential to study human diseases (dificult,
unethical to be studied in human)
– to study the diseases mechanisms (patophysiology)
– to developes: diagnostic tools, therapeutic and preventive
interventions.
Can be used small or large animal
• The relevance of small animal (mice)
– to natural human in vivo physiologic and metabolic kinetics
remains unclear.
• Large animal : most common: non-human Primates
 may provide more appropriate preclinical models
 closely reflect human physiological characteristics and
behavior.
• the best : has a closer phylogenetic relationship to
humans.
 Pathologic models in monkey :

1. Spontaneous disease model :

 occure from the breeding colony
2. Development of experimental animal models
for chronic diseases.
3. Infectious diseases models in the highly
controlled biosafety laboratory facility.
1. Spontaneous diseases model in monkeys:

• Spontaneous diseases (resemble human diseases) may

develop during breeding and colonisation of animal. .

• There are also several age-related or congenital disorder that

quite similar to human diseases.

• Using those spontaneous animal models:

– enable to investigate:
• the mechanism of age-related or congenital diseases,
and
• establishment of therapy.
 Post-menopausal osteoporosis
• Sexually mature
cynomolgus monkeys
show menstrual cycles
and menopause similar to
those in humans.
• Bone mass and density 
can be measured to
determine post-
menopausal osteoporosis
 to study prevention or
treatment methods for
osteoporosis in humans.
 Diabetes mellitus
• Some monkeys can showed
type II diabetes.
• Progression of the symptoms
of this disease was similar to
that in humans.

• As shown in the figure, a

diabetic monkey gradually
became obese over several
years. Then, suddenly they lost
weight rapidly, and at the same
time their blood glucose levels
significantly increased.
 Alzheimer’s disease
• In aged cynomolgus monkey
brains, we can find
spontaneous senile plaque
formation: one of the major
histopathological features in
Alzheimer’s disease.
• Thus cynomolgus monkey is
considered as an useful
animal model for
Alzheimer’s disease and
other aging-related
neurodegenerative
disorders.
Hyperlipemia
(Genetic diseases)
• Some monkeys can showed
hyperlipemia even in strict
nutritionally controlled colony.
• A genetic diseases gene
that control energy production
in adipocyte tissue
• An obese monkey and lacteal
serum collected from this
monkey are shown in this
figure.
Development of experimental animal
model of human diseases
 Hepatitis C virus
• Hepatitis C virus (HCV) is known
to infect to human and
chimpanzee
• However, chimpanzees are
endangered and are hardly
available, and if possible, the
use is extraordinarily expensive.
• To break through the difficulty,
now can be used a surrogate
(substitute) model of HCV
infection using tamarins and
marmosets
Fig: Marmoset.
 Parkinson’s disease
• A model of Parkinson’s disease in
cynomolgus monkeys can be
developed by chronic injection of
MPTP (1-metil-4-phenil-1,2,3,6-
tetrahydropyridine).
• MPTP monkeys show rigidity of
limbs, bradykinesia, facial
expressionless (see figure), and
severe tremors in their limbs.
• When tremors in the forelimbs
become severe, MPTP monkeys
have trouble gripping their food
and thus sometimes drop it.
 Hemodyscrasia
(Hematopoietic disorders)
• Cytokine-mobilized
peripheral blood stem cells
are widely used for
autologous & allogeneic
transplantation therapies to
treat hematological
malignancies (leukemia and
lymphoma).

• safe and efficient method

has been established for
collecting peripheral blood
stem cells (leukapheresis) in
monkeys and human
 Laboratory Mice for genetic studies

• The DNA (genes) are similar to those of humans,

(evolutionary distance of 75 million years to human).
• Very handy:
– mice are small,
– fecund: female mouse in two months can
produce10 babies.
– Mice live only 2-3 years, allowing researchers to
follow disease processes from beginning to end in
a relatively short time.
– Mouse genes : prize tools for studying human
genetic disease .
Molecular techniques to study genetic
diseases
• There are technique to create gene
mutationcreated by:
• Inserting foreign genes into its embryos,
"transgenic" animals, the cells follow the
instructions
- the interloper genes as well as
- the ancestral genes.
• The advantage gene manipulation techniques:
 gene theraphy, DNA vaccine, etc
Example of transgenic mouse: Giant mice

• A giant mouse (left)

grown from an
embryo inserted
with rat growth-
hormone genes,
weighs nearly twice
as much as its
normal sibling.
 Laboratory Cell culture/tissue culture

• Definition : Tissue culture: propagation or culture of cell

originated from tissue or organ in culture medium (within a
culture vessel) in the laboratory
• Primary culture : propagation or culture of intact or dissociated
tissue directly from organ fragment.
• A culture is considered as primary culture until it is sub-cultured
(or passaged).
• Sub-culture (passage) : to transfer or transplant cells from an
ongoing culture to a new culture vessel, so as to propagate the
cell population or set up replicate culture for study.
Primary subculture
Laboratory cell culture can be used
• To study the effect of: extract, metabolites,
synthetic protein, new drugs, certain infection,
etc (invivo/invitro) on :
– Cell morphology
– Cell activities (phagocytosis, cytokine secretion,
etc.
– Cell proliferation
– Cell migration
– etc
Animal cell usually used for experiment:

HEPAR

LIEN

Peritoneal macrophages
LIEN
Isolation of human macrophages
Culture of peritoneal cells
Cell activities:
- Phagocytosis
- Reactive Oxygen
Intermediate (ROI)
Cell line/Clone cell

• Cell HeLa (cervical cancer)

• Myeloma cell line (human multiple myeloma)
• T47D cell line (human breast cancer)
• L929 & 3T3 cell line (mouse fibroblast)
• C6/36 cell line (Ae. Albopictus mosquito)
• A549 cell line (Lung cancer)
• HT-29 cell line (Adeno Ca Colorectal)
• HepG2 cell line (Liver Hepatocellular Ca)
Hybridoma technology

short live

Myeloma cells

Produce Ab
Immortal
Production of monoclonal antibody using cell culture
 Basic components of cell culture environment

• To promote cell survival & proliferation, the in vitro

environment must meet the fundamental physiological
requirements of the cells.
• Component of culture environment
– factors associated with medium,
• nutrient composition,
• pH,
• osmolality,
• the volume &
• frequency of media replenishment.
– Other factor that also can be controlled :
• incubation temperature,
• gas composition &
• relative humidity.
• Culture medium :
– is a growth media to establish condition to support optimal
cell proliferation.
– Culture medium must provide essential nutrient, vitamin,
metabolic substrate, amino acid.
– Essential component:
• Fetal Calf Serum/Human Serum : 10%
• Optimal condition of mammalian cells culture
environment:
– pH : 7,3 – 7,5
– Osmolality: 280-320 mOsmol/kg
– CO2 : 2 – 5% in air
– Temperature 35 – 37oC
Some exception do occur
All cell manipulation for culture must be
performed in sterile condition

•In modern Laboratory sterile handling

cell culture is carried out in a laminar
air flow cabinet or ‘hoods’

 - Clean bench
- Biosafety Cabinet/Purifier Class 2
Clean Bench

Non-infectious
Biosafety cabinet Class II
 Refference
• Culture of Animal Cells: A manual of
Basic Technique and Specialized
Application.
Outhor: R. Ian Freshney.
Sixth edition 2007. Blackwell Scientific
Publication, Oxford, England.