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Laboratory experimental

research
Evy Sulistyoningrum
Histology department
Outlines
Introduction
Animal experimental
Culture experimental
Introduction
Laboratory experimental research : biomedical
research
In vivo : animal study
In vitro culture study
Confounding variables were strictly controlled
Research subject:
Certain Animals
Certain cell/tissue/organism culture
Pre-clinical trial
Steps in biomedical research
1. Choose the appropriate subject
2. Choose research design models
3. Sample size
4. Animal/sample welfare guarantee
5. Treatments/intervention
6. Data Collection
7. Analysis


Subjects
Animals
Custom animal
Animal model for human condition
Genetically modified animal
Human cells/ cell model for human cells
Pathogens
Microbes
Paracytes
Fungus, etc



Research designs
Post test-only with control group
Pre and post test with control group
Post test-only without control
Pre and post test with control group
Complete Randomized Design

Control?
No treatment at all
Disease model
Gold standard
Post test-only with control group
scheme
T : (X) O1
C : (-) O2


T : treatment group
C : Control group
(X) : given treatment
(-) : no treatment or given placebo
O : observation in treated group
Pre and post test with control group
scheme
T : O1 (X) O3

C : O2 (-) O4

T : treatment group
C : Control group
(X) : given treatment
(-) : no treatment or given placebo
O : observation
Simple Complete randomized design
N animals
C
(-)
O
A
Xa
O
B
Xb
O
C
Xc
O
C : Control group (-) : no treatment or given placebo

(X) : given treatment O : observation
Pre and post test CRD
N animals
C
O1
(-)
O2
A
O1
Xa
O2
B
O1
Xb
O2
C
O1
Xc
O2
C : Control group (-) : no treatment or given placebo
(X) : given treatment O 1 : observation before treatment
O2 : observation after treatment
Sample size
Animal
Federer : (n-1)(t-1) > 15
WHO
Minimal number Reduction
Cells
Literature based
Pathogens
Literature based
Treatments
Variant depend on research intention
Route based on research intention
Dosing :
Toxicology : LD50 , etc
Other dosing: literature-based
Human dose need conversion

Data collection
Based on research design
Based on data specification
Source:
Animal behaviors
Parameters of animal specimen
Animal tissue
Specific products
Can be measured research variables
Types of Observations
Activity Level
e.g., hypoactivity, hyperactivity, restlessness, lack of
inquisitiveness

Attitude
e.g., arousal, depression, awareness of surroundings

Behavior, Spontaneous
e.g., vocalization, self-trauma, isolation from cage mates
without disturbing the animal

Behavior, Provoked
e.g., vocalization, hiding, aggressiveness, minimal response
with disturbing the animal
Types of Observations
Body Condition
e.g., missing anatomy

Food and Fluid Intake
e.g., elimination of feces and urine

Skin
e.g., cyanotic, pale, or congested mucous
membranes or skin (ears, feet, tail); skin lesions

Eyes
e.g., clarity/condition of lens, cornea; position of
globe; condition of eyelids, encrustation

Posture
e.g., hunched back, tucked abdomen; prostrate;
head tucked down
Types of Observations
Locomotion
e.g., gait, ataxia, action of each limb, position of tail when ambulating

Neurological
e.g., tremor, convulsion, circling, paralysis, head tilt, coma

Vital Signs
e.g., respiratory distress (open mouth breathing, pronounced chest
movement)

Other clinical parameters that are relevant
e.g., presence and status of tumors, infection, or surgical wounds

Examination
Behavior
Body weight
Surface lesions (wounds, masses)
Hydration status
Body temperature (telemetric methods)

Blood parameters
Other specimens parameters

Tissue specimen


Variables & Analysis
Variables
Based on research designs and observation needed
Scales based on research preference
Specimen source must be stated
International unit or other common measurement units


Analysis
Based on research objective
Based on variables scales
Descriptive

Examples
Nodul size
Blood level of AST, ALT, ALP reflect liver
function
Blood level of ureum, creatinin reflect renal
function
Fasting glucose level, HbA1c
Inhibition zone reflect antibacterial activity
Balooning degeneration
Johnsons criteria for spermatogenic level


Animal experiments
Why perform animal experiments?
Some experiments cannot be performed on humans
or better performed on animals
Organs and body systems similar to humans and
other animals
Susceptible to the same diseases that affect humans
Short life span allows to be studied throughout
entire life
Environment easily controllable to keep
experimental variables to a minimum
Benefits of Animal Research
Penicillin
Mice

Blood Transfusions
Dogs

Tuberculosis Medicine
Guinea pigs

Meningitis Vaccine
Mice

Kidney Transplants
Dogs and Pigs
Breast Cancer Treatments
Mice, Rats and Dogs

Asthma Inhalers
Guinea Pigs and Rabbits

Polio Vaccine
Mice

Insulin for Diabetics
Dogs

Deep Brain Stimulation for
Parkinson's Disease
Monkeys
Benefits Continued
Vaccine for Smallpox
Vaccine for Anthrax
Rabies Vaccine
Typhoid Vaccine
Cholera Vaccine
Treatment for Beriberi
Treatment for Rickets
Corneal Transplants
Local Anaesthetics
Discovery of Vitamin C
Canine Distemper Vaccine
Coronary Bypass Operation
German Measles Vaccine
MMR Vaccine
Antidepressants and Antipsychotic
CT Scanning for Improved Diagnosis
Chemotherapy for Leukaemia
Medicines to Treat Ulcers
Inhaled Asthma Medication
Combined Therapy for HIV infection
Medicines for Type 2 Diabetes
Cervical Caner Antibodies
Avian Flu Vaccine
Malaria Vaccine
Modern Anaesthetics
Tetanus Vaccine
Diphtheria Vaccine
Anticoagulants
Streptomycin
Kidney Dialysis
Whooping cough Vaccine
Heart Lung Machine
Hip replacements
Cardiac Pacemakers
High Blood Pressure Medicines
Replacements of Heart Valves
Chlorpromazine Psychiatric Medicine
MRI Scanning for improved Diagnosis
Prenatal Corticosteroids for Premature Babies
Treatment for River Blindness
Life Support for premature Babies
Medicines to control Transplant Rejection
Hepatitis B Vaccine
Leprosy Treatment
Oral and Inhaled Insulin for Type 1 Diabetes
Angiogenesis Inhibitors for Cancer and Blindness
Gene Therapy for Muscular Dystrophy
Alzheimers Vaccine
Limitations of animal experimentation
Species differences
Some symptoms are hard to discover in animals
In some cases, genetically modified animals is
better
discovery of gene functions
treatment and knowledge of genetic disease
minimisation of rejection following
xenotransplantation
development and production of therapeutic
protections

Hazards of Working with animal


Injuries
Allergies
Zoonoses

Two Major Points of View
(1) Animal Rights - ending all animal use
no food, clothing, entertainment, medical research or hunting

(2) Animal Welfare - animals must be treated and
used humanely

Rules for animal experiments
Animal Welfare Act, 1966 [USC Title 7, Sections 2131 to 2156] as amended in
1970, 1976, 1985 and 1990.
Animal Welfare Regulations [Title 9 CFR, Subchapter A, Animal Welfare, Parts 1, 2
and 3]
Health Research Extension Act, 1985 [Public Law 99-158, November 20, 1985,
Section 495]
US Government Principles for the Utilization and Care of Vertebrate Animals Used
in Testing, Research, and Training, 1985
PHS Policy on Humane Care and Use of Laboratory Animals, 1986
2000 Report of the AVMA Panel on Euthanasia [JAVMA, Vol. 218, No. 5, March 1,
2001]
Guide for the Care and Use of Laboratory Animals (Guide) [NRC, 5th Ed., 1996]
NIH Grants Policy Statement (03/01), Part II: Terms and Conditions of NIH Grant
Awards Subpart A: General -- Part 2 of 7
The 3 Rs
Russel & Burch (1959):
(a) Refinement: Improve the experiments for
minimising animal pain and suffering
(b) Reduction: Use statistical methods so that a
smaller number of animals are required
(c) Replacement: Use alternative, non-animal methods to achieve
the same scientific aim

Refinements
Proper animal living conditions
Qualified and trained personnel
Medical care available and provided by a qualified
veterinarian
Procedures avoid or minimize discomfort, distress, and
pain
Procedures that may cause pain or distress :
Analgesia unless well justified
Unrelieved pain euthanize
Surgical procedures performed aseptically and
appropriate
Animals not used in more than one major, survival
operative procedure
Acceptable methods of euthanasia
Examples of Refinement
Reduction
Animal Numbers : minimum possible to achieve
statistically significant data and are well justified :
Citation of previous research: sufficient information to
indicate that the previous research is similar enough in
concept and methodology
Power analysis: enough information to show ability in
analyzing data and using a power analysis
If need animal specimen: state clearly how many
amount of material is needed
Replacement
In Vitro Testing











Computer Modelling

MRI Scanning








Micro dosing
Animal models
Animal that biologically or habitually normative, can
be spontaneously or patogenetically investigated,
and human phenomenon mimicking

Mice: most frequently used (mice + rodents :+ 90 %)
Other rodents:
Rats
Guinea pig
Hamster etc

Dogs, cats, rabbits, farm animals, fish, frogs, birds,
nonhuman primates, etc : 10%
Animal models
Human condition mimicking:
Diabetes
Dislipidemic
Hepatotoxic
Ulcers
Cancers

Dosing
Literature based
Conversion from human dose




Animal handling
Acclimation & sexing
Acclimation
Period of time allows animals to adapt to a new
environment biological stabilization

Effects of transportation stress :
various blood parameters
food intake and animal behavior

Period of time necessary : depend on the
parameters to be studied.

Sexing
Genital anatomy
Sexing

Sexing
scrotum
Mouse Pups
1 day old mice 2 days old pups day 3
4 days old pups
6 days old pups
5 days old pups
and more pups
Day 7 Day 8
Day 9
Days 12,13 and 14
Day 11 Day 10
Blood Collection from Tail
TOOLS
Alcohol cotton ball for
surface disinfection
Small plastic bottle
with 1/2 cm diameter
holes in both ends as
mouse restrainer
Scissors
Micropipette and tips
A vial for blood
collection
For collection of small amount of
blood (Approximate 0.1 ml )
Leave the tail of the mouse
outside the cover of the
restrainer
Amputate the tip of the
mouse tail by scissors
Massage the tail and collect blood by pipette
Blood Collection from Orbital Sinus in
Mouse
TOOLS
Alcohol cotton ball for
surface disinfection
Kloroform for general
anesthetic
27 G needle with 1 ml
syringe for injection
Glass capillary tube
and vial for blood
collection
Anesthetic before blood withdraw
Collect amount up to 0.5 ml
Anesthetize a mouse by
intraperitoneal injection
Use a glass capillary tube to
penetrate the orbital conjunctiva
and rupture the orbital sinus
Collect blood with a vial
Cardiac puncture in Mouse
TOOLS
Alcohol cotton ball for
surface disinfection
Chloroform used as
anesthetic
27G needle with 1 ml
syringe for injection
24G needle with 3 ml
syringe for blood
withdraw
For collect up to 1 ml of blood within a short
period of time
Must be performed under general anesthetic
Anesthetize a mouse by
intraperitoneal injection
Disinfect the thorax area
with 75% alcohol cotton
ball
Search for the maximum
heart palpitation
Cardiac puncture in 90
angle
Withdraw blood slowly by right hand
Blood collection from Saphenous vein in mice
TOOLS
Alcohol cotton ball
for surface
disinfection
50 ml syringe tube
with small holes at
the end as
restrainer
a scalpel and shaver
for remove of hair
24 G 1 needle for
release of blood
tips and pipette for
blood collection

Multiple samples taken in the course of a
day
Place the mouse in the
restainer
Pull out the leg and
removed the hair by a
assistant
Hair also be shaved by
using a small scalpel
The saphenous vein
is seen on the surface
of the thigh
Apply vaseline to the
surface area to reduce
clotting and coagulation
during blood collection
The saphenous vein
is ruptured by scalpel
Blood is aspirated
through pipette
Injection in Mouse
TOOLS
Alcohol
cotton ball
for surface
disinfection
25G 1/2
needle with
1 ml syringe
for injection
Handle a mouse
properly
The injection site : the lower left quadrant of the abdomen
because vital organs are absent from this area. Only the tip of
the needle should penetrate the abdominal wall to prevent
injection into the intestine.
Intraperitoneal injection
Pick up a nude mouse and spin its tail
Subcutaneous injection
Oral Feeding in Mouse
TOOLS
A 18 G
stainless
steel, ball
tipped
needle
a glove
Gastric intubation ensures that all the material was
administered
Feeding amount limited to 1% of body weight
Gastric intubation
Dissection
Genetically modified animal
Transgenic mice
Transgenic: an organism that has had DNA
introduced into one or more of its cells artificially
transgenic: DNA is integrated in a random
fashion by injecting it into the pronucleus of a
fertilized ovum
Random (+ 10% disrupt an endogenous gene important
for normal development)
multiple copies

Knock-out mice
knockout: DNA is introduced first into embryonic
stem (ES) cells
ES cells that have undergone homologous
recombination are identified and injected into a 4 day
old mouse embryo - a blastocyst
targeted insertion


Pronuclear injection in transgenic
technology
Implanting 1(or 2) cell embryos

(cont.)

Transgenic mice

Trangenic mouse embryo in which the promoter for a gene
expressed in neuronal progenitors (neurogenin 1)
drives expression of a beta-galactosidase reporter gene.

Neural structures expressing the reporter transgene are dark
blue-green. (Dr. Anne Calof)
Tail tip
9.5 day embryos -
GFP and wt
GFP transgenic mouse (Nagy)
GFP transgenic mouse (Nagy)
ES cells growing in culture in knock out
technology
Successfully transformed ES cells are
injected into blastocysts
Implanting blastocysts
1
2
Implanting blastocysts (cont.)
3 4
Littermates
Black mouse -
no apparent ES cell
contribution
Chimeric founder -
strong ES cell
contribution

Chimeric founder -
weaker ES cell
contribution
Cell culture
Introduction
Cell culture is the process by which prokaryotic,
eukaryotic or plant cells are grown under controlled
conditions
In practice it refers to the culturing of cells derived
from animal cells.
Cell culture was first successfully undertaken by Ross
Harrison in 1907
Genetic engineering can be done to cells produce
desired substance (recombinant)
Examples of recombinant
products
Viral vaccine
Hybridoma capable of secreting a monoclonal
antibody.
Human insulin recombinant protein
Human growth hormone produced from
recombinant bacteria
Lymphoblastoid IFN
Tissue-type plasminogen activator (tPA) from
recombinant animal cells
Recombinant erythropoietin in trial.
HBsAg, factor VIII, HIVgp120, CD4, GM-CSF, EGF,
mAbs, IL-2

Why is cell culture used for?
Model systems for:
Studying basic cell biology
Interactions between agents and cells
Effects of drugs on cells
Toxicity testing
Study the effects of new drugs on cells
Cancer research
Study the function of various chemicals, virus & radiation
to convert normal cultured cells to cancerous cells
Study of the effects of drugs in cancer cells
Contd.
Virology
Cultivation of virus for vaccine production
Genetic Engineering
Production of commercial proteins
Gene therapy
Cells having a functional gene can be replaced
to cells which are having non-functional gene
Primary culture
Cells when surgically or enzymatically removed
from an organism and placed in suitable culture
environment will attach and grow
Primary culture contains heterogeneous
population of cells sub culturing of primary
cells leads to generation of cell lines
Cell lines have limited life span, they passage
several times before they become senescent

Some Common cell lines
Human cell lines
MCF-7 breast cancer
HL 60 Leukemia
HEK-293 Human embryonic kidney
HeLa Henrietta lacks cervical cells infected by HPV 16
HUVEC human Umbilical Vein Endothelial cells

Primate cell lines
Vero African green monkey kidney epithelial cells
Cos-7 African green monkey kidney cells

Contaminants of cell culture
Chemical:
difficult to detect invisible
caused by endotoxins, plasticizers, metal ions or traces of
disinfectants

Biological:
cause visible effects
Mycoplasma, yeast, bacteria or fungus or cross-
contamination of cells from other cell lines
Basic equipments used in cell culture
Laminar cabinet
Incubation
Refrigerators
Inverted microscope
Tissue culture ware- Culture plastic ware
treated by polystyrene
Pathogen cultures
Similar with cell culture
Larger organism
Proper & specific media
McConkey
Sabouroud
etc
Can be treated with various substance
produce specific effect that can be
measurable

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