BIOL2264…

LECTURE 3

Applications of Tissue Culture technology

 In this week and next lecture…

o Drug Discovery & In vitro Toxicity

o Stem Cells

o Monoclonal antibody production and hybridoma technology

o Vaccine production

o Recombinant protein production

o Artificial organs, transplantation , regenerative medicine

o Nanobiotechnology and nanomedicine

Stem Cells
Potency
 This cell
Can form the
Embryo and placenta Cells from early (1-3 days) embryos

This cell
Can just form the
Embryo and germ layers Some cells of blastocyst (5 to 14 days)

Kinds of
Stem
Cells Fetal tissue, cord blood,
and adult stem cells
Fully mature
 Stem Cell – Definition
A cell that has the ability to continuously divide (self renew) and differentiate (develop) into various
other kind(s) of cells/tissues

All stem cells—regardless of their source have

3 distinct properties

• they are capable of dividing and renewing themselves for long periods

• they are unspecialized

• they can give rise to specialized cell types

 Stem Cell – Types

1. Embryonic
2. Adult or Somatic
3. Induced pluripotent (Reprogramed genetically
from adults cells)
Embryonic Stem Cell Culture

Embryonic Stem cells are grown over Feeder layer

What laboratory tests are used to identify embryonic stem cells?

At various points during the process of generating embryonic stem cell lines,
researchers test the cells to see whether they exhibit the fundamental properties that
make them embryonic stem cells. This process is called characterization.
1. Growing and subculturing the stem cells for many months in undifferentiated
state (microscopic observation)

2. Using specific techniques to determine the presence of transcription factors that

are typically produced by undifferentiated cells (Oct4 and Nanog)

3. Examining the chromosomes under a microscope. This is a method to assess

whether the chromosomes are damaged or if the number of chromosomes has
changed. It does not detect genetic mutations in the cells.

4. Cryopreservation and revival

5. Testing whether the human embryonic stem cells are pluripotent by

1. allowing the cells to differentiate spontaneously in cell culture;
2. manipulating the cells so they will differentiate to form cells
characteristic of the three germ layers;
3. injecting the cells into a mouse with a suppressed immune system to
test for the formation of a benign tumour called a teratoma.
 Adult stem cells

Adult stem cells typically generate the cell types of the

tissue in which they reside

For example, a blood-forming adult stem cell in the bone

marrow normally gives rise to the many types of blood cells.
It is generally accepted that a blood-forming cell in the bone
marrow—which is called a hematopoietic stem cell—cannot
give rise to the cells of a very different tissue, such as nerve
cells in the brain.

The adult stem cell can renew itself and can differentiate to yield some or all of the
major specialized cell types of the tissue or organ.
Where are adult stem cells found, and what do they normally do?

• Skin
• Fat Cells
• Bone marrow
• Peripheral blood
• Brain
• Many other organs & tissues

They are thought to reside in a specific area of each tissue (called a "stem cell niche")

Stem cells may remain quiescent (non-dividing) for long periods of time until they are
activated by a normal need for more cells to maintain tissues, or by disease or tissue
injury.
What are the key questions about adult stem cells research?

1. How many kinds of adult stem cells exist, and in which tissues do they
exist?
2. How do adult stem cells evolve during development and how are they
maintained in the adult? Are they "leftover" embryonic stem cells, or do
they arise in some other way?
3. Why do stem cells remain in an undifferentiated state when all the cells
around them have differentiated? What are the characteristics of their
“niche” that controls their behavior?
4. Do adult stem cells have the capacity to transdifferentiate, and is it possible
to control this process to improve its reliability and efficiency?
5. If the beneficial effect of adult stem cell transplantation is a trophic effect,
what are the mechanisms? Is donor cell-recipient cell contact required,
secretion of factors by the donor cell, or both?
6. What are the factors that control adult stem cell proliferation and
differentiation?
7. What are the factors that stimulate stem cells to relocate to sites of injury or
damage, and how can this process be enhanced for better healing?
What laboratory tests are used to identify adult stem cells?

At various points during the process of culturing and using adult stem cells,
researchers test the cells to see whether they exhibit the fundamental properties that
make them adult stem cells.

(1) label the cells in a living tissue with molecular markers and then determine the
specialized cell types they generate;

(1) remove the cells from a living animal, label them in cell culture, and transplant
them back into another animal to determine whether the cells replace (or
"repopulate") their tissue of origin.
What is known about adult stem cell differentiation?

http://stemcells.nih.gov/StaticResources/images/figure2_lg.jpg
http://www.stem-cell-marker.com/
Isolation and culture of Haematopoietic Stem Cells from Mouse Bone Marrow

http://www.jove.com/video/1026/the-preparation-primary-hematopoietic-cell-
cultures-from-murine-bone

Isoltation of Mesenchymal Stem Cells from Mouse Bone

https://www.youtube.com/watch?v=Dnvq_wMZ2fI
 Stem Cells in Regenerative
Medicine
• Tissue regrowth and future potentials
• Restoring sight to the blind
 Vaccine production
• A vaccine is a biological preparation.

• Used to produce or improve immunity against a particular disease.

• By inoculating killed or weakened disease-causing

microorganisms (or crucial fragments, products or derivatives) the
production of antibodies is stimulated.

• If and when our immune system encounters the disease-causing

microorganism, it then itself prevents the disease through reacting
rapidly and effectively.
Immunity is the body’s successful
defence against a pathogen Secondary immune
response,
rapid and stronger
Antibody response

Primary immune response,

slow to build up
and not very strong

Memory cells remain

Time

Vaccination Infection by pathogen

 Types of Vaccine
Depending on the disease-causing agent, on how it infects the cell and on how the immune
system responds to it, scientists decide the best approach to design a vaccine.

1.Live-attenuated (weakened) vaccines

• The varicella-zoster vaccine,
• Oral poliovirus (OPV) vaccine,
• Yellow fever virus vaccine
• Cowpox (heterologus)

2.Killed-inactivated vaccines
• Inactivated poliovirus (IPV) vaccine,
• Pertussis vaccine,
• Rabies vaccine,
• Hepatitis A virus vaccine

3. Sub-unit vaccines
• Haemophilus influenzae type B
• Acellular pertussis vaccine
• Toxoids
Cell Cultures for Vaccine Production

Cytopathic effect

The Parkinson's Institute and Clinical Center (2012)

 Cell Cultures are used either for
propagation or attenuation or
combination of both for pathogen
Cell Cultures for Vaccine Production

The Parkinson's Institute and Clinical Center (2012)

Monoclonal antibody production
and hybridoma technology
 What is MAb ?
Antigenic determinants

B
Cell

Antibodies in blood= Polyclonal

 What is hybridoma ?

Antibody Producing cells : B Cells

(limited life span)

Antibody producing hybridoma

Genetically engineered myloma cells

(indefinite life span)
 Steps Involved

– Immunisation
– Fusion
– Selection/ Screening
– Cloning
– Expansion
Monoclonal antibody production and
hybridoma technology
The HAT selection
Aminopterin

DNA Synthesis
De Novo pathway

Salvage pathway

HGPRT
Hypoxanthine
TK TK-
Thymidine
Recombinant protein production
Cell Cultures for Material Science
Nanobiotechnology and nanomedicine
 Goal !!!
Design and Development of Cancer Theranostic Agents with following attributes…

Biocompatible

Aqueous

Non-immunogenic Proteins and peptides humanized proteins, Nucleic acids and Small Molecules

Room temperature synthesis

Less or NO byproduct generation (Environment Friendly)

Readily injectable kit formulation (Clinician Friendly)

Ease of application through currently available clinical procedures

Cell and tumor specific toxicity/ targeting capabilities

Low cost

Multifunctional (Single agent for diagnosis, therapy and response to therapy)

Less regulatory issues (Complexity is proportional to scrutiny and rigorous characterizations for FDA approval)

Ease of specific radioactivity manipulations

School of Applied Sciences CAMIC-NanoBiotechnology Research Laboratory (NBRL) RMIT University

 Cell Biology Meets Nanomaterials

Biocompatibility of Gold Nanoparticles and Their Endocytotic Fate Inside the Cellular
Compartment: A Microscopic Overview

Untreated
Nuclear region 50Site of Active Nanomaterial Endocytosis
µM AuNP 100 µM AuNP
Peri-nuclear region

Nucleus Nucleus
Un

Macrophage cells

• The synthesized AuNP are spherical, 3±1 nM in size, polycrystalline and showed absorbance maxima at 530 nM.

• Gold nanoparticles are non-cytotoxic at high concentrations on a variety of cell lines and primary cultures

• Gold nanoparticles do not adversely effect actin cytoskeleton and also do not hamper islet functionality indicating their bio-compatibility

• Gold nanoparticles do not induce ROS and NO indicating its non – cytotoxicity and non induction of pro-inflammatory cytokines

• Gold nanoparticles exposure does not lead to secretion of proinflammatory cytokines

• Gold nanoparticles are internalized in the cells and remain sequestered in the extra-nuclear region

Langmuir, 2005, 21 (23), pp 10644–10654

School of Applied Sciences & Health Innovation Research Institute

 Understanding Material-Cell-Interactions and Nanoparticles
surface corona

Cells/ Animal Biomaterials

Nanomaterials
Models

• Stability • Cellular phenotype and function • Strength

Research
• AggregationQuest : • Cellular adherence and motility • Modification
• Ligand exchange and • Cell differentiation • Composition
Can We Regulate Cellular
surface modification
Responses
• Endocytosis (mode and extent) Through Materials ?
• Leaching
• Subcellular localization
• Subcellular signalling
• Cell cycle progression
• Cellular toxicity and apoptosis
• Biodistribution
• Targeting**
Applications: NANO MEDICINE
 Targeted Drug delivery, Imaging and Therapy  Microencapsulation and artificial organs
 Biocompatible materials and surfaces  Stem Cells Differentiation

School of Applied Sciences CAMIC-NanoBiotechnology Research Laboratory (NBRL) RMIT University

 EGCG Conjugated Theranostic Nanoparticles
Clinical CT Image In vivo Tumor Radiotherapy

NaAuCl4
Water

EGCG

Green Tea

L Change in Hounsfield UnitsAqua regia

ΔHU
Change in Hounsfield Units Δ HU
Neutrons

150
2 min
100
50 25 oC
0
25 50 100198AuFoil
Au-Foil 250 198Au Carrier Au EGCG EGCG-198AuNP
EGCG-AuNP
EGCG-AuNP
Laminin Endocytosis
Treatment
67 Receptors takes
(µg/mL)
mediated place through
selective Laminin
uptake of 67 Receptors
biocompatible overexpressed
EGCG-AuNP in
makes them
prospective Imaging and therapy Prostate
agentsCancer Cells
for clinical management of Prostate Cancer
Kit Components Shukla et al. 2012 Proc. Natl. Acad. Sci. USA
US Patent Pending

RMIT University©2014 School of Applied Sciences & Health Innovation Research Institute 40
Material Science applications

Epithelial Cells

The goal of this exercise is to be able to study, in a controlled way, the

behaviours of individual cells as well as groups of cells and their
interactions.

Questions:
• Can these patterns on transparent substrate be analysed by Phase
Contrast Microscopy ? (Subject to thickness (a) and spacing (s))

• Is there specific pattern cells follow to grow on these patterns ?

(Subject to thickness (a) and spacing (s))

• Toxicity issues of polymer/Metal/ patterns ?

Pattern Cells on Pattern
• Can the cells grown on patterns be analysed by confocal microscopy ?

Actin Vinculin Nucleus

RMIT University©2012 School of Applied Sciences & Health Innovation Research Institute 41
 Molecular Biosensing …
A biosensor is a device that - in some way - makes use of a biological detection system.
Biosensing is the process of using biosensors to gather information about living
systems.

http://iysn.org/2012/06/04/new-wonderkid-on-the-block-nanobiotechnology/

• Molecular recognition element

(Enzyme, Antibodies, receptors, cells, membranes, tissues, organisms, nucleic acids, organic molecules)

• Signal transducer
(Electrochemical, Optical, Calorimetric, Acoustic)
Colorimetric Biosensing Platform
• nanozyme activity of gold nanoparticles for kanamycin

Limit of detection (LoD): 1.49 nM

Limit of quantification (LoQ): 4.52 nM
Linearity: 1–100 nM
Dynamic range: 1–100 nM
Precision: 95.7%
Accuracy: 90% accuracy at 5%
confidence level and
95% accuracy at
10% confidence level

Shukla and Coworkers, Chem. Commun., 2014, 50, 15826