Reviewer Co1

1 STEM CELL REVIEWER CO1
LECTURE 1: SCOPE OF TISSUE ENGINEERING o growth factors, steroids, hormones,

cytokines, chemokines
Tissue Engineering
bind to membrane receptors usually with high affinity
Tissue Engineering is the in vitro development (low binding constants: 10-100 pM)
(growth) of tissues or organs to replace or support the can diffusion long distances
function of defective or injured body parts.
2. Cell-to-Cell Contact:
Tissue Organization some membrane receptors are adhesive molecules
Before a tissue can be developed in vitro, first we o adherent junctions and desmosomes
other serve to create junctions between adjacent cells
must understand how tissues are organized. The
basic tenant here is that: allowing for direct cytoplasmic communication
all tissues are comprised of several levels of o gap junctions
o 1.5-2 nm diameter and only allow transport
structural hierarchy
of small molecules ~1 kDa
These structural levels exist from the macroscopic
level (centimeter range) all the way down the
molecular level (nanometer range) there can be as 3. Cell-ECM Interactions:
many as 7-10 distinct levels of structural organization ECM is multifunctional and also provides a substrate
in some tissues or organs that cells can communicate
since cells synthesize the ECM, they can modify the
Organization of the Tendon ECM to elicit specific cellular responses
several specialized receptors that allow for cell-ECM
interactions
o integrins, CD44, etc.
o also a mechanism by with cells respond to
external stimuli (mechanical transducers)
Tissue Engineering Scaffolds
1. Biomaterial Scaffolds Materials:
Polymeric
o chitosan, alginate, etc.
Organization of the Kidney o foams, hydrogels, fibres, thin films
natural
o collagen, elastin, fibrin, etc.
o hydrogels
ceramic
o calcium phosphate based for bone tissue
engineering
o porous structures
permanent versus resorbable
o degradation typically by hydrolysis
o must match degradation rate with tissue
growth
2. Chemical and Physical Modifications:
Cellular Communications attachment of growth factors, binding sites for
integrins, etc.
1. Soluble Signals: nanoscale physical features
small proteins (15-20 kDa) which are chemically
stable with long half-lives (unless specifically Culturing of Cells
degraded) Types of Cell Culture
monolayer (adherent cells)
suspension (non-adherent cells) Stem Cells

three-dimensional (scaffolds or templates)
Stem cells naturally exist in some tissues (especially those that
rapidly proliferate or remodel) and are present in the
circulation.
There are two predominant lineages of stem cells:
Sterilization Methods
mesenchymal
ultra-violet light, 70% ethanol, steam autoclave,
o give rise to connective tissues (bone,
gamma irradiation, ethylene oxide gas
cartilage, etc.)
Growth Conditions o although found in some tissues, typically
isolated from bone marrow
simulate physiological environment hematopoietic
o pH 7.4, 37C, 5% CO2, 95% relative o give rise to blood cells and lymphocytes
humidity o isolated from bone marrow, blood (umbilical
o culture (growth) media replenished cord)
periodically
Stem cells are rare; bone marrow typically has:
Culture (Growth) Media
a single mesenchymal stem cell for every 1,000,000
appropriate chemical environment myeloid cells
o pH, osmolality, ionic strength, buffering a single hematopoietic stem cell for every 100,000
agents myeloid cells
appropriate nutritional environment
o nutrients, amino acids, vitamins, minerals, Stem Cells (Mesenchymal)
growth factors, etc.
Cell Sources
Since the ultimate goal of tissue engineering is to develop
replacement tissue (or organs) for individuals, the use of
autologous cells would avoid any potential immunological
complications.
Various classifications of cells used in tissue engineering
applications:
primary cells
o differentiated cells harvested from the patient
(tissue biopsy) Stem Cells (Hematopoietic)
o low cellular yield (can only harvest so much)
o potential age-related problems
passaged cells
o serial expansion of primary cells (can
increase population by 100-1000X)
o tendency to either lose potency or de-
differentiate with too many passages
stem cells
o undifferentiated cells
o self-renewal capability (unlimited?)
o can differentiate into functional cell types
o very rare
Colony-Forming Units (CFUs) d) Perfusion

media flows directly through construct
e) Controlled Mechanics
to apply physiological forces during culture
LECTURE 2: TISSUES
Tissue Structure and Function
Tissue - An aggregation of cells and cell products of similar
structure and embryonic origin that perform a common function
Histology - The study of tissues, especially their structure and
arrangement
Basic Tissue Types
Epithelial
Connective
Muscle
Nervous
Bioreactors
a) Spinner Flask:
semi-controlled fluid shear
Organ- An aggregation of tissues organized into a functional
can produce turbulent eddies which could be
unit
detrimental
System - Organs working together as functional units
b) Rotating Wall
Epithelial Tissue
low shear stresses, high mass transfer rate
can balance forces to stimulate zero gravity Sheet-like layer of cells
Cover surfaces or line cavities
c) Hollow Fibre
Often has glandular cells for fluid secretion
used to enhance mass transfer during the culture of Endothelium
highly metabolic cells o Epithelium that lines heart, blood vessels
and lymphatic vessels
The Study of Tissues

Epithelial Types
200 Different cell types
Simple Epithelium Four primary tissue classes
o Simple squamous inside blood vessels o epithelial tissue
o Simple cuboidal lines ducts, ex. Kidney o connective tissue
tubules, mucous glands o muscular tissue
o Simple columnar lining of small intestine o nervous tissue
o Pseudostratified Columnar trachea, Histology (microscopic anatomy)
bronchi o study of tissues organ formation
Organ = structure with discrete boundaries
o composed of 2 or more tissue types
Stratified Epithelium
o Stratified squamous - skin
o Transitional urinary tract and bladder Features of Tissue Classes
Tissue = similar cells and cell products
Connective Tissue o arose from same region of embryo
Differences between tissue classes
Tissue that connects
o types and functions of cells
o Loose connective tissue
o characteristics of matrix (extracellular
adipose (fat)
material)
o Dense connective tissue
fibrous proteins
cartilage
ground substance
Bone
clear gels (ECF, tissue
o Vascular tissue
fluid, interstitial fluid, tissue
Erythrocytes: Red Blood Cells
gel)
carry oxygen
rubbery or stony in
Leucocytes: White Blood Cells
cartilage or bone
part of the immune system
o space occupied by cells versus matrix
Platelets - clotting
connective tissue cells are widely
Muscle Tissue Types separated
little matrix between epithelial and
Smooth Muscle (=Involuntary Muscle) muscle cells
o Ex. Small intestine
Skeletal Muscel (=Voluntary Muscle)
o Ex. Large muscles of body Embryonic Tissues
Cardiac Muscle
Embryo begins as single cell
Nervous Tissue o divides into many cells and layers (strata)
3 Primary germ layers
Includes Nerves, Spinal Cord, Brain o ectoderm (outer)
Cells are called neurons
forms epidermis and nervous
LECTURE 2B: HISTOLOGY system
o endoderm (inner)
Histology forms mucous membrane lining GI
Study of Tissues tract and respiratory system and
Epithelial Tissue digestive glands
Connective Tissue o mesoderm (middle) becomes mesenchyme
Nervous and Muscular Tissue wispy collagen fibers and
Intercellular Junctions, Glands and Membranes fibroblasts in gel matrix
Tissue Growth, Development, Death and Repair gives rise to muscle, bone, blood
o named by shape of apical cells

Tissue Techniques and Sectioning
Simple Squamous Epithelium
Preparation of histological specimens o Single row of flat cells
o fixative prevents decay (formalin) o Permit diffusion of substances
o sliced into thin sections 1 or 2 cells thick o Secretes serous fluid
o mounted on slides and colored with o Alveoli, glomeruli, endothelium, and serosa
histological stain
stains bind to different cellular Simple Cuboidal Epithelium
components o Single row cube-shaped cells with microvilli
Sectioning reduces 3-dimensional structure to 2- o Absorption and secretion, mucus production
dimensional slice o Liver, thyroid, mammary and saliva glands,
bronchioles, and kidney tubules
Sectioning Solid Objects
Sectioning a cell with a centrally located nucleus Simple Columnar Epithelium
Some slices miss the cell nucleus
Single row tall, narrow cells
In some the nucleus is smaller
o oval nuclei in basal half of cell
Absorption and secretion; mucus secretion
Sectioning Hollow Structures
Lining of GI tract, uterus, kidney and uterine tubes
Cross section of blood vessel, gut, or other tubular
organ.
Pseudostratified Epithelium
Longitudinal section of a sweat gland. Notice what a
single slice could look like. Single row of cells some not reaching free surface
o nuclei give layer stratified look
Types of Tissue Sections Secretes and propels respiratory mucus
Longitudinal section
o tissue cut along longest direction of organ Stratified Epithelia
Cross section More than one layer of cells
o tissue cut perpendicular to length of organ Named for shape of surface cells
Oblique section o exception is transitional epithelium
o tissue cut at angle between cross and Deepest cells on basement membrane
longitudinal section Variations
o keratinized epithelium has surface layer of
Epithelial Tissue dead cells
Layers of closely adhering cells o nonkeratinized epithelium lacks the layer of
Flat sheet with upper surface exposed to the dead cells
environment or an internal body cavity
No blood vessels Keratinized Stratified Squamous
o underlying connective tissue supplies Multilayered epithelium covered with dead squamous
oxygen cells, packed with keratin
Rests on basement membrane o epidermal layer of skin
o thin layer of collagen and adhesive proteins Retards water loss and barrier to organisms
o anchors epithelium to connective tissue
Nonkeratinized Stratified Squamous
Simple Versus Stratified Epithelia
Multilayered surface epithelium forming moist,
Simple epithelium
slippery layer
o contains one layer of cells
Tongue, oral mucosa, esophagus and vagina
o named by shape of cells
Stratified epithelium
o contains more than one layer
Stratified Cuboidal Epithelium

Connective Tissue Ground Substance
Two or more cell layers; surface cells square
Secretes sweat; produces sperm and hormones Gelatinous material between cells
Sweat gland ducts; ovarian follicles and seminiferous o absorbs compressive forces
tubules Consists of 3 classes of large molecules
o glycosaminoglycans chondroitin sulfate
Transitional Epithelium disaccharides that attract sodium
and hold water
Multilayered epithelium surface cells that change from
role in regulating water and
round to flat when stretched
electrolyte balance
o allows for filling of urinary tract
o Proteoglycan (bottlebrush-shaped molecule)
o ureter and bladder
create bonds with cells or
extracellular macromolecules
Connective Tissue
o adhesive glycoproteins
Widely spaced cells separated by fibers and ground protein-carbohydrate complexes
substance bind cell membrane to collagen
Most abundant and variable tissue type outside the cells
Functions
o connects organs Fibrous Connective Tissue Types
o gives support and protection (physical and Loose connective tissue
immune) o gel-like ground substance between cells
o stores energy and produces heat o types
o movement and transport of materials areolar
reticular
Cells of Connective Tissue adipose
Dense connective tissue
Fibroblasts produce fibers and ground substance o fibers fill spaces between cells
Macrophages phagocytize foreign material and o types vary in fiber orientation
activate immune system dense regular connective tissue
o arise from monocytes (WBCs) dense irregular connective tissue
Neutrophils wander in search of bacteria Areolar Tissue
Plasma cells synthesize antibodies
o arise from WBCs Loose arrangement of fibers and cells in abundant
Mast cells secrete ground substance
o heparin inhibits clotting Underlies all epithelia, between muscles,
o histamine that dilates blood vessels passageways for nerves and blood vessels
Adipocytes store triglycerides
Reticular Tissue
Fibers of Connective Tissue
Loose network of reticular fibers and cells
Collagen fibers (white fibers) Forms supportive stroma (framework) for lymphatic
o tough, stretch resistant, yet flexible organs
o tendons, ligaments and deep layer of the Found in lymph nodes, spleen, thymus and bone
skin marrow
Reticular fibers
o thin, collagen fibers coated with glycoprotein Adipose Tissue
o framework in spleen and lymph nodes
Empty-looking cells with thin margins; nucleus
Elastic fibers (yellow fibers) pressed against cell membrane
o thin branching fibers of elastin protein
Energy storage, insulation, cushioning
o stretch and recoil like rubberband (elasticity) o subcutaneous fat and organ packing
o skin, lungs and arteries stretch and recoil
o brown fat (hibernating animals) produces Bone

heat
Spongy bone - spongy in appearance
o delicate struts of bone
Dense Regular Connective Tissue
o covered by compact bone
Densely, packed, parallel collagen fibers o found in heads of long bones
o compressed fibroblast nuclei Compact bone - solid in appearance
Tendons and ligaments hold bones together and o more complex arrangement
attach muscles to bones o cells and matrix surround vertically oriented
blood vessels in long bones
Dense Irregular Connective Tissue
Bone Tissue (compact bone)
Densely packed, randomly arranged, collagen fibers
and few visible cells Calcified matrix in lamellae around central canal
o withstands stresses applied in different Osteocytes in lacunae between lamellae
directions Skeletal support; leverage for muscles; mineral
o deeper layer of skin; capsules around organs storage
Cartilage Blood
Supportive connective tissue with rubbery matrix Variety of cells and cell fragments; some with nuclei
Chondroblasts produce matrix and some without
o called chondrocytes once surrounded Nonnucleated pale pink cells or nucleated white blood
No blood vessels cells
o diffusion brings nutrients and removes Found in heart and blood vessels
wastes
o heals slowly Nerve Tissue
Types of cartilage vary with fiber types
Large cells with long cell processes
o hyaline, fibrocartilage and elastic cartilage
o surrounded by smaller glial cells lacking
processes
Hyaline Cartilage
Internal communication between cells
Rubbery matrix; dispersed collagen fibers; clustered o in brain, spinal cord, nerves and ganglia
chondrocytes in lacunae
o supports airway, eases joint movements Muscle Tissue
Ends of bones at movable joints; sternal ends of ribs;
supportive material in larynx, trachea, bronchi and Elongated cells stimulated to contract
Exert physical force on other tissues
fetal skeleton
o move limbs
o push blood through a vessel
Elastic Cartilage
o expel urine
Hyaline cartilage with elastic fibers Source of body heat
Provides flexible, elastic support 3 histological types of muscle
o external ear and epiglottis o skeletal, cardiac and smooth
Fibrocartilage Skeletal Muscle

Hyaline cartilage with extensive collagen fibers (never Long, cylindrical, unbranched cells with striations and
has perichondrium) multiple peripheral nuclei
Resists compression and absorbs shock o movement, facial expression, posture,
o pubic symphysis, meniscus and breathing, speech, swallowing and excretion
intervertebral discs
Cardiac Muscle
Short branched cells with striations and intercalated Unicellular glands endo or exocrine
discs o goblet or intrinsic cells of stomach wall
o one central nuclei per cell
Pumping of blood by cardiac (heart) muscle Exocrine Gland Structure
Stroma = capsule and septa divide gland into lobes
Smooth Muscle and lobules
Parenchyma = cells that secrete
Short fusiform cells; nonstriated with only one central
Acinus = cluster of cells surrounding the duct draining
nucleus
those cells
o sheets of muscle in viscera; iris; hair follicles
and sphincters Types of Exocrine Glands
o swallowing, GI tract functions, labor
Simple glands - unbranched duct
contractions, control of airflow, erection of Compound glands - branched duct
hairs and control of pupil
Shape of gland
o acinar - secretory cells form dilated sac
Intercellular Junctions
o tubuloacinar - both tube and sacs
All cells (except blood) anchored to each other or
their matrix by intercellular junctions Types of Secretions
Serous glands
Tight Junctions o produce thin, watery secretions
Encircle the cell joining it to surrounding cells sweat, milk, tears and digestive
o zipperlike complementary grooves and juices
ridges Mucous glands
Prevents passage between cells o produce mucin that absorbs water to form a
o GI and urinary tracts sticky secretion called mucus
Mixed glands contain both cell types
Cytogenic glands release whole cells
Desmosomes o sperm and egg cells
Patch between cells holding them together
Holocrine Gland
o cells spanned by filaments terminating on
Secretory cells disintegrate to deliver their
protein plaque
accumulated product
cytoplasmic intermediate filaments
o oil-producing glands of the scalp
also attach to plaque
Uterus, heart and epidermis
Merocrine and Apocrine Secretion
Merocrine glands release their product by exocytosis
Gap Junctions
o tears, gastric glands, pancreas, etc.
Ring of transmembrane proteins form a water-filled
Apocrine glands are merocrine glands with confusing
channel
appearance (apical cytoplasm not lost)
o small solutes pass directly from cell to cell
o mammary and armpit sweat glands
o in embryos, cardiac and smooth muscle
Mucous Membranes
Endocrine and Exocrine Glands
Epithelium, lamina propria and muscularis mucosae
Secrete substances
Lines passageways that open to the exterior:
o composed of epithelial tissue
reproductive, respiratory, urinary and digestive
Exocrine glands connect to surface with a duct
o Mucous (movement of cilia) trap and remove
(epithelial tube)
foreign particles and bacteria from internal
Endocrine glands secrete (hormones) directly into
body surfaces
bloodstream
Mixed organs do both
Membrane Types
o liver, gonads, pancreas
Cutaneous membrane = skin o disuse atrophy from lack of use (leg in a

o stratified squamous epithelium over cast)
connective tissue Necrosis = pathological death of tissue
o relatively dry layer serves protective function o gangrene - insufficient blood supply
Synovial membrane lines joint cavities o gas gangrene - anaerobic bacterial infection
o connective tissue layer only, secretes o infarction - death of tissue from lack of blood
synovial fluid o decubitus ulcer - bed sore or pressure sore
Serous membrane (serosa) internal membrane Apoptosis = programmed cell death
o simple squamous epithelium over areolar o cells shrink and are phagocytized (no
tissue, produces serous fluid inflammation)
o covers organs and lines walls of body
cavities Tissue Repair
Regeneration
Tissue Growth o replacement of damaged cells with original
Hyperplasia = tissue growth through cell multiplication cells
Hypertrophy = enlargement of preexisting cells o skin injuries and liver regenerate
o muscle grow through exercise Fibrosis
Neoplasia = growth of a tumor (benign or malignant) o replacement of damaged cells with scar
through growth of abnormal tissue tissue
function is not restored
Changes in Tissue Types healing muscle injuries,
Tissues can change types scarring of lung tissue in
Differentiation TB or healing of severe
o unspecialized tissues of embryo become cuts and burns of the skin
specialized mature types o keloid is healing with excessive fibrosis
mesenchyme to muscle (raised shiny scars)
Metaplasia
o changing from one type of mature tissue to Tissue Engineering
another Production of tissues and organs in the lab
simple cuboidal tissue before o framework of collagen or biodegradable
puberty changes to stratified polyester fibers
squamous after puberty o seeded with human cells
o grown in bioreactor (inside of mouse)
Stem Cells supplies nutrients and oxygen to
Undifferentiated cells with developmental plasticity growing tissue
Embryonic stem cells Skin grafts already available
o totipotent (any cell type possible) o research in progress on heart valves,
source = cells of very early embryo coronary arteries, bone, liver, tendons
o Pluripotent (tissue types only possible)
source = cells of inner cell mass of Wound Healing of a Laceration
embryo Damaged vessels leak blood
Adult stem cells (undifferentiated cells in tissues of Damaged cells and mast cells leak histamine
adults) o dilates blood vessels
o multipotent (bone marrow producing several o increases blood flow
blood cell types) o increases capillary permeability
o unipotent (only epidermal cells produced) Plasma carries antibodies, clotting factors and WBCs
into wound
Wound Healing of a Laceration
Tissue Shrinkage and Death Clot forms
Atrophy = loss of cell size or number Scab forms on surface
Macrophages start to clean up debris

New capillaries grow into wound
Fibroblasts deposit new collagen to replace old
material
Fibroblastic phase begins in 3-4 days and lasts up to
2 weeks
Epithelial cells multiply and spread beneath scab
Scab falls off
Selected growth factors important to wound healing
Epithelium thickens
EGF (epidermal growth factor).
Connective tissue forms only scar tissue (fibrosis)
o Stimulates wound re-epithelialization and
Remodeling phase may last 2 years
stimulates blood vessels and fibroblasts.
FGF (fibroblast growth factor).
LECTURE 3: PHYSIOLOGY OF WOUND HEALING
o Stimulates new blood vessel and collagen
formation.
Physiology of wound healing
PDGF (platelet derived growth factor).
Wound healing is:
o Attracts/stimulates smooth muscle cells,
Complicated process that involves at least 4 distinct
fibroblasts, and other cells. Important in ECM
cell types
formation.
Commonly referred to as occurring in PHASES
TGF- (transforming growth factor-beta).
Affected by several factors
o Slows buildup of epithelial cells, suppresses
immunoglobulin secretion and is helpful in
Phases of wound healing process (WHP)
ECM formation.
Where does a chronic wound get stuck
TNF-a (tumor necrosis factor-alpha).
o Haemostasis
o Activates neutrophils, causes fibroblasts to
o Inflammation
multiply, causes bone/cartilage resorption.
o Proliferation
IL-1 (interleukin-1).
o Maturation
o Attractant for epithelial cells, neutrophils,
mono and lymphocytes; also stimulates
Platelet Activity
collagen synthesis.
Chronic wounds characteristics
Increased inflammatory cytokines

Altered fibroblast phenotype
Abnormalities growth factors
Increased proteases
Altered keratinocyte function
Senescent cells (increased number)
Wound bed preparation

Debridement
Role of keratinocytes in wound healing Bacterial balance
Dressing therapies
o (f.i. silver dressings prevent of infections,
help reduce healing time)
Tissue engineering - Biology, medicine and technology are

today closely interleaved with each other
Tissue engineering combining cells and biomaterials into Allogeneic cultivated human skin keratinocytes
functional tissues
Make rapid healing of the ulcers particularly those
Cells are seeded onto a biomaterial scaffold to be integrated that are difficult to heal
into a specific tissue No clinical or laboratory evidence of rejection
No evidence of preexisting cytotoxic antibodies
Tissue engineering specific fort the HLA class I antigens expressed on
Advances HSE cells
o Biological wound dressings A fibrin-based skin substitute produced in the defined
o Material scaffolds and cell material keratinocyte medium could be safely used to threat a
interactions number of skin defect
o The use of stem cells for tissue engineering
o Combination of stem cells and material Methods of tissue bioengineering
scaffolds into tissue engineered Autologous platelet rich plasma product (platelet gel)
replacements of tissues and organs Allogeneic platelet gel
o The effect is attributed to the growth factors
Tissue engineering implants
Synthetic polymeric biomaterials Accepted methods of treatment
Nonbiodegradable Autologous bone transplants
o Is required to provide and maintain optimal o Cancellous bone graft (contains all
cellular function -> e.g. alginate, liposome, necessary characteristics of bone
Biodegradable substitutes)
o To restore the histological structure and o Corticocancellous graft (possibly
replace the cellular function of recipients -> vascularized limited amount)
e.g. poly L-lactic acid, poly glycolic acid, Homologous (allogeneic) graft
o Bone banks, treated (no rejection), contains
Wound healing promoting anti-adhesive matrix only osteoconductive properties
The collagen grafting is also applied to produce a healing / Ilizarow intercallary bone transport (traction method)
promoting antiadhesive membrane
Properties of bone grafts
Particularly necessary in peritoneal surgery to prevent Osteogenesis (bone marrow, cancellous bone)
postoperative adhesion Osteoinduction
To produce skin wound dressing membranes o Demineralized bone matrix
o Growth factors (platelet rich plasma, bone
Methods of tissue bioengineering morphogenic proteins BMPs)
Skin replacement Osteoconduction
o Cultured epidermal graft o Ceramics
o Cultured human autologous and allogeneic o Collagen
keratinocytes
o Semi synthetic materials (composed of Alternatives
human neonatal dermal fibroblasts cultured Bone substitute (biomaterials for scaffold):
onto a bioabsorbable mesh) Demineralized bone matrix
o Active dressings (f.i.: with maggots excret, Biocompatible ceramics
with honey) Synthetic Calcium phosphate
o Photobiomodulation (modulate cellular Mineral bone
activity in red to near infrared light) Collagen
o Hyperbaric oxygen therapy: as therapeutic Composite grafts
benefit in WT Osteoinductive collagen
o Growth factors (from blood)
Role of GROWTH FACTORS LECTURE 4A: STEM CELLS

Role of STEM CELLS
Stem Cells and Regenerative Biology
Collagen based matrices in tissue engineering Major Topics for Discussion:
Skin equivalent 1) What are Stem Cells?
Cartilage repair 2) What are the major types of Stem Cells and where
Bone repair are they found?
Matrices are also prepared from synthetic polymers 3) Advantages & Disadvantages to ESCs and ASCs
4) Why are Stem Cells so very important?
Fracture healing promoting molecules 5) The Controversy over Stem Cells?
Growth and different factors
o The transforming growth factor- (TGF-) What are Stem Cells?
superfamily Stem Cells are extraordinary because:
Bone morphogenetic proteins They can divide and make identical copies of
(osteoprogenitors, mesenchymal themselves over and over again through mitotic cell
cells, osteoblasts and chondrocytes division (Self-Renewal)
within the extracellular matrix They have the potential to differentiate into a diverse
produce BMPs.) range of specialized cell types (Potency)
BMP-2, BMP-4
BMP-5, BMP-6, BMP-7 The Major Types of Stem Cells
GDF-5 (BMP-14), GDF-6 (BMP-13), Embryonic Stem Cells
GDF-7 (BMP-12) o From blastocysts left over from In-vitro
BMP-3 (Osteogenin), GDF-10 fertilization (IVF) laboratory
(BMP-3b) o From aborted fetuses
o Platelet-derived growth factor (PDGF) Adult Stem Cells
o Fibroblast growth factor (FGFs) o Stem cells have been found in the blood,
o Insulin-like growth factor (IGFs) bone marrow, liver, kidney, cornea, dental
pulp, umbilical cord, brain, skin, muscle,
Mesenchymal stem cell: the promise for treating skeletal salivary gland
disorders Function: repair system for the
body, replenishing specialized cells,
Adult stem cell are being isolated from various tissues but also maintaining the normal
turnover of regenerative organs,
Adult stem cells such as blood, skin or intestinal
Bone marrow contains tissues.
o Hematopoietic stem cells (HSCs)
All types of blood cells Potency Definitions
o Bone marrow mesenchymal stem cells Totipotent - cells can differentiate into embryonic and
(MSCs) extraembryonic cell types
Generating bone, cartilage, fat, o stem cells are produced from the fusion of
fibrous connective tissue an egg and sperm cell. Cells produced by
the first few divisions of the fertilized egg are
Bone tissue formation also totipotent.
Osteogenic progenitor cells Pluripotent - stem cells that are the descendants of
Locations: totipotent cells and can differentiate into each of the
Periost more than 200 cell types of the adult body
Peritrabecular soft tissue o can develop when given sufficient and
Cancellous bone and bone marrow (in BM aspirate up necessary stimulation for a specific cell type.
to 40x less stem cells then in cancellous bone) They do not contribute to the extra-
embryonic membranes or the placenta.
Multipotent - stem cells can produce only cells of a

closely related family of cells Why the Controversy Over Stem cells?
o (e.g. hematopoietic stem cells differentiate Embryonic Stem cells are derived from extra
into red blood cells, white blood cells, blastocysts that would otherwise be discarded
platelets, etc.). following IVF.
Unipotent - cells can produce only one cell type, but Extracting stem cells destroys the developing
have the property of self-renewal which distinguishes blastocyst (embryo).
them from non-stem cells
o (e.g. muscle stem cells). ALCHEMY
Alchemists 300 years ago tried, unsuccessfully, to
Advantages and Disadvantages to Embryonic and Adult turn base LEAD into valuable GOLD
Stem Cells. Cellular Alchemy:
o Normally, stem cells give rise to somatic
cells of the adult organism
o Recent developments have resulted in
reversing this process with the production of
stem cells from adult somatic cells, eg. skin
cells
o These stem cells have been termed
Induced Pluripotent Stem (iPS) Cells
What is a Stem Cell?- Properties

An unspecialized cell with a unique capacity for
- indefinite or prolonged self-renewal and
- ability to give rise to differentiated cells
What is Self Renewal? Differentiation?

Self-renewal - ability to undergo numerous cycles of
cell division while maintaining the undifferentiated or
unspecialized state
Clonality ability of a single cell to form many similar
cells
Differentiation - process by which a less specialized
Why is Stem Cell Research So Important to All of Us? cell becomes a more specialized one.
Stem cells allow us to study how organisms grow and Potency- the potential for differentiation to specialized
develop over time. cell types
Stem cells can replace diseased or damaged cells
that can not heal or renew themselves. Potency of stem cells
We can test different substances (drugs and Pluripotent give rise to cells of all 3 germ layers
chemicals) on stem cells. (ectoderm, endoderm, mesoderm and germ cells)
We can get a better understanding of our genetic Multipotent ability to differentiate into many, related
machinery. cell types
What Human Diseases are Currently Experimentally Being Progenitors
Treated with Stem Cells? o oligopotent few cell types
Parkinsons Disease o unipotent one cell type but can self renew
Leukemia (Bone Marrow Transplants)
Skin Grafts resulting from severe burns Where are stem cells found?
Stem cells have been isolated from the embryo, fetus
Stem Cell Therapy has the Potential to: and adult
Regenerate tissues/organs
Cure diseases like diabetes, multiple sclerosis, etc.
Embryonic stem (ES) cells: derived from the inner cell Drugs and chemicals can be screened and tested on
mass of the blastocyst (4-5 day embryo) patients stem cells and their differentiated tissues
Adult stem cells : from adult tissues
Embryonic Stem (ES) cells
Division of Stem Cells Derived from inner cell mass of blastocyst
Capacity for almost unlimited symmetrical divisions
without differentiation
Give rise to endoderm, ectoderm, mesoderm
Clonogenic- derived from a single ES cell
Capable of colonizing germline and forming egg and
sperm cells
Characterization of human and mouse ES cells

Expression of cell surface markers SSEA-3,SSEA-4 (hESC)
SSEA-1 (mESC),TRA-1-60, TRA-1-81, alkaline phosphatase,
GTCM-2
Timeline of Stem Cell Research Pluripotency transcription factors Oct-4, Sox-2,
1960s - Joseph Altman and Gopal Das present Nanog, Rex1
scientific evidence of adult neurogenesis, ongoing High Telomerase activity
stem cell activity in the brain; their reports are largely Karyotype- normal (46 XX or XY)
ignored. In vitro pluripotency- embryoid body formation
1978 - Haematopoietic stem cells in human cord Teratoma formation in immune-incompetent mice-
blood. tumour contains tissues from all 3 germ layers
1981 - Mouse embryonic stem cells are derived from Pluripotency in vivo Chimera formation (mESC)
the inner cell mass by scientists Martin Evans,
Matthew Kaufman, and Gail R. Martin. Gail Martin Embryonic or Adult Stem Cells for Cell
is attributed for coining the term "Embryonic Stem ReplacementTherapy: Advantages and Disadvantages
Cell".
1996 - Cloning of Dolly the sheep by somatic cell Embryonic SC
nuclear transfer Pluripotent
1997 - Leukemia is shown to originate from a Stable. Can undergo many cell divisions.
haematopoietic stem cell, the first direct Easy to obtain but blastocyst is destroyed (Ethics)
evidence for cancer stem cells. Possibility of immune rejection
1998 - James Thomson and coworkers derive the first High potential for tumours
human embryonic stem cell line
2000s Several reports of adult stem cell plasticity Adult SC
2004-2005 Korean researcher Hwang Woo-Suk Multipotent
human embryonic stem cell line from unfertilized Less Stable. Capacity for self-renewal is limited.
human oocytes by SCNT. The lines were later shown No ethical concerns
to be fabricated. Difficult to isolate in adult tissue.
August 2006- Mouse induced pluripotent stem cells; Host rejection minimized or absent
journal cell publishes Takahashi and Yamanakas Less tumorigenic potential
work.
The Ideal Stem cell the Holy Grail of Cell Replacement
Whats so special about Stem Cells? Therapy
They have the potential to replace cell tissue that has o Ability to differentiate into many cell types
been damaged or destroyed. o Easily accessible
They can replicate themselves over and over for a o Individual-specific i.e. personalized or non-
very long time- nearly inexhaustible source immunogenic
Understanding how stem cells develop into healthy o Vastly renewable
and diseased cells will assist the search for cures.
o Demonstrably safe Cultures of animal cells are usually divided into 3

o Non-tumorigenic classes:
1. Primary cells
Re-programming the nucleus stem cell 2. Cell strains
3. and cell lines
1- Primary Culture
When cells are surgically removed from an organism
Nuclear reprogramming- functional or molecular and placed into a suitable culture environment they
changes in cell undergoing fate changes will attach, divide and grow
Most of the primary culture cells have a finite lifespan
Transcription factors for reprogramming of 5-10 divisions in vitro
Transcription factors are proteins that bind to DNA Due to their limited lifespan, one cannot do long-term
and regulate gene expression experiments with these cells
Oct3/4 and Sox2: transcription factors that function in Primary cells are considered by many researchers to
maintaining pluripotency in both early embryos and be more physiologically similar to in vivo cells
ES cells. There are two basic methods for obtaining primary
c-Myc and Klf4: transcription factors that modify culture:
chromatin structure so that Oct3/4 and Sox2 can bind 1. Explant cultures:
to their target; proto-oncogenes Small pieces of tissue are attached
(using plasma clots or fibrinogen) to
Were these iPS cells identical to the ES cells? a glass or treated plastic culture
NO vessel and immersed in culture
o The transcriptional profile was somewhere medium
between fibroblasts and ES cells After a few days individual cells will
o No live chimeras produced move from the tissue explant out
o So these iPS cells were somewhat similar onto the culture vessel surface or
but not identical to ES cells substrate where they will begin to
WHY? divide and grow
o Because fbx15 was selected for. Fbx15 is a 2. Enzymatic dissociation:
factor that is expressed in ES cells but is not More widely used
essential for the maintainence of speeds up the process by adding
pluripotency digesting (proteolytic) enzymes
such as trypsin or collagenase to
LECTURE 4B: CELL CULTURE the tissue fragments to dissolve the
cement holding the cells together
What is Cell & Tissue Culture This creates a suspension of single
Tissue culture is the general name for the removal of cells that are then placed into
cells, tissues or organs from an animal or plant and culture vessels containing culture
their subsequent placement into artificial environment medium and allowed to grow and
conductive to growth divide
This environment usually consists of a suitable glass
or plastic culture vessel containing a liquid or semi- Hayflicks Phenomenon
solid support medium that supplies the nutrients Cells will continue to grow and divide normally for a
essential for survival and growth limited number of passages
When the cells are removed from the organ When they get to a certain point even if they are given
fragments, thus disrupting their normal relationship the appropriate nutrients, they simply stop dividing
with neighboring cells, it is called cell culture and will eventually die
There appears to be a correlation between the
maximal number of passages and aging
Classes of Culture Cells
The number of passages decreases when cells are

harvested from older individuals
2- Cell Strains Cell Culture Systems

Cell strains are cells that have been adapted to Cells may be loosely divided into two main types:
culture but, unlike cell lines, have a finite division 1- Suspension cell culture (Anchorage-independent)
potential o derived from cells which can divide
Upon serial transfers of primary cells, a gradual and survive without being attached
selection may occur until a particular cell type to a substrate,
becomes predominant o e.g. cells of haemopoietic lineage
If these cells continue to grow at a constant rate over o Can be maintained in culture
successive passages, these primary cells are referred vessels that are not tissue-culture
to as a cell strain treated,
These cells have a finite lifespan of 40-60 divisions in o requires agitation for adequate gas
vitro exchange
They are useful in vaccine production o Easier to passage
o Cell Culture Systems
3- Cell Lines 2- Adherent cell culture (Anchorage-dependent)
If the cells in a cell strain undergo a transformation must adhere to a surface to survive
process (spontaneous or induced changes in Form monolayers
karyotype, morphology or growth properties) that e.g. cells derived from different
makes them "immortal (able to divide indefinitely) tissue (breast, liver)
they are called a cell line Growth is limited by surface area
It is not known how a diploid cell strain becomes a Will cease proliferating once they
cell line, although this event may be mimicked by become confluent (completely
infection with oncogenic viruses or by exposure to cover the surface of cell culture
chemical carcinogens vessel)
Cell Lines often have abnormal chromosome Cells are dissociated enzymatically
numbers and maybe tumorigenic when inoculated or mechanically from surface
into susceptible animals
Cell lines that have been derived from tumors often Growth Cycle in Attachment Culture
do not exhibit contact-inhibition (inhibition of growth Eukaryotic cells in attachment culture have a
under crowded conditions), but rather continue to pile- characteristic growth cycle similar to bacteria
up The growth cycle is typically divided into three
phases:
1- Lag Phase
Transformation of Cells This is the time following subculture
Transformed, Infinite or Established Cells and reseeding during which there is
Changed from normal cells to cells with many of the little evidence of an increase in cell
properties of cancer cells number
Some of these cell lines have actually been derived It is a period of adaptation during
from tumors or are transformed spontaneously in which the cell replaces elements
culture by mutations lost during trypsinization, attaches
Chemical or gamma ray treated cells can become to the surface, and spreads out
infinite with loss of growth factors 2- Log Phase
Viral infection with SV40 T antigen can insert This is the period of exponential
oncogenes and lead to gene alteration increase in cell number
No matter how transformation occurred, the result is a The length of the log phase
cell with altered functional, morphological, and growth depends on the seeding density,
characteristics the growth rate of the cells
It is the optimal time for sampling growth can also be useful if warming of
since the population is at its most media prior to use is desired
uniform and viability is high 2. The second requirement for successful cell culture is
the practice of sterile technique
Prior to beginning any work, the biological
3- Plateau Phase safety cabinet should be turned on and
Toward the end of the log phase, allowed to run for at least 15 min to purge
the culture becomes confluent the contaminated air
All the available growth All work surfaces within the cabinet should
surface is occupied and all be decontaminated with an appropriate
the cells are in contact solution;
with surrounding cells 70% ethanol or isopropanol are
Following confluence the growth routinely used for this purpose
rate of the culture is reduced, and in Any materials required for the procedure
some cases, cell proliferation should be similarly decontaminated and
ceases almost completely placed in or near the cabinet
At this stage, the culture enters the This is especially important if solutions have
plateau (or stationary) phase, and been warmed in a water bath prior to use
the growth fraction falls The worker should put on appropriate
personnel protective equipment for the cell
Morphology of Cells type in question
Cultured cells are usually described based on their Gloved hands should be sprayed with
morphology (shape & appearance), there are two decontaminant prior to putting them into the
basic morphologies: cabinet and gloves should be changed
1. Epithelial-like: regularly if something outside the cabinet is
cells that appear flattened and polygonal in touched
shape Care should be taken to ensure that
2. Fibroblast-like: anything coming in contact with the cells of
cells that appear thin and elongated interest, or the reagents needed to culture
Culture conditions paly an important role in and passage them, is sterile (either
determining shape and that many cell cultures are autoclaved or filter-sterilized)
capable of exhibiting multiple morphologies 3. A third necessity for successful cell culture is
appropriate, quality controlled reagents and supplies
Basic Requirements For Successful Cell Culture There are numerous suppliers of tissue
1. The first necessity is a well-established and properly culture media and supplements
equipped cell culture facility. All facilities should be Examples include:
equipped with the following: Invitrogen (www.invitrogen.com),
A certified biological safety cabinet SigmaAldrich
protects both the cells in culture (www.sigmaaldrich.com),
and the worker from biological BioWhittaker (www.cambrex.com),
contaminants and StemCell Technologies Inc.
A centrifuge, preferably capable of (www.stemcell.com).
refrigeration Similarly, there are numerous suppliers of
A microscope for examination of cell cultures the plasticware needed for most cell culture
and for counting cells applications (i.e., culture dishes and/or
And a humidified incubator set at 37C with flasks, tubes, disposable pipets)
5% CO2 in air Basic Requirements For Successful Cell Culture
A 37C water bath filled with water 4. The final necessity for successful cell culture is the
containing inhibitors of bacterial and fungal knowledge and practice of the fundamental
techniques involved in the growth of the cell type of L-Glutamine

interest L-Glutamine is an essential amino acid
The majority of cell culture carried out by required by virtually all mammalian cells
investigators involves the use of various non- grown in culture
adherent or adherent continuously growing It is used for protein production, as an
cell lines energy source, and in nucleic acid
These cell lines can be obtained from metabolism
reputable suppliers such as: It is also more labile in liquid cell culture
the American Tissue Type media than other amino acids
Collection (ATCC; www.atcc.org) The rate and extent of L-glutamine
or DSMZ (the German Collection of degradation are related to storage
Microorganisms and Cell Cultures) temperatures, age of the product, and pH
(www.dsmz.de/mutz/mutzhome.htm
l) Buffering in Cell Culture
Alternatively, they can be obtained A pH indicator may be Included in the original
from collaborators formulation to permit direct observation of the pH of
Regardless of the source of the cells, it is the medium
advisable to verify the identity of the cell line Optimum pH between 7.2 to 7.4 is generally needed
and to ensure that it is free of mycoplasma for mammalian cells
contamination Generally in the cell culture medium pH indicator,
commonly phenol red is used to analyze the pH of
Cell Culture Medium environment in which cells are growing
Cells have complex nutritional requirements that must Phenol red is:
be met to permit their propagation in vitro yellow in acidic medium (pH 6.8),
Different types of cells have different growth tomato red at neutral pH (7.0),
requirements and a number of chemically-defined red at an alkaline pH (7.4)
formulations have been developed that support the and blue at increased basicity (pH 7.6)
growth of a variety of established cell lines and finally purple at high pH
Although some serum-free media are available and
some cell lines have been adapted to growing in such Supplements to Medium: Antibiotics
a medium, most cell lines require the addition of 5- Prevention of contamination by the different
10% serum as a supplement to promote cellular microorganisms (bacteria, mycoplasma and fungi) is
multiplication the most important part of all animal cell culture
Fetal Bovine Serum (FBS) is often the best to use The risk of contamination during culture can be
avoided by adding different antibiotics, such as:
Cell Culture Medium penicillin (100 U/ml) for bacteria,
1- The various nutrients required are: streptomycin (100 mg/ml) for bacteria,
glucose, or gentamycin (50mg/ ml) for bacteria,
fats and fatty acids, and nystatin (50mg/ml) for fungi and yeast
lipids, phospholipids and sulpholipids, The routine use of antibiotics is generally not
ATP and amino acids recommended because:
Vitamins it may lead to a relaxation of aseptic
Minerals technique
2- Serum: resistant microorganisms may develop
Serum can provide various growth factors, microbial growth may be controlled but
hormones and other factors needed by the biochemical alteration may be produced
most mammalian cells for their long term
growth and metabolism
Temperature & Humidity The vessel surface is treated to render it hydrophilic

Temperature Most cell lines are cultivated on treated plastic
Optimum temperature is also required for the surfaces in dishes or flasks
proper growth of the cell Some fastidious cell lines require further treatment of
The optimum temperature of mammal is the growth surface before they will attach and
37oC proliferate
Humidity The most common techniques include coating the
Proper humidity is also essential for cell surface with serum, collagen, laminin, gelatin, poly-L-
growth as humidity distribution indirectly also lysine, or fibronectin
has effect on temperature
For cell growth 100% humidity is essential to LECTURE 4C: TISSUE CULTURE
reduce evaporation
Introduction to Tissue culture
Storage of Medium What is tissue culture?
Once prepared, the cell culture medium has to be In vitro culture (maintain and/or proliferate) of cells,
properly stored tissues or organs
For long-term storage, it should be frozen without Types of tissue culture
NaHCO3 o Organ culture
On a short-term basis the medium should be kept at o Tissue culture
4C and warmed up to 37C only for the time o Cell culture
necessary to perform a given experiment
Culture Vessels Organ culture
Culture vessels provide a contamination barrier to The entire embryos or organs are excised from the
protect the cultures from the external environment body and culture
while maintaining the proper internal environment Advantages
For anchorage-dependent cells, the vessels provide a o Normal physiological functions are
suitable and consistent surface for cell attachment maintained.
Other characteristics of vessels include easy access o Cells remain fully differentiated.
to the cultures and optically clear viewing surfaces Disadvantages
o Scale-up is not recommended.
Culture Vessels o Growth is slow.
Flasks o Fresh explantation is required for every
Plastic flasks are available with a range of experiment.
growing areas, a variety of shapes, with
several different neck designs Tissue Culture
Flasks surfaces are specially treated for Fragments of excised tissue are grown in culture
growing anchorage-dependent cells media
Cell culture dishes Advantages
Cell culture dishes offer the best economy o Some normal functions may be maintained.
and access to the growth surface o Better than organ culture for scale-up but not
Cell culture dishes surfaces are specially ideal.
treated for growing anchorage-dependent Disadvantages
cells o Original organization of tissue is lost.
Multiwell plates
Multiwell plates offer significant savings in Cell Culture
space, media, and reagents when compared Tissue from an explant is dispersed, mostly
to an equal number of dishes enzymatically, into a cell suspension which may then
Surface Coatings be cultured as a monolayer or suspension culture.
Most tissue culture work uses disposable polystyrene Advantages
vessels
o Development of a cell line over several Outgrowth of excised tissue in

generations culture
o Scale-up is possible Dissociation into single cells (by
Disadvantages enzymatic digestion or mechanical
o Cells may lose some differentiated dispersion)
characteristics. o Advantages:
usually retain many of the
Why do we need Cell culture? differentiated characteristics of the
Research cell in vivo
o To overcome problems in studying cellular o Disadvantages:
behavior such as: initially heterogeneous but later
confounding effects of the become dominated by fibroblasts.
surrounding tissues the preparation of primary cultures
variations that might arise in is labor intensive
animals under experimental stress can be maintained in vitro only for a
o Reduce animal use limited period of time.
Commercial or large-scale production 2. Continuous Cultures
o Production of cell material: vaccine, o derived from subculture (or passage, or
antibody, hormone transfer) of primary culture
Subculture = the process of
Cell culture application dispersion and re-culture the cells
after they have increased to occupy
all of the available substrate in the
culture
o usually comprised of a single cell type
o can be serially propagated in culture for
several passages
o There are two types of continuous cultures
Cell lines
Continuous cell lines
Types of continuous culture

1. Cell lines
Advantages of Cell culture finite life, senesce after
Advantages: approximately thirty cycles of
o Absolute control of physical environment division
o Homogeneity of sample usually diploid and maintain some
o Less compound needed than in animal degree of differentiation.
models it is essential to establish a system
Disadvantages: of Master and Working banks in
o Hard to maintain order to maintain such lines for long
o Only grow small amount of tissue at high periods
cost 2. Continuous cell lines
o Dedifferentiation can be propagated indefinitely
o Instability, aneuploidy generally have this ability because
they have been transformed
Types of Cell culture tumor cells.
1. Primary Cultures
viral oncogenes
o Derived directly from excised tissue and
chemical treatments.
cultured either as
the disadvantage of having retained the incubation temperature

very little of the original in vivo the cell-cell and cell-matrix
characteristics interaction
Cell cycle
Transformation VS Transfection Interphase:
Transformation o generally lasts at least 12 to 24 hours in
o Spontaneous or induced permanent mammalian tissue
phenotypic changes resulting from change in o the cell is constantly synthesizing RNA,
DNA and gene expression producing protein and growing in size
growth rate Gap 0 (G0): cell will leave the cycle
mode of growth (loss of contact and quit dividing temporary or more
inhibition) permanent
specialized product formation Gap 1 (G1): Cells increase in size,
longevity RNA and protein synthesis, there is
loss of need for adhesion a G1 Checkpoint
Transfection S Phase: The DNA replication
o Introduction of DNA into a cell (like viral occurs
DNA) Gap 2 (G2): The cell will continue to
grow and produce new proteins.
Cell Culture Morphology There is a G2 Checkpoint
Morphologically cell cultures take one of two forms: Mitosis or M Phase:
o growing in suspension (as single cells or o Cell growth and protein production stop
small free-floating clumps) o the cell cycle divides into two similar
cell lines derived from blood daughter cell
(leukaemia, lymphoma) o Mitosis last perhaps only one to two hours
o growing as a monolayer that is attached to o there is a Checkpoint in the middle of mitosis
the tissue culture flask. (Metaphase Checkpoint) that ensures the
cells derived from solid tissue cell is ready to complete cell division.
(lungs, kidney), endothelial,
epithelial, neuronal, fibroblasts Factors affecting cell proliferation
Promotion of cell proliferation
Special types of Cell culture o low cell density (leaves the cell with free
Cells in the culture can be grown to adopt in vivo edge)
characteristic o signals from environment: Growth factors
Histotypic culture Inhibition of cell proliferation
o Single cell lineage o Density limitation: high cell density
Organotypic culture o Contact inhibition: cell contact
o Multiple cell lineages o signals from environment: p53 gene product
Biology of Culture cells Factors affecting cell diferentiation

Cell growth and differentiation in the culture depends Cell differentiation is important for normal cell
on: functions
o The nature of cells Factors promoting cell differentiations
o The culture environment o high cell density
the nature of the substrate on which o cell-cell and cell-matrix interaction
cell grow o inducers: hydrocortisone, retinoid, matrix
the physicochemical and
physiological constitution of culture Factors affecting cell adhesion
medium Cell adhesion is important for cell proliferation and
the constitution of gas phase differentiation (signaling through cytoskeleton)
Cell adhesion molecule regulate membrane potential by

o Cell-cell interaction: CAMs, cadherins provision of sodium, potassium and
o Cell-matrix interaction: integrin, calcium ions.
transmembrame proteoglycan are required in the cell matrix for
Tight junctional complex in epithelial cells for cell-cell cell attachment and as enzyme
interaction cofactors.
o Carbohydrates
Factors affecting cell adhesion Most media contain 4-20 mM
Enzymatic disaggregation digests the adhesion glucose
molecule and extracellular matrix main source of energy from
Most cells from solid tissues grow as adherent glycolysis
monolayer Liquid phase
Matrix-coated surface promotes cell proliferation and o Proteins and Peptides
differentiation are used to replace those normally
present in serum eg. transferrin,
Factors affect cell culture success fibronectin
Appropriate cells o Amino acids
Suitable environment important for cell proliferation and
o Solid phase differentiation
substrate or phase on which the cell glutamine can enter Krebs cycle
grow eg. glass, plastic, collagen, o Fatty Acids and Lipids
agar important in serum free media e.g.
o Liquid phase cholesterol and steroids essential
physicochemical and physiological for specialized cells.
constitution of the medium o Vitamins
o Gaseous phase vitamins B are necessary for cell
o Temperature growth and proliferation
o Aseptic environment precursors for numerous co-factors
The vitamins commonly used in
Solid phase media include thiamine, riboflavin
Anchorage dependent cells require a nontoxic, and biotin
biologically inert to attach and allow movement for o Trace Elements
growth zinc, copper, selenium and
The most convenient vessels are polystyrene plastic tricarboxylic acid intermediates.
other growth surface such as glass, filter wells Selenium is a detoxifier and helps
The surface can be treated by remove oxygen free radicals.
o coated with matrix substrate eg. Collagen, o Buffering Systems
poly-l-lysine, matrigel most cells need optimal pH
o Feeder layers: monolayer of supporting cells, conditions in the range 7.2 - 7.4
perhaps promote cell growth and close control of pH is essential for
differentiation by cell contact and substance optimum culture conditions
secreted bicarbonate/CO2 buffering
Neurons on glial cell feeder layers systems
Chemical buffering:
Liquid phase HEPES
Components of culture media Most commercial culture media
o Inorganic Salts include phenol red as a pH indicator
retain the osmotic balance of the yellow (acid) or purple
cells (alkali)
o Osmolarity Culture vessels

similar to plasma osmolarity 290
mOsm Cryopreservation of Cell Lines
o Serum The aim of cryopreservation is to enable stocks of
Undefined factors: complex mix of cells to be stored to prevent the need to have all cell
albumins, growth factors and lines in culture at all times
growth inhibitors Reduced risk of microbial contamination
increase the buffering capacity of Reduced risk of cross contamination with other cell
cultures lines
able to bind and neutralize toxins Reduced risk of genetic drift and morphological
can be important for slow growing changes
cells or where the seeding density Work conducted using cells at a consistent passage
is low number
Subject to batch to batch variation Reduced costs (consumables and staff time)
Heat inactivation of serum
(incubation at 56C for 30 minutes) Risk Assessment
can help to reduce the risk of Risks depend on:
contamination o Source of material
o the nature of operation being carried out
Gaseous phase Assesment:
Carbondioxide o Pathogenicity
o important for buffering system o Route of transmission
5-10% CO2 o Agent stability
Endogenous production: pyruvate o Infectious dose
Oxygen o Concentration
o most cells in culture require low oxygen o Availability of data from animal studies
tension o Availability of an effective prophylaxis
o anaerobic glycolysis o Medical surveillance
o high oxygen can produce toxic free radical o Experience and skill level of at-risk
personnel
Temperature
The optimum temperature depends on Risk groups for animal cell culture
o the body temperature of animals from which The level of risk depends on the cell line to be used
the cells were obtained and is based on whether the cell line is likely to cause
o anatomical variation of temperature (skin harm to humans.
temperature may be lower than the rest of Low risk
the body) o Non human/non primate continuous cell lines
and some well characterized human diploid
Aseptic techniques lines of finite lifespan
Microorganism remains a major problem in cell Medium risk
culture o Poorly characterized mammalian cell lines.
prevention of contamination High risk
o Antibiotics o Cell lines derived from human/primate tissue
o improvement of laboratory condition or blood.
o Aseptic techniques o Cell lines with endogenous pathogens (the
Clean and tidy work surface precise categorization is dependent upon the
Personal hygiene pathogen)
hand washing o Cell lines used following experimental
caps, gowns, face mask infection where the categorization is
Reagents and media dependent upon the infecting agent
LECTURE 5: THE HUMAN IMMUNE SYSTEM

Safety aspects of cell culture
SAFETY CONSIDERATIONS What is the immune system?
Assume all cultures are hazardous since they may The bodys defense against disease causing
harbor latent viruses or other organisms organisms, malfunctioning cells, and foreign particles
The following safety precautions should also be
observed: The Immune Response
o pipetting: use pipette aids to prevent Immunity: Free from burden. Ability of an organism
ingestion to recognize and defend itself against specific
o keep aerosols down to a minimum pathogens or antigens.
o no eating, drinking, or smoking Immune Response: Third line of defense. Involves
o wash hands after handling cultures and production of antibodies and generation of specialized
before leaving the lab lymphocytes against specific antigens.
o decontaminate work surfaces with Antigen: Molecules from a pathogen or foreign
disinfectant (before and after) organism that provoke a specific immune response.
o autoclave all waste
o use biological safety cabinet (laminar flow The First Line of Defense
hood) ~Skin~
o use aseptic technique The dead, outer layer of skin, known as the
o dispose of all liquid waste after each epidermis, forms a shield against invaders and
experiment and treat with bleach secretes chemicals that kill potential invaders
You shed between 40 50 thousand skin cells every
Risk Group (RG) day!
Classification is based on the potential effect of biological As you breathe in, foreign particles and bacteria
agent on healthy human adult bump into mucus throughout your respiratory system
RG1-agents are not associated with disease and become stuck
RG2-agents are associated with human disease Hair-like structures called cilia sweep this mucus into
which is rarely serious and for which preventive or the throat for coughing or swallowing
therapeutic interventions are often available
RG3-agents are associated with serious or lethal Whats the first thing you do when you cut your finger?
human disease for which preventive or therapeutic Swallowed bacteria are broken down by incredibly
interventions may be available strong acids in the stomach that break down your
RG4-agents are likely to cause serious or lethal food
human disease for which preventive or therapeutic The stomach must produce a coating of special
interventions are not usually available mucus or this acid would eat through the stomach!
Biosafety cabinets Escherichia coli - is common and plentiful in all of our

The Class I BSC provides personnel and digestive tracts. Why are we all not sick?
environmental protection, but no product protection.
The Class I BSC is hard-ducted to the building The Second Line of Defense
exhaust system, thimble-connected, or recirculated ~White Blood Cells~
back into the room depending on use. If invaders actually get within the body, then your
The Class II (Types A, B1, B2, and B3)24 biological white blood cells (WBCs) begin their attack
safety cabinets provide personnel, environmental and WBCs normally circulate throughout the blood, but
product protection. will enter the bodys tissues if invaders are detected
These white blood cells are responsible for eating
foreign particles by engulfing them
Once engulfed, the phagocyte breaks the foreign
particles apart in organelles called ________
Viruses Proteins that latch onto, damage,

Viruses enter body cells, hijack their organelles, and clump, and slow foreign particles
turn the cell into a virus making-factory. The cell will Each antibody binds only to one
eventually burst, releasing thousands of viruses to specific binding site, known as an
infect new cells. antigen
The Second Line of Defense The Immune System is the Third Line of Defense Against
~Interferon~ Infection
Virus-infected body cells release interferon when an
invasion occurs
Interferon chemical that
interferes with the ability to viruses
to attack other body cells
White Blood Cells
~T-Cells~
T-Cells, often called natural killer cells, recognize
infected human cells and cancer cells Innate or Genetic Immunity: Immunity an organism is
T-cells will attack these infected cells, quickly kill born with.
them, and then continue to search for more cells to o Genetically determined.
kill o May be due to lack of receptors or other
The Second Line of Defense molecules required for infection.
~The Inflammatory Response~ Innate human immunity to canine
Injured body cells release chemicals called distemper.
histamines, which begin inflammatory response Immunity of mice to poliovirus.
Capillaries dilate Acquired Immunity:Immunity that an organism develops
Pyrogens released, reach during lifetime.
hypothalamus, and temperature o Not genetically determined.
rises o May be acquired naturally or artificially.
Pain receptors activate Development of immunity to
WBCs flock to infected area like measles in response to infection or
sharks to blood vaccination.
Two Divisions of the Immune System Components of Human Immune System

The efforts of the WBCs known as phagocytes and T-
cells is called the cell-mediated immune system.
Protective factor = living cells
Phagocytes eat invaders
T-cells kill invaders
The other half of the immune system is called
antibody-mediated immunity, meaning that is
controlled by antibodies
This represents the third line of defense in the
immune system
Types of Acquired Immunity
The Third Line of Defense I. Naturally Acquired Immunity: Obtained in the course of
~Antibodies~ daily life.
Most infections never make it past the first and A. Naturally Acquired Active Immunity:
second levels of defense Antigens or pathogens enter body naturally.
Those that do trigger the production and release of Body generates an immune response to
antibodies antigens.
Immunity may be lifelong (chickenpox or B cells that are stimulated will actively
mumps) or temporary (influenza or intestinal secrete antibodies and are called plasma
infections). cells.
B. Naturally Acquired Passive Immunity: Antibodies are found in extracellular fluids
Antibodies pass from mother to fetus via (blood plasma, lymph, mucus, etc.) and the
placenta or breast feeding (colostrum). surface of B cells.
No immune response to antigens. Defense against bacteria, bacterial toxins,
Immunity is usually short-lived (weeks to and viruses that circulate freely in body
months). fluids, before they enter cells.
Protection until childs immune system Also cause certain reactions against
develops. transplanted tissue.
II. Artificially Acquired Immunity: Obtained by receiving a
vaccine or immune serum. Antibodies are Produced by B Lymphocytes
1. Artificially Acquired Active Immunity:
Antigens are introduced in vaccines
(immunization).
Body generates an immune response to
antigens.
Immunity can be lifelong (oral polio vaccine)
or temporary (tetanus toxoid).
2. Artificially Acquired Passive Immunity:
Preformed antibodies (antiserum) are
introduced into body by injection.
Snake antivenom injection from
horses or rabbits. Antibodies are Proteins that Recognize Specific Antigens
Immunity is short lived (half life three weeks).
Host immune system does not respond to
antigens.
Serum: Fluid that remains after blood has
clotted and cells have been removed.
Antiserum: Serum containing antibodies to a
specific antigen(s). Obtained from injecting
an animal (horse, rabbit, goat) with antigen
(snake venom, botulism or diphtheria toxin). Duality of Immune System (Continued)
Serology: The study of reactions between II. Cell Mediated Immunity
antibodies and antigens. Involves specialized set of lymphocytes
Gamma Globulins: Fraction of serum that called T cells that recognize foreign antigens
contains most of the antibodies. on the surface of cells, organisms, or
Serum Sickness: Disease caused by tissues:
multiple injections of antiserum. Immune Helper T cells
response to foreign proteins. May cause Cytotoxic T cells
fever, kidney problems, and joint pain. Rare T cells regulate proliferation and activity of
today. other cells of the immune system: B cells,
macrophages, neutrophils, etc.
Duality of Immune System Defense against:
I. Humoral (Antibody-Mediated) Immunity Bacteria and viruses that are inside
Involves production of antibodies against host cells and are inaccessible to
foreign antigens. antibodies.
Antibodies are produced by a subset of Fungi, protozoa, and helminths
lymphocytes called B cells. Cancer cells
Transplanted tissue Antibody Structure

Monomer: A flexible Y-shaped molecule with four
Antigens protein chains:
Most are proteins or large polysaccharides from a o 2 identical light chains
foreign organism. o 2 identical heavy chains
o Microbes: Capsules, cell walls, toxins, viral Variable Regions: Two sections at the end of Ys
capsids, flagella, etc. arms. Contain the antigen binding sites (Fab).
o Nonmicrobes: Pollen, egg white , red blood Identical on the same antibody, but vary from one
cell surface molecules, serum proteins, and antibody to another.
surface molecules from transplanted tissue. Constant Regions: Stem of monomer and lower parts
Lipids and nucleic acids are only antigenic when of Y arms.
combined with proteins or polysaccharides. Fc region: Stem of monomer only. Important
Molecular weight of 10,000 or higher. because they can bind to complement or cells.
o Hapten: Small foreign molecule that is not
antigenic. Must be coupled to a carrier Immunoglobulin Classes
molecule to be antigenic. Once antibodies I. IgG
are formed they will recognize hapten. Structure: Monomer
Epitope: Percentage serum antibodies: 80%
o Small part of an antigen that interacts with Location: Blood, lymph, intestine
an antibody. Half-life in serum: 23 days
o Any given antigen may have several Complement Fixation: Yes
epitopes. Placental Transfer: Yes
o Each epitope is recognized by a different Known Functions: Enhances phagocytosis,
antibody. neutralizes toxins and viruses, protects fetus and
newborn.
Epitopes: Antigen Regions that Interact with Antibodies II. IgM
Structure: Pentamer
Percentage serum antibodies: 5-10%
Location: Blood, lymph, B cell surface (monomer)
Half-life in serum: 5 days
Complement Fixation: Yes
Placental Transfer: No
Known Functions: First antibodies produced during an
infection. Effective against microbes and agglutinating
antigens.
Antibodies III. IgA
Proteins that recognize and bind to a particular Structure: Dimer
antigen with very high specificity. Percentage serum antibodies: 10-15%
Made in response to exposure to the antigen. Location: Secretions (tears, saliva, intestine, milk),
One virus or microbe may have several antigenic blood and lymph.
determinant sites, to which different antibodies may Half-life in serum: 6 days
bind. Complement Fixation: No
Each antibody has at least two identical sites that Placental Transfer: No
bind antigen: Antigen binding sites. Known Functions: Localized protection of mucosal
Valence of an antibody: Number of antigen binding surfaces. Provides immunity to infant digestive tract.
sites. Most are bivalent. IV. IgD
Belong to a group of serum proteins called Structure: Monomer
immunoglobulins (Igs). Percentage serum antibodies: 0.2%
Location: B-cell surface, blood, and lymph
Half-life in serum: 3 days
Complement Fixation: No oMany virus infected cells will undergo

Placental Transfer: No apoptosis, to help prevent spread of the
Known Functions: In serum function is unknown. On infection.
B cell surface, initiate immune response. Clonal Selection
V. IgE o Clonal Selection: B cells (and T cells) that
Structure: Monomer encounter stimulating antigen will proliferate
Percentage serum antibodies: 0.002% into a large group of cells.
Location: Bound to mast cells and basophils o Why dont we produce antibodies against our
throughout body. Blood. own antigens? We have developed
Half-life in serum: 2 days tolerance to them.
Complement Fixation: No o Clonal Deletion: B and T cells that react
Placental Transfer: No against self antigens appear to be destroyed
Known Functions: Allergic reactions. Possibly lysis of during fetal development. Process is poorly
worms. understood.
How Do B Cells Produce Antibodies? Consequences of Antigen-Antibody Binding

B cells develop from stem cells in the bone Antigen-Antibody Complex: Formed when an antibody binds
marrow of adults (liver of fetuses). to an antigen it recognizes.
After maturation B cells migrate to lymphoid Affinity: A measure of binding strength.
organs (lymph node or spleen). 1. Agglutination: Antibodies cause antigens (microbes) to
Clonal Selection: When a B cell encounters clump together.
an antigen it recognizes, it is stimulated and IgM (decavalent) is more effective
divides into many clones called plasma cells, that IgG (bivalent).
which actively secrete antibodies. Hemagglutination: Agglutination of
Each B cell produces antibodies that will red blood cells. Used to determine
recognize only one antigenic determinant. ABO blood types and to detect
influenza and measles viruses.
Clonal Selection of B Cells is Caused by Antigenic 2. Opsonization: Antigen (microbe) is covered with antibodies
Stimulation that enhances its ingestion and lysis by phagocytic cells.
Consequences of Antibody Binding
Humoral Immunity (Continued)
3. Neutralization: IgG inactivates viruses by binding to their
surface and neutralize toxins by blocking their active sites.
4. Antibody-dependent cell-mediated cytotoxicity: Used to
destroy large organisms (e.g.: worms). Target organism is
coated with antibodies and bombarded with chemicals from
nonspecific immune cells.
5. Complement Activation: Both IgG and IgM trigger the
complement system which results in cell lysis and
inflammation.
Humoral Immunity (Continued)

Apoptosis
o Programmed cell death (Falling away). Immunological Memory
o Human body makes 100 million lymphocytes Antibody Titer: The amount of antibody in the serum.
every day. If an equivalent number doesnt Pattern of Antibody Levels During Infection
die, will develop leukemia. Primary Response:
o B cells that do not encounter stimulating o After initial exposure to antigen, no
antigen will self-destruct and send signals to antibodies are found in serum for several
phagocytes to dispose of their remains. days.
oA gradual increase in titer, first of IgM and Recognize antigen on the surface
then of IgG is observed. of antigen presenting cells (e.g.:
o Most B cells become plasma cells, but some macrophage).
B cells become long living memory cells. Activate macrophages
o Gradual decline of antibodies follows. Induce formation of cytotoxic T cells
Immunological Memory Stimulate B cells to produce
(Continued) antibodies.
Secondary Response: 2. Cytotoxic T (Tc) Cells: Destroy target cells.
o Subsequent exposure to the same antigen Most are CD4 negative (CD4 -).
displays a faster and more intense antibody Recognize antigens on the surface
response. of all cells:
o Increased antibody response is due to the Kill host cells that are
existence of memory cells, which rapidly infected with viruses or
produce plasma cells upon antigen bacteria.
stimulation. Recognize and kill cancer
cells.
T Cells and Cell Mediated Immunity Recognize and destroy
Antigens that stimulate this response are mainly transplanted tissue.
intracellular. Release protein called perforin
Requires constant presence of antigen to remain which forms a pore in target cell,
effective. causing lysis of infected cells.
Unlike humoral immunity, cell mediated immunity is Undergo apoptosis when
not transferred to the fetus. stimulating antigen is gone.
Cytokines: Chemical messengers of immune cells. Cytotoxic T Cells Lyse Infected Cells
o Over 100 have been identified. 3. Delayed Hypersensitivity T (TD) Cells:
o Stimulate and/or regulate immune Mostly T helper and a few cytotoxic T cells that are
responses. involved in some allergic reactions (poison ivy) and
Interleukins: Communication rejection of transplanted tissue.
between WBCs. 4. T Suppressor (Ts) Cells: May shut down immune
Interferons: Protect against viral response.
infections.
Chemokines: Attract WBCs to Nonspecific Cellular Components
infected areas. 1. Activated Macrophages: Stimulated phagocytes.
Cellular Components of Immunity: Stimulated by ingestion of antigen
o T cells are key cellular component of Larger and more effective
immunity. phagocytes.
o T cells have an antigen receptor that Enhanced ability to eliminate
recognizes and reacts to a specific antigen intracellular bacteria, virus-infected
(T cell receptor). and cancerous cells.
o T cell receptor only recognize antigens 2. Natural Killer (NK) Cells:
combined with major histocompatability Lymphocytes that destroy virus
(MHC) proteins on the surface of cells. infected and tumor cells.
MHC Class I: Found on all cells. Not specific. Dont require antigen
MHC Class II: Found on stimulation.
phagocytes. Not phagocytic, but must contact
o Clonal selection increases number of T cells. cell in order to lyse it.
Relationship Between Cell-Mediated and Humoral
Types of T cells Immunity
1. T Helper (TH) Cells: Central role in immune response. 1. Antibody Production
Most are CD4+ T-Dependent Antigens:
Antibody production requires assistance Active Immunity

from T helper cells. You produce the antibodies
A macrophage cells ingest antigen and o Your body has been exposed to the antigen
presents it to TH cell. in the past either through:
TH cell stimulates B cells specific for antigen Exposure to the actual disease
to become plasma cells. causing antigen You fought it, you
Antigens are mainly proteins on viruses, won, you remember it
bacteria, foreign red blood cells, and hapten- Planned exposure to a form of the
carrier molecules. antigen that has been killed or
T-Independent Antigens: weakened You detected it,
Antibody production does not require eliminated it, and remember it
assistance from T cells. Vaccine
Antigens are mainly polysaccharides or Antigens are deliberately introduced into the immune
lipopolysaccharides with repeating subunits system to produce immunity
(bacterial capsules). Because the bacteria has been killed or weakened,
Weaker immune response than for T- minimal symptoms occur
dependent antigens. Have eradicated or severely limited several diseases
2. Antibody Dependent Cell Mediated Cytotoxicity from the face of the Earth, such as polio and smallpox
Target cell is covered with antibodies,
leaving Fc portion sticking outwards. How long does active immunity last?
Natural killer and other nonspecific cells that It depends on the antigen
have receptors for Fc region are stimulated Some disease-causing bacteria multiply into new
to kill targeted cells. forms that our body doesnt recognize, requiring
Target organism is lysed by substances annual vaccinations, like the flu shot
secreted by attacking cells. Booster shot - reminds the immune system of the
Used to destroy large organisms that cannot antigen
be phagocytosed. Others last for a lifetime, such as chicken pox
Antibody Production Think the flu is no big deal?

WBCs gobble up invading particles and break them Think again
up In 1918, a particularly deadly strain of flu, called the
They show the particle pieces to T-cells, who identify Spanish Influenza, spread across the globe
the pieces and find specific B-cells to help It infected 20% of the human population and killed
B-cells produce antibodies that are equipped to find 5%, which came out to be about 100 million people
that specific piece on a new particle and attach
Do we get all the possible vaccines we can?
Immunity Although the Center for Disease Control (CDC)
New particles take longer to identify, and a person recommends certain vaccines, many individuals go
remains ill until a new antibody can be crafted without them
Old particles are quickly recognized, and a person Those especially susceptible include travelers and
may never become ill from that invader again. This students
person is now immune. Consider the vaccine for meningitis, which is
recommended for all college students and infects
What is immunity? 3,000 people in the U.S., killing 300 annually
Resistance to a disease causing organism or harmful
substance Passive Immunity
Two types You dont produce the antibodies
Active Immunity o A mother will pass immunities on to her baby
Passive Immunity during pregnancy - through what organ?
o These antibodies will protect the baby for a Types of Transplant

short period of time following birth while its Autograft is self-tissue transferred from one body to
immune system develops. What endocrine another site in the same individual.
gland is responsible for this? Isograft is tissue between genetically identical
o Lasts until antibodies die individuals
Allograft is tissue transferred between genetically
Immune Disorders different members of the same species.
~Allergies~ Xenograft is tissue transferred between different
Immune system mistakenly recognizes harmless species
foreign particles as serious threats
Launches immune response, which causes sneezing, Immunology of Transplant Rejection
runny nose, and watery eyes Components of the Immune system involved in graft Rejection
Anti-histamines block effect of histamines and bring 1) Antigen presenting cells
relief to allergy sufferers Dendritic cells
Macrophages
Aquired Immune Deficiency Syndrome Activated B Cells
Caused by the Human Immunodeficiency Virus 2) B cells and antibodies
Discovered in 1983 Preformed antibodies
Specifically targets and kills T-cells Natural antibodies
Because normal body cells are unaffected, immune Preformed antibodies from prior
response is not launched sensatization
Induced antibodies
AIDS 3) T cells
~The Modern Plague~ 4) Other cells
The HIV virus doesnt kill you it cripples your Natural killer cells
immune system T cells that express NK cell
With your immune system shut down, common associated Markers
diseases that your immune system normally could Monocytes/Macrophages
defeat become life-threatening
Can show no effects for several months all the way The Immunology of Allogeneic Transplantation
up to 10 years Recognition of transplanted cells that are self or
AIDS foreign is determined by polymorphic genes (MHC)
~The Silent Spread~ that are inherited from both parents and are
Transmitted by sexual contact, blood transfusions, expressed co-dominantly.
contaminated needles Alloantigens elicit both cell-mediated and humoral
As of 2007, it affects an estimated 33.2 million people immune responses.
Recognition of Alloantigens
LECTURE 8: TRANSPLANTATION IMMUNOLOGY Direct Presentation
o Recognition of an intact MHC molecule
Introduction displayed by donor APC in the graft
Transplantation immunology - sequence of events o Basically, self MHC molecule recognizes the
that occurs after an allograft or xenograft is removed structure of an intact allogeneic MHC
from donor and then transplanted into a recipient. molecule
A major limitation to the success of transplantation is o Involves both CD8+ and CD4+ T cells.
the immune response of the recipient to the donor Indirect Presentation
tissue. o Donor MHC is processed and presented by
recipient APC
o Basically, donor MHC molecule is handled
like any other foreign antigen
o Involve only CD4+ T cells. Xenograft Response

o Antigen presentation by class II MHC
molecules Acute Rejection
Vascular and parenchymal injury mediated by T cells
and antibodies that usually begin after the first week
of transplantation if there is no immunosuppressant
Activation of Alloreactive T cells and Rejection of therapy
Allografts Incidence is high (30%) for the first 90 days
Donor APCs migrate to regional lymph nodes and are
recognized by the recipients TH cells. Acute Rejection
Alloreactive TH cells in the recipient induce generation 1. T-cell, macrophage and Ab mediated,
of TDTH cell and CTLs then migrate into the graft and 2. myocyte and endothelial damage,
cause graft rejection. 3. Inflammation
Role of CD4+ and CD8+ T Cells Chronic Rejection

CD4+ differentiate into cytokine producing effector Occurs in most solid organ transplants
cells o Heart
o Damage graft by reactions similar to DTH o Kidney
CD8+ cells activated by direct pathway kill nucleated o Lung
cells in the graft o Liver
CD8+ cells activated by the indirect pathway are self Characterized by fibrosis and vascular abnormalities
MHC-restricted with loss of graft function over a prolonged period.
Role of Cytokines in Graft Rejection Chronic Rejection

IL 2, IFN , and TNF - are important mediators 1. Macrophage T cell mediated
of graft rejection. 2. Concentric medial hyperplasia
IL promotes T-cell proliferation and generation of 3. Chronic DTH reaction
T Lymphocytes.
IFN - is central to the development of DTH Tissue and Organ Transplantation
response. Today it is possible to transplant many different
TNF - has direct cytotoxic effect on the cells of organs and tissues including.
graft. o Most common transplantation is blood
A number of cytokines promote graft rejection by transfusion.
inducing expression of class I or class II MHC o Bone Marrow transplantation
molecule on graft cell. o Organs : Heart, kidneys, pancrease, lungs,
The interferon (, and ), TNF and TNF - all liver and intestines.
increases class I MHC expression, and IFN - o Tissues : include bones, corneas, skin,
increases class II MHC expression as well. heart values, veins, cartilage and other
connective tissues.
Effector Mechanisms of Allograft Rejection
Hyperacute Rejection Bone Marrow Transplantation
Acute Rejection Used for Leukemia, Anemia and immunodeficiency,
Chronic Rejection especially severe combined immunodeficiency
(SCID).
Hyperacute Rejection About 109 cells per kilogram of host body weight, is
Characterized by thrombotic occlusion of the graft injected intravenously into the recipients.
Begins within minutes or hours after anastamosis Recipient of a bone marrow transplant is
Pre-existing antibodies in the host circulation bind to immunologically suppressed before grafting.
donor endothelial antigens
Activates Complement Cascade
Eg. Leukemia patients are often treated with cyclo- Liver one year survival exceeds 75% and
phosphamide and total body irradiation to kill all five year is 70%.
cancerous cells.
Because the donor bone marrow contains Pancreas Transplantation :
immunocompetent cells, the graft may reject the host, Offers a cure for diabetes mellitus.
causing graft versus host disease (GVHD). Graft survival is 72% at one year.
Further improved if a kidney is transplanted
Graft vs. Host Disease simultaneously.
Caused by the reaction of grafted mature T-cells in Overall goal - to prevent the typical diabetic
the marrow inoculum with alloantigens of the host secondary complications.
Acute GVHD
o Characterized by epithelial cell death in the Skin grafting :
skin, GI tract, and liver It is used to treat burn victims.
Chronic GVHD In severe burn, grafts of foreign skin may be used and
o Characterized by atrophy and fibrosis of one rejection must be prevented by the use of
or more of these same target organs as well immunosuppressive therapy.
as the lungs
Xenogeneic Transplantation
Heart Transplantation : A major barrier to xenogeneic transplantation is the
First heart transplant in South Africa by Dr. presence of natural antibodies that cause hyperacute
Christian Barnard in 1964. rejection.
One year survival rate is >80%.
HLA matching is desirable but not often Immunosuppressive Agents
possible, because of the limited supply of Immunosuppression can be brought about by 3 different ways :
heart and the urgency of the procedure. Surgical ablation
Total Lymphoid Irradiation
Lung Transplantation : Immunosuppressive drugs
First attempt in 1963 by Hardy and Co -
workers. Immunosuppressive Drugs
First successful transplantation by Toronto Three main immunosuppressant drugs
group in 1983. o Cyclosporins act by inhibiting T-cell
In conjunction with heart transplantation, to activation, thus preventing T-cells from
treat diseases such as cystic fibrosis and attacking the transplanted organ.
emphysema or acute damage to lungs. o Azathioprines disrupt the synthesis of DNA
First year survival rate is about 60%. and RNA and cell division.
o Corticosteroids such as prednisolone
Kidney Transplantation : suppress the inflammation associated with
Diseases like diabetes and various type of transplant rejection.
nephritis can be elleviated by kidney
transplantation. Immunosuppressants can also be classified depending on
Survival rate after one year transplantation the specific transplant:
is >90%. Basiliximab in combination with cyclosporin
25,000 candidates are waiting for kidney and corticosteroids, in kidney transplants.
transplantation. Daclizumab in combination with cyclosporin
and corticosteroids, in kidney transplants.
Liver transplantation : muromonab CD3 (Orthoclone OKT3) along
It treat congenital defects and damage from with cyclosporin, in kidney, liver and heart
viral (hepatitis) or chemical agents. (Chronic transplants.
alcoholism). Tacrolimus is used in liver transplants and is
under study for kidney, bone marrow, heart,
pancreas, pancreatic island cell, and small modified animal may become a new source of organ
bowel transplantation. supply.
Side effects of immunosuppressive agent use for graft
Some immunosuppressants are also used to treat a need a change of specificity in action and avoiding
variety of autoimmune diseases: general immune suppression.
Azathioprine in treatment of rheumatoid Techniques such as transgenic animal production and
arthritis , chronic ulcerative colitis but limited wide range of research in this field hope to result in
value. opening a new window for the process of
Cyclosporin is used in heart, liver, kidney, transplantation immunology.
pancreas, bone marrow and heart/lung
transplantation. Also used to treat psoriasis
and rheumatoid arthritis, multiple sclerosis,
diabetes and myesthenia gravis.
Glatiramer acetate is used in treatment of
relapsing-remitting multiple sclerosis.
Mycophenolate is used along with
cyclosporin in kidney, liver and heart
transplants. Also used to prevent the kidney
problems associated with lupus
erythematosus.
Sirolimus in combination with cyclosporin
and corticosteroids, in kidney transplants.
The drug is also used for the treatment of
psoriasis.
Immunosuppressive Therapy
Monoclonal antibodies
To suppress the activity of subpopulation of T-cells.
To block co-stimulatory signals.
Ab to the CD3 molecule of TCR (T cell receptor)
complex results in a rapid depletion of mature T-cells
from the circulation.
Ab specific for the high-affinity IL-2 receptor is
expressed only on activated T-cell, blocks
proliferation of T-cells activated in response to the
alloantigens of the graft.
To treat donors bone marrow before it is
transplanted.
Molecules present on particular T-cells subpopulation
may also be targeted for immunosuppressive therapy.
Antibody to CD4 shown to prolong graft survival.
Ab specific for implicated cytokine can prolong the
survival of graft.
Conclusion
More than 50,000 people, waiting for compatible
donor. For ethical an practical reasons, species
closely related to human such as Chimpanzee have
not been widely used.
Xenogenetic transplantation may be major issue of
research xenograft technology including genetically

Reviewer Co1

Uploaded by

Copyright:

Available Formats

Reviewer Co1

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Reviewer Co1

Uploaded by

Copyright:

Available Formats

1 STEM CELL REVIEWER CO1

LECTURE 1: SCOPE OF TISSUE ENGINEERING o growth factors, steroids, hormones,

suspension (non-adherent cells) Stem Cells

Colony-Forming Units (CFUs) d) Perfusion

The Study of Tissues

o named by shape of apical cells

Stratified Cuboidal Epithelium

o brown fat (hibernating animals) produces Bone

Fibrocartilage Skeletal Muscle

Cutaneous membrane = skin o disuse atrophy from lack of use (leg in a

Macrophages start to clean up debris

Chronic wounds characteristics

Increased inflammatory cytokines

Wound bed preparation

Tissue engineering - Biology, medicine and technology are

Role of GROWTH FACTORS LECTURE 4A: STEM CELLS

Multipotent - stem cells can produce only cells of a

What is a Stem Cell?- Properties

What is Self Renewal? Differentiation?

Characterization of human and mouse ES cells

o Demonstrably safe Cultures of animal cells are usually divided into 3

The number of passages decreases when cells are

2- Cell Strains Cell Culture Systems

techniques involved in the growth of the cell type of L-Glutamine

Temperature & Humidity The vessel surface is treated to render it hydrophilic

o Development of a cell line over several Outgrowth of excised tissue in

Types of continuous culture

the disadvantage of having retained the incubation temperature

Biology of Culture cells Factors affecting cell diferentiation

Cell adhesion molecule regulate membrane potential by

o Osmolarity Culture vessels

LECTURE 5: THE HUMAN IMMUNE SYSTEM

Biosafety cabinets Escherichia coli - is common and plentiful in all of our

Viruses Proteins that latch onto, damage,

Two Divisions of the Immune System Components of Human Immune System

Transplanted tissue Antibody Structure

Complement Fixation: No oMany virus infected cells will undergo

How Do B Cells Produce Antibodies? Consequences of Antigen-Antibody Binding

Humoral Immunity (Continued)

Antibody production requires assistance Active Immunity

Antibody Production Think the flu is no big deal?

o These antibodies will protect the baby for a Types of Transplant

o Involve only CD4+ T cells. Xenograft Response

Role of CD4+ and CD8+ T Cells Chronic Rejection

Role of Cytokines in Graft Rejection Chronic Rejection

You might also like