Download Immunoisolation

Immunoisolation
In 1964, T.M.S. Chang1 proposed the idea of using ultrathin
polymer membrane microcapsules for the immunoprotection of
transplanted cells and introduced the term ‗artificial cells‘ to
define the concept of bioencapsulation.
This was implemented 20 years later to immobilize xenograft
islet cells.
When implanted into rats, the microencapsulated islets
corrected the diabetic state for several weeks2.,
1.
2.
Chang, T.M.S. Semipermeable microcapsules. Science 146, 524–525 (1964).
Lim, F. & Sun, A.M. Microencapsulated islets as bioartificial endocrine pancreas. Science 210, 908–
909 (1980).
Cell/Tissue Transplantation
•Diabetes (insulin)
•Parkinson’s disease (dopamine producing cells) is a degenerative
disorder of the central nervous system that often impairs the sufferer's
motor skills, speech, and other functions
•Chronic pain
•Alzheimer’s, common form of dementia, degenerative, and terminal
disease
•Huntington’s disease is a progressive neurodegenerative genetic
disorder,
•CNS malignancies
Cell/tissue sources:
Autogeneic: arising from self; pertaining to an autograft
Allogeneic: denoting individuals (or tissues) that are of the same species
but antigenically distinct
Xenogenic: Originating outside the organism or from a foreign substance
introduced into the organism
Genetically engineered: they could potentially lead to generate tumor
Immunoisolation
Cells and tissue are enclosed in a semipermeable membrane.
The function of this semipermeable membrane;
•Must prevent immune attack by host
•Must allow diffusion of nutrients metabolic products and
therapeutic agent
Cell microencapsulation. a, Nutrients, oxygen and stimuli diffuse across the
membrane, whereas antibodies and immune cells are excluded. b, Pre-vascularized
solid support system to facilitate optimal nutrition of the enclosed cells.
Immune System
Cellular Component
•T-cells
•Macrophages, etc.
Humoral Component
•Antibodies (IgGs, IgMs)
•Complement (C3, C5a, etc.)
•Cytokines (interleukin 1 , IL1 , etc.)
•These will interact with the capsule if the membrane does not
have proper characteristics.
Immunoisolation
Membranes can be made
•microporous, pore size ~0.6 micronmeter
•Nanoporous ( molecular weight cut off ~ 30,000 Da )
IgG MW ~ 150 kDa
IgM MW ~ 900 kDa
C3 MW ~ 200 kDa
IL1- MW ~ 17 kDa
Bigger Challenge : Macrophages can generate small
molecular weight species that can affect cell viability (free
radicals, H2O2, NO)
Microencapsulation of islets
Glucose
Insulin
islet
O2 & nutrients
Immune cells
Antibodies
Membrane Function
Allow diffusion of:
•Nutrients, O2, waste products
•Metabolic products, therapeutic agent
•Signal that triggers production of therapeutic agent.
Prevent diffusion of
immune rejection by host
fibrous formation (important one, because if you get
fibrous capsule forming around the implant, it‘ll ellicit an
inflammatory response, fibroblasts will compete for
nutrients.
viral transfer to host (in the case of
xenotransplantation)
invasion of the host by the transplanted cells
Challenges for Cell Transplantation
1.Cell/Tissue Source
•Tissue donors
•Xenogeneic cells
•Cell lines
For the treatment of conditions affecting the CNS (alzheimer‘s),
~106 cells may be required.
For the treatment of diabetes, ~109 cells may be required.
Enough tissue may not be available because of lack of donors.
2.Semipermeable Membrane
•The precise molecular weight cut off of the membrane has not
been established.
C5 MW 10 kDa
IL-a MW 17 kDa
Some studies suggest that diffusion of molecules with MW
~75 kDa are necessary to sustain long term survival of the
transplanted cells
Challenges for Cell Transplantation
3.Oxygen supply to transplanted cells
This will depend on the metabolic activity of the cells
Site of implantation
• pO2 ~100 torrs in arterous blood
• pO2 ~ 40 torrs in microvasculature
Approaches for cell encapsulation:
Hallow fibers/capsules: cells are on the outside and blood is
circulating in the hallow fiber.
Cell encapsulation with hydrogel (high water content). This will
allow fast diffusion of nutrients, most important property of the
hydrogel will be the crosslink density and the thickness.
Types of Bioartificial Pancreas
Diffusion chamber, Hollow
fiber unit and Microcapsule
•The diffusion chamber and hollow fiber types of bioartificial pancreas can be
classified as a macrocapsule type.
•In diffusion chamber type pancreases, Nuclepore™ membranes with a pore size
of 0.05–1 μm have been used as an immunoisolation membrane.
•It was reported that normoglycemia was maintained for 30 weeks in
streptozotocin (STZ)-induced diabetic rats by implanting a device that
encapsulated mice pancreatic beta cell lines (MIN6) (xenotransplantation).
•Hallow fiber device consists of a chamber passing a semi-permeable tubular
membrane that is connected to vascular grafts. Islets are placed between the
housing chamber and the tubular membrane.
Types of Bioartificial Pancreas
Bioartificial pancreas. (a) The concept of the immunoisolation membrane. (b)
Islets enclosed in agarose microcapsules.
Types of Bioartificial Pancreas
Islets can be encapsulated by forming a suspension in alginate, consists of
polysaccharide followed by exposure to Ca+2 ions
Microencapsulation of islets
with alginate–
polylysine.
Techniques of Encapsulation
Interfacial polymerization:The technique involves physical adsorption of the
initiator eosin Y on the islet cell surface, which then allows polymerization to
occur on the islet–prepolymer interface. The interfacial photoinitiation process
employed with this technique results in conformal coating of cross-linked PEGbased hydrogel on the islet cell surface.
The process of selective withdrawal is another
novel method reported recently for the purpose
of coating cell clusters, such as islet cells.171
Briefly, the process involves the insertion of a
tube into a container such that its tip
is suspended at a specific height above an
interface separating two immiscible fluids.
Air-jet syringe pump droplet generator:
The device consists of an air jacket surrounding
an alginate nozzle through which alginate
solution is injected. As alginate droplets are forced
out of the end of the needle by the syringe pump,
the droplets are pulled off by the shear forces set up
by the flowing air stream. The size of the spherical
droplets is controlled by adjusting the flow rate
of the air jacket.
Techniques of Encapsulation
Layer-by-layer Assembly of Conformal Nano-thin PEG Coating
poly(l-lysine)-g-poly(ethylene
glycol)biotin
and streptavidin (SA),
Wilson, Cui, Chaikof, Nanoletters, 2008; 8(7):1940-1948.
Fluorescent Staining Assay
NHS-PEG-biotin
rinse
Streptavidin
Biotin-PEG-FITC
Dynamic insulin response to glucose stimulation of coated
and uncoated rat islets
Insulin secretion is enhanced after formation of biotin-PEGNHS/SA/biotin-PEG-GLP-1 coating on islet surface.
Insulin secretion is higher after third step of
surface coating, which binds GLP-1 to islet
surface.
0.001
biotin-PEG
SA-biotin-PEG
GLP-1-PEG-biotin-SA-biotin-PEG
Control
6.0
5.0
4.0
3.0
2.0
1.0
0.0
0
10
3.3 mM
glucose
20
30
Time (min)
16.7 mM
glucose
40
50
60
Insulin Released per Minute as a Fraction of Total
Insulin
Insulin Concentration ( g/L)
7.0
Insulin is normalized as a fraction of total
insulin.
Control
0.0008
Modified
0.0006
0.0004
0.0002
0
0
3.3 mM
glucose
10
20
30
Time (min)
40
50
60
Microencapsulation of islets
Interfacial polymerization
Xenotransplantion of Porcine
Islets into rats. Mice maintained
normoglycemia for up to 110
days. (~100 micron)
Cruise GM et al., Cell
Transplantation, Vol. 8
pp. 293-306, 1999.
Selective withdrawal
(~20 micron)
Alginate encapsulation
(~800 micron)
Wyman, Kizilel, Mrksich,
Nagel, Garfinkel, Small,
2007 Apr;3(4):683-90.
Kim S, Kim SW, Bae YH,
Biomaterials 26, 35973606, 2005.
Stem Cells
•Stem cells offer the potential to replicate
indefinetely in culture but also to generate many or
most of the cells in the body (including those cells
that normally do not regenerate such those found in
the central nervous system and cardiac muscle).
•A stem cell can replicate and produce cells that
take more specialized functions.
•Development potential or potency of a stem cell
refers to the breadth of functions that more
differentiated daughter cells and their progeny can
adopt.
Stem Cells
•To generate cultures of specific
types of differentiated cells—heart
muscle cells, blood cells, or nerve
cells—scientists try to control the
differentiation of embryonic stem
cells.
•The chemical composition of the
culture medium, the surface of the
culture dish may be changed or the
cells can be altered by inserting
specific genes.
•Scientists have established some
basic protocols or ―recipes‖ for the
directed differentiation of
embryonic stem cells into some
specific cell types.
http://stemcells.nih.gov/info/basics/
Stem Cells
•Unipotent stem cells give rise to only one type of
differentiated cells
•The terms oligopotent, pluripotent, and multipotent
represent and increase in the number of
differentiated cells that can be derived from a stem
cell (from few to many to most)
•A totipotent cell can generate all of the different cell
types that comprise an organism.
Normal differentiation pathways of adult Stem Cells
In a living animal, adult stem cells are available to divide for a long period, when
needed, and can give rise to mature cell types that have characteristic shapes and
specialized structures and functions of a particular tissue.
http://stemcells.nih.gov/info/basics/
Normal differentiation pathways of adult Stem Cells
Hematopoietic stem cells give rise to all the types of blood cells: red blood cells, B
lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils,
eosinophils, monocytes, and macrophages.
Mesenchymal stem cells have been reported to be present in many tissues. Those
from bone marrow (bone marrow stromal stem cells, skeletal stem cells) give rise
to a variety of cell types: bone cells (osteoblasts and osteocytes), cartilage cells
(chondrocytes), fat cells (adipocytes), and stromal cells that support blood
formation. However, it is not yet clear how similar or dissimilar mesenchymal cells
derived from non-bone marrow sources are to those from bone marrow stroma.
Neural stem cells in the brain give rise to its three major cell types: nerve cells
(neurons) and two categories of non-neuronal cells—astrocytes and
oligodendrocytes.
Epithelial stem cells in the lining of the digestive tract occur in deep crypts and
give rise to several cell types: absorptive cells, goblet cells, Paneth cells, and
enteroendocrine cells.
Skin stem cells occur in the basal layer of the epidermis and at the base of hair
follicles. The epidermal stem cells give rise to keratinocytes, which migrate to the
surface of the skin and form a protective layer. The follicular stem cells can give
rise to both the hair follicle and to the epidermis.
http://stemcells.nih.gov/info/basics/
Stem Cells
Specialized
cells
Stem Cells
•Only a few stem cell types can be classified as
pluripotent/totipotent:
•Embryonal carcinoma cells (EC); derived from a
teratocarcinomas that arose from transfer of a
postimplantation embryo to an ectopic site.
•Ebryonic stem cells (ES); derived from the inner
cellular mass (ICM) of preimplantation embryos.
•Embryonic germ cells (EG); derived from primordial
germ cells (PGC) that migrate to and colonize the
gonad.
Stem Cells
•Multipotent stem cells can be obtained from fetal
and adult sources. The best known are cells derived
from bone marrow which contains hematopoietic
stem cells.
•Pluripotent cells posses high levels of alkalyne
phosphatase (AP) activity and present specific cell
surface glycolipids and glycoproteins. However,
these are not sufficient and/or necessary conditions
for pluripotency.
Stem Cells
•The only direct measurement of developmental
potential is the analysis of cell differentiation.
•Most EC and EG require leukemia inhibitory factor
(LIF; ~1000 U/ml) in the culture media to remain
pluripotent.
•Withdrawal of LIF enables cell differentiation if the
cells are put in an environment conductive to
aggregation rather than adhesion to a substrate.
Stem Cells
•ES/EG can generate cells of hemapoietic lineage
and cardiomyocytes if the proper growth and
differentiation factors are supplemented to the
culture media.
•Mouse ES have been used to generate in vitro
cultures of neurons, skeletal muscle, vascular
endothelial cells, adipocytes, and visceral
endoderm.
Stem Cells
•Exogenous factors can also be used to direct
differentiation (retinoic acid, dimethyl sulfoxide,
hexamethyl bisacetamide, dibutryl cAMP, forskolin,
3-isobutyl-1-methyl-xanthine.
•The modes of action by which these compounds
act are not fully understood.
•Growth factors can also be used to induce
differentiation of pluripotent stem cells.
Stem Cells
•The exact components that cause a desired
differentiation effect are often unknown.
•The nature of the substrate that the cell attaches to
also has a strong impact in its characteristics.
•Collagens I and IV have been used to promote
adhesion and cell division in a number of cells
(vascular and endothelial cells). Laminin has been
shown to promote neurite outgrowth in many culture
systems including ES cell-derived neuronal cells.
Stem Cells
•Cell derived extracellular matrices and human ECM
have also been used to support differentiation of
cells.
•Human mesenchymal stem cells comprise only
0.01-0.0001 % of total nucleated cells in bone
marrow.
•Genetic selection, transcription factor expression,
fluorescence activated cell sorting can be used
identify and generate pure populations of specific
cell lines.
Stem Cells
•When bone marrow derived from a variety of species
is placed in a diffusion chamber that is implanted into
the peritoneum of immunotolerant hosts, bone and
cartilage are synthesized inside the diffusion
chambers from the marrow derived MSCs and their
descendents. (Hematopoietic cells expire within the
chamber while mesenchymal cells divide and
differentiate into cartilage and bone forming cells.
Adv Drug Del Rev 1998, 33,3.
Stem Cells
•Bone develops along the inner surface closer to the
overlying vasculature.
•Cartilage forms in the central region, away from
vasculature in an envirenment that is not as rich in
environmental stimuli.
•If the host is irradiated prior to implantation of cell
loaded diffusion chamber, neither bone, nor
cartilage develops, instead, hematopoietic cells
multiply.
Adv Drug Del Rev 1998, 33,3.
Adv Drug Del Rev 1998, 33,3.
Adv Drug Del Rev 1998, 33,3.
Stem Cells
•The in vivo environment of old rats supports the
differentiation of MSCs into osteoblasts at levels
similar to those observed in young rats.
•The osteogenic potential of MSCs from young and
old rats is identical.
•The number of marrow-derived osteoprogenitors
declines as a function of donor age.
Adv Drug Del Rev 1998, 33,3.