Studied by 2 peopleWhat are Cell-Based Therapies?
delivering cells as therapeutics instead of drugs
Examples of Cell-Based Therapies
-blood transfusion -bone marrow transplant -organ transplants
What is it called when a cell recipient is also the donor?
Autograft
What is it called when a cell recipient is a different organism from the cell donor?
Allograft
What is the main advantage of autograft therapies?
no immune rejection
Why are transplants insufficient?
There is an organ shortage --> people die on the waiting list for organs
What is a kidney transplant chain?
-one person is a match for a stranger instead of the person they are checked against -they donate to that second recipient -those whoa re checked for the second recipient may be a match for a third recipient -they donate to that third recipient, and so on
What is the term for the first donor in a kidney transplant chain?
Altruistic donor
Tissue Engineering: Therapeutics for tissue function
-restore -maintain -improve
Tissue Engineering: General Steps
cell sourcing from donor
manipulation or expansion of cells
cell seeding and extracellular matrix expression
culture cells
implantation into the recipient
Tissue Replacement: Three Ends of Therapeutic Context
Whole Organ Transplantation
Engineered Tissues
Medical Devices
Tissue Replacement: What type of transplant do engineered tissues involve?
-not a whole organ -interfacing device
Tissue Replacement: Engineered Tissues
biomaterials and cells
Tissue Replacement: Engineered Tissues - Types of Biomaterials
-acellular -hybrid -biomaterial-free systems w/cell derived ECM
Tissue Replacement: Engineered Tissues - acellular
-biomaterials that recruit cells -biomaterial by itself -cells from the body may get recruited to the biomaterial
Tissue Replacement: Engineered Tissues - hybrid
-biomaterials that house cells -example: ear grown in mouse
Tissue Replacement: Engineered Tissues - biomaterial-free systems w/cell-derived ECM
-cells can make a material themselves -cells do not absolutely require biomaterial
Tissue Replacement: Most common acellular engineered tissue
artificial skin
What is the most successful artificial skin development?
Integra
Integra
-bovine collage and shark GAG -silicone surface layer to provide barrier -host cells infiltrate and remodel matrix over 3 weeks -silicone is peeled off and replaced by ultra-thin split-thickness graft
What is the purpose of silicone in integra?
it serves as a synthetic outer barrier
What is an example of a hybrid engineered tissue?
Hybrid Apligraf from Organogenesis -this has a scaffold and cells because it is a hybrid
Hybrid Apligraf from Organogenesis
-dermal cells put onto scaffold for bottom layer -epidermal layer added afterwards -fed from underneath like actual skin with blood vessels -exposing top part to air to simulate barrier function
Key Question of Designing an Engineered Tissue
What function are you replacing?
Function of Tissue Replacements
-fulfill a biomechanical role -replace a physiological function (i.e. metabolism) -deliver a secretory product -a combination of the above
Where do primary cells originate?
-directly from an animal -can be from an adult organism or from early-stage cells
What are the two terms for cells no longer duplicating anymore?
-senescence -Hayflick Limit
Hayflick Limit
-after enough passages, you often reach a limit where cells cannot proliferate anymore -somtimes, transformation beyond the Hayflick Limit can occur -if you get primary cells from an animal source, there is likely a limit to how much they can go
What is an example of cells that grow beyond their Hayflick Limit?
cancer cells
Do embryonic and adult stem cells reach the Hayflick limit?
if they are cultured the right way, they will not reach it (or at least not nearly as quickly)
What is the idea of developing patient-specific pluripotent stem cells?
-solve the exact issue at hand -more easily avoid immune rejection
What is a natural biomaterial?
something made by cells and tissues (ex. ECM)
Examples of synthetic biomaterial scaffolds
-PLGA, PCL -PEG -ePTFE -glycans -titanium
What is Fabrication?
the idea of creating a specific structure that dictates function
What is stereolithography?
3-D fabrication --> you can have a scaffold made that has a specific structure to serve a specific function
Scaffolds from Decellularized Organs: Heart
-rate hearts treated with detergents to lyse cells -matrix is there as a scaffold -if you add autograft cells, maybe this could be used -takes a long time, so timing can be complicated -it is hard to get cells inside the heart
Tissue Classifications
Epithelial Tissue
Connective Tissue
Muscle Tissue
Neural Tissue
Tissue Classifications: Epithelial Tissue
-tightly packed continuous cell sheets -serve as linings and barriers -examples: outer layer of skin, inside of mouth
Tissue Classifications: Connective Tissue
-support and structure -fewer cells -larger ECM -examples: inner layer of skin, tendons, blood, fat, bone
Tissue Classifications: Muscle Tissue
-optimized for contraction
Tissue Classifications: Neural Tissue
-neurons and glial cells -examples: brain, spinal cord, peripheral nerves
Epithelial cells
-tightly packed -asymmetric (polarized) -lots of cell-cell interactions -form junctions
Two sides of epithelial cells
-apical (facing inside of organ) -basal (attached to connective tissue)
Epithelial Sheets: Types of Junctions
-tight junction -adherens junction -desmosome -gap junction -hemidesmosome
Tissue Classifications: Types of Junctions - tight junction
-seals neighboring cells together -prevents leakage
Tissue Classifications: Types of Junctions - adherens junction
-joins an actin bundle in one cell to a similar bundle in another cell -actin cytoskeleton is connected
Tissue Classifications: Types of Junctions - desmosome
-joins intermediate filaments in one cell to those of a neighbor
Tissue Classifications: Types of Junctions - gap junction
-forms channels -allow small water-soluble molecules (including ions) to pass from cell to cell -important with muscle
Tissue Classifications: Types of Junctions - hemidesmosome
-anchors intermediate filaments to basal lamina
Adherens Junctions
-linker protein bound to actin to maintain tension -linker also connected to cadherin molecule -cadherins of several cells bind together
What is a cadherin?
An extracellular molecule attached to a cell and used to bind to other cadherins for cell-cell adhesion
Connective Tissues
-more matrix -cell density is lower
Examples of connective tissue component
collagen
Connective Tissues Component: Collagen
-provides tensile strength like a rope -connective tissues make lots of collagen
Connective Tissues Components: Fibroblasts
-good at making/remodeling matrix -prevent tissue from being pulled apart -secrete and reorganize collagen
Connective Tissues Components: Protecoglyans
-subclass of glycoproteins -provide compressive strength -long unbranched repeating polysaccharide -sugars are hydrophilic --> water helps to maintain structure and compressive strength
Muscle Tissue
-very close to each other -gap junctions --> important for calcium
Hepatocytes
-epithelial cells in liver -apical surface on inside between cells -basal surface on both sides for blood to interact
What was the difference between single-gel collagen growth of hepotocytes in comparison to sandwich growth within 2 gels?
-cells almost just died with single gels -hepatocytes make more proteins when sandwiched
Why are cells the size that they are?
-most efficient size for transport from blood -if too big, O2 may not diffuse into the center cells -things may not be able to get out, either -at their size, O2 and nutrients can interact most efficiently
What is a liposome?
-lipid bilayer with hollow interior -inside can be aqueous
What is a micelle?
-single spherical layer of lipids -can be printed into oil -can later be made into liposomes
How would liposomes be used as model cells/tissues?
-printing aqueous micelle droplets -assembly into a bilayer with an open interior
If a column of two types of cells are made, with the right side having low concentrations of solute and the left side having high concentrations of solute, what happens?
What drives this system?
-right cells shrink -left cells swell -overall structure starts to bend/close
A difference in osmolarity (gradient of something) drives this system
History of cell sorting
-dissociated cells reconstruct in the same pattern -mimic events in vivo -sorting vs invasion is a fundamental concept in tissue engineering
Work of Adhesion
-concept that underlays the interface of liquids -this is the work required to inject air at the interface -it is the energy required to separate it from another thing
What does it mean if something has a high work of adhesion?
-it takes a lot of work to separate them -high level of adhesion
Three Types of Tissue Sorting
sorting
random
separation
Three Types of Tissue Sorting: Sorting
Waa > Wab ≥ Wab
aa wants to bind itself and binds ab just as much, so a is completely on the inside with b surrounding it
Three Types of Tissue Sorting: Random
Waa ≤ Wab > Wbb
they can interact with each other or themselves, so they are randomly organized
Three Types of Tissue Sorting: Separation
Waa ≥ Wbb >>Wab
a binds to itself more than it does to b, b binds to itself more than it does to a, so the two molecules will separate
How to Measure Surface Free Energy?
-surface tension for a liquid droplet between two parallel plates -parallel plate tensiometer -medium surrounding cell -calculate interfacial tension between droplet and its surrounding medium, which depends on compression force and different radii of curvature of the droplet's surface
Surface Tension in Cell Spreading
-spreading behavior of embryonic tissues is different due to surface tension -continuously record force and aggregate's profile as it attempts to return its shape from a parallel plate tissue surface tensiometer -essentially "cell squishing"
Surface Tension Findings in Embryonic Tissues
higher surface tension --> goes to inside
Describe mechanisms of cell-cell adhesion
-use cadherins and other adhesion molecules on surface -interactions can occur due to calcium ions -cell adhesion zippers can be made between two sets of fibers that meet in alternating fashion
Morphogenesis
-describes evolution and development of how tissues and organs form -refers to structural and functional changes observed during development and elucidation of underlying mechanisms -can be broader in tissue engineering
Broader view of morphogenesis for tissue engineering
formation and differentiation of tissue and rogans
Morphagen
-signal that causes morphogenesis
Morphogenetic Spatiotemporal Scales
-space constraints --> limited by how much they can communicate -time constraints --> influenced by cellular fate process
Embryogenesis: Steps and differentiation abilities
-zygote: single cell -totipotent: can become any cell type of embryo or extraembryonic (i.e. placenta) -blastocyst: inner cell mass is pluripotent --> can become embryonic stem cells or any embryonic cells
What is a trophoblast?
the outer layer of the blastocyst that becomes the placenta
As the inner cell mass develops, what happens?
-splits into two layers -yolk sac cavity forms where blood cells form in the embryo over time
What are the two layers of the inner cell mass once it splits?
-epiblast -hypoblast
What is the streak of cells on the epiblast?
line of cells that starts to separate dorsal from ventral
Developmental Order of Tissues
Blastocyst -->
inner cell mass & trophoblast (extraembryonic) -->
inner cell mass becomes epiblast (all tissues in the body) and hypoblast (extraembryonic)
What happens to the epiblast after it is formed?
it goes through gastrulation
Gastrulation
formation of three tissue layers from epiblast
Three Tissue Layers
Ectoderm
Mesoderm
Endoderm
Three Tissue Layers: Endoderm
-digestive tube -internal organs
Three Tissue Layers: Mesoderm
-muscle -skeleton -heart -kidneys
Three Tissue Layers: Ectoderm
-skin -brain -spinal cord
Self-Organization Epithelial Cell Paper: MEPs and LEPs
-two kinds of epithelial cells -LEPs secrete milk proteins and reside on the inside of MEPs
Self-Organization Epithelial Cell Paper: How did they track how LEPs and MEPs organized?
authors used K14 (keratin) to stain surface markers on the outside of the cells
Self-Organization Epithelial Cell Paper: How did they separate LEPs from MEPs for labeling?
used FACS --> single-cell sorting
Self-Organization Epithelial Cell Paper: How/why can FACS be done on LEPs and MEPs?
-they express different proteins -this way, they can be individually labeled and tracked
Self-Organization Epithelial Cell Paper: Describe the microwell structure used by the authors
-you can make a 3D structure -can define dimensions and number of cells -made from non-adhering plastic --> proteins can bind
Self-Organization Epithelial Cell Paper: Why were HMECs with each label used?
-served as controls -LEPs and MEPs in this population (no sorting) -arbitrarily labeled them to see if the labels had an effect
Self-Organization Epithelial Cell Paper: What did the authors find?
LEPs started to centralize at the core with more MEPs around the outside
Self-Organization Epithelial Cell Paper: Why did the authors visualize cadherin for MEPs and LEPs?
-they found that LEPs had higher E-cadherin -E-cadherin may be the reason why the cells are sorting
more adhesion molecules = better cell-cell adhesion = higher surface tension (higher work of adhesion) --> may cause those cells to move inside
Self-Organization Epithelial Cell Paper: What happened when anti-E-cadherin molecules were added?
the cells did not sort
Note 
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