BIOE 476: Exam 1 (copy) (super big set)

What are Cell-Based Therapies? fdfd

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

1. cell sourcing from donor

2. manipulation or expansion of cells

3. cell seeding and extracellular matrix expression

4. culture cells

5. implantation into the recipient

Tissue Replacement:
Three Ends of Therapeutic Context

1. Whole Organ Transplantation
2. Engineered Tissues
3. 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

1. Epithelial Tissue
2. Connective Tissue
3. Muscle Tissue
4. 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

1. sorting
2. random
3. 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

1. Ectoderm
2. Mesoderm
3. 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