MSE 104 Midterm 2
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166 Terms
What are the advantages of using CRISPR/Cas9 over similar systems like TALEN or ZFN?
CRISPR/Cas9 allows for the targeting of multiple DIFFERENT sequences simultaneously as well as limiting off target effects compared to TALEN and ZFN
What is a Bioreactor
A bioreactor is a vessel in which biological processes take place vie the culturing of cells.
A space in which we can grow cells for an experiment that allows us to control parameters of the cells environment.
What main processes are required of bioreactors?
Temperature Control
Nutrient input and output of waste products
Allow or facilitate gas exchange (Mainly of O2 and CO2)
Sealed from the outside
Introduce agitation to the system to prevent biofilm formation
Have other sensors that allow for further control of the system.
Cells usually uptake oxygen around what rate?
0.5-1.0 micromol/10^6 cells / hour
What is the common concentration of oxygen in the body?
0.2 mM
Explain the tradeoff between height of a liquid and diffusivity in the context of oxygen uptake by cells
The higher the level of liquid in a dish of cells, the longer it takes for oxygen or any other gases to diffuse through the liquid to reach the bottom of the wells where cells are located, thus limiting the uptake rate of oxygen by the cells.
The maximum height of the liquid possible therefore correlates to the minimum oxygen uptake required for survival since there is an inverse relationship between the two.
What equation is used to calculate the diffusion of oxygen through a liquid?
Fick’s Equation
O2 Flux = (D/R)*[Cstar - Cbed]
where
D = Diffusivity of oxygen through the medium
R = The height of the medium
C star = The concentration of oxygen in the air above the liquid
C bed = Concentration of oxygen at the bed (where the cells are).
How can Ficks equation be related to the Michaelis Menten Equation and what does it mean?
The Michaelis menten equation is used to solve for the maximum consumption rate of oxygen per cell. Therefore, at the maximum height of a liquid, we can measure the corresponding maximum oxygen consumption of the cells (v), as all of the oxygen diffusing to the bottom (O2 Flux) will be taken up by the oxygen and consumed.
What is the Michaelis menten Equation used for and what is its formula?
The Michaelis menten equation is used to calculate the maximum oxygen consumption rate per cell at a specific concentration of oxygen at the bed.
v = (Vmax[O2])/(K’M + [O2])
where
v = the rate of oxygen consumption per cell
Vmax = The maximum oxygen uptake rate by cells
K’M = Partial pressure of oxygen at which uptake rate is half that of the maximum.
[O2] = concentration of oxygen at the bed/available to cells.
What is a critical volume for media?
A critical volume is the maximum volume of media allowed for cells to still survive under minimum rate of oxygen uptake.
Any added volume will kill the cells
What are the 3 main types of cell signaling?
Autocrine: Self signaling
Paracrine Signaling: Cell signals only travel a short distance to neighboring cells
Ex: Neurotransmitters traveling through the synaptic cleft
Endocrine Signaling: Cell signals travel long distance through the blood stream to get to the target
Ex: Hormones from the pituitary gland traveling to the leg.
What are the 2 types of signals?
Soluble and Insoluble SignalsS
Insoluble Signals
Signals that cannot move freely and cannot physically move into the cytosol but instead trigger a transduction pathway through receptors
Ex: Cell-Cell contact signaling through Cadherins, Extracellular Matrix signaling through Integrins
The goal of all signaling pathways is to control what?
Gene expression through regulation of transcription or translation.
What limits a cells response to a given signal?
The amount of transcription and translation machinery they have AVAILABLE to express a gene.
The time is takes for certain genes to be expressed.
The amount of protein it is able to produce as stimulated by the signal
Because of these, signals must compete with one another for the cells resources and time.
Soluble Signals examples
Signals that are able to travel into the cytosol or move freely to induce a signal transduction pathway
Ex: Growth factors, cytokines, etc.
Cell binding to the ECM can also allow the transfer of?
Signals from the extracellular matrix about the cells environment.
What is a Signal Transduction Cascade?
A signal transduction cascade describes the process in which an extracellular signal is able to stimulate a cellular response by binding to a receptor.
This binding induces a conformational change which then activates a series of conformational changes in cytosolic proteins that eventually results in an impact to gene expression
The rate of change of the concentration of a given ECM protein that can be bound (soluble) is a function of _______?
The rate of change in the concentration of a given bindable ECM protein is a function of the rate that ECM proteins are being formed by fibroblasts, and the rate at which the ECM protein is being bound and degraded by cells
What types of models are used to model cell differentiation?
Mathematical models: The Compartment model (discrete) and the Continuous Model
Both are phenomenological models
What does it mean for a model to be phenomenological
A Phenomenological model relies solely on the relationship of phenomena to one another and does not require actual molecular data to formulate but follows the theory.
What factors play into and therefore must be considered when modeling cell differentiation?
Number of Cells
Ex: Initial # of cells
Number of Cell states
Ex: differentiated or undifferentiated or mildly differentiated
Rates of movement of a cell between states
Ex: The rate of differentiation
The Compartment Model is used to model?
Cellular Differentiation
What “mathematical” expression does the compartment model use?
F → X1 —(k1)→ X2 —(k2)→ Xi —(ki)→ XN
where
F = The # of cells committing to differentiation
Xi = The # of cells at a given state
ki = The rate of transition of cells from one state to another
i = stage of differentiation
The rate of change in the concentration of bound ECM proteins is a function of what?
The rate at which soluble ECM protein is being bound minus the rate at which bound ECM protein is being degraded.
rb - rd
What are the benefits of modeling
Allows us to better formulate a hypothesis and provide expectations
Can predict an outcome that was not thought of
Allows us to analyze a process without actually having to experimentally test it.
What are the 4 cellular fate processes that can be modeled mathematically?
Cell differentiation, proliferation, migration, and apoptosis
Why might the Continuous Model of cell differentiation be considered as more accurate?
The continuous model takes into account many other impacts to the # of cells including time, rate of differentiation, rate of apoptosis, rate of cell growth, and cell maturity
What is α in the Continuous Model?
α represents the cell maturity or the state that it is in and ranges from 0 to 1 in which 0 is an undifferentiated cell and 1 is a fully differentiated cell
What is the equation used in the Continuous Model?
where
X = # of cells → dependent on time (t) and cell maturity (a)
δ = Rate of Differentiation
a = State of differentiation of a cell
σ(a) = Apoptosis function
μ(a) = Growth function
When cells grow to full differentiation a = 1.
By saying “the # of cells at steady state….” we are saying what?
The number of cells in the system does not change over time
In a realistic physiological situation, the rate of (differentiation/growth) is slower than that of (differentiation/growth)
differentiation; growth
What are the assumptions of the Compartment model?
All of the cells in a given compartment or state are homogenous
Cell number at a given state is a function of time ONLY → nothing else impacts the # of cells
Differentiation is irreversible → Cells do not travel in the opposite direction of states
What are the assumptions of the “cell walk” model?
Cells only move in 2 dimensions (up/down and left/right)
Cells do not replicate during this time
What parameters can be measured from a “cell walk” graph and what are their units?
Persistence time (time)
Speed (distance/time)
Random motility coefficient (distance²/time)
What is persistence and persistence time?
Persistence is a unitless number describing the movement of a cell in a direction in which the cell does not significantly change direction.
Persistence time is the longest time that a cells travels in 1 distance without significantly changing direction
How is speed calculated from a cell walk graph?
Initial position (x1,y1) Final position (x2,y2)
Speed in the X direction: (x2-x1)/total time
Speed in the Y direction: (y2-y1)/total time
Speed = (speedX + speedY) / 2
What is the equation for random motility coefficient
σ = ½(s²)(p)
where
s = speed
p = persistence time
The Mathematical model for cell migration is a function of ____?
The Mathematical model for cell migration is a function of the random cell walk, how responsive a cell is to chemical factors, and movement of cells along a concentration gradient (chemotaxis)
What is Chemokinesis
Chemokinesis is the change in speed of movement of a cell that is correlated to increases in chemical concentrations.
What is the equation to model cell proliferation?
X out = Xin*e^(u*t)
where
u = growth rate
What is the growth rate a function of?
u = (umax*[C])/(Km + [C])
The growth rate is a function of the maximum growth rate for a given concentration of a growth influencing compound (umax*[C]), and the concentration at which half of the maximal growth is achieved (Km)
How can we model different phases of the cell cycle?
Using age-time structured descriptions which describes the number of cells as bot ha function of time and cell state
What are the 3 phases of apoptosis?
Induction: Apoptosis is induced by soluble signals
Effector: Apoptosis taking place as proteins are degraded and the mitochondria stops working
Degradation: Biochemical and morphological changes
What is the simple way to model cell apoptosis only as a function of time
dX/dt = aX → Xout = Xin*e^(a*t)
where a = the rate of apoptosis
This equation is the exact same as the growth rate equation except the growth rate would be negative for apoptosis.
How can the compartmental model incorporate cell differentiation, division, and apoptosis?
dX/dt = I - (u+a-f)X
where
I = # of cells entering from another compartment
X = # of cells
u = rate of cell growth
a = rate of apoptosis
f = probability of self renewal rather than differentiation.
What are the limitations of the compartment model that incorporates differentiation, apoptosis and growth?
The function only considers time as a factor that changes in the system
What is cell delivery?
Cell delivery is an interdisciplinary application of engineering and biology to allow for the delivery of stem cells into some area in the body to allow for some therapeutic response.
What are some parameters that constitute a successful cell delivery
The cells must survive through delivery
The cells should be delivered to the appropriate location
The cells do not cause harm upon arrival at the site of delivery
Temperature, pH, etc. ov the environment must all be optimal
Having an appropriate scaffold at the site of delivery.
What are the 3 types of cell delivery methods
Homing
Magnetic Cell Delivery
Encapsulation
The Mathematical model for cell migration is a function of ____?
The Mathematical model for cell migration is a function of the random cell walk, how responsive a cell is to chemical factors, and movement of cells along a concentration gradient (chemotaxis)
What are the 2 main types of homing mechanisms?
Systemic and Non-systemic
Systemic mechanisms of homing involve the recruitment of endogenous or injected mesenchymal stromal cells into the bloodstream where they must then go through a multistep process to travel to the target location.
Non-systemic mechanisms of homing involve the transplantation of MSCs into the target tissue then guided to the site of injury with a chemokine gradient.
What are some strategies to improve homing?
Genetic Modification
Cell-Surface Engineering
Ultrasound
How can genetic modification improve homing?
Modifying genes for homing factors to be overexpressed can lead to increased homing ability of cells to site of injury
How can the compartmental model incorporate cell differentiation, division, and apoptosis?
dX/dt = I - (u+a-f)X
where
I = # of cells entering from another compartment
X = # of cells
u = rate of cell growth
a = rate of apoptosis
f = probability of self renewal rather than differentiation.
What are the disadvantages of homing?
To accurately target the delivery, the MSCs must be injected into the specific region.
Homing abilities of cell are naturally not great so different additional steps must be taken to improve them.
What is Magnetic Cell delivery?
Magnetic cell delivery refers to a method of cell delivery in which the cells to be delivered are labeled and tagged with magnetic particles, then are injected into the tissue and are guided to the specific area of injury by using a magnet.
Can be visualized by MRIs
What are some parameters that constitute a successful cell delivery
The cells must survive through delivery
The cells should be delivered to the appropriate location
The cells do not cause harm upon arrival at the site of delivery
Temperature, pH, etc. ov the environment must all be optimal
Having an appropriate scaffold at the site of delivery.
What are the 3 types of cell delivery methods
Homing
Magnetic Cell Delivery
Encapsulation
What is Homing in the context of cell delivery
Homing is the use of soluble signals, growth factors, and other bioactive compounds to attract and guide endogenous host cells or inserted exogenous cells to specific locations in tissue
Relies on the intrinsic properties and behavior of cells to move them into the appropriate location.
What are the 2 main types of homing mechanisms?
Systemic and Non-systemic
Systemic mechanisms of homing involve the recruitment of endogenous or injected mesenchymal stromal cells into the bloodstream where they must then go through a multistep process to travel to the target location.
Non-systemic mechanisms of homing involve the transplantation of MSCs into the target tissue then guided to the site of injury with a chemokine gradient.
What are the limitations of cell delivery systems?
Reproducibility of results of a cell delivery are limited due to the different environments and factors that impact the delivery
Low survival rate of the implanted cells
Proliferation of the implanted cells is low
Target of delivery must be very specific
Very specific chemical and physical properties must be taken into account
Cell Delivery is based on ________ and _________ environments
Extrinsic and Intrinsic
What is genetic manipulation?
The process of inducing changes to the expression of genes and expression of new genes.
What are the different ways genes are manipulated?
Genes can be subject to:
Knock-ins: Introduction of specific small changes to a gene
Introducing mutations
Introduction of new genes
Knock-outs: Silencing the expression or removing a gene
Duplications
Modifications
In general these can lead to:
Modification of an endogenous gene
Introduction of new gene
What are the 2 types of delivery methods for genetic manipulation?
Viral and Non-viral Methods
What are the different viral vector delivery methods
All of the methods involve the inclusion of the desired gene into the genome of a viral vector
Lentiviral vectors:
Have a small insert size (8kb)
Integrate into the genome
Adenoviral vectors:
Can transfect virtually all cell types and dividing and non-dividing cells
Does not integrate into the host genome
Small insert size
Subject to immune response
Adeno-Associated vectors (AAVs)
Most commonly used
Small insert size (5kb)
Possible immune response
Integrates into chromosome 19
What is one of the biggest problems with using viral vectors as a means for gene delivery?
They can cause death due to the fact that the vectors can cause intense immune responses in hosts.
What are the different nonviral methods of gene delivery?
Naked DNA Injection:
Ballistic DNA: Use of DNA gun
Electroporation: Use of electric pulse to make pores
Sonoporation: Use of sound to make pores
Photoporation: Use of a laser to make pores
Nanoparticles: Small encapsulating nanoparticles
What is electroporation?
Electroporation is the use of an electrical pulse to create pores in the membrane of a cell that allow DNA to pass through
What are the advantages and disadvantages of the different methods of nonviral gene delivery?
Naked DNA Injection:
Only works for tissue accessible by direct injection
Only allows temp gene expression
Transfection efficiency is low
Ballistic DNA:
Use of a Gene gun to deliver large amounts of DNA on coated gold particles
Physically damages the cell
Electroporation:
Very effective and can deliver large amounts
Damages cell membrane for delivery
Non-specific and low efficiency
Requires surgery for internal organs
Sonoporation:
Harmless to the cell
Non-specific and low efficiency
Photoporation
Noninvasive but still damage the cell membrane less
Low efficiency
Nanoparticles
What are the different Gene modification Machinery?
Meganuclease
Zinc Finger Nuclease
Transcription activator-like Effector Nucleases
CRISPR/Cas9 System
PASTE genome editing
What are the advantages/disadvantages of using a meganuclease?
Advantages
Naturally occurring and non toxic
Very specific binding sequences limit off target effects
Disadvantages
Efficiency is extremely variable (2-70%)
What are some advantages of magnetic cell targeting?
By using a magnet, cell diffusion of the transplanted cells is inhibited
What are some disadvantages of magnetic cell targeting?
Physiologically trapping cells with a magnet is not easy.
Can’t be used in patients with magnetic implants due to MRI use
The magnetic tracer is diluted when the cell divides, leading to less effective cell targeting over time.
What is Encapsulation as a cell delivery system?
Encapsulation involves the encapsulation of an extracted stem cell in a hydrogel polymer membranous capsule, followed by reinjection of the capsule into injured tissue
What are the advantages of encapsulation?
Injection of stem cells allows for specific targeting
Due to the encapsulation, the stem cells are able to access all the nutrients they need and are immune isolated (able to escape detection by the immune system).
What is a biomaterial?
All substances other than food and drugs that are utilized for biomedical purposes.
Can be synthetic or natural in origin and can partially or completely replace the function of a tissue
What are the 4 characteristics of a biomaterial?
Biocompatibility
Biological Functionality
Chemical Stability
Machinability
What is Biocompatibility
The ability of a material to function with a specific application in a specific area of the body without causing an immunological or toxic response
What is the characteristic of biological functionality
The biomaterial must be able to complete the job the same was as the native tissue would and must be biodegradable
Chemical Stability
The biomaterial should be chemically stable and should not degrade while or before completing its intended function.
Machinability
Biomaterial should be able to be mass produced.
What are the advantages/disadvantages of using a meganuclease?
Advantages
Naturally occurring and non toxic
Very specific binding sequences limit off target effects
Disadvantages
Efficiency is extremely variable (2-70%)
What are the advantages/disadvantages of using Zinc Finger Nucleases?
Advantages:
Relatively small = easy to deliver and low cost
Disadvantages
May be subject to rejection
Low specificity since each zinc finger domain only recognizes 3 bases
Leads to off-target effects
What are the advantages/disadvantages of using TALEN?
Advantages
Fewer off target effects than ZFN
High specificity
Disadvantages
May be subject to rejection
Can be large in size so hard to deliver.
What are the advantages/disadvantages of using CRISPR/Cas9?
Advantages
High Specificity and low cost
Disadvantages
Large Size = hard to deliver
Can lead to off target effects
What is PASTE genome editing?
The use of a fused Cas9, reverse transcriptase, and integrase, to nick and insert precise sequences of DNA into the host genome.
Chemical Stability
The biomaterial should be chemically stable and should not degrade while or before completing its intended function.
Machinability
Biomaterial should be able to be mass produced.
What are the 3 necessary structural characteristics of a biomaterial?
Surface chemistry: Should be able to adhere cells
Microscopic Geometry: Must be porous to allow for cell survival, networking, and cell signaling
3-D Structure: Should be a 3D scaffold to accurately mimic the native environment of cells
Why is important for biomaterials to be biodegradable?
Biomaterials that support the regeneration of tissue must biodegrade so that space becomes available for the new regenerated tissue to grow.
The rate at which biomaterials degrade must match the rate of __________?
Regeneration of tissue for maximal regeneration.
Material must allow for cells to do what functions?
Adhere, Migrate, Secrete their own ECM, and then function normally.
This is a chronological process in which they must first establish their ECM surrounding before functioning
All non-toxic biomaterials are considered biocompatible True or False
False because biocompatibility also includes a requirement for adequate function. Must be nontoxic AND fulfill the job requirement.
Explain the different sections of the Stress/Strain Curve
The y axis represents stress and the x axis represents strain
Elastic Region: The first region before the proportional limit is referred to as the Elastic region where a material can undergo a certain amount of strain in response to applied stress without undergoing a PERMANENT deformation
Signified by a linear line whose slope is defined by the modulus of elasticity.
Plastic Region: The second region after the proportional limit in which any deformation as a result of applied stress is permanent
Nonlinear curve
List what each # signifies on the stress strain curve depicted
Stress
Strain
Elastic Region
Plastic Region
Proportional Limit
Ultimate Strength
Fracture Point
Modulus of Elasticity
Strain
Strain refers to the amount of deformation that an object undergoes in response to a given amount of stress.
Biomaterials must usually be highly porous, made up of fibers, and highly structured, why?
The main role for biomaterials is to mimic the ECM for cells and to do so accurately, they must be able to mimic the highly fibrous nature of the ECM and its many pores as well as structure.
What characteristics of pores in a biomaterial make a good biomaterial?
Small, interconnected pores, and higher porosity allow for more cell adhesion, migration, and activity.
Pore sizes too large lower the mechanical stability of the biomaterial.
Note 
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