Stem cell test 2

Lecture 7: Cell Culture Basics: General Passaging rules: passage at _ % to _% confluence, do not over _, abdicate neutralize _ properly

70 80 trypsonize trypsin

Lecture 7: Cell Culture Basics: General Passaging rules: Feeding

you should change the media every _ to _ days

check under microscope every _ to _ days

keep _ and do not let _

2 3 1 2 warm dry

Lecture 7: Cell Culture Basics: Lab 1 what are the phases in a cell growth curve

lag log stationary death

Lecture 7: Cell Culture Basics: Lab 1: Proliferation affects what 3 things

differentiation metabolism drug response

Lecture 7: Cell Culture Basics: Lab 1 Growth curves are a __

baseline measurement

Lecture 7: Cell Culture Basics: Lab 1 Growth Curve Lag Phase: Cells adapt to new _

attachment, spreading, and __

little to no increase in __

lag DNE dead cells

environment metabolic adjustment cell number

Lecture 7: Cell Culture Basics: Lab 1 Log phase

cells divide at a __

nutrients are _

__ is measurable

constant rate sufficient doubling time

Lecture 7: Cell Culture Basics: Lab 1 stationary phase

Nutrients _

_ accumulates

Division rate = __

depleted waste death rate

Lecture 7: Cell Culture Basics: Lab 1 Death phase

Cell death exceeds _

_, nutrient depletion, and _

division stress overcrowding

Lecture 7: Cell Culture Basics: Not all cells grow the same: Stem cells

Slower _

sensitive to density, __, and passage number

Balance between __ and _

Growth is Regulated

proliferation media consumption self renewal differentiation

Lecture 7: Cell Culture Basics: Not all cells grow the same Cancer cells:

Faster _

reduced contact inhibition

Less dependence on external _

Often metabolically _

Growth is deregulated

proliferation signals aggressive

Lecture 7: Cell Culture Basics: How do we measure cell growth

Cell count

_ + _

measures actual cell _

distinguishes _ vs _ cells

hemocytometer trypan blue number live dead

Lecture 7: Cell Culture Basics: How do we measure cell growth

What are the pro and con of hemocytometer

accurate labor intensive

Lecture 7: Cell Culture Basics: How do we measure cell growth Metabolic Assay

_, XTT, Resazurin, etc

measures cell __

__ reduction of a reagent

used as an _ proxy for viable cell number

MTT metabolic activity enzyme mediated indirect

Lecture 7: Cell Culture Basics: How do we measure cell growth

what are the pros and cons of metabolic assay

fast scalable does not directly measure cell number

Lecture 7: Cell Culture Basics: Cell growth and differentiation

Stem cell differentiation depends on the what of the cells

growth state

Lecture 7: Cell Culture Basics: Cell growth and differentiation

Growth phase influences

__ activity

_ state

sensitivity to __

cell cycle metabolic differentiation cues

Lecture 7: Cell Culture Basics: Cell growth and differentiation

Growth phase matters

Lag phase: cells adapting —> poor _ response

log phase: healthy, responsive cells → __

Stationary phase: stress and nutrient depletion → __

differentiation optimal induction altered fate

Lecture 7: Cell Culture Basics: Adipogenic differentiation timeline

what are the phases of Adipogenic differentiation

proliferation and commitment early differentiation intermediate differentiation mature adipocytes

Lecture 7: Cell Culture Basics: Adipogenic differentiation timeline

what days do early differentiation, intermediate differentiation, and mature adipocytes occur

2-4 5-7 10-14

Lecture 8: Biomechanics basics: Mechanobiology

Paradigm: mechanical forces modulate _, structure, and _ of tissues, particularly _ tissues such as bone cartilage ligaments and tendons

morphology functions skeletal

Lecture 8: Biomechanics basics: Mechanobiology

Traditional biomechanics is focused on how these tissue perform the _ and _ functions of the _

structural locomotory skeleton

Lecture 8: Biomechanics basics: Mechanobiology

Mechanobiology is more concerned with the theme of how these __ tissue are produced, _, and adapted by cells as responses to __ (tissue homeostasis)

load bearing maintained biophysical stimulus

Lecture 8: Biomechanics basics: Mechanobiology

Goal- understand the _ adaptation to benefit _ and human health (e.g., congenital deformities, osteoporosis) and to enhance __

functional medicine tissue engineering

Lecture 8: Biomechanics basics: Mechanotransduction

Cells and tissues are known to respond to mechanical stimuli such as _, _, and _ (gravity is one of the best examples)

shear stress compression tension

Lecture 8: Biomechanics basics: Mechanotransduction

Ultimately, all _ stimuli must be converted to either _ and _ signals. but the exact mechanisms remain to be elucidated

external bioelectrical biochemical

Lecture 8: Biomechanics basics: Mechanotransduction

One of the key mechanotransduction mechanisms is now believed to be mediated by __ bindings via _ → _ transmit the force

cell ecm integrins directly

Lecture 8: Biomechanics basics: Mechanotransduction

Other potential mechanisms could include __ biochemical signaling, _ activated ion channels, _ activation, and altered _ (e.g., altered affinity binding)

integrin triggered mechanically G protein conformation

Lecture 8: Biomechanics basics: Mechanical Language

In order to understand mechanical aspects of cell/tissue _ and _, one has to become familiar with the language of mechanics

responses regulation

Lecture 8: Biomechanics basics: Mechanical language

Stress (σ) is defined as the _ per area _ to the _ of force applied (tensile or compressive force)

force perpendicular direction

Lecture 8: Biomechanics basics: Mechanical language

Stress has units of pascals which are what and what is the other unit

N/m² dynes/cm²

Lecture 8: Biomechanics basics: Mechanical language

Strain (𝜺) is what? (unitless)

deformation due to stress

Lecture 8: Biomechanics basics: Mechanical language

Strain is defined as what

deviation from the unstressed dimension

Lecture 8: Biomechanics basics: Mechanical language

Strain is defined as deviation from the unstressed dimension

for example, in 1D bar, 𝜺=𝜹L/Lo, where Lo is the original length and 𝜹L is the _

deviation

Lecture 8: Biomechanics basics: Stress and Strain

Intuitive to suggest that, as stress becomes _, the _ (i.e. deformation) should _

large strain increase

Lecture 8: Biomechanics basics: Stress and Strain

Intuitive to suggest that, as stress becomes large, the strain (i.e. deformation) should increase

If in fact a material is considered _, there is a _ relationship between _ and _

elastic linear stress strain

Lecture 8: Biomechanics basics: Stress and Strain

Intuitive to suggest that, as stress becomes large, the strain (i.e. deformation) should increase

If in fact a material is considered elastic, there is a linear relationship between stress and strain

σ=E𝜺, this is called what law? and E is a _ and known as what?

hookes constant youngs modulus

Lecture 8: Biomechanics basics: Other Mechanical Models

• Not all biological systems could or should be described by the linear relationship of __

• What are other models that are more sophisticated and complicated? these are collectively referred to as _ models

• In addition to _ behavior of the system, incorporation of the _ behavior provides a more _ description of how a biological system may respond to a stimulus

hookes law viscoelastic elastic viscous realistic

Lecture 8: Biomechanics basics: Mechanical Tissue Response

Linear relationship between stress and strain applicable in a _ range

Proper mechanical description of such response based on _

limited viscoelasticity

Lecture 8: Biomechanics basics: Mechanical Tissue Response

Linear relationship between stress and strain applicable in a limited range

Proper mechanical description of such response based on viscoelasticity

Relaxation: _ relaxes while _ is continuously applied

Creep: _ increase after _ is removed

Strain stress strain stress

Lecture 8: Biomechanics basics: Mechanical Tissue Response

What is relaxation?

strain relaxes while stress continuously applied

Lecture 8: Biomechanics basics: Mechanical Tissue Response

What is creep?

strain increase after stress removed

Lecture 8: Biomechanics basics: Mechanical Tissue Response

what tissues are these curves depicting

bone skin cartilage

Lecture 8: Biomechanics basics: Maxwell Model

A dashpot (e.g., shock absorber) is added to the system, Unlike a spring the dashpot displacement is proportional to dx dashpot/dt.

Total displacement of the system, of course is

x(t)= x (_) + x(_)

dashpot spring

Lecture 8: Biomechanics basics: Maxwell Model

A dashpot (e.g., shock absorber) is added to the system, Unlike a spring the dashpot displacement is proportional to dx dashpot/dt.

Total displacement of the system, of course is

x(t)= x_spring + x_spring

Note that his is entirely analogous to an electrical circuit in series

F(t)= F_spring = F_dashplot

Finally, one can show, without too much difficulty that

x(t)/F=1/K₀+(_)t

1/n0

Lecture 8: Biomechanics basics: Kelvin Model

A slightly more complicated model that has the Maxwell body in _ with another spring (k1). In analogous to a parallel electric circuit, force can be _ distributed but the _ (x) of each branch must be the same

where 𝝉 is the ___ and is given by n₀ {(k₀+k₁)/k₀k₁

relaxation time constant

Lecture 8: Biomechanics basics: Structural Organization of Articular Cartilage

what are the 3 zones

superficial middle lower

Lecture 8: Biomechanics basics: Structural Organization of Articular Cartilage

what percent of collagen and chondrocyte distribution do the superficial zone, middle zone, and lower zone have?

10 45 45

Lecture 8: Biomechanics basics: Mechanical Requirements for Articular Cartilage

What are the four zones from top to bottom?

surface transitional radial tidemark

Lecture 8: Biomechanics basics: Structural Organization of Articular Cartilage

What are the requirements for the surface zone

__ in/out

_ pressure/tension

large __ (consolidation)

_ surface strains

high flow fluid compressive strain tensile

Lecture 8: Biomechanics basics: Structural Organization of Articular Cartilage

What are the requirements for the transitional zone

_ fluid flow

mainly _ pressure

_ compressive strains (consolidation)

low fluid moderate

Lecture 8: Biomechanics basics: Structural Organization of Articular Cartilage

What are the requirements for the radial zone

_ fluid flow

_ pressure

_ compressive strain (consolidation)

little fluid small

Lecture 8: Biomechanics basics: Structural Organization of Articular Cartilage

What are the requirements for the tidemark zone

_ fluid flow

_ pressure

_ compressive strain (consolidation)

interface __

no fluid no shear strains

Lecture 8: Biomechanics basics: AFM image analysis

__ (nm heights) on the surface MDCK cells (5×5 um)

A: control cell

B: control cell + P188

C: .01% Saponin without P188

D: .01% Saponin + P188

topographical measurements

Lecture 8: Biomechanics basics: Biomechanics properties

What is the youngs modulus of human bone marrow stem cell, human osteoblast, bovine aortic EC, human adipocyte (stem cell derived), mouse myotube, and human vascular EC

3.2 1.8 1.7 .82 2.8 2.6

Lecture 8: Biomechanics basics: Cell Detachment by Shear Stress

_ dynes/cm² (3.3 pa) induced _% cell detachment

33 50

Lecture 8: Biomechanics basics: Shear Stress or Stretching

One of the best known examples in mechanobiology is the _ responses by _ or _.

cellular shear stress stretching

Lecture 8: Biomechanics basics: Shear Stress or Stretching

One of the best known examples in mechanobiology is the cellular responses by shear stress or stretching. Postulate is that _ forces act on the __ sites. At least some, but certainly not all, of the _ pathways have been identified

external focal adhesion signaling

Lecture 8: Biomechanics basics: Shear Stress

Shear stress is the what?

frictional force exerted by blood flow on vessel walls

Lecture 8: Biomechanics basics: Shear Stress

frictional force exerted by blood flow on vessel walls

Endothelial cells line the interior surface of blood vessels and directly sense __ and transduce it into __ that regulate gene expression, cell _, and _

shear stress biochemical signals morphology function

Lecture 8: Biomechanics basics: Shear Stress

frictional force exerted by blood flow on vessel walls

Endothelial cells line the interior surface of blood vessels and directly sense shear stress and transduce it into biochemical signals that regulate gene expression, cell morphology, and function

Normal _ shear stress promotes _ whereas disturbed (turbulent) or _ shear stress contributes to _ dysfunction such as atherosclerosis

laminar homeostasis low endothelial

Lecture 8: Biomechanics basics: Shear Stress

Shear stress at the wall in a laminar flow is T_w=what/what

4uq piR³

Lecture 8: Biomechanics basics: Shear Stress

what are Q, u, and R

volume flow rate fluid viscosity radius of tube

Lecture 8: Biomechanics basics: Shear Stress and Stem cells

Some stem cells typically do interact with shear stress such as

• _ progenitor cells

• _ stem cells in bone marrow

• _ progenitor cells

endothelial hematopoietic cardiac

Lecture 8: Biomechanics basics: Shear Stress and Stem cells

Some stem cells typically do interact with shear stress such as

• endothelial progenitor cells

• hematopoietic stem cells in bone marrow

• cardiac progenitor cells

Shear stress has been used as a tool to manipulate stem cell responses

• through __ that connect the ECM to the cytoskeleton

• __ ion channels on the cell surface

focal adhesions mechanosensitive

Lecture 8: Biomechanics basics: Shear Stress and Differentiation

Shear stress influences MSCs differently depending on what and what

magnitude pattern

Lecture 8: Biomechanics basics: Shear Stress and Differentiation

Low/physiological shear levels (1-10 dyne/cm²) encourages what kind of cell responses

High shear levels (>10 dyne/cm²) encourages what kind of cell responses

Oscillatory/disturbed shear levels encourages what kind of cell response

endothelial osteogenic osteogenic chondrogenic pro inflammatory phenotype

Lecture 8: Biomechanics basics: Shear Stress and Differentiation

Challenges

1. How to _ precise shear stress

2. how to determine the complexity of _

3. how to scale from in _ to in _

4. how to apply shear stress for a _ period of time

control mechanotransduction vitro vivo long