Cell Biomaterial Interactions

Question 1

Tissue culture

Answer 1

Harvest of cells, tissues, or organs and their growth or maintenance in an artificial envirionment

Question 2

What is blood replaced by in IN VITRO cultures

Answer 2

Culture media
Changed periodically to replenish constituents to maintain cell function

Question 3

What is an issue with culture conditions?

Answer 3

• Susceptible to bacterial and fungal contamination
• strict aseptic conditions must be maintained
• anti-bacterial/ anti-fungal are added to the medium

Question 4

Components of culture media

Answer 4

• Provides energy source (Glucose- Low: 1g/L High 4.5 g/L)
• Essential nutrients (salts and amino acids)
• Proteins and hormones (serum, generally from fetal bovine)
• Buffer ( to maintain neutral pH)

Question 5

What are cultured cells held in

Answer 5

Culture dishes and flasks at physiological conditions

Question 6

What are physiological conditions

Answer 6

37 degrees Celsius
7.4 pH
Controlled humidity of 95%

Question 7

Why are culture lids not sealed

Answer 7

To allow for gas exchange

Question 8

Why is CO2 added?

Answer 8

5%
interacts with bicarb buffer to maintain pH levels

Question 9

How is pH maintained?

Answer 9

Phenol red is added as an indicator dye to visually confirm pH maintenance
red-orange: 6.8-7.4
yellow: acidic
pink/purple: basic

Question 10

Why is a water dish placed at the bottom of the incubator

Answer 10

Maintenance of humidity

Question 11

Primary Cells

Answer 11

Harvested from tissue, limited length of division time

Question 12

Cell lines

Answer 12

Genetically modified to be immortalized
Easier to work with

Question 13

How are primary cells cultured?

Answer 13

• Passing media through media cavities of long bones
• Using collagenase to enzymatically digest pieces of tissues (collagen digested leaving cells)
• Harvest cells are plated and allowed to attach to the surface; after some time, unattached and dead cells are washed away
• Once 70-90% confluence, cells are subcultured by using trypsin/EDTA to degrade cell adhesion proteins allowing for cells to life
• Sells are reseeded at low density to allow cell proliferation

Question 14

Confluence

Answer 14

Area occupied by cells

Question 15

Subculture/Passaging

Answer 15

Taking cells off the original culture and reculture on more plates to prevent cellular issues and give enough space for cells to continue to divide

Question 16

What happens if primary cells reach 100% confluence

Answer 16

Proliferation may cease
Cell death due to contact inhibition

Question 17

How many times can primary cells be passaged? What happens when cells are excessively passaged?

Answer 17

Only a few times
excessive passaging can result in loss of cell phenotype

Question 18

How are cell lines formed?

Answer 18

By treating or transforming normal cells with viruses, oncogenes, drugs, or chemicals in transgenic mice

Question 19

How many times call cell lines be passaged?

Answer 19

Many times with no loss of phenotype
Divided cells are genetic clones of parent cells
No contact inhibition issues

Question 20

Why are cell lines used in experiments?

Answer 20

Provides better reproducibility

Question 21

What can impact reproducibility of an experiment that uses primary cells?

Answer 21

Genetic variations
different animals
Unintentional isolation of unwanted cell types

Question 22

Can cell lines be used to confirm findings of an experiment?

Answer 22

No, they can be used initially however they exhibit different morphologies and metabolisms in comparison to primary cells
Findings using cell lines must be confirmed with PRIMARY CELLS

Question 23

What are some characteristics of cultured cells

Answer 23

Contact inhibition
Cell morphology

Question 24

Contact inhibition of cultured cells

Answer 24

Cells grow in mono layers in vitro
Upon confluence

Question 25

Cell Morphology of cultured cells

Question 26

Cell adhesion

Answer 26

The initial step in cell biomaterial interactions
important for cell guidance, proliferation, and differentiation
Tissue culture polystyrene (TCP) is commonly used
Cells bind to the absorbed protein layer from serum that is applied on the TCP surface

Question 27

Mechanisms of cell attachment

Answer 27

A slightly amphipathic material for cells/ serum to be applied to
Integrins of cell membrane bind to specific ligand sequences on the surface to facilitate cell attachment
Focal Contacts -> focal adhesion
Cell adhesion triggers biochemical signaling pathways within the cell influencing cell proliferation and differentiation

Question 28

Integrin

Answer 28

Transmembrane proteins of cells consisting of alpha and beta subunits that act as receptors

Question 29

Ligands

Answer 29

Short peptide sequences on the TCP surface

Question 30

Integrin

Answer 30

Transmembrane proteins of cells consisting of alpha and beta subunits that act as receptors

Question 31

What would happen if the surface is overly Hydrophobic/Hydrophilic

Answer 31

Inhibits cell attachment

Question 32

Focal adhesions

Answer 32

Complex assembly of intra- and extracellular proteins coupled through transmembrane integrins

Individual focal adhesions: focal contacts

Question 33

How do you increase cell attachment on polystyrene (PS) (Hydrophobic)

Answer 33

Gas plasma treatment generates energetic oxygen ions that oxidize
Oxidation creates OH ions (polar), making the surface more hydrophilic allowing for more attachment of proteins

Question 34

How is chemical treatment of sulfuric acid used to make surface modifications

Answer 34

Introduces sulfonic groups (SO3-)
Changes conformation of proteins absorbed on surface inhancing integrin mediated cell adhesin

Question 35

How is chemisorption used to perform surface modification for cell adhesion?

Answer 35

Incorporation of RGD peptides on surface via chemical bonds
immobilization of growth factors on biomaterial surface

Question 36

Dynamic control of cell surfaces

Answer 36

Development of surfaces that are responsive to thermal, chemical, or electrical/magnetic stimuli

Question 37

How can Poly[NIPAAm] be used for dynamic control of cell surfaces

Answer 37

T>32 hydrophobic: readily absorbs cell adhesive proteins resulting in cell monolayer
T<32 hydrophilic: detachment of cell sheet

Question 38

pHEMA and cell adhesion

Answer 38

In air: Hydrophobic due to non polar methyl group
In water: forms hydrogel due to polar OH group

Question 39

How can pHEMA be used to prevent cellular attachment?

Answer 39

TCP surfaces coated with pHEMA hydrogel inhibit cell attachment by minimizing protein absorption from serum

Question 40

Propose a method for increasing cell adhesion

Answer 40

Collagen/fibronectin/Poly-L-lysine coated substrates enhance cell adhesion
RGD peptide immobilization
growth factor tether
Fundamental strategy: enhance absorption of serum and proteins in culture medium improving cell adhesion

Question 41

How do growth factors tether on the surface of materials influence cell proliferation and differentiation?

Answer 41

Physical absorption results in burst release of growth factors
Chemical immobilization needed for sustained release

Question 42

Surface topography: Isotropic vs ansiotropic

Answer 42

Isotropic: Uniform in all directions
Ansiotropic: Uniform in 1 direction

Question 43

How do cells respond to surface elasticity

Answer 43

Cells respond to mechanical properties on the surface

Question 44

What can be used to study stiffness effects?

Answer 44

Hydrogels
The stiffness of hydrogels can be modulated by:
- controlling the water content in the gel
- Changing polymer concentration
- modulating the extent of crosslinking

Question 45

What happens to cell spreading on softer substrates?

Answer 45

Reduced cell spreading

Question 46

What is mechanotaxis?

Answer 46

Cell migration via mechanical cues
Cells migrate towards surfaces of greater stiffness

Question 47

Cell differentiation and Surface mechanics

Answer 47

Neuronal: soft
Myogenic: Stiff
Osteogenic: Stiffest