Cell Biomaterial Interactions Flashcards by Rebecca Fitch

Tissue culture

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

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What is blood replaced by in IN VITRO cultures

Culture media
Changed periodically to replenish constituents to maintain cell function

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What is an issue with culture conditions?

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

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Components of culture media

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)

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What are cultured cells held in

Culture dishes and flasks at physiological conditions

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What are physiological conditions

37 degrees Celsius
7.4 pH
Controlled humidity of 95%

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Why are culture lids not sealed

To allow for gas exchange

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Why is CO2 added?

5%
interacts with bicarb buffer to maintain pH levels

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How is pH maintained?

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

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

Maintenance of humidity

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Primary Cells

Harvested from tissue, limited length of division time

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Cell lines

Genetically modified to be immortalized
Easier to work with

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How are primary cells cultured?

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

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Confluence

Area occupied by cells

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Subculture/Passaging

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

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What happens if primary cells reach 100% confluence

Proliferation may cease
Cell death due to contact inhibition

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

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

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How are cell lines formed?

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

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How many times call cell lines be passaged?

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

Why are cell lines used in experiments?

Provides better reproducibility

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

Genetic variations
different animals
Unintentional isolation of unwanted cell types

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

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

What are some characteristics of cultured cells

Contact inhibition
Cell morphology

Contact inhibition of cultured cells

Cells grow in mono layers in vitro
Upon confluence

Cell Morphology of cultured cells

Cell adhesion

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

Mechanisms of cell attachment

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

Integrin

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

Ligands

Short peptide sequences on the TCP surface

Integrin

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

What would happen if the surface is overly Hydrophobic/Hydrophilic

Inhibits cell attachment

Focal adhesions

Complex assembly of intra- and extracellular proteins coupled through transmembrane integrins Individual focal adhesions: focal contacts

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

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

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

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

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

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

Dynamic control of cell surfaces

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

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

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

pHEMA and cell adhesion

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

How can pHEMA be used to prevent cellular attachment?

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

Propose a method for increasing cell adhesion

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

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

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

Surface topography: Isotropic vs ansiotropic

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

How do cells respond to surface elasticity

Cells respond to mechanical properties on the surface

What can be used to study stiffness effects?

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

What happens to cell spreading on softer substrates?

Reduced cell spreading

What is mechanotaxis?

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

Cell differentiation and Surface mechanics

Neuronal: soft Myogenic: Stiff Osteogenic: Stiffest