Cel Material Interactions and how to test them

Question 1

What can we test for using cell culture?

Answer 1

Cell adhesion
Cell toxicity (necrosis/ apoptosis)
Cell proliferation
Cell phenotype (differentiation/ dedifferentiation)

Question 2

Who are the standards set by for cell testing of biomaterials?

Answer 2

BSi – British Standards specific 
ASTM – American Society for Testing and Materials. American specific but widely used
ISO – International Organisation for Standardisation

Question 3

What are the advantages to standards being performed on cell lines?

Answer 3

1) immortalised, don’t need to keep getting fresh cells
2) no aging effects
3) can be standard in labs around the world
4) reproducible data
5) predictable responses

Question 4

What are the disadvantages to standards being performed on cell lines?

Answer 4

1) respond differently to primary cells
2) can’t look at human specific effects (there are human cell lines but developed from cancers with not respond like normal cells)
3) can’t look at effects on cell cycle

Question 5

What are the three main standards for in vitro toxicity testing for biomaterials?

Answer 5

1) On surface – Direct contact test
2) Through gel – Agar diffusion test
3) Soluble factors – Elution test

Question 6

What is the direct contact test?

Answer 6

grow cells on material, culture, see how many cells we have left

1) cells in suspension are seeded onto material of interest
2) culture in incubator in media (pH balanced solution with salts/sugars/growth factors added)
3) count /analyse examine cells

Question 7

What is the problem with the direct contact test?

Answer 7

Number of cells left does not tell you how many cells adhered, what proliferation rate is or whether cells died. ONLY final number of cells present at time of analysis

Question 8

What is the agar diffusion test?

Answer 8

grow cells on tissue culture well, add agar, place material on top of agar, stain for live cells

1) cells In well are poured on agar and let set
2) material of interest added
3) incubate with dye that distinguishes live from dead cells

Question 9

What is the problem with the agar diffusion test?

Answer 9

BUT Need to standardise agar, how soluble is product of material?

Question 10

What is the elution test?

Answer 10

grow cells on tissue culture well, dissolve material, add solution to cells

1) material of interest in a solvent (NaCl or media)
2) add dissolved substances to cells
3) count analyse examine cells as per direct contact

Question 11

What is the problem with the elution test?

Answer 11

BUT solvent may affect cells and not represent body fluids?

Question 12

What does the elution test show?

Answer 12

Get dose response effect can see if soluble products are toxic at a distance form material

Question 13

What does the agar diffusion test show?

Answer 13

Can see ratio live/ dead cells Have concentration gradient.

Question 14

What are the advantages to methods that involve counting remaining cells?

Answer 14

can fix sample no time constrain

Question 15

What are the disadvantages to methods that involve counting remaining cells?

Answer 15

assume dead cells have floated off can’t distinguish live from dead (not appropriate for 3D scaffolds)

Question 16

What can methods that involve counting remaining cells be used for?

Answer 16

Simple histological stain e.g. HandE
Metabolic activity analysis
Total DNA analysis
Nuclear stains

Question 17

What are methods that assay cell viability?

Answer 17

MTT/MTS - metabolic activity

LIVE/DEAD staining - compromised membranes

Question 18

How is metabolic activity measured?

Answer 18

Resazurin reduction assay

Question 19

What is a MTT assay?

Answer 19

solution is yellow
converted to purple by live cells
- read purpleness using colorimeter

Question 20

What is a LIVE/DEAD assay?

Answer 20

use principal of membrane permeability

Propidium iodide

Question 21

What are the problems with translation in vitro toxicity results to in vivo?

Answer 21

-Dose: would the body cells receive that dose?
• Cell type: using fibroblasts indicates response of connective tissue but what about immune cells?
• Solubility/ degradablity: would it degrade that way and give those products in vivo?

Question 22

How do you measure the number of focal adhesions?

Answer 22

Measuring area over which cell spreads
(But does not necessarily directly correlate with cell strength)
Sometimes will want to encourage cell spreading (endothelial cells creating a capillary) Some times will not want spreading – cartilage cells DEDIFFERENIATE if flatten out. Current trend in biomaterials is to manipulate spreading by manipulating surface

Question 23

How can a biomaterial orientate cells?

Answer 23

Grooved or fibrous scaffolds will orientate cells

Question 24

How do you test cell adhesion?

Answer 24

use of fluid flow

Parallel Plate flow chambers apply shear stress to cells

Question 25

What are the advantages for using fluid flows as a test of adhesion?

Answer 25

Adhesion occurs under well defined flow conditions.
Use of well defined adhesive substrates.
Use of well defined test particles.
Ability to look at molecule-molecule interactions.
Versatility.
Low cost.

Question 26

What are the disadvantages for using fluid flows as a test of adhesion?

Answer 26

Other cell types are lacking which may influence adhesion (i.e. red blood cells).
Other soluble factors may be missing which could influence adhesion.
Typically flow is not pulsatile flow (as in blood). – However it is possible to deliver pulsatile flow.
Geometry of the flow chamber may not reflect that of in vivo environment

Question 27

How do you look at cell migration?

Answer 27

1) Watch them move: video-time lapse photography
2) Create artificial wound for cell to grow across 
3) Create barrier they have to squeeze through (transwell) 
4) Add fluorescent proteins to visualise specific molecules involved

Question 28

How can cell motion be tracked?

Answer 28

Random in absence of chemoattractant
Attractant chemical can direct cell ‘walk’
Can measure number of cells per area at distance from chemoattractan