Cel Material Interactions and how to test them Flashcards
What can we test for using cell culture?
Cell adhesion
Cell toxicity (necrosis/ apoptosis)
Cell proliferation
Cell phenotype (differentiation/ dedifferentiation)
Who are the standards set by for cell testing of biomaterials?
BSi – British Standards specific
ASTM – American Society for Testing and Materials. American specific but widely used
ISO – International Organisation for Standardisation
What are the advantages to standards being performed on cell lines?
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
What are the disadvantages to standards being performed on cell lines?
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
What are the three main standards for in vitro toxicity testing for biomaterials?
1) On surface – Direct contact test
2) Through gel – Agar diffusion test
3) Soluble factors – Elution test
What is the direct contact test?
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
What is the problem with the direct contact test?
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
What is the agar diffusion test?
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
What is the problem with the agar diffusion test?
BUT Need to standardise agar, how soluble is product of material?
What is the elution test?
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
What is the problem with the elution test?
BUT solvent may affect cells and not represent body fluids?
What does the elution test show?
Get dose response effect can see if soluble products are toxic at a distance form material
What does the agar diffusion test show?
Can see ratio live/ dead cells Have concentration gradient.
What are the advantages to methods that involve counting remaining cells?
can fix sample no time constrain
What are the disadvantages to methods that involve counting remaining cells?
assume dead cells have floated off can’t distinguish live from dead (not appropriate for 3D scaffolds)
What can methods that involve counting remaining cells be used for?
Simple histological stain e.g. HandE
Metabolic activity analysis
Total DNA analysis
Nuclear stains
What are methods that assay cell viability?
MTT/MTS - metabolic activity
LIVE/DEAD staining - compromised membranes
How is metabolic activity measured?
Resazurin reduction assay
What is a MTT assay?
solution is yellow
converted to purple by live cells
- read purpleness using colorimeter
What is a LIVE/DEAD assay?
use principal of membrane permeability
Propidium iodide
What are the problems with translation in vitro toxicity results to in vivo?
-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?
How do you measure the number of focal adhesions?
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
How can a biomaterial orientate cells?
Grooved or fibrous scaffolds will orientate cells
How do you test cell adhesion?
use of fluid flow
Parallel Plate flow chambers apply shear stress to cells
