Cellular and Molecular Assays

Totipotent Cells

can form any cell type, as well as extra-embryonic (placental) tissue (e.g. zygote)

Pluripotent

can form any cell type (e.g. embryonic stem cells)

Multipotent

can differentiate into a number of closely related cell types (e.g. haematopoeitic adult stem cells)

Unipotent

can not differentiate, but are capable of self renewal (e.g. progenitor cells, muscle stem cells)

Cytotoxicity (survival)

evaluate cytotoxicity of both direct surface interactions and/or leachables from material

Direct Contact Assay

place materials directly on top of monolayer cells (Cytotoxicity)

Qualitative Analysis: score cell death/damage underneath material

Quantitative Analysis:

1. Measure release of intracellular enzyme in media

2. Measure viable cells with viability dyes

   1-2 days after exposure to material

Agar Diffusion Assay

layer of agar in between cells and material (Cytotoxicity)

Qualitative Analysis: Score cel ldeath damage relative to distance from material

Quantitative Analysis: Incorporate viability dyes in media

1-2 days after exposure to material

Elution Assay

culture cells in the presence of media previously exposed to material of interest (Cytotoxicity)

Qualitative Analysis: Score cell death damage

Quantitative Analysis: same as direct contact assay

1-2 days after exposure to material

Simple Wash Assay

allow cells to attach to surface for short time (min-hr) then wash away unattached cells (Adhesion Assay)

Limitations: Wash force may vary = high experimental error

Controlled Force Assays

parallel plate flow chamber (shear stress is proportional to volumetric flow rate and media viscosity) (Adhesion Assay)

centrifugal assays

DNA Synthesis (direct)

specifically labels dividing/proliferating cells (Proliferation Assay)

• most accurate method

• timely and not high-throughput

Cell Metabolism (indirect)

respiring cells convert MTT to a purple formazan dye RNa- measure with spectrophotometer (Proliferation Assay)

• simple, high-throughput

• metabolic activity may not accurately measure proliferation

Immunohistochemistry (indirect)

evaluate proliferation proteins such as Ki67 (nuclear antigen expressed in cell cycle phases G1, S, G2, and M) (Proliferation Assay)

• accurate/reliable

• low-throughput

RNA Analysis Techniques

Reverse-transcription polymerase chain reaction (RT-PCR); amplification of target RNA to detectable limit (measuring changes in gene expression)

Western Blots, ELISAs, Immunohistochemistry

quantitative and qualitative methods for measuring changes in protein expression (differentiation)

Bioadhesive substrates - proliferation/differentiation

• RGD functionalization

• RGD-alginate surface