Table 2 Parameters of interest for characterisation of hPSC culture surfaces and analytical methods for their assessment
From: Defining synthetic surfaces for human pluripotent stem cell culture
Parameter | Analysis method | Pros | Cons |
|---|---|---|---|
Surface topography | Atomic force microscopy (tapping mode) | Compatible with an aqueous environment, can view individual proteins that have absorbed to the surface, modern instruments acquire images at a faster rate. | Images are generally of a small area, therefore may not be representative. |
Scanning or transmission electron microscopy | Widely available | Resolution is not as high, significant sample preparation is required, unable to quantify topography. | |
Ligand density | ELISA assays | Straightforward assay | Not very sensitive for adsorbed protein, requires antibodies to specific proteins or molecules. |
Fluorescence from adsorbed or covalently attached fluorophore | Relatively straightforward assay | Microenvironment and dye-dye quenching effects from surface anchored species introduces artefacts, construction of calibration curve difficult. | |
Fluorescence from fluorophore released into solution | Quantitative, sensitive, relatively straightforward assay | Cleavable fluorophore needs to be synthesised and chemically attached to ligand/CAM. | |
Lanthanide (e.g. Eu-chelate) labelling of ligand | Quantitative, sensitive, relatively straightforward assay | Need to carry out chemical coupling of Eu-chelate to ligand. | |
Radio-labelling of ligand | Quantitative, sensitive, relatively straightforward assay | Complex chemistry required to either radio-label pre-synthesised ligands or synthesise ligand with radioisotope-containing precursors. | |
Chemical properties | Nuclear magnetic resonance (NMR) | Straightforward sample preparation | Solid-state NMR generally not sensitive enough, complex spectra. |
Wettability | Water contact angle | Simple | Very non-specific - many adsorbed species can modify wettability, |
Chemical composition (directly detecting protein adsorption) | X-ray photoelectron spectrometry | Elemental composition quantitative, sample preparation is very simple (removal of buffer salts and drying). | Elemental composition is straightforward but high resolution spectra complex, amide bond-containing materials generate false positives, no specificity in relation to protein type, ultra high vacuum technique (can cause structural rearrangements). |
Time-of-flight secondary ion mass spectrometry | Minimally-destructive, minimal sample preparation, efficient, | Analysis generally not quantitative, produces large data sets often requiring statistical methods, no specificity in relation to protein type, ultra high vacuum technique (can cause structural rearrangements) | |
Fourier transform infrared spectroscopy | Widely available, can be powerful if coupled with synchrotron | Not “surface-sensitive” enough, no specificity in relation to protein type. | |
Indirect assessment of protein adsorption | Embryoid body adhesion assay | Straightforward if embryoid bodies are being generated in house | Expensive, time-consuming |
HeLa or other e.g. L929 cell adhesion assay | Reliable, cheap if cell lines are available in laboratory | Cell attachment for cells other than hPSCs may be mediated by different ligands. |