Table 2.

Parameters of interest for characterisation of hPSC culture surfaces and analytical methods for their assessment

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.

Surface topography, ligand density, chemical properties, wettability and protein adsorption can be tested by the methods listed. Pros and cons are listed for each method in this non-comprehensive list.