High Content Analysis of 3D cell cultures and other disease models

There is increasing recognition that cell biology in “2D cell cultures” (i.e. monolayers or cells on a coverslip) can be quite different from what is observed in vivo, suggesting that drug discovery assays and other research questions may be better addressed in more complex models of the disease in question that can recapitulate, e.g., cell signalling and cell dynamics in three dimensions, interactions between different cell types and different cell states.  For cancer and other diseases, this has led to the development of more complex "3D disease models" including 3D cell cultures, such as tumour spheroids, co-cultures in tissue matrix, patient-derived organoids or tissue slices.  The observations of different phentotypes in assays of such disease models frequently requires extended time-lapse live cell imaging (~hours to days) and it is often desirable to study the fates of individual cells, e.g. to address heterogeneity in the response to drugs or other pertubations.  

The use of 3D disease models makes the imaging much more challenging compared to imaging monolayers of cells – particularly for HCA where it is important to achieve a reasonable throughput to enable assays of 100’s of samples - and 3D (depth-resolved) imaging is required to study single cells. Unfortunately, the more complex 3D disease models also recapitulate many of the challenges of optical imaging in vivo - heterogeneous optical properties, multiple scattering, attenuation and background (e.g., autofluorescence) contributions to measured signals. In manual microscopy experiments, these challenges might be addressed using laser scanning confocal or multiphoton microscopy. For long time-lapse live cell experiments, it can also be challenging to ensure that the measurement (imaging) process itself does not perturb the experiment. For fluorescence-based assays, phototoxicity is a significant challenge and the excitation intensities associated with standard laser scanning microscopy techniques are usually too high for extended time-lapse imaging of live cells. Some degree of parallelisation is required, e.g. using multiple excitation beams and detection channels or light sheet microscopy. 

The commercial state-of-the-art for HCA of 3D cell cultures is most commonly represented by multiwell plate spinning (Nipkow) disc confocal microscopy or by line scanning confocal microscopy, both of which provide rapid optical sectioning and lower phototoxicity than standard confocal microscopy at the expense of interpixel cross-talk. These techniques can be used to acquire single optical sectioned images at each field of view or full z-stacks for 3D image data (at necessarily slower acquisition rates).  We have implemented both spectral FRET and FLIM HCA with a spinning disc confocal scanner.   

Light sheet microscopy offers 3D imaging with lower phototoxicity than laser scanning confocal or spinning disc confocal microscopy. Oblique plane microscopy (OPM) provides a convenient approach to implement automated multiwelll plate light sheet microscopy in a standard motorised inverted epifluorescence microscope. OPM is a single objective light sheet microscopy technique that projects an oblique light sheet up into the sample.  By translating samples arrayed in a multiwell plate through this light sheet, OPM HCA can be realised, providing volumetric 3D image-based assays of multiple samples over extended time lapse experiments. 

We are developing a suite of instruments for HCA of 3D cancer models as part of a CRUK Accelerator project: www.MACH3Cancer.org. These HCA capabilities include OPM, confocal spinning disc FLIM/FRET and multibeam mulitphoton multiwell plate microscopy.