Imaging-Based Machine Learning Analysis of Patient-Derived Tumor Organoid Drug Response

Three-quarters of compounds that enter clinical trials fail to make it to market due to safety or efficacy concerns. This statistic strongly suggests a need for better screening methods that result in improved translatability of compounds during the preclinical testing period. Patient-derived organoids have been touted as a promising 3D preclinical model system to impact the drug discovery pipeline, particularly in oncology. However, assessing drug efficacy in such models poses its own set of challenges, and traditional cell viability readouts fail to leverage some of the advantages that the organoid systems provide. Consequently, phenotypically evaluating complex 3D cell culture models remains difficult due to intra- and inter-patient organoid size differences, cellular heterogeneities, and temporal response dynamics. Here, we present an image-based high-content assay that provides object level information on 3D patient-derived tumor organoids without the need for vital dyes. Leveraging computer vision, we segment and define organoids as independent regions of interest and obtain morphometric and textural information per organoid. By acquiring brightfield images at different timepoints in a robust, non-destructive manner, we can track the dynamic response of individual organoids to various drugs. Furthermore, to simplify the analysis of the resulting large, complex data files, we developed a web-based data visualization tool, the Organoizer, that is available for public use. Our work demonstrates the feasibility and utility of using imaging, computer vision and machine learning to determine the vital status of individual patient-derived organoids without relying upon vital dyes, thus taking advantage of the characteristics offered by this preclinical model system.

Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the “solution effect damage” during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

Cooperativity between Dimerization and Binding Equilibria in the Ternary System Laponite-Indocyanine Green-Water

Laponite is an artificial nanoclay available in large quantities and at low cost. This marterial represents an efficient and suitable way of delivering hydrophobic vital dyes without the need for chemical functionalization. Laponite is available in large quantities and at low cost, then it would be an efficient way of delivering hydrophobic vital dyes without the need for chemical functionalization. The hydrodynamic diameter of laponite extrapolated to infinite dilution indicates that this clay is completely exfoliated. Furthermore, the hydrodynamic diameter in the laponite-Indocyanine green-water ternary system, at a fixed laponite concentration (2% (m/m)) exhibits a saturation curve. It was found that the extrapolated diameter at dye zero concentration is smaller than in pure water. Absorption spectra with fixed concentration of dye exhibit a red shift of 10–13 nm. On the contrary, the spectra acquired at a constant concentration of laponite do not undergo any displacement. The deconvolution of the spectra with two Gaussian peaks allows to calculate the concentration of the monomeric and dimeric species. The results were interpreted as a synergy between the dye dimerization balance and the dye-laponite binding.

The use of fluorescein in ophthalmic practice

The structure of the primary ophthalmologist appoint¬ment at the outpatient department has changed over the last 10-20 years. Universal computerization, the use of tablets and smartphones, being in air-conditioned rooms are accompanied by an increase in the number of patients with complaints of eye redness, dryness and discomfort. The use of vital dyes for biomicroscopy of the eye anterior segment is a simple and informative method for the early detection of diseases of conjunctiva and cornea. Practical recommendations regarding the use of fluorescein in the daily ophthalmic practice, diagnostic features of obtained patterns in diseases of the eye anterior segment as well features that help to assess the fit of contact lenses are giv¬en in the article.