b Representative images (10×) of gemcitabine (100 nM) treated organoids for a 4-h drug pulse followed by normal growth media, continuous treatment of paclitaxel (10 nM) for 72-h, continuous treatment of gemcitabine (100 nM) for 72-h, a combination dose of gemcitabine (100 nM) + paclitaxel (10 nM) for 72-h, and negative and positive controls (staurosporine 10 mM). Organoids can be analyzed for growth, morphology changes, or death. Drug treatments on each channel can be changed on demand, creating time-varying drug treatments. Each color represents a different drug formulation. e Multiple Z image slices or stacks of a group of fixed and fluorescently stained organoids with DAPI and phalloidin (scale bar 100 μm).Ī On-platform drug treatment and stimulation with continuous fluorescence and phase imaging of organoids for the treatment duration. Nuclei staining (DAPI) and F-actin (Phalloidin) are demonstrated (scale bar 100 μm). d Long-term culture, growth, and fluorescent staining of fixed PDAC organoids on the platform. Patient 1 (blue), Patient 2 (red), Patient 3 (green). Each dot represents the cross-sectional area of an individual organoid. c Organoid growth curves of PDAC organoid samples derived from three patients grown from single cells for 7 days on the platform. Organoids from patient 4 demonstrated a well-differentiated morphology with simple spherical organoids and uniform nuclear and cytoplasmic features with little or no accumulation of necrotic luminal cells. In both platforms, organoids from patient 1 exhibited back-to-back glands with a high degree of nuclear atypia and pleomorphism with an accumulation of apoptotic luminal necrotic cells.
After mature organoid formation, organoids were harvested, H&E stained, and their morphologies compared and analyzed. b Organoids from two patients were grown in parallel in a traditional 24-well plate and on our microfluidic platform (scale bar 100 μm). The 3D culture chamber device can also be disassembled for cell harvesting and further cellular assays.Ī On-platform growth of organoids: three separate patient-derived pancreatic ductal adenocarcinoma (PDAC) organoids in Matrigel from single cells to formation of differentiated morphology of complex 3D structures (scale bar 100 μm). e, f Organoids or 3D cellular structures are continuously observed through time-lapse imaging for quantification fluidic culture conditions can be changed on demand. d 30 chemical inputs and 30 outlets of the multiplexer control device ( a) are preprogrammed to provide combinatorial and time-varying stimulations to the 3D culture chamber device ( b). c A cross-section of the two-layer multichambered PDMS-based 3D culture chamber device. The channel layer is reversibly clamped on top of the chamber layer to provide media and other chemical stimulation without leakage. b, c The 3D culture chamber platform contains 200 individual chambers that are compatible with temperature-sensitive gels (i.e., Matrigel), and an overlaying channel layer enables 20 independent fluidic conditions (scale bar 100 μm). This integrated platform advances organoids models to screen and mirror real patient treatment courses with potential to facilitate treatment decisions for personalized therapy.Ī A programmable membrane-valve-based microfluidic chip (multiplexer control device) provides automated stimulation profiles to various chambers of a separate 3D culture platform ( b) to produce many parallel and dynamical culture experiments. We observe significant differences in the response of individual patient-based organoids to drug treatments and find that temporally-modified drug treatments can be more effective than constant-dose monotherapy or combination therapy in vitro. We validate our system by performing individual, combinatorial, and sequential drug screens on human-derived pancreatic tumor organoids. Our system provides combinatorial and dynamic drug treatments to hundreds of cultures and enables real-time analysis of organoids. Here, we present an automated, high-throughput, microfluidic 3D organoid culture and analysis system to facilitate preclinical research and personalized therapies. Automated microfluidics offers advantages in high-throughput and precision analysis of cells but is not yet compatible with organoids. Three-dimensional (3D) cell culture technologies, such as organoids, are physiologically relevant models for basic and clinical applications.
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