phase contrast images  (Nikon)

Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: The automated OsciSphere bioassembly platform and workflow. a 3D model of the OsciSphere platform. The inset shows the core 8-channel droplet generator, which integrates an oscillator, syringe pump, disposable pipette tips, and a cooling block to maintain Matrigel in a liquid state. b Photograph of the automated workstation layout. c Schematic of the OsciSphere workflow. 1) Cells are suspended in Matrigel at 4 °C. 2) The platform’s droplet generator dispenses uniform droplets into a 96-well plate containing a biphasic carrier oil/culture medium overlay. 3) Droplets are solidified at 37 °C for 10 min. 4) Solidified microspheres are transferred into the underlying medium using an antistatic gun or gentle agitation. 5) This automated process yields arrays of µMCTs or µTDOs ready for high-throughput screening applications. d Time-lapse imaging (left) demonstrates the “pull-break-sediment” cycle of droplet formation. The platform reliably produces approximately 100 uniform 30 nL droplets from only 3 µL of Matrigel (right), achieving excellent size uniformity. Scale bar, 500 µm. e Encapsulation uniformity demonstrated by fluorescent beads within Matrigel microspheres (60.9 ± 5.1 beads per droplet, CV = 7.67%, n = 28). f Platform versatility is shown by forming uniform microspheres from agarose and HAMA. g Radar chart comparing OsciSphere’s performance metrics (e.g., uniformity, throughput, automation) against conventional 3D culture methods. OsciSphere enables rapid and uniform 3D culture, including ( h ) µMCTs and i µTDOs. j Dome TDOs exhibit significant spatial heterogeneity, leading to diffusion-limited regions and apoptotic cores by Day 3

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Transferring, Blocking Assay, Gentle, High Throughput Screening Assay, Imaging, Encapsulation, Diffusion-based Assay

Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: OsciSphere operates in a deterministic regime to produce uniform µMCTs that recapitulate key physiological tumor features. a Scaffold-free ULA culture (Day 4) yields a multimodal population, with a single large central spheroid and numerous highly variable satellite aggregates. b Conventional Matrigel dome culture (Day 3) exhibits significant spatial heterogeneity driven by diffusion gradients, with smaller spheroids in the nutrient-poor center and larger ones at the periphery. c OsciSphere-generated µMCTs (~600 cells/droplet, Day 3) exhibit structural isotropy and consistency. d Quantification of spheroid diameters confirms the superior monodispersity of µMCTs ( n = 100, CV = 4.2%) compared to the high variability of ULA ( n = 244) and dome ( n = 237) cultures (mean ± SD, **** p < 0.0001). e Bright-field time-lapse shows rapid self-assembly of compact HCT116 spheroids by Day 1. f Live/Dead staining at 72 h confirms high cell viability. g Histological analysis (H&E) reveals a dense 3D tissue architecture, with immunohistochemistry for Ki67 confirming robust proliferative activity within the spheroid. h Optimization of formation efficiency reveals a critical density threshold at 600–900 cells per microsphere, achieving 99.6% successful formation by Day 2 ( n = 10, mea n ± SD, red star indicates the optimal 600-cell condition). i Immunofluorescence (IF) of 2D HCT116 cultures shows basal expression of Vimentin and N-cadherin. Scale bar, 10 µm. j µMCTs display pronounced, organized expression of mesenchymal markers (Vimentin, N-cadherin), indicative of EMT. Scale bar, 50 µm. k This invasive phenotype is validated by RT-qPCR, showing significant upregulation of key EMT-associated genes ( N-cadherin , Snail , Slug ) and cancer stem cell-associated genes ( Sox2 , Oct4 ) in µMCTs ( n = 3, mea n ± SD, **** p < 0.0001). l µMCTs exhibit a physiologically relevant reduced proliferation rate compared to 2D cultures, mimicking in vivo tumor kinetics ( n = 5, mean ± SEM, **** p < 0.0001). m Flow cytometry analysis reveals elevated intracellular ROS levels in µMCTs (51.2%) versus 2D cultures (30.2%), consistent with the establishment of metabolic gradients and a hypoxic tumor microenvironment. Statistical significance was analyzed by using one-way analysis of variance (ANOVA)

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Diffusion-based Assay, Generated, Staining, Immunohistochemistry, Activity Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, In Vivo, Flow Cytometry

Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: OsciSphere-derived µTDOs resolve diffusion limitations to drive superior growth and maturation. a Comparative time-lapse microscopy reveals the impact of culture geometry. Conventional Matrigel domes exhibit severe spatial heterogeneity: while organoids at the nutrient-rich “Edge” grow, those in the diffusion-limited “Core” undergo apoptosis (red arrow) by Day 3. In contrast, OsciSphere µTDOs (bottom row) exhibit uniform, necrosis-free growth independent of spatial position. b Quantification of projected surface area demonstrates significantly accelerated expansion kinetics for µTDOs compared to dome cultures ( n = 58, mean ± SEM, **** p < 0.0001). c Morphogenic analysis reveals enhanced maturation in the OsciSphere format, with a significantly higher frequency of multi-budded organoids observed by Day 2 ( n = 58, mea n ± SEM). d Viability imaging (Calcein-AM/PI) on Day 3 confirms that µTDOs maintain high cell survival without the central necrosis observed in static hydrogel cultures. e Histological validation against native murine intestine. H&E staining demonstrates that µTDOs recapitulate the polarized crypt-villus architecture of the in vivo epithelium. Immunohistochemistry for Ki67 (brown) confirms the preservation of active proliferative zones in the crypt domains of both µTDOs and native tissue. Statistical significance was analyzed by using one-way ANOVA

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Derivative Assay, Diffusion-based Assay, Time-lapse Microscopy, Imaging, Biomarker Discovery, Staining, In Vivo, Immunohistochemistry, Preserving

Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: OsciSphere enables high-fidelity modeling of the patient-specific tumor-immune microenvironment . a Schematic of the precision immuno-oncology workflow. Patient-derived hCRC tissues are processed into µPDOs via OsciSphere, creating physically permissive scaffolds that support autologous PBMC infiltration. b Bright-field micrographs of source PDO lines established from three independent hCRC patients. c Transcriptomic validation confirms high-fidelity modeling: established PDOs (O) maintain strong gene expression correlations ( R > 0.87) with their matched parental tumor tissue (T). d Genomic profiling demonstrates that µPDOs preserve the patient-specific mutational landscape across a panel of key oncogenic drivers. e Histological comparison reveals that µPDOs (bottom) recapitulate the native tumor architecture (top). H&E staining shows comparable morphology, while Ki67 staining confirms the maintenance of robust proliferative zones in both the parent tissue (brown) and the µPDOs (red). f Failure mode of conventional culture: endpoint imaging reveals that the dense, large-volume Matrigel dome acts as a physical barrier, excluding PBMCs (red) from the tumor core. g OsciSphere overcomes the barrier effect: time-lapse imaging captures the active migration of PBMCs (red) into the µPDO (dashed circle), facilitating sustained tumor-immune interactions (yellow arrows) over 72 h. Scale bar, 100 µm. h Flow cytometry analysis quantifies IFN-γ levels. i Quantif i cation of the mean fluorescence intensity of CD8 + IFN-γ + PBMCs from PBMC-only, PBMCs + µPDOs, PBMC + µPDOs + sintilimab groups ( n = 3, mea n ± SD; * p < 0.05, ** p < 0.01, ns, not significant). Statistical significance was analyzed by using one-way ANOVA

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Derivative Assay, Biomarker Discovery, Gene Expression, Comparison, Staining, Imaging, Migration, Flow Cytometry, Fluorescence

Journal: Oncogenesis

Article Title: Transcriptomic profiling of co-cultured cancer-host cells identifies hypoxia as a driver of the skeletal muscle cell’s anti-proliferative effect on cancer cells

doi: 10.1038/s41389-026-00601-9

Figure Lengend Snippet: A Schematic representation of co-cultures. C2C12 and MLg cells were submitted to similar culture conditions for 6 days. At day 0 (D0), when C2C12 myotubes and MLg cells were fully confluent, EO771 cancer cells were seeded onto the cells. B Representative phase-contrast microscopy images of C2C12 and MLg stained with May-Grünwald and Giemsa at D0. Scale bar = 200 µm. C Number of EO771 cells per mm 2 attached onto C2C12 myotubes (C2C12 + EO771) and MLg cells (MLg + EO771) 3 h post-seeding at D0. D Outgrowth of EO771 cells (100 cells per well in 96-well plate) co-cultured with C2C12 myotubes (C2C12 + EO771) or MLg cells (MLg + EO771) for 2 days. EO771 area represents the percentage of fluorescent cells normalized to D0 ( n = 21–24 from three independent experiments). Representative fluorescence microscopy images of EO771 cell outgrowth on C2C12 or MLg at day 2 (D2). Scale bar = 200 µm. E Outgrowth of EO771 cells in co-cultures with C2C12 myotubes or MLg cells when seeded at 500, 1000, 4000, and 8000 cells per well in a 96-well plate at day 0. Green fluorescence of EO771 was measured over 2 days without normalization ( n = 24 from two independent experiments). F Representative fluorescence microscopy images of EO771 cell outgrowth (4000 cells per well in 96-well plate) on C2C12 or MLg. Scale bar = 200 µm. Data are mean ± SEM. Normality was tested with the Shapiro–Wilk test. Comparison between groups was done using the Mann–Whitney test (comparing two groups with one variable) or two-way ANOVA adjusted for multiple testing with the Šidák method (comparing two groups with two variables). ****P < 0.0001.

Article Snippet: Phase-contrast microscopy images were acquired using a CKX53 microscope with a UC90 color camera (Olympus, Tokyo, Japan) and a 10x objective, operated with Olympus cellSens software (version 2.2, Olympus).

Techniques: Microscopy, Staining, Cell Culture, Fluorescence, Comparison, MANN-WHITNEY