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: Molecular Biology of the Cell

Article Title: DYN-1 regulates SPD-2 and PLK-1 localization and mitotic spindle pole organization

doi: 10.1091/mbc.E25-07-0337

Figure Lengend Snippet: PLK-1 persists abnormally at centrosomes during mitotic progression in dyn-1(RNAi) embryos. (A) Time-lapse analysis of different mitotic stages in control RNAi and dyn-1(RNAi) embryos. White arrows: anterior centrosome-localized PLK-1::sfGFP. (B) Percentage of centrosomes retaining PLK-1 at each stage. Fisher's Exact Test; ns = not significant; n = centrosomes analyzed. Experimental replicates: three for all assays. All fluorescence images include a 10 µm scale bar.

Article Snippet: Time-lapse imaging was performed using the cellSens software (Olympus America) by taking 1.5 μm Z-stacks every ∼75 s at a 60X magnification with a 1.42 numerical aperture (NA) objective using a Prime 95B CMOS camera.

Techniques: Control, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: DYN-1 regulates SPD-2 and PLK-1 localization and mitotic spindle pole organization

doi: 10.1091/mbc.E25-07-0337

Figure Lengend Snippet: DYN-1 depletion disrupts Intercellular Bridge (IB) assembly. (A) Time-lapse images of mCherry::tubulin showing normal IB formation in control RNAi embryos (top panel) and abnormal (middle panel) or missing (bottom panel) IBs in dyn-1(RNAi) embryos. White arrows: IB (B) Classification of IB morphology (normal, abnormal, absent). n = embryos analyzed. Experimental replicates: three for all assays. All fluorescence images include a 10 µm scale bar.

Article Snippet: Time-lapse imaging was performed using the cellSens software (Olympus America) by taking 1.5 μm Z-stacks every ∼75 s at a 60X magnification with a 1.42 numerical aperture (NA) objective using a Prime 95B CMOS camera.

Techniques: Control, Fluorescence

Journal: bioRxiv

Article Title: Spindle pole proteins confine chromosomes to ensure their expulsion during female meiosis

doi: 10.64898/2026.03.24.713942

Figure Lengend Snippet: (A) Time-lapse imaging of in utero oocytes during meiosis I (left) and II (right). Each spindle pole protein was endogenously labeled with GFP or mNeonGreen (green). mCherry::tubulin was co-visualized to monitor spindle shortening and rotation during the anaphase transition (magenta). Spindle shortening was set as 0 s. (B) Diagram of ex utero oocytes forming the first polar body facing toward the microscope objective (top). GFP::ZYG-9 (green) encapsulated chromosomes indicated by mCherry::H2B (magenta) during the anaphase transition (middle). Enlarged images of each signal show voids of GFP::ZYG-9 signals occupied by chromosomes at the anaphase transition (bottom). Onset of chromosome segregation was set as 0 s. All scale bars are 5 µm.

Article Snippet: Time-lapse images were taken using an inverted Nikon ECLIPSE Ti2 equipped with Yokogawa Spinning Disk Field Scanning Confocal System (CSU-W1), piezo Z stage, and iXon Ultra EMCDD camera (Andor) controlled by NIS-Elements software.

Techniques: Imaging, In Utero, Labeling, Microscopy

Journal: bioRxiv

Article Title: Spindle pole proteins confine chromosomes to ensure their expulsion during female meiosis

doi: 10.64898/2026.03.24.713942

Figure Lengend Snippet: (A) Super-resolution microscopy of ex utero C. elegans oocytes expressing GFP::ZYG-9 (spindle poles, green) and mCherry::H2B (chromosomes, magenta) in meiosis I (top). Chromosome regions are cropped (bottom). (B) Total fluorescence of spindle-associated GFP::ZYG-9. (C) Distance between the spindle pole and the chromosome bivalent. Data are normalized to maxima and represent mean ± 95% C.I. (n= 10). (D) Super-resolution microscopy of endogenously labeled spindle pole proteins (green) and chromosomes (Sir-DNA, magenta) during chromosome segregation in anaphase (left). Line scans of normalized signal intensity across the long axis of the spindle (right). (E) Time-lapse imaging of spindle pole proteins after nocodazole treatment. (F) Normalized total fluorescence of each spindle pole proteins in (E). Data represent mean ± 95% C.I. of GFP::ASPM-1 (n= 6), CMD-1::GFP (n= 8), LIN-5::mNeonGreen (n= 6), GFP::TAC-1 (n= 5), GFP::ZYG-9 (n= 5). (G) Super-resolution microscopy of ex utero oocytes treated with 20 µM nocodazole for 15 min (left). Enlarged images shown on the right. All scale bars are 5 µm.

Article Snippet: Time-lapse images were taken using an inverted Nikon ECLIPSE Ti2 equipped with Yokogawa Spinning Disk Field Scanning Confocal System (CSU-W1), piezo Z stage, and iXon Ultra EMCDD camera (Andor) controlled by NIS-Elements software.

Techniques: Super-Resolution Microscopy, Expressing, Fluorescence, Labeling, Imaging

Journal: bioRxiv

Article Title: Spindle pole proteins confine chromosomes to ensure their expulsion during female meiosis

doi: 10.64898/2026.03.24.713942

Figure Lengend Snippet: (A) Co-visualization of GFP::ZYG-9 (green) and mCherry::tubulin (magenta) during metaphase I (left). Morphology of the spindle indicated by the tubulin signal (right). (B) Time-lapse imaging of GFP::ZYG-9 (green) and mCherry::histone H2B (magenta) throughout anaphase I. Yellow allow heads indicate regressed chromosomes from the polar body to the oocyte cytoplasm. (C-G) Normalized total fluorescence of spindle-associated GFP::ZYG-9 (C, E), lateral distance of the farthest chromosomes (D, F), and population of oocytes with regressed chromosomes (G) during anaphase I and II. Data are mean ± 95% C.I. (WT, n= 20; 5RG, n= 23). (H) Co-visualization of GFP::ZYG-9 (green) and mCherry::histone H2B (magenta) during interphase (left) and mitosis (right) in 2-cell stage embryos. Yellow arrowheads indicate extra nucleus and ectopic chromosomes. (I) Population of 1-4 cell stage embryos with abnormal chromosomes in (H). Data represent mean ± SD from four independent experiments (WT, n= 63; 5RG, n= 72). p-value was determined by unpaired t-test. (J) Number of hatched eggs from single mother expressing GFP::ZYG-9 WT (n= 8) or GFP::ZYG-9 5RG (n= 8). Data represent mean + SD. p-value was determined by unpaired t-test. (K) Time-lapse imaging of ex utero oocytes treated with 20 µM nocodazole. Yellow arrows indicate de-clustered chromosomes. (L-P) Quantification of total fluorescence of GFP::ZYG-9 condensate (L), distance between the farthest chromosomes (M), number of de-clustered chromosomes (N), normalized mCherry::Histone H2B intensity (O), and chromosome size (P) after nocodazole treatment. Data are mean ± 95% C.I. (WT, n= 8; 5RG, n= 10). All scale bars are 5 µm.

Article Snippet: Time-lapse images were taken using an inverted Nikon ECLIPSE Ti2 equipped with Yokogawa Spinning Disk Field Scanning Confocal System (CSU-W1), piezo Z stage, and iXon Ultra EMCDD camera (Andor) controlled by NIS-Elements software.

Techniques: Imaging, Fluorescence, Expressing