Accelerated maturation of branched organoids confined in collagen droplets

Abstract

Droplet-based organoid culture offers several advantages over conventional bulk organoid culture, such as improved yield, reproducibility, and throughput. However, organoids grown in droplets typically display only a spherical geometry and lack the intricate structural complexity found in native tissue. By incorporating singularised pancreatic ductal adenocarcinoma cells into collagen droplets, we achieve the growth of branched structures, indicating a more complex interaction with the surrounding hydrogel. A comparison of organoid growth in droplets of different diameters showed that while geometrical confinement improves organoid homogeneity, it also impairs the formation of more complex organoid morphologies. Thus, only in 750 μm diameter collagen droplets did we achieve the consistent growth of highly branched structures with a morphology closely resembling the structural complexity achieved in traditional bulk organoid culture. Moreover, our analysis of organoid morphology and transcriptomic data suggests an accelerated maturation of organoids cultured in collagen droplets, highlighting a shift in developmental timing compared to traditional systems.

Fig. 1. Microfluidic setup for organoid production. (a) Singularised cells are encapsulated in collagen droplets using droplet-based microfluidics. After breaking the emulsion, the droplets are transferred to cell culture medium, and the organoids are cultured for 8 days. In large droplets, the seeded cells develop into branched organoids. (b) The microfluidic chip was operated with three different temperature gradients throughout the pipeline to establish optimal collagen polymerisation conditions. (c) At room temperature, the collagen droplets polymerise heterogeneously because of collagen fibre aggregation (n = 2 independent experiments). (d) The microfluidic chip is operated at room temperature, and the reservoir is cooled during droplet collection. Afterward, polymerisation is induced at 37 °C. The collagen polymerised prematurely as the temperature control was disrupted during droplet production (n = 2 independent experiments). (e) Maintaining the whole setup at 4 °C and the collecting reservoir at 37 °C allowed for homogeneous collagen polymerisation throughout the droplet (n = 2 independent experiments).

Fig. 2. Characterisation of organoid morphology in small versus large droplets. (a) Morphology of organoids grown in large and small droplets over time (n_Large ≥ 34 and n_Small ≥ 24 for each day). (b) Probability of obtaining spheroids versus branched structures in small (red) versus large (blue) droplets (N = 3 independent experiments with n_Small ≥ 116 and n_Large ≥ 153 structures evaluated for each experiment). Error bars indicate the standard deviation. (c) The major axis measured for organoids grown in small (red) and large (blue) droplets (n_Large ≥ 34 and n_Small ≥ 24 for each day). (d) The distribution of the area for 2D projected organoids on day 6 for organoid culture in small (red) and large droplets (blue) and in bulk (green) (n_Small = 24, n_Large = 60, n_Bulk = 13).

Fig. 3. Organoids contract the collagen droplets. (a) The droplets' surface is homogeneous 2 days after organoid seeding as the growing organoid does not yet exert sufficient mechanical force to deform the collagen matrix (n = 32 droplets). The segmented collagen droplet is visualised in blue. (b) The droplet's surface is increasingly irregular 6 days after organoid seeding, as the branched organoid pulls on the surrounding matrix and causes increasing droplet deformation, resulting in a reduced diameter (n = 29 droplets). (c) For large droplets, branched structures cause a 12% reduction in droplet diameter because of collagen deformation. In contrast, small droplets only undergo 5% of diameter reduction on day 6 (n_Large ≥ 23 and n_Small ≥ 23 for each time point). Error bars indicate the 95% confidence interval. (d) The deformation of a collagen droplet by an organoid cultivated for 5 days leads to the formation of a collagen cage. The collagen deformation is the highest at the tips of the organoid's branches (n = 2 independent experiments).

Fig. 4. Branched organoids in large droplets display similar developmental stages to those in bulk. (a) PDAC organoids cultured in bulk display local heterogeneous expression levels of E-cadherin and F-actin that indicate lumen formation sites on day 13 (n = 3 independent experiments). (b) PDAC organoids grown in large droplets display local heterogeneous expression levels of E-cadherin and F-actin, indicating lumen formation sites already on day 6 (n = 3 independent experiments). (c) A fully formed lumen in a branched organoid grown in a large droplet on day 7 (n = 3 independent experiments).

Fig. 5. PDAC organoids expression pattern in different growth conditions. (a) K-means clustering with five clusters (k = 5) using 2000 genes with the most variable expression level between different growth conditions: droplets day 4, 7, 9 and bulk day 7, 9, 13 (n = 4 individual experiments), FDR = 0.05. (b) GO biological processes enrichment analysis from cluster 3 for selected conditions (droplets day 4, 9 and bulk day 7, 13) highlights genes involved in MAPK signaling and apoptosis-related processes. (c) Gene expression heatmap (cluster 2) for selected genes regulating tube morphogenesis and cell migration. (d) Gene expression heatmap (cluster 1) of a subset of genes involved in lipid metabolism and small molecule metabolism. (e) Heat map for selected genes (cluster 4) involved in transcription regulation and anti-apoptotic pathways facilitating cancer survival.