A process engineering approach to increase organoid yield

Abstract

Temporal manipulation of the in vitro environment and growth factors can direct differentiation of human pluripotent stem cells into organoids - aggregates with multiple tissue-specific cell types and three-dimensional structure mimicking native organs. A mechanistic understanding of early organoid formation is essential for improving the robustness of these methods, which is necessary prior to use in drug development and regenerative medicine. We investigated intestinal organoid emergence, focusing on measurable parameters of hindgut spheroids, the intermediate step between definitive endoderm and mature organoids. We found that 13% of spheroids were pre-organoids that matured into intestinal organoids. Spheroids varied by several structural parameters: cell number, diameter and morphology. Hypothesizing that diameter and the morphological feature of an inner mass were key parameters for spheroid maturation, we sorted spheroids using an automated micropipette aspiration and release system and monitored the cultures for organoid formation. We discovered that populations of spheroids with a diameter greater than 75 μm and an inner mass are enriched 1.5- and 3.8-fold for pre-organoids, respectively, thus providing rational guidelines towards establishing a robust protocol for high quality intestinal organoids.

Keywords: Aggregates; Intestinal; Organoids; Process engineering; Sorting; Yield.

Fig. 1.

Hindgut spheroid characterization. (A) DAPI staining allows visualization of spheroids. White dashed cross shows major and minor axes used to obtain an estimate for the diameter of a spheroid, which is an average of the major and minor axes. (B) Whole spheroids stain positive for hindgut marker CDX2 (green), whereas subpopulations stain for epithelial marker E-cadherin (white) and mesenchymal marker vimentin (red). Solid arrow: entirely epithelial; arrowhead: entirely mesenchymal; hollow arrow: inner mesenchyme, outer epithelial. (C) Scatter plots of spheroid diameter (µm) versus the number of cells per spheroid. Standard deviation is indicated. (D) Percentage of spheroids with an inner cell mass from time points during hindgut induction. The number on top of each bar indicates the number of spheroids analyzed. (E) Average diameter of the inner mass (blue) and the size of the inner mass relative to the spheroid (green). The number on top of each bar indicates the number of spheroids analyzed. (F) Percentage of all analyzed spheroids and buds in hindgut cultures that did or did not display the morphology of an inner mass with an outer ring, separated based on a threshold of a 75 µm diameter. The number on top of each bar indicates the number of spheroids analyzed. (G) Staining of spheroids for E-cadherin (white) and vimentin (red) allows for visualization of spatial organization of the cells. 3D renderings. (H) Polarized of epithelial cells was visualized with stains for apical marker ezrin (white) and basal marker laminin (green). Scale bars: 10 µm in A,B,G; 5 μm in H.

Fig. 2.

Hindgut culture characterization. (A) Phalloidin (green) staining of hindgut cultures for F-actin illustrates the global 3D morphology over a 1.2 mm×1.2 mm region of the culture surface. (B) Hindgut cultures stained for the epithelial marker E-cadherin (white) and mesenchymal marker vimentin (red). (C) Hindgut cultures co-stained for nuclei (DAPI, blue), the epithelial cytoplasmic membrane protein ezrin (white), and basement membrane protein laminin (green). Insets show high-resolution images of the budding spheroid region co-stained for (i) DAPI and laminin and (ii) ezrin and laminin, to illustrate spatial organization within the bud. (D) Immunostaining for fibronectin (red) and collagen IV (Col IV, white). Scale bars: 100 μm in B,C,D; 50 μm in Ci,ii.

Fig. 3.

Spheroid sorting. (A) System overview, three-axis positioning unit schematic. (B) Propidium iodide staining to mark dead cells in spheroids; (i) harvested spheroids, (ii) non-harvested spheroids. The red arrows indicate dead cells. Insets show magnification of marked regions. (C-E) Spheroids from hindgut induction days 5-8 went through the sorting process but were not separated by any parameters (C), were sorted by diameter (D), or were sorted by the morphological feature of an inner mass with an outer ring (E). The percentage in each pie chart is the percent of matured spheroids. The number below each pie graph indicates the total number of spheroids sorted for that condition.