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. 2024 Nov 18;4(11):100903.
doi: 10.1016/j.crmeth.2024.100903.

Accelerated production of human epithelial organoids in a miniaturized spinning bioreactor

Shicheng Ye  1 , Ary Marsee  1 , Gilles S van Tienderen  1 , Mohammad Rezaeimoghaddam  2 , Hafsah Sheikh  1 , Roos-Anne Samsom  1 , Eelco J P de Koning  3 , Sabine Fuchs  4 , Monique M A Verstegen  5 , Luc J W van der Laan  5 , Frans van de Vosse  2 , Jos Malda  6 , Keita Ito  2 , Bart Spee  1 , Kerstin Schneeberger  7
Affiliations

Accelerated production of human epithelial organoids in a miniaturized spinning bioreactor

Shicheng Ye et al. Cell Rep Methods. .

Abstract

Conventional static culture of organoids necessitates weekly manual passaging and results in nonhomogeneous exposure of organoids to nutrients, oxygen, and toxic metabolites. Here, we developed a miniaturized spinning bioreactor, RPMotion, specifically optimized for accelerated and cost-effective culture of epithelial organoids under homogeneous conditions. We established tissue-specific RPMotion settings and standard operating protocols for the expansion of human epithelial organoids derived from the liver, intestine, and pancreas. All organoid types proliferated faster in the bioreactor (5.2-fold, 3-fold, and 4-fold, respectively) compared to static culture while keeping their organ-specific phenotypes. We confirmed that the bioreactor is suitable for organoid establishment directly from biopsies and for long-term expansion of liver organoids. Furthermore, we showed that after accelerated expansion, liver organoids can be differentiated into hepatocyte-like cells in the RPMotion bioreactor. In conclusion, this miniaturized bioreactor enables work-, time-, and cost-efficient organoid culture, holding great promise for organoid-based fundamental and translational research and development.

Keywords: CP: Biotechnology; CP: Stem cell; RPMotion; bioreactor; organoid; stem cell; suspension culture.

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Conflict of interest statement

Declaration of interests S.Y., G.S.v.T., B.S., and K.S. hold a Dutch patent, NL2029095B1, and a PCT patent application, WO2023031329A1, on the organoid bioreactor with royalties paid. K.S. and B.S. are founders, employees, and shareholders of the Utrecht University spin-off company Orgonex B.V.

Figures

None
Graphical abstract
Figure 1
Figure 1
Design of the RPMotion bioreactor and computational fluid dynamics analysis (A) Five designs of rotors (R0, R1, R2, R3, R4) were tested for the RPMotion bioreactor, and R4 was eventually chosen for further experiments. (B) The control enclosure was placed outside of the incubator, with wires connecting to the bioreactors inside the incubator. The hardware enclosure contains an Arduino Uno, an LCD screen, a push-button, a 12-V power source, motor connections, and associated components. (C and D) Top (C) and (D) side view of one assembled RPMotion unit. Each unit can run up to four bioreactors, which sit in a custom 3D-printed holder. One bioreactor consists of one 50-mL Falcon tube, one rotor connected to one motor, which was controlled by an individual wire from the control unit, and two 0.22-μm filters on the side of the lid to allow for gas exchange. (E and F) Detailed designs of R0 and R4. Parameters include the lengths of the rotors, the widths of the rotors and their blades, the numbers of blades, and the distance between blades. (G and H) Vector field visualization for R0 and R4. Computational results, rotor tip, and middle closed views are also shown as well as the cut plane velocity streamlines.
Figure 2
Figure 2
ICOs expand faster in the RPMotion bioreactor (A) Morphology of ICOs expanded in static culture (SC) and in the bioreactor (RP) at 60 rpm. Bright-field images were taken on D4, D7, D11, and D14 after single-cell seeding. Scale bars, 400 μm (D4 and D7) and 1,000 μm (D11 and D14). (B) Fold changes of cell proliferation in static culture and in the bioreactor at 60 rpm. Four donors were used (with numbers 1, 2, 3, and 4). (C) Gene expression of ICOs cultured in static culture and in the bioreactor with expansion medium. Šídák’s multiple comparisons test was applied. Graphs indicate mean ± SD. ∗p < 0.05; ∗∗∗p < 0.0005. (D) Characterization of ductal (KRT19), epithelial (ECAD), and proliferative (Ki67 and PCNA) markers of ICOs expanded in expansion medium by immunofluorescent (IF) staining. Scale bar, 200 μm.
Figure 3
Figure 3
ICOs can be differentiated into hepatocyte-like cells in the RPMotion bioreactor (A) Morphology of ICOs differentiated in static culture and in the bioreactor at 100 rpm. Bright-field images were taken at D4 and D9 of differentiation. Scale bar, 400 μm. (B and C) Gene expression of ICOs cultured in static culture and in the bioreactor with differentiation medium. Šídák’s multiple comparisons test was applied. Graphs indicate mean ± SD. ∗p < 0.05. (D) Characterization of ECAD, KRT19, and hepatic (ALB and MRP2) and proliferative (Ki67 and PCNA) markers in differentiated organoids by IF staining. Scale bar, 100 μm. (E–H) Intracellular levels of (D) albumin, (E) GLDH, (F) ALT, and (G) AST of differentiated organoids. (I) Rhodamine 123 transport assay to assess the function (transporter protein, MDR1) of differentiated ICOs. Scale bar, 200 μm. (J) Ammonia elimination of differentiated ICOs; n = 4.
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