A Modular Microfluidic Organoid Platform Using LEGO-Like Bricks

Abstract

The convergence of organoid and organ-on-a-chip (OoC) technologies is urgently needed to overcome limitations of current 3D in vitro models. However, integrating organoids in standard OoCs faces several technical challenges, as it is typically laborious, lacks flexibility, and often results in even more complex and less-efficient cell culture protocols. Therefore, specifically adapted and more flexible microfluidic platforms need to be developed to facilitate the incorporation of complex 3D in vitro models. Here, a modular, tubeless fluidic circuit board (FCB) coupled with reversibly sealed cell culture bricks for dynamic culture of embryonic stem cell-derived thyroid follicles is developed. The FCB is fabricated by milling channels in a polycarbonate (PC) plate followed by thermal bonding against another PC plate. LEGO-like fluidic interconnectors allow plug-and-play connection between a variety of cell culture bricks and the FCB. Lock-and-play clamps are integrated in the organoid brick to enable easy (un)loading of organoids. A multiplexed perfusion experiment is conducted with six FCBs, where thyroid organoids are transferred on-chip within minutes and cultured up to 10 d without losing their structure and functionality, thus validating this system as a flexible, easy-to-use platform, capable of synergistically combining organoids with advanced microfluidic platforms.

Keywords: LEGO®; microfluidics; modular; organoids; organ‐on‐a‐chip; thyroid.

Figure 1

A modular microfluidic platform for dynamic culture of organoid models. A) 3D design concept of the FCB and the cell culture bricks. Plug‐and‐play insertion of bricks in the FCB is made by LEGO‐inspired fluidic interconnectors. B) The FCB is made from a top PC layer (which features the fluidic interfaces) thermally bonded against a bottom layer with engraved channels. The bottom layer can be customized to create different fluidic pathways. C) Schematics of different fluidic pathways using distinct bottom FCB layers. Photographs of FCBs plugged with reservoir and OoC bricks and injected with a red and blue food dye. A porous, membranous carrier can be integrated in the OoC device for various studies, such as transepithelial transport and cell migration assays across biological barriers as well as for studies of gradients of bioactive factors (Video S2, Supporting Information). D,E) Photographs of six FCBs in parallel before and during cell culture experiments.

Figure 2

Micromilling of fluidic channels and thermal bonding of PC–PC FCB plates. A) Photograph of a PC test sample with an engraved fluidic channel (W = 1 mm, H = 0.5 mm). On the right, optic laser images of cross sections of channels with different geometries (round, square, and triangular). Scale bar, 500 µm. B) Topography analysis of engraved channels. The dimensions of the engraved channels were quantified by an optical profilometer (Keyence VK‐X100K). C) Quantification of the surface roughness (Ra) for test slots milled at different spindle speeds and feed rates (mean ± SD). Data are shown from three milled replicas (n = 3), and each point represents the average of multiple line Ra measurements across the samples surface area. D) Optic photographs of engraved microchannels and burrs formation. Images were taken with a 10× magnification. Scale bar, 250 µm. E) Schematic illustration of the system apparatus for bonding the two FCB plates (Al, aluminum). F) Fluorescent images of enclosed channels perfused with a FITC dye and prebonded at different bonding temperatures. Profile lines of the fluorescence intensity (a.u., arbitrary units) are presented below for each bonding temperature. For each image, three lines were drawn perpendicular to the channels length and placed equidistant from each other. G) Photograph of an enclosed channel of a FCB sample. I) Stereomicroscope images of two cross sections from channels bonded at 120 and 140 °C. Scale bar, 500 µm. J) Quantification of height and width changes of channels bonded at different bonding temperatures. All data are tested for statistical significance using Kruskal–Wallis test. ns, nonsignificant; p > 0.5; *p < 0.1; **p < 0.01. Data are shown from three measurements across four channel cross sections from two independent bonded samples (n = 12).

Figure 3

Development of LEGO‐like fluidic interconnections. A) Photograph of a modular brick bottom part containing four studs. Studs are equidistant to each other similar to those of commercially available LEGO bricks. B) 3D model of the fluidic interface. A modular brick is inserted into a circular recess of the FCB. An O‐ring is located radially on the stud. Flow connection is realized between the modular brick and the FCB via centric and aligned microchannels. C) Illustrations of O‐ring failure scenarios. A high clearance gap (S) and low compression over the O‐ring can lead to leakages, while excessive compression may cause O‐ring extrusion and damage. D) Quantification of the maximum burst pressure values as measure of the sealing strength of studs either made from 3D printed resin (MED610) or from machined PC (mean ± S.E.M.). E) Photograph of a modular brick plugged in a FCB and being perfused with a food dye at 0.5 mL min⁻¹. F) Percentage of successful plugging of O‐ring studs made from 3D printed material and machined PC. G) Photographs of the sidewalls and the O‐ring grooves with and without O‐rings for 3D printed and machined PC studs. Scale bar, 1.5 mm. H) 3D heat map of O‐ring groove surfaces for 3D printed and PC machined studs. J) Measurement of the surface finishing of 3D printed and machined studs (mean ± S.E.M.).

Figure 4

Cell culture modular bricks and development of a LnP chip device. A) 3D model views of an organoid‐on‐a‐chip, a cell culture medium reservoir module, a sensor module for continuous measurement of dissolved oxygen and pH values, and a mini‐microscope. B) 3D explosive model view of the LnP organoid‐on‐a‐chip device. The device consists of a PC housing (grey), fluidic chips (cyan), and a thermoformed membranous carrier (yellow). The carrier can be easily transferred in/out of the chip with tweezers. Brightfield picture of Matrigel‐embedded mouse ESC‐derived thyroid organoids cultured in the membranous carriers. Image taken with a 4× magnification. Scale bar, 250 µm. C) Photographs of the clamped versus unclamped organoid‐on‐a‐chip. D) 3D view of the fluidic pathway through the organoid‐on‐a‐chip device. E) Extracted still images from a time‐lapse recording of the filling of the LnP organoid‐on‐a‐chip with a passive bubble trap tank. The device was perfused with a blue food dye at 0.5 mL min⁻¹.

Figure 5

Validation of the developed microfluidic platform for organoid culture. A) Photograph of the experiment set‐up at day 0. FCBs were connected with six reservoirs, six OoC module bricks, and one sensor module brick for monitoring of DO₂ and pH. B) Top view of most of the OoC module bricks at day 3 of perfusion. Some bubbles were entrapped inside the bubble trap. C) Side view of the fluidic system at day 10. The photograph shows no leakages at the fluidic interfaces. D) Bottom view of the sensor module brick. Yellow arrows indicate the flow direction, which passes through a bubble trap, a pH sensor dot (white), and finally through a black dot (DO₂). E) Continuous oxygen and pH measurement. F) Brightfield and live/dead assay pictures of thyroid organoids exposed to flow or kept in static wells for 3 and 10 d of culture. In green, Tg promoter‐driven GFP. Brightfield images taken with a 4× magnification. Scale bar, 150 µm. Fluorescence images of a live/dead staining of organoids cultured in the OoC module brick versus static controls. In red, dead cells stained with ethidium homodimer‐1 (EthD). Scale bar, 250 µm. G) Measurement of the equivalent diameter of thyroid organoids. All data are presented as mean (n = 35) and tested for statistical significance using Kruskal–Wallis test. ns, nonsignificant; p > 0.05. H) Confocal Z‐projections of thyroid organoids cultured for 3 d in OoC module bricks. Immunofluorescence images of organoids marked with GFP (green), and stained for Tg (red), ZO‐1 (white), and counterstained with DAPI (blue). Images taken with a magnification of 25×. Scale bar, 100 µm and insets, 25 µm.