A High-Throughput Platform for Culture and 3D Imaging of Organoids

Summary

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm², in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

Abstract

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM).

The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

Introduction

In organotypic 3D cell cultures, hereafter referred to as organoids, stem cells differentiate and self-organize into spatial structures that share strong morphological and functional similarities with real organs. Organoids offer valuable models to study human biology and development outside the body^1,2,3. A growing number of models are being developed that mimic the liver, brain, kidney, lung, and many other organs^2,4,5. Differentiation in organoids is directed by the addition ....

Protocol

NOTE: The first part of this protocol details the microfabrication of the cell culture device. An original primary mold with pyramidal cavities can be produced in-house-if micro-fabrication facilities are available-or outsourced to external companies. The primary mold used in this work is produced in-house, with fabrication process steps described elsewhere^11,13. A basic protocol for the microfabrication of the mold is available in Supplementary File 1

Discussion

The procedure for the fabrication of the microwell culture dish, which allows high-density organoid culture and differentiation, has been described in this paper. Owing to the geometry and arrangement of the microcavities, thousands of spheroids can be cultured and stained in a single plate for long periods of time (several weeks or more) with nearly no loss of material. As a comparison, an area of 4 mm x 2 mm on the cell culture plate can contain as many spheroids as a single 384-well plate with an area of 12 cm x 8 cm........

Materials

Name	Company	Catalog Number	Comments
2-Propanol	Thermofisher scientific	AA19397K7
Acetone	Thermofisher scientific	AA19392K7
BC43 Benchtop Confocal Microscope	Andor Technology		spinning disk confocal microscope
bovine serum albumin	Thermofisher scientific	37525
Buffered oxide etching solution	Merck	901621-1L
CEE Spin Coater	Brewer Science	200X
DAPI	Thermofisher scientific	62248
Developer AZ400K	Merck	18441223164
DI Milliq water	Millipore
Fetal Bovine Serum (FBS)	Invitrogen	10082147
Glass coverslips	Marienfled	117650	1.5H, round 25 mm diameter
Hepes	Invitrogen	15630080
Imaris software	BitPlane		image analysis software
Inverted Transmission optical microscope	Nikon	TSF100-F
Labsonic M	Sartorius Stedium Biotech		Ultrasonic homogenizer
Lipidure	NOF America	CM5206	bio-mimetic copolymer
NOA73	Norland Products	17-345	UV curable adhesive
Penicillin-Streptomycin	Invitrogen	15070063
Phalloidin	Thermofisher scientific	A12379	Alexa Fluor
Phosphate Buffer Solution	Thermofisher scientific	10010023
Photo Resist AZ5214E	Merck	14744719710
Pico Plasma tool	Diener Electronic GmbH + Co. KG	Pico Plasma	For O2 plasma treatment
RapiClear 1.52	Sunjin lab	RC 152001	water-soluble clearing agent
RCT Hot Plate/Stirrer	IKA (MY)
Reactive Ion Etching tool	Samco Inc. (JPN)	RIE-10NR
RPMI 1640	Invitrogen	11875093	culture medium for HCT116 cells
Sylgard 184 Silicone Elastomer Kit	Dow Corning	4019862	Polydimethylsiloxane or in short, PDMS
Trichloro(1H,1H,2H,2H-perfluorooctyl)silane	Sigma Aldrich	448931-10G
Triton X-100	Sigma Aldrich	T9284	surfactant
Trypsin EDTA	Thermofisher scientific	15400054
Ultrasonic Cleaner	Bransonic	CPX2800
UV-KUB 2	KLOE		UV-LED light source, 365 nm wavelength, 35 mW/cm2 power density
UV mask aligner	SUSS Microtec Semiconductor (DE)	MJB4