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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 body1,2,3. A growing number of models are being developed that mimic the liver, brain, kidney, lung, and many other organs2,4,5. 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 elsewhere11,13. A basic protocol for the microfabrication of the mold is available in Supplementary File 1

Representative Results

Figure 8F shows the typical aspect of a cell culture coverslip after successful fabrication. The UV-curable adhesive layer appears flat and adheres well to the coverslip. The transfer of the adhesive layer on the coverslip might fail if the layer on the coverslip is overcured, or if the removal of the flat PDMS substrate is done incorrectly (as shown in Figure 8G,H). In both cases, the failure is evident as no textured film is transferred to the.......

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........

Acknowledgements

The research is supported by the CALIPSO project supported by the National Research Foundation, Prime Minister's Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) programme. V.V. acknowledges the support of NRF investigator NRF-NRFI2018-07, MOE Tier 3 MOE2016-T3-1-005, MBI seed funding, and ANR ADGastrulo. A.B. and G.G. acknowledge the support from MBI core funding. A.B. acknowledges Andor Technologies for the loan of the BC43 microscope.

....

Materials

NameCompanyCatalog NumberComments
2-PropanolThermofisher scientificAA19397K7
AcetoneThermofisher scientificAA19392K7
BC43 Benchtop Confocal MicroscopeAndor Technologyspinning disk confocal microscope
bovine serum albumin Thermofisher scientific37525
Buffered oxide etching solutionMerck901621-1L
CEE Spin CoaterBrewer Science200X
DAPIThermofisher scientific62248
Developer AZ400KMerck18441223164
DI Milliq waterMillipore
Fetal Bovine Serum (FBS)Invitrogen10082147
Glass coverslipsMarienfled1176501.5H, round 25 mm diameter
HepesInvitrogen15630080
Imaris softwareBitPlaneimage analysis software
Inverted Transmission optical microscopeNikonTSF100-F
Labsonic MSartorius Stedium BiotechUltrasonic homogenizer
LipidureNOF AmericaCM5206bio-mimetic copolymer
NOA73Norland Products17-345UV curable adhesive
Penicillin-StreptomycinInvitrogen15070063
PhalloidinThermofisher scientific A12379Alexa Fluor
Phosphate Buffer SolutionThermofisher scientific10010023
Photo Resist AZ5214EMerck14744719710
Pico Plasma toolDiener Electronic GmbH + Co. KGPico PlasmaFor O2 plasma treatment
RapiClear 1.52Sunjin labRC 152001water-soluble clearing agent
RCT Hot Plate/StirrerIKA (MY)
Reactive Ion Etching toolSamco Inc. (JPN)RIE-10NR
RPMI 1640Invitrogen11875093culture medium for HCT116 cells
Sylgard 184 Silicone Elastomer KitDow Corning4019862Polydimethylsiloxane or in short, PDMS
Trichloro(1H,1H,2H,2H-perfluorooctyl)silaneSigma Aldrich448931-10G
Triton X-100Sigma AldrichT9284surfactant
Trypsin EDTAThermofisher scientific15400054
Ultrasonic CleanerBransonicCPX2800
UV-KUB 2KLOEUV-LED light source, 365 nm wavelength, 35 mW/cm2 power density
UV mask alignerSUSS Microtec Semiconductor (DE)MJB4

References

  1. Kim, J., Koo, B. -. K., Knoblich, J. A. Human organoids: model systems for human biology and medicine. Nature Reviews Molecular Cell Biology. 21 (10), 571-584 (2020).
  2. Takebe, T., Wells James, M. Organoids by design. Science. 364....

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