Name: a high-throughput platform for culture and 3d imaging of organoids
Uploaded: 2022-10-14T14:00:00
Description: Watch this Scientific Journal Video about A High-Throughput Platform for Culture and 3D Imaging of Organoids at JoVE.com

The research is supported by the CALIPSO project supported by the National Research Foundation, Prime Minister&#39;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.

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</s...

<ol>
	<li>Kim, J., Koo, B. -. K., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organoids:+model+systems+for+human+biology+and+medicine.">Human organoids: model systems for human biology and medicine.</a> Nature Reviews Molecular Cell Biology. 21 (10), 571-584 (2020).</li><li>Takebe, T., Wells James, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+by+design.">Organoids by design.</a> Science. 364 (6444), 956-959 (2019).</li><li>Kratochvil, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+materials+for+organoid+systems.">Engineered materials for organoid systems.</a> Nature Reviews Materials. 4 (9), 606-622 (2019).</li><li>Rossi, G., Manfrin, A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+potential+in+organoid+research.">Progress and potential in organoid research.</a> Nature Reviews Genetics. 19 (11), 671-687 (2018).</li><li>O'Connell, L., Winter, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids:+past+learning+and+future+directions.">Organoids: past learning and future directions.</a> Stem Cells and Development. 29 (5), 281-289 (2020).</li><li>Vives, J., Batlle-Morera, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+developing+human+3D+organoids+into+medicines.">The challenge of developing human 3D organoids into medicines.</a> Stem Cell Research &amp; Therapy. 11 (1), 72 (2020).</li><li>Busslinger, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+and+challenges+of+patient-derived+organoids+in+guiding+the+multimodality+treatment+of+upper+gastrointestinal+malignancies.">The potential and challenges of patient-derived organoids in guiding the multimodality treatment of upper gastrointestinal malignancies.</a> Open Biology. 10 (4), 190274 (2020).</li><li>Lukonin, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+landscape+of+intestinal+organoid+regeneration.">Phenotypic landscape of intestinal organoid regeneration.</a> Nature. 586 (7828), 275-280 (2020).</li><li>Rios, A. C., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+organoids:+a+bright+future+ahead.">Imaging organoids: a bright future ahead.</a> Nature Methods. 15 (1), 24-26 (2018).</li><li>Dekkers, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+3D+imaging+of+fixed+and+cleared+organoids.">High-resolution 3D imaging of fixed and cleared organoids.</a> Nature Protocols. 14 (6), 1756-1771 (2019).</li><li>Galland, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+high-+and+super-resolution+imaging+using+single-objective+SPIM.">3D high- and super-resolution imaging using single-objective SPIM.</a> Nature Methods. 12 (7), 641-644 (2015).</li><li>Beghin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+3D+imaging+method+for+quantitative+characterization+of+organoid+development+and+phenotype.">High content 3D imaging method for quantitative characterization of organoid development and phenotype.</a> bioRxiv. , (2021).</li><li>Beghin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+high-speed+3D+imaging+of+organoid+cultures+with+multi-scale+phenotypic+quantification.">Automated high-speed 3D imaging of organoid cultures with multi-scale phenotypic quantification.</a> Nature Methods. 19 (7), 881-892 (2022).</li></ol>

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....

In organotypic 3D cell cultures, hereafter referred&#160;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 of soluble growth factors and an extracellular matrix in a precise time sequence. However, in marked contrast to organs, the development of organoids is quite heterogeneous.
Beyond numerous biological challenges6,7, organoid cultures also pose technological challenges in terms of cell culture methods, characterization of transcriptomics, and imaging. In vivo organ development occurs in a biological environment that results in a highly stereotypical self-organization of cell arrangements. Any phenotypic alteration can be used as a proxy to diagnose a diseased state. In contrast, organoids develop in vitro in minimally controlled microenvironments compatible with cell culture conditions, resulting in large variability in the development path and shape formation for each individual organoid.
A recent study8 demonstrated that quantitative imaging of organoid shape (phenotype descriptors) coupled to the assessment of a few genetic markers allow the definition of phenotypic development landscapes. Arguably, the ability to relate the diversity of genomic expression in organoids with their phenotypic behavior is a major step toward unleashing the full potential of organotypic cultures. Thus, it begs for the development of dedicated, high-content imaging approaches allowing the characterization of organoid features at subcellular, multicellular, and whole-organoid scales in 3D9,10.
We developed a versatile high-content screening (HCS) platform allowing streamlined organoid culture (from isolated human embryonic stem cells [hESCs], human induced pluripotent stem cells [hIPSCs], or primary cells to 3D, multicellular, differentiated organoids) and fast, non-invasive 3D imaging. It integrates a next-generation, miniaturized, 3D cell culture device, called the JeWells chip (the chip hereafter), that contains thousands of well-arrayed microwells flanked with 45&#176; mirrors that allow fast, 3D, high-resolution imaging by single-objective light-sheet microscopy11. Compatible with any standard, commercial, inverted microscope, this system enables the imaging of 300 organoids in 3D with subcellular resolution in &#60;1 h.
The microfabrication of the cell culture device starts from an existing micro structured mold, which contains hundreds of micropyramids (Figure 1A) with a square base and sidewalls at 45&#176; with respect to the base. Figure 1C shows electron microscope (EM) images of such structures. The mold itself is made of poly(dimethylsiloxane) (PDMS) and can be made as a replica cast of a primary mold (not shown here) with corresponding features (as cavities) using standard soft-lithographic procedures. The primary mold can be produced by different procedures. The one used for this protocol was made using silicon wet etching as reported in Galland et al.11; the procedure for the fabrication of the primary mold is not critical for this protocol. The pyramids are arranged in a squared array, with the same pitch for the X and Y directions (in this case the pitch is 350 &#181;m).
As an illustration, proof-of-concept experiments12 were published to demonstrate that the chip allows long-term culture (months) and differentiation protocols while precisely defining the number of initial cells in the wells. Individual development of a large number of organoids can be automatically monitored live using standard brightfield and 3D light-sheet fluorescence microscopes. Moreover, organoids can be retrieved to perform further biological investigations (e.g., transcriptomic analysis). This paper outlines detailed protocols for the fabrication of the cell culture coverslips, the seeding and staining procedure for fluorescence microscopy, as well as the retrieval of the organoids.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Propanol</td>
			<td>Thermofisher scientific</td>
			<td>AA19397K7</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Thermofisher scientific</td>
			<td>AA19392K7</td>
			<td></td>
		</tr>
		<tr>
			<td>BC43 Benchtop Confocal Microscope</td>
			<td>Andor Technology</td>
			<td></td>
			<td>spinning disk confocal microscope</td>
		</tr>
		<tr>
			<td>bovine serum albumin&#160;</td>
			<td>Thermofisher scientific</td>
			<td>37525</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffered oxide etching solution</td>
			<td>Merck</td>
			<td>901621-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>CEE Spin Coater</td>
			<td>Brewer Science</td>
			<td>200X</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermofisher scientific</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer AZ400K</td>
			<td>Merck</td>
			<td>18441223164</td>
			<td></td>
		</tr>
		<tr>
			<td>DI Milliq water</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Invitrogen</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Marienfled</td>
			<td>117650</td>
			<td>1.5H, round 25 mm diameter</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Invitrogen</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>BitPlane</td>
			<td></td>
			<td>image analysis software</td>
		</tr>
		<tr>
			<td>Inverted Transmission optical microscope</td>
			<td>Nikon</td>
			<td>TSF100-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Labsonic M</td>
			<td>Sartorius Stedium Biotech</td>
			<td></td>
			<td>Ultrasonic homogenizer</td>
		</tr>
		<tr>
			<td>Lipidure</td>
			<td>NOF America</td>
			<td>CM5206</td>
			<td>bio-mimetic copolymer</td>
		</tr>
		<tr>
			<td>NOA73</td>
			<td>Norland Products</td>
			<td>17-345</td>
			<td>UV curable adhesive</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>Thermofisher scientific&#160;</td>
			<td>A12379</td>
			<td>Alexa Fluor</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution</td>
			<td>Thermofisher scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo Resist AZ5214E</td>
			<td>Merck</td>
			<td>14744719710</td>
			<td></td>
		</tr>
		<tr>
			<td>Pico Plasma tool</td>
			<td>Diener Electronic GmbH + Co. KG</td>
			<td>Pico Plasma</td>
			<td>For O2 plasma treatment</td>
		</tr>
		<tr>
			<td>RapiClear 1.52</td>
			<td>Sunjin lab</td>
			<td>RC 152001</td>
			<td>water-soluble clearing agent</td>
		</tr>
		<tr>
			<td>RCT Hot Plate/Stirrer</td>
			<td>IKA (MY)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reactive Ion Etching tool</td>
			<td>Samco Inc. (JPN)</td>
			<td>RIE-10NR</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Invitrogen</td>
			<td>11875093</td>
			<td>culture medium for HCT116 cells</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>4019862</td>
			<td>Polydimethylsiloxane or in short, PDMS</td>
		</tr>
		<tr>
			<td>Trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td>
			<td>Sigma Aldrich</td>
			<td>448931-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T9284</td>
			<td>surfactant</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Thermofisher scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Cleaner</td>
			<td>Bransonic</td>
			<td>CPX2800</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-KUB 2</td>
			<td>KLOE</td>
			<td></td>
			<td>UV-LED light source, 365 nm wavelength, 35 mW/cm2 power density</td>
		</tr>
		<tr>
			<td>UV mask aligner</td>
			<td>SUSS Microtec Semiconductor (DE)</td>
			<td>MJB4</td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput platform for culture and 3d imaging of organoids

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).

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...

Watch this Scientific Journal Video about A High-Throughput Platform for Culture and 3D Imaging of Organoids at JoVE.com

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

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.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

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