Name: culturing, freezing, processing, and imaging of entire organoids and spheroids while still in a hydrogel
Uploaded: 2022-12-23T14:10:00
Description: Watch this Scientific Journal Video about Culturing, Freezing, Processing, and Imaging of Entire Organoids and Spheroids While Still in a Hydrogel at JoVE.com

We are grateful to Dale Mertes from the University of Chicago for the preparation of diagrams, to Dr. Mehmet Serif Aydin for his technical support at Istanbul Medipol University Research Institute for Health Sciences and Technologies, and to Dr. Rana Kazemi from Maltepe University for editing the manuscript.

1. Culturing organoids and spheroids
<ol>
	<li>Place the hydrogel on ice overnight (in a refrigerator or a cold room) to thaw.</li>
	<li>Place the commercially available multipurpose device (MD; see Table of Materials) in the incubator 1 day before the experiment (37 &#176;C, 5% CO2) to warm.</li>
	<li>Place sterile wide-ended pipette tips in the refrigerator at 4 &#176;C. 
	NOTE: Steps 1.1-1.3 are to be performed on Day 0, and steps 1.4-1.11 are to be performed on Day ...

The MD, complementing formulations and protocols described here, facilitates rapid and spontaneous 3D growth of organoids and spheroids in a more controlled environment and continues the experiment in the same conditions. The specimen stays in the same environment during the entire process, and nearly 100% of the 3D growing architectures remain intact in the container. This improves the homogeneity during the sequential experiments and allows for an extended culture period. In addition, the number of steps during organoi...

The future of cell research and therapy lies within 3D cell cultures1,2,3. Organoid and spheroid models close the gap between in vitro experiments and animal models by creating better models that mimic human body development, physiology, and diseases4,5,6,7,8,9. However, the reproducibility and repeatability of these models remain challenging. Furthermore, handling, harvesting, transferring, and centrifuging these structures with current technologies results in loss or damage of the organoids and spheroids in many conditions, significantly affecting the results.

Despite many protocols for histological staining, immunohistochemical staining, immunofluorescence labeling, and cryopreservation, there is no universal approach related to standardizing experimental conditions, handling, and processing these delicate structures without losing or damaging them. Current protocols are also incredibly long, alternating from a few days to several weeks, and include complex procedures with various reagents10,11,12,13,14. Additionally, harvesting, pipetting, centrifuging, and transferring 3D growing structures between cell culture vessels and cryovials cause changes in the positioning of the structures and mechanical forces, and ultimately affect the differentiation and maturation of the organoids and spheroids. It has been reported that tissue topology, positioning of the cells, and mechanical forces significantly impact cell differentiation and maturation6,15,16,17.

Therefore, it is desirable to improve the current conventional technologies to generate organoids and spheroids with stable quality. A method/device that will skip the centrifugation and other steps described above and provide the material in a single safe environment from the beginning to the end of the multiple processes would be beneficial to reach the most consistent and reliable data. Additionally, this will reduce time, labor, and cost constraints.
The multipurpose device (MD) described here provides a single safe environment for multiple processes of organoids and spheroids (Supplementary&#160;Figure 1). This device and complementing protocols eliminate the harvesting, pipetting, transferring, and centrifuging steps. The organoids and spheroids remain in their in vitro environment during the sequential processes. This environment mainly comprises natural or synthetic extracellular matrix components, like commercially available hydrogels. In other words, the methods described here allow a whole-mount sample of organoids/spheroids to be processed, examined, and frozen while still in a hydrogel drop.
The biocompatible device is resistant to temperatures between 60 &#176;C and -160 &#176;C, which makes it feasible to restore the organoids/steroids in a liquid nitrogen tank at -160 &#176;C or to prepare resin blocks for electron microscopy at 60 &#176;C. The niche in the device has been designed to define a limited space for 3D growing structures and stimulate the formation of spheroids or organoids based on the previous studies18,19,20,21,22,23. That part of the device is transparent and contains a specific plastic that provides high optical quality (refractive index: 1.43; abbe value: 58; thickness: 7.8 mil [0.0078 in or 198 &#181;m]). Both the niche and the surrounding &#39;side&#39; part cause autofluorescence. The transparent niche in the center has an 80 mm2 area, while the side part is 600 mm2. The depth of the container is 15 mm, and the thickness is 1.5 mm. These features, in addition to the size and design of the device, make it possible to make observations under different types of high-tech microscope and prepare the samples for electron microscopic examinations (Figure 2). The closing system of the device provides two positions, one sealed in the freezer and the other allowing gas flow in the incubator. CCK8 proliferation and cytotoxicity assays demonstrate similar effects on cells compared to the traditional cell culture dishes (Supplementary&#160;Figure 2). Trypan blue exclusion test demonstrates high cell viability (94%) during the cell culture in the MD (Figure 3).
The processes that can be performed for one sample in the single device comprise (1) culturing, (2) histological staining, (3) immunostaining, including immunohistochemical and immunofluorescence labeling, (4) freezing, (5) thawing, (6) examining under optical microscopes, such as brightfield, darkfield, fluorescence, confocal, and super-resolution microscopes, (7) coating and examining directly under a scanning electron microscope, or (8) preparing for transmission electron microscopy (Figure 2).
Different methodologies exist for histological staining, immunohistochemical labeling, or fluorescently labeling organoids and spheroids10,11,12,13,14,24,25. Harvesting them from the hydrogel is the first and principal step of the current technology. After this step, some methods allow whole-mount immuno-labeling. The harvested organoids are embedded in paraffin, sectioned, and labeled for staining and immunostaining in others. However, the sections may not present the whole sample and provide only limited data related to the 3D architecture of the structure. Furthermore, damage to these 3D structures and loss of antigenicity are well-known side effects of these technologies.
The complementing new protocols for microscopic examinations in this article allow an analysis of whole-mount samples still in a hydrogel. The protocols described here include two newly developed formulations: solution for immunohistochemistry (S-IHC) and solution for immunofluorescence labeling (S-IF). The methods with these solutions allow researchers to gain more accurate data, since there are no harmful effects of traditional workflows, such as centrifuging, pipetting, and transferring of the delicate structures. The protocol described here also eliminates the need for harvesting, blocking, clearing, and antigen retrieval steps, and shortens the whole procedure to 6-8 h. Furthermore, the methodology allows simultaneously adding one to three antibodies to the same S-IF. Therefore, it is possible to get the results on the same day even after the multiple labeling experiments, which is another advantage of the protocol described here; traditional whole-mount immunofluorescence labeling protocols typically take between 3 days and several weeks10,11,12,13,14.
Paraffin embedding, another harmful step that reduces antigenicity, is also omitted. The 3D structure remains in its in vitro environment from the beginning to the end of the microscopic examination. Since the 3D structure remains in its growing conditions, the protein expression and localization data mimic in vivo conditions better. More accurate results are expected, since the methodology eliminates the steps affecting the sample&#39;s antigen expression. Table 1&#160;and Table&#160;2&#160;demonstrate how these new protocols eliminate steps, save time and labor in the lab, and reduce costs and waste products compared to traditional workflows.
In addition to the crucial steps described above, another problem is providing a cryopreservation medium and method to preserve the 3D structure of the sample with higher cell viability rates26,27,28,29,30,31. Cryopreservation is essential to creating a stable model system and enabling the biobanking of organoids and spheroids32,33. Biobanking the entire original 3D structure will allow for a more faithful recapitulation of the natural state of health or disease. The key considerations are the convenience and reliability of cryopreservation and the thawing of organoids/spheroids. Post-thaw organoid recovery is very low in most current technologies, often less than 50%. However, recent studies have shown promising results with improved survival rates26,27,28,29. Lee et al. demonstrated that 78% of spheroid cells survived after cryopreservation when they used the University of Wisconsin solution containing 15% DMSO28. The cell survival ratio increased to 83% in the study of Arai et al.29. However, the results after cryopreservation are significantly affected since the 3D structures cannot maintain the same characteristics and quality. In addition, serum-free reagents are required for good manufacturing practice in pharmaceutical and diagnostic settings. Traditional workflows use a medium containing fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) for the slow-freezing method, both of which are associated with handicaps. FBS is an animal-derived product and can have batch variations. DMSO is a very successful cryoprotectant, but long-term exposure, especially during thawing, might cause cytotoxic effects30,31.
This article also describes the freezing/thawing methodology of entire organoids or spheroids while still in a hydrogel. Two formulas for freezing organoids and spheroids are used in the study: (1) 10% DMSO containing traditional freezing solution (FS) and (2) a serum- and DMSO-free cryopreservation medium. This cryopreservation medium contains extracellular matrix components, which are different from current formulas. The extracellular matrix comprises two main classes of macromolecules, proteoglycans, and fibrous proteins, which are essential for physical scaffolding for the cellular constituents, but also initiate processes required for tissue morphogenesis, differentiation, and homeostasis34,35,36,37,38,39,40. Collagens provide tensile strength, regulate cell adhesion, support chemotaxis and migration, and direct tissue development37. In addition, elastin fibers provide recoil to tissues that undergo repeated stretch38. A third fibrous protein, fibronectin, directs the organization of the interstitial extracellular matrix and has a crucial role in mediating cell attachment and functions as an extracellular mechano-regulator39. Du et al. have demonstrated the cryoprotective effect of chicken collagen hydrolysate on the natural actomyosin model system41. Their results suggest that collagen hydrolysate can inhibit ice crystal growth, reduce protein freeze-denaturation and oxidation similarly to commercial cryoprotectants, and provide a better gel structure after freeze-thaw cycles. Therefore, adding extracellular matrix components to the cryopreservation media provides a more secure and protective environment for the sample and supports the living structures to heal after freezing-thawing.
Additionally, the present study describes a straightforward protocol to label cytoplasmic membranes and nuclei of live organoids and spheroids while they are still in the hydrogel.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absolute Ethanol (EtOH)</td>
			<td>Merck</td>
			<td>8187602500</td>
			<td>Dilute in dH2O to make 30%, 50%, 70%, 80%, 90% and 96% solution and store at RT</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Merck</td>
			<td>8222512500</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Alexa fluor wheat germ agglutinin and Hoechst&#160; in Hank&#39;s balanced salt solution (HBSS) &#160;</td>
			<td>Invitrogen</td>
			<td>I34406</td>
			<td>Image-IT LIVE Plasma Membrane and Nuclear Labeling Kit, Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Alpha-1-Fetoprotein (AFP) Concentrated and Prediluted Polyclonal Antibody</td>
			<td>Biocare Medical</td>
			<td>CP 028 A</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-albumin antibody</td>
			<td>Abcam</td>
			<td>EPR20195</td>
			<td>Store at +4 &#176;C, Dilution: 1:50</td>
		</tr>
		<tr>
			<td>Anti-beta galactosidase antibody, Chicken polyclonal</td>
			<td>Abcam</td>
			<td>134435</td>
			<td>Store at +4 &#176;C, Dilution: 1:25</td>
		</tr>
		<tr>
			<td>Anti-cytokeratin 5</td>
			<td>Abcam</td>
			<td>53121</td>
			<td>Store at +4 &#176;C, Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Arginase-1 Concentrated and Prediluted Rabbit Monoclonal Antibody</td>
			<td>Biocare Medical</td>
			<td>ACI 3058 A, B</td>
			<td>Store at +4 &#176;C, Dilution: 1:50</td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>Sigma</td>
			<td>C1016-500G</td>
			<td>Dissolve in Karnovsky&#39;s fixative to make 2 mM CaCl2; store at RT</td>
		</tr>
		<tr>
			<td>Cell Counting Kit 8 (WST-8 / CCK8)</td>
			<td>Abcam</td>
			<td>ab228554</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 15 mL&#160;</td>
			<td>Nest</td>
			<td>601051</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 50 mL&#160;</td>
			<td>Nest</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II Microbiological Safety Cabinet Bio II Advance Plus</td>
			<td>Telstar</td>
			<td>EN12469</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Panasonic</td>
			<td>KM-CC17RU2</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper Grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>G100-Cu</td>
			<td>Ultra-thin sections put on the grids; 100 lines/inch square mesh</td>
		</tr>
		<tr>
			<td>Critical Point Dryer</td>
			<td>Leica</td>
			<td>EM CPD300</td>
			<td>For drying biological samples for SEM applications in absolute acetone</td>
		</tr>
		<tr>
			<td>DAB/AEC chromogen solution mixture &#160;</td>
			<td>Sigma Aldrich</td>
			<td>AEC101</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Diamond knife</td>
			<td>Diatome</td>
			<td>Ultra 45&#176;, 40-US</td>
			<td>Use for ultra-thin sections for TEM</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide for molecular biology</td>
			<td>Biofroxx</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Plastic Pasteur Pippettes</td>
			<td>Nest</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>41966-029</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Eosin Y Solution Alcoholic</td>
			<td>Bright Slide</td>
			<td>2.BS01-105-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Epon resin&#160;</td>
			<td>Sigma</td>
			<td>45359-1EA-F</td>
			<td>Epoxy Embedding Medium kit, Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum with Additive Fortifier</td>
			<td>Pan Biotech</td>
			<td>P30-3304</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Freezing Solution (FS)</td>
			<td>Cellorama</td>
			<td>CellO-F</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Glass knife maker</td>
			<td>Leica</td>
			<td>EM KMR3</td>
			<td>For make glass knives in 8 mm thickness</td>
		</tr>
		<tr>
			<td>Glass knife strips (Size 8 mm x 25.4 mm x 400 mm)</td>
			<td>Leica</td>
			<td>7890-08</td>
			<td>Use for ultra- or semi-thin sections for TEM</td>
		</tr>
		<tr>
			<td>Glutaraldehyde Aqueous Solution, EM grade, 25%&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>16210</td>
			<td>Dilute in dH2O to make 2.5% solution and store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Glycerol solution</td>
			<td>Sigma Aldrich</td>
			<td>56-81-5</td>
			<td>Store at -20 C, Dilution :1:100</td>
		</tr>
		<tr>
			<td>Goat anti-chicken IgY (H+L) Secondary Antibody,Alexa, 647</td>
			<td>Invitrogen</td>
			<td>A32933</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight, 488</td>
			<td>Invitrogen</td>
			<td>35502</td>
			<td>Store at +4 &#176;C,&#160; Dilution :1:50</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight, 550</td>
			<td>Invitrogen</td>
			<td>84540</td>
			<td>Store at +4 &#176;C,&#160; Dilution :1:50</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight, 488</td>
			<td>Invitrogen</td>
			<td>35552</td>
			<td>Store at +4 &#176;C,&#160; Dilution :1:50</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight, 550</td>
			<td>Invitrogen</td>
			<td>84541</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Hematoxylin Harris&#160;</td>
			<td>Bright Slide</td>
			<td>2.BS01-104-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>HepG2 cells</td>
			<td>ATCC</td>
			<td>HB-8065</td>
			<td>Store in nitrogen tank</td>
		</tr>
		<tr>
			<td>Human/Rat OV-6 Antibody Monoclonal Mouse IgG1 Clone # OV-6</td>
			<td>R&#38;D Systems</td>
			<td>MAB2020</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Hydrogel</td>
			<td>Corning</td>
			<td>354248</td>
			<td>Matrigel, Basement Membrane Matrix High Concentration (HC), LDEV-free, 10 mL, Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Hydrogel</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Matrigel, Basement Membrane Matrix, LDEV-free, 10 mL, Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Hydrogel</td>
			<td>ThermoFischer Scientific</td>
			<td>A1413201</td>
			<td>Geltrex, LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
		</tr>
		<tr>
			<td>Hydrogel</td>
			<td>Biotechne, R&#38;D Systems</td>
			<td>BME001-01</td>
			<td>Cultrex Ultramatrix RGF BME, Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Karnovsky&#39;s fixative</td>
			<td></td>
			<td></td>
			<td>%2 PFA, %2.5 Glutaraldehyde in 0.15 M Cacodylate Buffer, 2 mM CaCl2; prepare fresh; use for TEM &#38; SEM samples</td>
		</tr>
		<tr>
			<td>L-Aspartic acid</td>
			<td>Sigma</td>
			<td>11189-100G</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Lead aspartate solution</td>
			<td></td>
			<td></td>
			<td>Dissolve 40 mg aspartic acid in 10 mL ddH2O and add 66 mg lead nitrate. Solution stabilize at 60 &#176;C and adjust pH to 5; prepare fresh</td>
		</tr>
		<tr>
			<td>Lead nitrate</td>
			<td>Electron Microscopy Sciences</td>
			<td>17900</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Leica Confocal Microscope</td>
			<td>Leica</td>
			<td>DMi8</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 700 Laser Scanning Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Biotek Synergy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose Device (MD)</td>
			<td>Cellorama</td>
			<td>CellO-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear-DNA stain</td>
			<td>Invitrogen</td>
			<td>H3569</td>
			<td>Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg/mL Solution in Water, Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Nuclear-DNA stain</td>
			<td>ThermoFischer Scientific</td>
			<td>62248</td>
			<td>DAPI solution, Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Osmium Tetroxide (OsO4) ,4%</td>
			<td>Electron Microscopy Sciences</td>
			<td>19190</td>
			<td>Dilute in dH2O to make 2% solution; store at +4 &#176;C and in airtight container; protect light</td>
		</tr>
		<tr>
			<td>Ov6 antibody</td>
			<td>R&#38;D systems</td>
			<td>MAB2020</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA) solution, 4%&#160;</td>
			<td>Sigma</td>
			<td>1.04005.1000</td>
			<td>Dissolve 4% PFA in dH2O and boil, cool and aliquot; store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4% in PBS, 1 L</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS), tablets</td>
			<td>MP Biomedicals, LLC</td>
			<td>2810305</td>
			<td></td>
		</tr>
		<tr>
			<td>Post-fixative solution</td>
			<td></td>
			<td></td>
			<td>%2 OsO4, %2.5 Potassium Ferrocyanide in dH2O; prepare fresh</td>
		</tr>
		<tr>
			<td>Potassium Ferrocyanide aqueous solution, 5%&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26603-01</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Primovert - Inverted Bright Field Microscope - ZEISS</td>
			<td>Zeiss</td>
			<td>Item no.: 491206-0001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom microcentrifuge tubes, 2 mL</td>
			<td>Nest</td>
			<td>620611</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscopy with STEM attachment</td>
			<td>Zeiss</td>
			<td>GeminiSEM 500</td>
			<td>We use Inlens Secondary Electron (SE) detector at 2-3 kV for scanning electron micrographs and aSTEM detector at 30 kV for transmission electron micrographs.</td>
		</tr>
		<tr>
			<td>SensiTek HRP Anti-Polyvalent Lab Pack</td>
			<td>ScyTek Laboratories</td>
			<td>SHP125</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Sodium Cacodylate Buffer, 0.4 M, pH 7.2</td>
			<td>Electron Microscopy Sciences</td>
			<td>11655</td>
			<td>Dilute in dH2O to make 0.2 M and store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Sodium/Potassium ATPase alpha 1 antibody [M7-PB-E9]</td>
			<td>GeneTex</td>
			<td>GTX22871</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Solution for Immunofluorescence Labeling (S-IF)</td>
			<td>Cellorama</td>
			<td>CellO-IF</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Solution for Immunohistochemistry (S-IHC)</td>
			<td>Cellorama</td>
			<td>CellO-P</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Specimen trimming device</td>
			<td>Leica</td>
			<td>EM TRIM2</td>
			<td>For prepare epon sample block to ultramicrotome</td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Leica</td>
			<td>EM ACE200</td>
			<td>Coat the SEM samples with 6 nm gold/palladium for 90 s</td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide (TCH)</td>
			<td>Sigma</td>
			<td>223220-5G</td>
			<td>Dilute in dH2O to make 0.5% solution and filter with 0.22 &#181;m membrane filter; store at RT; prepare fresh</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra gel super glue</td>
			<td>Pattex</td>
			<td>PSG2C</td>
			<td>For glue polymerized epon block with sample to holder epon block</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td>EM UC7</td>
			<td>For prepare high-quality ultra- or semi-thin sections for transmission electron microscopy (TEM)</td>
		</tr>
		<tr>
			<td>Universal Pipette Tips, 10 &#181;L</td>
			<td>Nest</td>
			<td>171215-1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Pipette Tips, 1000 &#181;L</td>
			<td>Isolab</td>
			<td>L-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Pipette Tips, 200 &#181;L&#160;</td>
			<td>Nest</td>
			<td>110919HA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td>Dilute in dH2O to make 2% solution and filter with 0.22 &#181;m membrane filter; keep tightly closed container store at RT</td>
		</tr>
	</tbody>
</table>

culturing, freezing, processing, and imaging of entire organoids and spheroids while still in a hydrogel

Organoids and spheroids, three-dimensional growing structures in cell culture labs, are becoming increasingly recognized as superior models compared to two-dimensional culture models, since they mimic the human body better and have advantages over animal studies. However, these studies commonly face problems with reproducibility and consistency. During the long experimental processes - with transfers of organoids and spheroids between different cell culture vessels, pipetting, and centrifuging - these susceptible and fragile 3D growing structures are often damaged or lost. Ultimately, the results are significantly affected, since the 3D structures cannot maintain the same characteristics and quality. The methods described here minimize these stressful steps and ensure a safe and consistent environment for organoids and spheroids throughout the processing sequence while they are still in a hydrogel in a multipurpose device. The researchers can grow, freeze, thaw, process, stain, label, and then examine the structure of organoids or spheroids under various high-tech instruments, from confocal to electron microscopes, using a single multipurpose device. This technology improves the studies&#39; reproducibility, reliability, and validity, while maintaining a stable and protective environment for the 3D growing structures during processing. In addition, eliminating stressful steps minimizes handling errors, reduces time taken, and decreases the risk of contamination.

The present article represents a multipurpose device (MD) and complementing methodologies for culturing, freezing, thawing, histological staining, immunohistochemical staining, immunofluorescence labeling, coating, and processing of entire organoids or spheroids while still in a hydrogel in a single uniquely designed environment. The current study was designed to prepare HepG2 liver cancer spheroids in 35 hydrogel drops in 35 MDs. Experiments were conducted in triplicate to ensure accuracy. Additionally, lung organoids i...

Watch this Scientific Journal Video about Culturing, Freezing, Processing, and Imaging of Entire Organoids and Spheroids While Still in a Hydrogel at JoVE.com

Culturing, Freezing, Processing, and Imaging of Entire Organoids and Spheroids While Still in a Hydrogel

The present study describes methodologies for culturing, freezing, thawing, processing, staining, labeling, and examining entire spheroids and organoids under various microscopes, while they remain intact in a hydrogel within a multipurpose device.

Organoids and spheroids, three-dimensional growing structures in cell culture labs, are becoming increasingly recognized as superior models compared to ...

Organoids and spheroids, being very fragile, get seriously damaged while handling. Our protocol ensures these 3D growing structures remain intact during various processes, thereby increasing accuracy, reproducibility, reliability, and repeatability. The researcher can culture, freeze, thaw, process, stain, label, and examine entire organoids and spheroids under various microscopes while they remain intact in a hydrogel within a single multi-purpose device.It enables creation of a disease model and tracking of sequential steps in one multi-purpose device. The technology brings more accuracy when examining biomarkers for diagnosis and prognosis of diseases. This method can be applied to any 3D growing system, including organoids, spheroids, individual cells, or living organisms.To begin the organoid or spheroid culture, place the properly thawed hydrogel on ice inside a laminar flow hood for 15 minutes. Also, keep the tube containing a pellet of the hepatocellular carcinoma, or HEPG2 cells, on ice. Next, plate 30 to 35 microliters of 100%hydrogel within the niche of the pre-warmed multipurpose device to create a gel drop, place 10, 000 HEPG2 cells in the center of the top of the hydrogel drop, and incubate the setup at 37 degrees Celsius for 15 minutes.After incubation, cover the hydrogel drop with 200 microliters of Dulbecco's Modified Eagle Medium, or DMEM, containing 10%fetal bovine serum, or FBS. Place the lid on the device properly, allowing gas flow, before placing it in the incubator. Every other day, feed the cells with 200 microliters of DMEM containing 10%FBS.Check the growth of the spheroids under an inverted microscope. Before starting immunofluorescence labeling of the organoids, warm up the required media and solutions to 37 degrees Celsius. Aspirate the cell culture medium surrounding the hydrogel drop before adding 200 microliters of 4%paraformaldehyde pre-warmed to 37 degrees Celsius and fix it by placing it in an incubator at 37 degrees Celsius for 15 to 30 minutes.Then, aspirate the fixative and wash the solution for immunofluorescence labeling, or SIF, pre-warmed to 37 degrees Celsius three times for 10 minutes each. Add distilled water to the area surrounding the niche to provide humidity before proceeding with the next steps. Aspirate the SIF surrounding the hydrogel drop and add 100 microliters of the pre-warmed primary antibody solution in SIF to the hydrogel before incubating it at 37 degree Celsius for 30 to 60 minutes.Then, aspirate the primary antibody solution and wash with SIF pre-warmed to 37 degrees Celsius three times for 10 minutes each. Following the wash, aspirate the remaining SIF and add 100 microliters of the pre-warmed secondary antibody solution in SIF to the hydrogel. Incubate the hydrogel at 37 degrees Celsius in the dark for 30 to 60 minutes.After the incubation, aspirate the secondary antibody solution and wash the hydrogel drop with PBS at 37 degrees Celsius three times in the dark for 10 minutes each. Next, aspirate the residual PBS and incubate the hydrogel with 100 microliters of nuclear DNA stain containing mounting medium or glycerol at 37 degrees Celsius in the dark. Fill the niche with glycerol to avoid drying before tightly closing the multi-purpose device containing the hydrogel.To examine the organoids by confocal microscopy, set the microscope parameters following the instructions given in the manuscript. To freeze the organoids, aspirate the cell culture media, add 200 microliters of the pre-warmed freezing solution, and incubate the sample at 37 degrees Celsius for one hour. Close the lid of the device tightly before placing it inside a foam box.Place this foam box in another foam box and close both foam boxes tightly. Keep the box at minus 20 degrees Celsius for two hours, then leave it overnight in a minus 80 degree Celsius freezer. To store the organoids for longer than six months, remove the multi-purpose device containing the organoids from the boxes and transfer it to the liquid nitrogen tank.To thaw the organoids, take out the multi-purpose device containing the sample from either the freezer or the liquid nitrogen tank and place it directly into an incubator at 37 degrees Celsius. Once thawed, gently aspirate the medium and freezing solution mixture before adding fresh medium and proceeding to image the whole-mount organoid hydrogel. In live imaging, the growing organoids inside the hydrogel became visible three to five days after inserting the cell pellet, especially at the periphery of the hydrogel dome.The time series of images at different levels of a hydrogel containing spheroids under an inverted phase contrast microscope is shown here. Confocal microscopy images of the whole-mount living organoids after live-staining the cell membrane and the nucleus showed similar labeling density in the periphery and the center. Further, the immunofluorescence labeling of the whole-mount steroids could be successfully performed with one antibody, two antibodies, as well as three antibodies, eliminating any need for additional treatment steps.The representative analysis demonstrates two immunofluorescent-labeled airway organoids in a device. SEM images of the entire spheroids in the multi-purpose device and 10 images of sectioned steroids indicated well-preserved 3D architecture of spheroids, cytoplasmic organelles, and other ultra-structural cellular features, demonstrating the effectiveness of the described protocol. Irregularity at the boundaries of the hydrogel dome was evident after freezing the samples.However, the size and roundness of the spheroids remained similar. Cell migration on the culture surface was observed after thawing the frozen samples. The technique is very simple.However, the researcher should be very gentle while aspirating or adding any liquids surrounding the hydrogel drop to prevent the damaging the sample. The researcher can look at the same sample in a single device using a high-tech microscope and conduct a correlative study.

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