Name: Culturing, Freezing, Processing, and Imaging of Entire Organoids and Spheroids While Still in a Hydrogel (Video) | JoVE Journal
Uploaded: 2022-12-23T14:10:00.000+00:00
Duration: 8 min 7 s
Description: Learn a complete protocol for culturing, freezing, and imaging organoids and spheroids in hydrogel using HEPG2 cells. This method preserves 3D structure integrity for reliable disease modeling and biomarker analysis.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

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 1.</li>
	<li>Place the hydrogel on ice in a laminar flow hood for 15 min.
	<ol>
		<li>Optional: Dilute the hydrogel in cold cell culture medium according to the manufacturer&#39;s recommendation.</li>
	</ol>
	</li>
	<li>Place the tube containing a pellet of HepG2 cells (commercially obtained hepatocellular carcinoma cell line; see Table of Materials) on ice.</li>
	<li>Plate 30-35 &#181;L of 100% hydrogel within the niche of the pre-warmed device to create a gel drop.</li>
	<li>Place 10,000 HepG2 cells in the middle of the top of each hydrogel drop (Figure 2), and incubate for 15 min at 37 &#176;C.</li>
	<li>Cover the hydrogel drop with 200 &#181;L of Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum.</li>
	<li>Cover the lid of the MD in the correct position to allow gas flow, and place the device in the incubator.</li>
	<li>Feed the cells with 200 &#181;L of DMEM with 10% FBS every other day.</li>
	<li>Check the growth of the spheroids under an inverted microscope (Figure 3). Spheroid formation starts after the 3rd day. Video 1 shows the location of spheroids at different levels in a hydrogel dome. 
	NOTES: It is strongly recommended to gently aspirate the liquid surrounding the hydrogel and slowly add the new liquid to the environment to prevent damaging the hydrogel drops.</li>
</ol>
2. Hematoxylin and Eosin staining of whole-mount organoids/spheroids in a hydrogel
<ol>
	<li>Warm the fixative (4% paraformaldehyde; PFA), PBS, and Hematoxylin (see Table of Materials) to 37 &#176;C.</li>
	<li>Aspirate the medium surrounding the hydrogel drop with a pipette, add 100-200 &#181;L of 4% PFA to fix, and incubate for 15-20 min at 37 &#176;C.</li>
	<li>Aspirate the 4% PFA and add 200 &#181;L of PBS to wash 3x for 5 min each at 37 &#176;C. Then, aspirate the PBS and incubate with 200 &#181;L of Hematoxylin solution for 15-20 min at 37 &#176;C.</li>
	<li>Aspirate the Hematoxylin and add 200 &#181;L of dH2O to wash 3x for 10 min each at 37 &#176;C. Aspirate the dH2O and incubate with 200 &#181;L of ethanol for 5-10 min at 37 &#176;C.</li>
	<li>Aspirate the ethanol and incubate with 200 &#181;L of Eosin (see Table of Materials) for 10 min at 37 &#176;C. Aspirate the Eosin and add 200 &#181;L of dH2O to wash for 5 min at room temperature (RT).</li>
	<li>Aspirate the dH2O and add 100 &#181;L of glycerol to cover the hydrogel drop as the mounting medium.</li>
	<li>Optional: Mount the niche with a coverslip. This step can be omitted to avoid squeezing organoids/spheroids of considerable size.</li>
	<li>Close the lid of MD firmly to avoid drying until examination. The sample is stable for examination for at least 6 months.</li>
</ol>
3. Immunohistochemistry of whole-mount organoids/spheroids in a hydrogel
<ol>
	<li>Warm the commercially obtained solution for immunohistochemistry (S-IHC; see Table of Materials) to 37 &#176;C.</li>
	<li>Incubate the organoids or spheroids in the hydrogel with 3% hydrogen peroxide (H2O2) in 200 &#181;L of dH2O for 5 min at 37 &#176;C.</li>
	<li>Aspirate the hydrogen peroxide solution and wash in dH2O for 5 min at 37 &#176;C. Aspirate the dH2O and incubate twice with 100 &#181;L of S-IHC at 37 &#176;C for 10 min each.</li>
	<li>Aspirate the S-IHC and incubate with 100 &#181;L of primary antibody (see Table of Materials) diluted in S-IHC for 1-2 h at 37 &#176;C (following the manufacturer&#39;s recommendations regarding working dilution).</li>
	<li>Aspirate the primary antibody solution and incubate with 100 &#181;L of S-IHC 3x for 5 min each at 37 &#176;C.</li>
	<li>Aspirate 100 &#181;L of S-IHC and incubate with a biotinylated secondary antibody (see Table of Materials) for 10 min at 37 &#176;C.</li>
	<li>Aspirate the secondary antibody solution and incubate with 100 &#181;L of S-IHC 3x for 5 min each at 37 &#176;C. Aspirate the S-IHC and incubate with 100 &#181;L of horseradish peroxidase (HRP) labeled streptavidin (see Table of Materials) for 10 min at 37 &#176;C.</li>
	<li>Aspirate the HRP labeled streptavidin and incubate with 100 &#181;L of S-IHC 3x for 5 min each at 37 &#176;C.</li>
	<li>Aspirate the S-IHC and incubate with 100 &#181;L of DAB/AEC chromogen solution mixture (see Table of Materials) for 5-10 min at 37 &#176;C.</li>
	<li>Monitor the intensity of the staining under a light microscope.</li>
	<li>Wash with dH2O 3x for 2 min each.</li>
	<li>Optional: Incubate with 100 &#181;L of Hematoxylin (see Table of Materials) for nuclear counterstaining for 5 min at 37 &#176;C.</li>
	<li>Aspirate the Hematoxylin and wash in dH2O for 5 min.</li>
	<li>Aspirate the dH2O and cover the hydrogel drop with 100 &#181;L of glycerol as the mounting media.</li>
	<li>Close the lid of the MD firmly until microscopic examination. 
	NOTES: Antigen retrieval and protein blocking steps are omitted in this protocol since S-IHC eliminates these steps.</li>
</ol>
4. Immunofluorescence labeling of whole-mount organoids/spheroids in a hydrogel
<ol>
	<li>Warm up the following materials to 37 &#176;C: 4% PFA, PBS, S-IF, primary antibody solution in S-IF, secondary antibody solution in S-IF, nuclear stain, and glycerol (see Table of Materials).</li>
	<li>Aspirate the cell culture medium and fix with 200 &#181;L of 4% PFA for 15-30 min at 37 &#176;C. Aspirate the fixative and wash in S-IF 3x for 10 min each at 37 &#176;C.</li>
	<li>Add dH2O to the side surrounding the niche to provide humidity during the following steps.</li>
	<li>Aspirate the S-IF surrounding the hydrogel drop and incubate the hydrogel drop with 100 &#181;L of primary antibody solution (see Table of Materials) in S-IF for 30-60 min at 37 &#176;C.</li>
	<li>Aspirate the primary antibody solution and wash in S-IF 3x for 10 min each at 37 &#176;C.</li>
	<li>Aspirate the S-IF and incubate with 100 &#181;L of secondary antibody solution (see Table of Materials) in S-IF for 30-60 min at 37 &#176;C in the dark.</li>
	<li>Aspirate the secondary antibody solution and wash with PBS 3x for 10 min each at 37 &#176;C in the dark.</li>
	<li>Aspirate the PBS and incubate with 100 &#181;L of nuclear-DNA stain containing mounting medium or glycerol at 37 &#176;C in the dark.</li>
	<li>Fill the niche with glycerol to avoid drying.</li>
	<li>Optional: Cover the niche with a coverslip. This step can be omitted to avoid squeezing the organoids/spheroids.</li>
	<li>Tightly close the MD. Samples in MDs can be stored at 4 &#176;C in the dark for at least 6 months with minimal loss of fluorescence.</li>
	<li>Use the following settings for confocal microscopic examination: set the pinhole value of all channels to 20.1, hold the gain master constant at 550 for 488 nm, 485 for 550 nm, and 450 for 594 nm, keep the laser power constant for all experiments, and the lowest percentage at 2.0. 
	NOTES: Primary antibodies: Anti-Na-K ATPase (1:100), Anti-Arginase (1:50), Anti-Albumin (1:50), Anti-Beta-galactosidase (1:25), Antimitochondrial antibody (1:100), Anti-Golgi Antibody (1:50), Anti-Cytokeratin 5 (1:100), Ov6 antibody (1:100). Secondary antibodies: Goat anti-Rabbit IgG (H+L)- 488, Goat anti-Rabbit IgG (H+L)-550, Goat anti-Mouse IgG (H+L)-488, Goat anti-Mouse IgG (H+L)-550, Goat anti-Chicken IgY(H+L)-647. The dilution for all secondary antibodies is 1:100. Additionally, FITC-Phalloidin (1:100), a conjugated antibody, is also used (see Table of Materials).</li>
</ol>
5. Plasma membrane and nucleus labeling of living organoids and spheroids in a hydrogel
<ol>
	<li>Prepare labeling solution containing Alexa fluor wheat germ agglutinin (5.0 &#181;g/mL) and Hoechst (2 &#181;M) in Hank&#39;s balanced salt solution (HBSS), according to the manufacturer&#39;s recommendations (see Table of Materials), and warm up to 37 &#176;C.</li>
	<li>Aspirate the cell culture medium and add 100 &#181;L of labeling solution to cover the hydrogel drop. Incubate for 15-30 min at 37 &#176;C.</li>
	<li>Remove the labeling solution and wash twice in PBS 2x for 10 min each at 37 &#176;C. Optional: Fix with 200 &#181;L of 4% formaldehyde for 15 min at 37 &#730;C.</li>
	<li>Cover the hydrogel drop with glycerol as a mounting medium. Optional: Mount the niche with a cover glass.</li>
	<li>Close the lid of the MD tightly and keep it refrigerated in the dark until fluorescence/confocal microscopic examination. It is stable for at least 6 months.</li>
</ol>
6. Freezing and thawing of whole-mount organoids/spheroids in a hydrogel
<ol>
	<li>Freeze the organoids following the steps below.
	<ol>
		<li>Warm up the commercially obtained freezing solution (FS; see Table of Materials) to 37 &#176;C.</li>
		<li>Aspirate the cell culture media surrounding the hydrogel dome gently.</li>
		<li>Add 200 &#181;L of FS gently. Incubate the sample with FS at 37 &#176;C for 1 h.</li>
		<li>Close the device&#39;s lid tightly and place it into a foam box. Place this foam box in another foam box, as shown in Supplementary Figure 3. Close both foam boxes tightly. Two foam boxes inside each other provide a temperature cooling gradient range of 1 to 2&#176; C/min of the sample in a -20 &#176;C freezer.</li>
		<li>Place the box at -20 &#176;C for 2 h. Transfer the box to a -80 &#176;C freezer and leave it overnight.</li>
		<li>Take out the sample from the boxes. The sample in the MD can be kept in the -80 &#176;C freezer for 6 months.</li>
		<li>Transfer the MD that contains the sample to the liquid nitrogen tank for longer-term storage.</li>
	</ol>
	</li>
	<li>Thaw the organoids following the steps below.
	<ol>
		<li>Take out the MD containing the sample from the freezer/nitrogen tank and place it directly into an incubator at 37 &#176;C. Incubate for 1 h at 37 &#176;C.</li>
		<li>Add 200 &#181;L of warm culture medium to the niche (the ratio of FS to cell culture medium is 1:1) and incubate for 30 min at 37 &#176;C.</li>
		<li>Add more warm culture medium to the niche (the ratio of FS to cell culture medium is 1:2) and incubate for 30 min at 37 &#176;C.</li>
		<li>Aspirate the medium and FS mixture gently. Proceed to scanning electron microscopy (step 7) and transmission electron microscopy (step 8) imaging of the whole-mount organoids/spheroids in the hydrogel.</li>
	</ol>
	</li>
</ol>
7. Scanning electron microscopy of whole-mount organoids/spheroids
<ol>
	<li>Warm up Karnovsky&#39;s fixative and post-fixative solution (see Table of Materials) to room temperature (RT).</li>
	<li>Aspirate the cell culture medium surrounding the hydrogel in the MD. Place the sample in the MD on ice in the laminar flow hood for 15 min.</li>
	<li>Aspirate the liquified hydrogel surrounding the organoids/spheroids very gently. A limited amount of matrigel can stay in the niche to avoid damaging and losing the sample.</li>
	<li>Fix with Karnovsky&#39;s fixative (2% PFA, 2.5% glutaraldehyde in 0.15 M Cacodylate buffer, and 2 mM CaCl2; see Table of Materials) at RT for 1 h.</li>
	<li>Aspirate the fixative gently and wash with distilled water (dH2O) 3x for 15 min each.</li>
	<li>Aspirate the dH2O gently and fix with post-fixative solution (1% aqueous osmium tetroxide [OsO4]; see Table of Materials) at RT for 1 h.</li>
	<li>Aspirate the post-fixative gently and wash with distilled water (dH2O) 3x for 15 min each.</li>
	<li>Dehydrate in a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 96%, 100%), mixture of ethanol/acetone (1:1; 1:2), and absolute acetone at RT for 15 min each.</li>
	<li>Dry with a critical point dryer.</li>
	<li>Coat the specimen in the device with 6 nm gold/palladium using a sputter coater (see Table of Materials) for 90 s.</li>
	<li>Observe under a scanning electron microscope with an in-lens secondary electron detector at 2-3 kV in vacuum mode (5 x 10-6 mA). Take images with a working distance of 8.1-8.2 mm and at 675x, 1050x, and 1570x magnifications. 
	NOTES: All steps are performed in the MD. Use 200-250 &#181;L of solution for each step.</li>
</ol>
8. Transmission electron microscopy of whole-mount organoids/spheroids in a hydrogel
<ol>
	<li>Warm up Karnovsky&#39;s fixative to 37 &#176;C. Aspirate the cell culture medium surrounding the hydrogel in the MD.</li>
	<li>Fix the sample with Karnovsky&#39;s fixative at RT for 1 h. Wash with dH2O 3x for 15 min each.</li>
	<li>Post-fix with 2% aqueous OsO4&#160;and 2.5% potassium ferrocyanide at RT for 45 min. Wash with dH2O 3x for 10 min each.</li>
	<li>Incubate in 0.5% Thiocarbohydrazide (TCH; see Table of Materials) at RT for 30 min. Wash with dH2O 3x for 10 min each.</li>
	<li>Incubate in 2% aqueous OsO4&#160;at RT for 30 min. Wash with dH2O 3x for 10 min each.</li>
	<li>Incubate in 2% uranyl acetate solution (see Table of Materials) at RT for 1 h. 
	NOTE: In this step, spheroids can be kept at 4 &#176;C overnight.</li>
	<li>Incubate in lead aspartate solution (see Table of Materials) at 60 &#176;C for 45 min. Wash with dH2O 3x for 10 min each.</li>
	<li>Dehydrate in a graded series of ethanol (50%, 70%, 90%, 100% [x2]) and absolute acetone at RT for 15 min each.</li>
	<li>Treat with a mixture of (1:1; 1:2) acetone/Epon resin and pure Epon resin (see Table of Materials) at RT for 2 h each.</li>
	<li>Polymerize the resin at 60 &#176;C overnight (minimum of 16 h). After the polymerization, remove the resin block from the MD, as shown in Supplementary Figure 4.</li>
	<li>Attach the resin block to a bigger one with a resin glue. Trim the block and reach the location of organoids or spheroids.</li>
	<li>Get semi-thin (1,000 nm) and ultra-thin sections (60 nm) using an ultramicrotome (see Table of Materials).</li>
	<li>Place the ultra-thin sections on 100 mesh copper grids and observe under a scanning electron microscope with a STEM detector at an accelerating voltage of 30 kV. Capture images with a working distance of 44 mm and at 2580x, 5020x, and 6060x magnifications. 
	NOTES: Use 200-250 &#181;L of solution for each step. Steps 8.1-8.10 are performed in the MD.</li>
</ol>

Ranan Gulhan Aktas owns MD, S-IHC, S-IF, and FS patent applications. Olgu Enis Tok was involved in the development of these products. Olgu Enis Tok and Gamze Demirel are R&amp;D team members of the company named Cellorama. Yusuf Mustafa Saatci, Zeynep Akbulut, and Ozgecan Kayalar do not have any conflicts of interest to declare.

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><tbody><tr><td>Absolute Ethanol (EtOH)</td><td>Merck</td><td>8187602500</td><td>Dilute in dH&lt;sub&gt;2&lt;/sub&gt;O 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&amp;nbsp; in Hank&#039;s balanced salt solution (HBSS) &amp;nbsp;</td><td>Invitrogen</td><td>I34406</td><td>Image-IT LIVE Plasma Membrane and Nuclear Labeling Kit, Store at -20 &amp;deg;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 &amp;deg;C</td></tr><tr><td>Anti-albumin antibody</td><td>Abcam</td><td>EPR20195</td><td>Store at +4 &amp;deg;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 &amp;deg;C, Dilution: 1:25</td></tr><tr><td>Anti-cytokeratin 5</td><td>Abcam</td><td>53121</td><td>Store at +4 &amp;deg;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 &amp;deg;C, Dilution: 1:50</td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>C1016-500G</td><td>Dissolve in Karnovsky&#039;s fixative to make 2 mM CaCl&lt;sub&gt;2&lt;/sub&gt;; 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&amp;nbsp;</td><td>Nest</td><td>601051</td><td></td></tr><tr><td>Centrifuge tubes, 50 mL&amp;nbsp;</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>CO&lt;sub&gt;2&lt;/sub&gt; 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 &amp;nbsp;</td><td>Sigma Aldrich</td><td>AEC101</td><td>Store at +4 &amp;deg;C</td></tr><tr><td>Diamond knife</td><td>Diatome</td><td>Ultra 45&amp;deg;, 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&#039;s Modified Eagle Medium</td><td>Gibco</td><td>41966-029</td><td>Store at +4 &amp;deg;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&amp;nbsp;</td><td>Sigma</td><td>45359-1EA-F</td><td>Epoxy Embedding Medium kit, Store at +4 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum with Additive Fortifier</td><td>Pan Biotech</td><td>P30-3304</td><td>Store at +4 &amp;deg;C</td></tr><tr><td>Freezing Solution (FS)</td><td>Cellorama</td><td>CellO-F</td><td>Store at +4 &amp;deg;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%&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>16210</td><td>Dilute in dH&lt;sub&gt;2&lt;/sub&gt;O to make 2.5% solution and store at +4 &amp;deg;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 &amp;deg;C,&amp;nbsp; 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 &amp;deg;C,&amp;nbsp; 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 &amp;deg;C,&amp;nbsp; 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 &amp;deg;C</td></tr><tr><td>Hematoxylin Harris&amp;nbsp;</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&amp;amp;D Systems</td><td>MAB2020</td><td>Store at -20 &amp;deg;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 &amp;deg;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 &amp;deg;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&amp;amp;D Systems</td><td>BME001-01</td><td>Cultrex Ultramatrix RGF BME, Store at -20 &amp;deg;C</td></tr><tr><td>Karnovsky&#039;s fixative</td><td></td><td></td><td>%2 PFA, %2.5 Glutaraldehyde in 0.15 M Cacodylate Buffer, 2 mM CaCl&lt;sub&gt;2&lt;/sub&gt;; prepare fresh; use for TEM &amp;amp; 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 ddH&lt;sub&gt;2&lt;/sub&gt;O and add 66 mg lead nitrate. Solution stabilize at 60 &amp;deg;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 &amp;deg;C</td></tr><tr><td>Nuclear-DNA stain</td><td>ThermoFischer Scientific</td><td>62248</td><td>DAPI solution, Store at +4 &amp;deg;C</td></tr><tr><td>Osmium Tetroxide (OsO&lt;sub&gt;4&lt;/sub&gt;) ,4%</td><td>Electron Microscopy Sciences</td><td>19190</td><td>Dilute in dH&lt;sub&gt;2&lt;/sub&gt;O to make 2% solution; store at +4 &amp;deg;C and in airtight container; protect light</td></tr><tr><td>Ov6 antibody</td><td>R&amp;amp;D systems</td><td>MAB2020</td><td>Store at +4 &amp;deg;C</td></tr><tr><td>Paraformaldehyde (PFA) solution, 4%&amp;nbsp;</td><td>Sigma</td><td>1.04005.1000</td><td>Dissolve 4% PFA in dH&lt;sub&gt;2&lt;/sub&gt;O and boil, cool and aliquot; store at -20 &amp;deg;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 &amp;deg;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 OsO&lt;sub&gt;4&lt;/sub&gt;, %2.5 Potassium Ferrocyanide in dH&lt;sub&gt;2&lt;/sub&gt;O; prepare fresh</td></tr><tr><td>Potassium Ferrocyanide aqueous solution, 5%&amp;nbsp;</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 &amp;deg;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 dH&lt;sub&gt;2&lt;/sub&gt;O to make 0.2 M and store at +4 &amp;deg;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 &amp;deg;C</td></tr><tr><td>Solution for Immunofluorescence Labeling (S-IF)</td><td>Cellorama</td><td>CellO-IF</td><td>Store at +4 &amp;deg;C</td></tr><tr><td>Solution for Immunohistochemistry (S-IHC)</td><td>Cellorama</td><td>CellO-P</td><td>Store at +4 &amp;deg;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 dH&lt;sub&gt;2&lt;/sub&gt;O to make 0.5% solution and filter with 0.22 &amp;micro;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 &amp;micro;L</td><td>Nest</td><td>171215-1101</td><td></td></tr><tr><td>Universal Pipette Tips, 1000 &amp;micro;L</td><td>Isolab</td><td>L-002</td><td></td></tr><tr><td>Universal Pipette Tips, 200 &amp;micro;L&amp;nbsp;</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 dH&lt;sub&gt;2&lt;/sub&gt;O to make 2% solution and filter with 0.22 &amp;micro;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

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...

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 ...

culturing-freezing-processing-imaging-entire-organoids-spheroids

Research

JoVE Journal

Biology

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 µm to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

Here, detailed methods for generating, maintaining, and characterizing human pluripotent stem cell-derived small intestinal and colonic organoids are described. These methods are designed to improve reproducibility, expand scalability, and decrease the working time required for plating and passaging of organoids.

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Culture and Imaging of Human Nasal Epithelial Organoids

Here, we describe a method of generating glioblastoma (GBM) organoids from primary patient specimens or patient-derived cell cultures and maintaining them to maturity. These GBM organoids contain phenotypically diverse cell populations and recreate tumor microenvironments ex vivo.

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

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.

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

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

We present a step-by-step protocol for high-resolution, label-free, and three-dimensional imaging of organoids using low-coherence holotomography. This protocol details organoid culture preparation, imaging acquisition, and computational image analysis, enabling real-time visualization of structural dynamics and drug responses in living organoids.

Label-free, High-Resolution 3D Imaging and Machine Learning Analysis of Intestinal Organoids via Low-Coherence Holotomography

Learn a complete protocol for culturing, freezing, and imaging organoids and spheroids in hydrogel using HEPG2 cells. This method preserves 3D structure integrity for reliable disease modeling and biomarker analysis.

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

Explore More Videos