Name: Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device (Video) | JoVE Journal
Uploaded: 2025-03-28T14:10:00.000+00:00
Duration: 5 min 40 s
Description: Learn a stamp-based microwell method for generating uniform 3D spheroids and organoids from 2D cell cultures. This cost-effective approach enhances cell interactions and reduces variability for biomedical research.

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5 minutes

We are especially grateful to the excellent technical assistance provided by Zizi de Mendon&#231;a (School of Medicine, University of S&#227;o Paulo, Brazil). This work was supported by grants from the following Brazilian research agencies: BNDES 09.2.1066.1, CAPES (PVE process number 88881.068070/2014-01), CNPq (grant numbers 457601/2013-2, 401430/2013-8, and INCT-Regenera number 465656/2014-5), FAPESP (Thematic project number 2016/05311-2), FINEP 01.08.06.05 and the Ministries of Science and Technology (MCTI) and Health (MS-DECIT).

This protocol follows the guidelines of the Human Research Ethics Committee of our Institution CEUA-FMUSP: 1699/2021, approved on September 8, 2021 -&#34;Isolation and Encapsulation of Porcine Pancreatic islets&#34; and is part of the Thematic Project of our Cell and Molecular Therapy NUCEL Group (www.usp.br/nucel), FAPESP Grant No. 2016/05311-2, entitled: &#8220;Regenerative Medicine aiming at therapy of chronic-degenerative diseases (cancer and diabetes)&#8221;.
1. Fabrication of the stamp device
NOTE: This Stamp is custom-made by the NUCEL group (https://w3nucel.webhostusp.sti.usp.br/). The stamp device was developed using the referenced software, widely recognized for its precision and advanced three-dimensional modeling capabilities.&#160;
<ol>
	<li>Ensure that the designed prototypes feature 663 micropins arranged to enable the generation of an equivalent number of three-dimensional cultures, such as spheroids or organoids. To ensure the stability of the stamp on T6 tray surfaces, position five support points strategically along the device&#39;s circumference.</li>
	<li>Ensure that the micropins have a conical design with adjustable dimensions, ranging from 1 to 3 mm in length. The base diameter of the cone varies between 0.7 and 1.5 mm, while the taper angle ranges from 5&#176; to 10&#176;.</li>
	<li>Make sure that the &#34;stop&#34; structure of the device, which defines the depth of the micropins, has an adjustable length of 10 to 19 mm and a diameter of 20 to 40 mm, with micropins uniformly distributed across its surface.</li>
	<li>Additionally, design the device to have an ergonomic handle, suitable for one-handed operation, to enhance practicality and precision during use. 
	NOTE: To facilitate large-scale production of the device, the digital file in .stl format is provided, being essential for the 3D printing process.</li>
	<li>Simply slice it using appropriate software and print it with a compatible 3D printer. Use the following printing dimensions: X - 68 mm; Y - 120 mm; Z - 150 mm, ensuring an ideal balance between functionality and structural quality.</li>
	<li>For the device&#39;s fabrication, use a photocurable 3D printer, which is compatible with the UV-curable polymer resin that must have high resolution and uniform surface finish, essential characteristics for optimal device&#39;s performance.</li>
	<li>Slice the digital model for printing using software compatible with the selected 3D printer.</li>
</ol>
2. Preparation of agarose microwells
<ol>
	<li>Prepare the agarose solution.
	<ol>
		<li>Weigh the pure agarose powder and dissolve it in 1x phosphate buffered saline (PBS) to achieve a final concentration of 1-2% (w/v).</li>
		<li>Heat the solution in a microwave or water bath until fully dissolved, ensuring it is homogeneous and transparent. 
		NOTE: Avoid overheating or excessive reheating, as this can degrade the agarose. Do not prepare large volumes of stock solution.</li>
		<li>Allow the solution to cool to ~40 &#176;C, the ideal temperature for pipetting .</li>
	</ol>
	</li>
	<li>Prepare the custom-made stamp.
	<ol>
		<li>Wash the stamp with a soft-bristle sponge and distilled water to remove residues.</li>
		<li>Expose the cleaned stamp to UV light for 5-10 min to ensure sterility.</li>
	</ol>
	</li>
	<li>Form the microwells.
	<ol>
		<li>Pour/pipette ~3 mL of the 40 &#176;C agarose solution into each well of a 6-well plate, ensuring a uniform depth of 2-3 mm.</li>
		<li>Position the custom-made stamp in the center of the well while the agarose is still warm. 
		NOTE: The stamp has lateral supports that ensure alignment and stability, eliminating the need for manual support. Each stamp creates ~663 microwells (~2 mm in height) per well. Ensure there are no bubbles on the micropins; if bubbles form, gently move the stamp to eliminate them.</li>
		<li>Allow the agarose to solidify for 5-10 min at room temperature without moving the plate. 
		NOTE: Do not disturb the agarose during solidification to avoid variations in the microwell structure.</li>
		<li>Remove the stamp with gentle back-and-forth motions to release the vacuum without damaging the microwells. 
		NOTE: This movement facilitates air entry, allowing smooth stamp removal without deforming the gel. The microwells must remain intact.</li>
		<li>Wash the microwells 3x with PBS solution to remove residues.</li>
		<li>Expose the plate to UV light for 10 min to eliminate microbial contamination.</li>
		<li>Add 3 mL of culture medium and incubate the plate in a CO2 incubator at 37 &#176;C overnight. 
		NOTE: This incubation verifies the absence of contamination before cell seeding.</li>
	</ol>
	</li>
</ol>
3. Cell seeding in microwells
<ol>
	<li>Prepare the cell suspension.
	<ol>
		<li>Collect the cells via trypsinization or another dissociation method to obtain a homogeneous suspension.</li>
		<li>Count the cells and adjust the concentration according to the desired number per microwell. 
		&#8203;NOTE: To seed 5,000 cells per microwell in 663 microwells, prepare a suspension containing at least 3.315 &#215; 106 total cells. Optimize the cell concentration to prevent central necrosis and to ensure adequate nutrient and oxygen diffusion.</li>
	</ol>
	</li>
	<li>Seed the cells.
	<ol>
		<li>Pipette ~3 mL of the prepared cell suspension over the microwells, ensuring even distribution by gently swirling the plate.</li>
		<li>Allow the cells to settle by gravity (~10-15 min) or perform gentle centrifugation at 100 &#215; g for 5 min. 
		&#8203;NOTE: Centrifugation can be used to ensure the cells concentrate in the microwells.</li>
		<li>Transfer the plate to a CO2 incubator at 37 &#176;C with a humidified atmosphere.</li>
	</ol>
	</li>
</ol>
4. Maintenance of 3D cell cultures
<ol>
	<li>Replace the culture medium.
	<ol>
		<li>Change the medium every 2-3 days, removing 50% of the old medium and adding fresh medium. 
		NOTE: Ensure the microwells remain submerged throughout the culture period to prevent dehydration.</li>
	</ol>
	</li>
	<li>Monitor the formation of 3D cultures.
	<ol>
		<li>Continue culturing until the 3D structures are fully formed and compact (e.g., 2-3 days for primary pancreatic islet cultures). 
		NOTE: For cultures requiring a longer formation time, perform partial medium changes every 2-3 days.</li>
	</ol>
	</li>
</ol>
5. Collection and characterization of 3D structures
<ol>
	<li>Collect the spheroids/organoids.
	<ol>
		<li>Remove the old medium and collect the 3D structures via vigorous pipetting using a wide-diameter or cut pipette tip to prevent compression or disaggregation. 
		NOTE: Vigorous pipetting must be controlled to dislodge the aggregates without deforming them.</li>
	</ol>
	</li>
	<li>Prepare for analysis.
	<ol>
		<li>Use the collected spheroids/organoids immediately or fix them for future analysis. 
		&#8203;NOTE: The association and stability of spheroids depend on cell type, formation time, and culture conditions. Take care is essential to avoid dissociation.</li>
	</ol>
	</li>
</ol>

<ol><li>Lian, J., Yue, Y., Yu, W., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunosenescence%3A+a+key+player+in+cancer+development%5BTitle%5D)+AND+%22J+Hematol+Oncol%22%5BJournal%5D)">Immunosenescence: a key player in cancer development.</a> J Hematol Oncol. 13 (1), 151(2020).</li>
<li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reciprocal+interactions+between+beta1-integrin+and+epidermal+growth+factor+receptor+in+three-dimensional+basement+membrane+breast+cultures%3A+a+different+perspective+in+epithelial+biology%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology.</a> Proc Natl Acad Sci USA. 95 (25), 14821-14826 (1998).</li>
<li>Dzobo, K., Dandara, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+extracellular+matrix%3A+its+composition%2C+function%2C+remodeling%2C+and+role+in+tumorigenesis%5BTitle%5D)+AND+%22Biomimetics%22%5BJournal%5D)">The extracellular matrix: its composition, function, remodeling, and role in tumorigenesis.</a> Biomimetics. 8 (2), 146(2023).</li>
<li>Sutherland, R. M., McCredie, J. A., Inch, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+of+multicell+spheroids+in+tissue+culture+as+a+model+of+nodular+carcinomas%5BTitle%5D)+AND+%22J+Natl+Cancer+Inst%22%5BJournal%5D)">Growth of multicell spheroids in tissue culture as a model of nodular carcinomas.</a> J Natl Cancer Inst. 46 (1), 113-120 (1971).</li>
<li>Lancaster, M. A., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organogenesis+in+a+dish%3A+modeling+development+and+disease+using+organoid+technologies%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Organogenesis in a dish: modeling development and disease using organoid technologies.</a> Science. 345 (6194), 1247125(2014).</li>
<li>Bialkowska, K., Komorowski, P., Bryszewska, M., Milowska, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spheroids+as+a+type+of+three-dimensional+cell+cultures-examples+of+methods+of+preparation+and+the+most+important+application%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Spheroids as a type of three-dimensional cell cultures-examples of methods of preparation and the most important application.</a> Int J Mol Sci. 21 (17), 6225(2020).</li>
<li>Razian, G., Yu, Y., Ungrin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+large+numbers+of+size-controlled+tumor+spheroids+using+microwell+plates%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Production of large numbers of size-controlled tumor spheroids using microwell plates.</a> J Vis Exp. (81), e50665(2013).</li>
<li>Andersen, T., Auk-Emblem, P., Dornish, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+Cell+culture+in+alginate+hydrogels%5BTitle%5D)+AND+%22Microarrays+%28Basel%29%22%5BJournal%5D)">3D Cell culture in alginate hydrogels.</a> Microarrays (Basel). 4 (2), 133-161 (2015).</li>
<li>Hou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+development+of+primary+pancreatic+organoid+tumor+models+for+high-throughput+phenotypic+drug+screening%5BTitle%5D)+AND+%22SLAS+Discov%22%5BJournal%5D)">Advanced development of primary pancreatic organoid tumor models for high-throughput phenotypic drug screening.</a> SLAS Discov. 23 (6), 574-584 (2018).</li>
<li>Napolitano, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scaffold-free+three-dimensional+cell+culture+utilizing+micromolded+nonadhesive+hydrogels%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Scaffold-free three-dimensional cell culture utilizing micromolded nonadhesive hydrogels.</a> Biotechniques. 43 (4), 494-500 (2007).</li>
<li>Maria-Engler, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Co-localization+of+nestin+and+insulin+and+expression+of+islet+cell+markers+in+long-term+human+pancreatic+nestin-positive+cell+cultures%5BTitle%5D)+AND+%22J+Endocrinol%22%5BJournal%5D)">Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures.</a> J Endocrinol. 183 (3), 455-467 (2004).</li>
<li>Mantovani, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immobilization+of+primary+cultures+of+insulin-releasing+human+pancreatic+cells%5BTitle%5D)+AND+%22Islets%22%5BJournal%5D)">Immobilization of primary cultures of insulin-releasing human pancreatic cells.</a> Islets. 1 (3), 224-231 (2009).</li>
<li>Wassmer, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+of+primary+pancreatic+islet+cell+spheroids+for+three-dimensional+culture+or+transplantation%3A+a+methodological+comparative+study%5BTitle%5D)+AND+%22Cell+Transplant%22%5BJournal%5D)">Engineering of primary pancreatic islet cell spheroids for three-dimensional culture or transplantation: a methodological comparative study.</a> Cell Transplant. 29, 963689720937292(2020).</li>
<li>Stuart, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Successful+low-cost+scaffold-free+cartilage+tissue+engineering+using+human+cartilage+progenitor+cell+spheroids+formed+by+micromolded+nonadhesive+hydrogel%5BTitle%5D)+AND+%22Stem+Cells+Int%22%5BJournal%5D)">Successful low-cost scaffold-free cartilage tissue engineering using human cartilage progenitor cell spheroids formed by micromolded nonadhesive hydrogel.</a> Stem Cells Int. 2017, 7053465(2017).</li>
<li>Charelli, L. E., Dernowsek, J. A., Balbino, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+tissue+spheroids+via+a+3D+printed+stamp-like+device%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Generation of tissue spheroids via a 3D printed stamp-like device.</a> J Vis Exp. (188), e63814(2022).</li>
<li>Micromold for the production of cellular spheroids and use. , Patent BR 10 2019 027360 7 A2 (2019).</li>
<li>Riffle, S., Pandey, R. N., Albert, M., Hegde, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Linking+hypoxia%2C+DNA+damage+and+proliferation+in+multicellular+tumor+spheroids%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">Linking hypoxia, DNA damage and proliferation in multicellular tumor spheroids.</a> BMC Cancer. 17, 338(2017).</li>
</ol>

The authors declare that they have no conflicts of interest.

Although various 3D culture protocols exist in the literature, a study conducted by Wassmer et al.13 tested several methodologies for generating 3D structures using pancreatic islets. The authors observed that native islets and self-aggregated spheroids exhibited considerable heterogeneity with respect to size and shape and were larger than those obtained using other methods. Based on their findings, they concluded that spheroids can be generated using different techniques, each with its own advan...

Over the past 50 years, numerous cell biology investigations have demonstrated that two-dimensional (2D) cultures fail to accurately replicate the in vivo conditions observed in animal models1. Structurally, 2D cell cultures do not allow cells to organize three-dimensionally and replicate the situation observed in in vivo systems. Furthermore, cellular signaling pathways are altered in 2D cultures compared to three-dimensional (3D) cultures, which could likely explain why certain types of drug screening using 2D cultures are so discrepant2. A significant advancement in cell culture techniques emerged with the introduction of 3D culture systems. 3D systems vary considerably in complexity depending on cellular composition and cytoarchitecture. Generally, two types of structures are generated, namely: spheroids and organoids. Spheroids are described as simple clusters of cells obtained from normal or tumor tissues, embryoid bodies, and cell lines. The formation of 3D structures is influenced by various factors, including cell-cell interactions and signaling pathways mediated by components of the extracellular matrix (ECM), which provide structural support and biochemical cues. These elements regulate interactions that contribute to tissue organization and function3. The spheroid culture system was first described in the early 1970s, using V79 Chinese hamster lung cell lines as a model of nodular carcinomas, growing under non-adherent conditions and forming perfect&#160;spheres4. Organoids are described as clusters of organ-specific cell types derived from stem or progenitor cells, which self-organize through processes, such as cell sorting and lineage specification, in a spatially confined manner, mirroring the in vivo development5.
Several available methods and materials are available to culture cells under 3D conditions. The main methods currently employed for generating 3D cultures are: 1) hanging drops; 2) rotating cell culture and low-attachment plastics; 3) pyramid plates containing conical wells; 4) macroporous scaffolds; 5) magnetic beads; and 6) scaffold-free hydrogels.
Hanging drops is the method used to obtain scaffold-free 3D cultures. This method presents certain limitations, including the need for extensive handling, low production efficiency, spherical geometry, and exposure to high shear forces. Moreover, specific procedures, such as medium replacement or compound addition, can be challenging and may result in material loss. Furthermore, literature reports indicate that some cell lines fail to produce tightly packed spheroids when employing this approach6.
Rotating cell culture and low-attachment plastics are used to prevent cells from attaching to the substrate, causing them to aggregate and form spheroids. This process requires specific flasks and/or agitation/rotation. Although this is one of the most straightforward approaches for large-scale spheroids or organoids production, it is not without drawbacks, such as the need for specific equipment, low culture longevity, size variation in spheroids, mechanical damage to cells, and low efficiency6.
Pyramid plates containing conical wells are commercially available plates impacting costs, in addition to the fact that some manipulations may hinder the formation of spheroids/organoids7.
Macroporous scaffolds are also employed for 3D culturing; however, a major obstacle lies in achieving effective cell seeding and uniform distribution. This issue arises because the pore sizes may either be too small for cell penetration or too large to securely retain the cells. To address this issue, several strategies have been explored8, which directly impact the complexity and cost of this technique.
The magnetic beads methodology generates a small number of spheroids/organoids, has a high cost, and may leave nanoparticle residues inside the cells9.
Among the systems for cultivating spheroids, non-adhesive agarose hydrogels are available, representing a scaffold-free hydrogel. This approach offers notable benefits, such as precise control over the size of the 3D structures and the capacity to generate a substantial number of these structures per plate. In this method, cells are introduced into a hydrogel with preformed wells, in which they sink and self-assemble into 3D spheroids10.
In this study, we present a device and methodology for generating agarose microwells using a micropattern mold in a simple, efficient, reproducible, and low-cost manner.
The use of this stamp as a mold to generate microwells in agarose, aided by gravity, aims to enhance cell interaction within the microwell and cellular organization, generating 3D structures (spheroids/organoids) in vitro in a simple, efficient, reproducible, and low-cost manner, thus saving research time and laboratory resources.

<table><tbody><tr><td>31L Microwave</td><td>Electrolux</td><td>78965840 6699 9</td><td>Equipment used to heat the agarose solution, facilitating its dissolution and ensuring greater homogeneity. It allows the solution to reach the ideal liquid state for the formation of the wells.</td></tr><tr><td>3DFila Gray Opaque Photosensitive 3D Resin</td><td></td><td></td><td>UV-curable polymer resin</td></tr><tr><td>3D Printer - Creality Halot One</td><td>Creality</td><td>N/A</td><td>3D printer used for printing the stamp device</td></tr><tr><td>Agarose</td><td>UNISCIENCE</td><td>UNI-R10111</td><td>To form the gel, dissolve 1 to 2% in Saline Phosphate Buffer (PBS) or appropriate medium.</td></tr><tr><td>Autodesk Fusion 360</td><td></td><td></td><td>3D modeling</td></tr><tr><td>BB15 CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Fisher</td><td>51023121</td><td>Equipment used to incubate cultured cells in a suitable and controlled environment.</td></tr><tr><td>Chitubox</td><td>Chitubox</td><td>N/A</td><td>Software used for slicing the part for printing</td></tr><tr><td>Class II Biological Safety Cabinet</td><td>Grupo VECO</td><td>N/A</td><td>Ensures a sterile environment for performing cell culture within established parameters and protocols.</td></tr><tr><td>Culture medium</td><td>USBiological/Life Sciences</td><td>C5900-03A</td><td>Contains additives for proper cell cultivation.</td></tr><tr><td>Culture plates (P6)</td><td>SARSTEDT</td><td>1023221</td><td>Used to shape the agarose and culture the cells.</td></tr><tr><td>Erlenmeyer Flask (25 mL)</td><td>Laborglas</td><td>91 216 14</td><td>A container used for dissolving 1&amp;ndash;2% agarose in Phosphate Buffered Saline (PBS) or another suitable medium, typically heated in a microwave.</td></tr><tr><td>Falcon 15 mL Polystyrene Centrifuge Tube</td><td>Corning</td><td>352099</td><td>Used to keep cells in suspension and perform possible dilutions.</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Vitrocell Embriolife</td><td>005/19</td><td>Additive for culture medium.</td></tr><tr><td>PBS solution (Saline Phosphate Buffer)</td><td>Lab made</td><td>N/A</td><td>Diluted 1x with MiliQ ultrapure water. Used to dissolve agarose 1 to 2% and to wash wells already produced.</td></tr><tr><td>Reagent bottle with blue cap - Schott</td><td>Laborglas</td><td>21801545</td><td>Used for preparing and storing culture medium.</td></tr><tr><td>Stamp device</td><td>NUCEL Group</td><td>N/A</td><td>Link- This link provides access to the .stl file of the stamp device. Simply slice it using appropriate software and print it with a compatible 3D printer. https://drive.google.com/drive/folders/1gTYComnJWzHpN6ZKOyK&lt;br/&gt; EChKS3Qns0rOA?usp=sharing</td></tr><tr><td>Treated culture flask with filter 25 cm&amp;sup2;</td><td>Corning</td><td>430639</td><td>Used for the cultivation and maintenance of adherent cells.</td></tr><tr><td>Trypsin</td><td>Merck</td><td>07-07-9002</td><td>For dissociation of cells before seeding.</td></tr><tr><td>Ultra violet light (UV)</td><td>N/A</td><td>N/A</td><td>Used to sterilize the stamp and plates.</td></tr></tbody></table>

generation of three-dimensional spheroids/organoids from two-dimensional cell cultures using a novel stamp device

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

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix.&#160;This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 &#176;C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization.&#160;The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

The cell culture used in this study was derived from porcine pancreatic islets. The islet preparation used in this study had an 80 ± 5% purity based on dithizone staining, and &gt;80% islet cell viability based on the detection of fluorescein diacetate in live cells or propidium iodide in dead cells (the Live/Dead fluorescent method). Ensure that the porcine pancreatic islet preparation is at least 80% pure (e.g., by dithizone staining) and &gt;80% viable. Upon isolation, maintain adherent cultures in CMRL 1066 medium, s...

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.

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) ...

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Research

JoVE Journal

Bioengineering

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Spheroids are a type of 3D cell culture model used to more accurately represent a cell’s environment compared to 2D cell culture monolayer models. In the example protocol, we will see scientists generate spheroids from a colorectal cancer cell line and apply live-cell imaging to track the spheroids’ growth.

Spheroid Generation from Cell Lines: A Three-Dimensional (3D) Cell Culture Method

This video demonstrates the transformation of human pluripotent stem cells (hPSCs) into spherical brain organoids. By using specific inhibitors, the hPSCs are guided to differentiate towards a neuronal lineage, forming neuroectodermal colonies. Special growth factors are then introduced, which encourage the self-organization and proliferation of the colonies, ultimately leading to the formation of spherical brain organoids.

Generation of Spheroids from Human Pluripotent Stem Cells

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

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

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

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

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.

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

Learn a stamp-based microwell method for generating uniform 3D spheroids and organoids from 2D cell cultures. This cost-effective approach enhances cell interactions and reduces variability for biomedical research.

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

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