Name: Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device (Video) | JoVE Journal
Uploaded: 2022-10-06T12:40:00.000+00:00
Duration: 6 min 39 s
Description: Learn to generate homogeneous tissue spheroids using a 3D printed stamp-like device for tissue engineering and drug development. This cost-effective method ensures high cell viability and consistent quality for biomedical research.

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5 minutes

This work was supported by the Foundation for Research Support of the State of Rio de Janeiro (FAPERJ, Brazil), the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil), and the Brazilian National Council for Scientific and Technological Development (CNPq, Brazil). We thank Bioedtech for providing the stamp-like devices used in this study and Professor Bartira Bergmann from the Immunopharmacology Laboratory for the use of their cell culture facilities.

The L929 cell line, mouse fibroblasts, was used for the present study. The stamp-like 3D printed biodevice was obtained from a commercial source (see Table of Materials). Good cell culture practice and sterile techniques were followed throughout the protocol. The fabricated device was sterilized by wiping it with 70% alcohol and exposing it to UV light for 15 min. The cell culture media and solutions were warmed to 37 &#176;C before contacting with the cells or tissue spheroids. A schematic representation of the protocol is shown in Figure 1.
1. Preparation of non-adherent molds from the stamp-like device
<ol>
	<li>Prepare 2% (w/v) agarose gel following the steps below.
	<ol>
		<li>Dilute the agarose powder in phosphate-buffered saline (1x PBS) and homogenize the resulting suspension with circular movements. 
		NOTE: This solution can be placed in a glass bottle. In this step, the agarose solution will not be translucent.</li>
		<li>Place the glass bottle into the microwave and set it for 30 s. Every 5 s, stop the microwave, remove the glass bottle, and manually homogenize the solution with circular movements. The heating process needs to be performed until the solution reaches a liquid-limpid state. 
		NOTE: A hot plate can also be used as a microwave alternative. After the heating process, the solution must be translucent/limpid.</li>
		<li>Add 1 mL of the agarose solution to each well of a 6-well plate planned for the experiment.</li>
		<li>Wait for ~15 min or until the agarose solidifies. 
		NOTE: A cooling plate can be used to decrease the solidification time.</li>
		<li>Add 1-2 mL of the agarose solution and gently insert the device above the liquid agarose. 
		NOTE: The placement of the device must be done carefully to prevent air bubbles in the agarose-device interface.</li>
		<li>Wait for ~30 min or until the agarose solidifies. 
		NOTE: A cooling plate can be used to decrease the solidification time.</li>
		<li>Gently remove the device from the agarose. 
		NOTE: The removal is critical. One should remove it carefully to maintain the agarose features intact; otherwise, the agarose can be disrupted.</li>
		<li>Add 2 mL of DMEM media, wait for 10 min, discard the media, and replace it with fresh DMEM. Repeat this three times to wash the well properly.</li>
		<li>Add 2 mL of DMEM and place the 6-well plate in an incubator (at 37 &#176;C in 5% CO2 and 80% humidity) until the cell seeding (Figure 2A-F, Supplementary Video 1).</li>
	</ol>
	</li>
</ol>
2. Generation of tissue spheroids
NOTE: Different cell lineages have different adhesion properties. Hence, using this methodology, some types of cells may not form the tissue spheroids properly.
<ol>
	<li>Grow the cells following traditional monolayer culture (i.e., grow the cells in cell culture flasks using DMEM with low glucose supplemented with 10% fetal bovine serum (FBS), 100 &#181;g/mL penicillin, and 100 &#181;g/mL streptomycin) (see Table of Materials). Maintain the cells at 37 &#176;C in a 5% CO2 incubator, and monitor until they reach 80% of confluence.</li>
	<li>After reaching the desirable confluence, wash the cells with 1x PBS. 
	NOTE: It is recommended to use 5 mL for 25 cm2 flasks , 10 mL for 75 cm2 flasks, and 15 mL for 150 cm2 flasks.</li>
	<li>Add the dissociation enzyme and incubate the cells for 2-5 min at 37 &#176;C in 5% CO2 and 80% humidity. 
	NOTE: The present study used 0.125% trypsin with 0.78 mM ethylenediamine tetraacetic acid (EDTA) as the dissociation enzyme (see Table of Materials).</li>
	<li>Observe the detachment of the cells from the cell culture flasks and add a growth medium supplemented with FBS to neutralize the cell dissociation enzyme. 
	NOTE: For the present study, DMEM with low glucose (see Table of Materials) was used supplemented with 10% FBS.</li>
	<li>Centrifuge the cell suspension at 400 x g for 5 min at room temperature. Then, count the cells manually.</li>
	<li>Take 50 x 105 cells per tube and add 5 mL of 1x PBS. 
	NOTE: The number of cells seeded influences the final tissue spheroid diameter. Hence, one can increase the cell number to generate tissue spheroids with larger diameters.</li>
	<li>Centrifuge the cell suspension at 400 x g for 5 min at room temperature.</li>
	<li>Remove the supernatant using a pipette, add 1 mL of the cell culture medium, and homogenize the solution. 
	NOTE: In the present study, a complete culture medium of DMEM with low glucose, supplemented with 10% FBS, 100 &#181;g/mL penicillin, and 100 &#181;g/mL streptomycin, was used.</li>
	<li>Remove 2 mL of medium from the 6-well plate (step 1.1.9) and add 1 mL of the cell suspension to the center of the agarose mold formed by the 3D-printed biodevice (step 1.1.7). Wait until the cells sediment in the micro resections (~20-30 min) and carefully add 1 mL of cell culture medium into the well. 
	NOTE: One needs to be extra careful in this step. It is recommended to gently add the medium, place the pipette tip close to the well wall, and dispense in small amounts.</li>
	<li>Place the 6-well plate in the incubator (at 37 &#176;C in 5% CO2 and 80% humidity) for the tissue spheroids to form (approximately 24-48 h, depending on the cell type) (Figure 3). 
	NOTE: Different cell types (e.g., cancer cells, primary cells) have different self-assembly kinetics11.</li>
</ol>

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<li>Baptista, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+stem+cells+spheroids+to+optimize+cell+colonization+in+scaffolds+for+cartilage+and+bone+tissue+engineering%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Adult stem cells spheroids to optimize cell colonization in scaffolds for cartilage and bone tissue engineering.</a> International Journal of Molecular Sciences. 19 (5), 1285(2018).</li>
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<li>Smyrek, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((microtubules+and+FAK+dominate+different+spheroid+formation+phases+and+important+elements+of+tissue+integrity%5BTitle%5D)+AND+%22Biology+Open%22%5BJournal%5D)">microtubules and FAK dominate different spheroid formation phases and important elements of tissue integrity.</a> Biology Open. 8 (1), 037051(2018).</li>
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</ol>

The 3D printed stamp-like devices were offered by the startup Bioedtech, in which Jana&#237;na Dernovsek is the cofounder and innovation director. The authors declare no competing financial interests.

The present protocol describes a simple, fast, and inexpensive method for the large-scale production of tissue spheroids. A stamp-like 3D printed device was used as a master mold, which generated up to 4,716 spheroids per 6-well plate. It has been shown that cells cultivated as spheroids have more realistic biological responses that closely resemble the in vivo environment1. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more pred...

Tissue spheroids are 3D micro-tissues formed by cell suspensions that undergo self-assembly without external forces1. These spheroids have been widely used in biofabrication protocols due to their resemblance with key features of the human physiological system2,3. Tissue spheroids provide more similar metabolism, cytoskeleton dynamics, cell viability, and metabolic and secretion activity than traditional monolayer cell culture1. Due to their fusion capability, they can also be used as building blocks (e.g., bioprinting protocols) to form complex tissue-engineered constructs with enhanced biological relevance4,5.
Due to their biological relevance, tissue spheroids have been used as a biotechnological tool for protocols ranging across tissue engineering, drug development, disease modeling, and nanotoxicological assessment, reducing time, space costs, and animal testing3,6,7,8. Nonetheless, to fully explore and leverage the potential of tissue spheroids, reliable and reproducible methods aiming at their large-scale production are highly necessary, and these remain an ongoing challenge.
Several methodologies produce spheroids, such as hanging drop, coated u-shaped bottom wells, microfluidics, and using a polymeric matrix9,10. Although these methodologies paved the way within the spheroid production market, they are still complex, time-consuming, labor-intensive, or expensive10.
The present protocol reports the large-scale production of homogeneous tissue spheroids using a low-cost and time-effective methodology. We have developed a 3D printed stamp-like device to generate up to 4,716 spheroids per 6-well plate. Moreover, the stamp-like device can be tailored to produce more spheroids per well, suitable for different cell culture plates. It is easily sterilizable and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to translate their use to the clinics, contributing to multiple areas of industry such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<table><tbody><tr><td>6 well plate</td><td>Merck</td><td>CLS3516</td><td></td></tr><tr><td>Agarose</td><td>Promega</td><td>V3121</td><td></td></tr><tr><td>Biodevice&amp;nbsp;</td><td>Bioedtech</td><td></td><td></td></tr><tr><td>Biological Safety Cabinet</td><td>ThermoFisher&amp;nbsp;</td><td>51029701</td><td></td></tr><tr><td>Centrifugue</td><td>ThermoFisher&amp;nbsp;</td><td>75004031</td><td></td></tr><tr><td>Corning 50 mL centrifuge tubes</td><td>Merck</td><td>CLS430829-500EA</td><td></td></tr><tr><td>Corning cell culture flasks surface area 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Merck</td><td>CLS430641</td><td></td></tr><tr><td>Draft Resin&amp;nbsp;</td><td>FormLabs</td><td>FLDRBL01</td><td></td></tr><tr><td>Dulbecco&amp;prime;s Modified Eagle&amp;prime;s Medium - low glucose</td><td>Merck</td><td>D6046</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher&amp;nbsp;</td><td>16000044</td><td></td></tr><tr><td>Form 2</td><td>FormLabs</td><td></td><td></td></tr><tr><td>Incubator</td><td>ThermoFisher&amp;nbsp;</td><td>51033782</td><td></td></tr><tr><td>L929 cell lines</td><td>Stablished in the lab&amp;nbsp;</td><td></td><td></td></tr><tr><td>Penicillin and Streptomycin (PS)</td><td>ThermoFisher&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Merck</td><td>806552</td><td></td></tr><tr><td>Trypsin with EDTA</td><td>Merck</td><td>T3924</td><td></td></tr></tbody></table>

generation of tissue spheroids via a 3d printed stamp-like device

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

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Generation of homogeneous micro resections using the 3D printed stamp-like device 
The 3D printed stamp-like device was successfully manufactured by the stereolithography method12 using a photocurable resin (Figure 2A). The final device was composed of cylindrical micropins with a height of 1.3 mm and a width of 650 µm (Figure 2A). Its use as a master mold to produce non-adherent micro resections was achieved by mainta...

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

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

generation-of-tissue-spheroids-via-a-3d-printed-stamp-like-device

Research

JoVE Journal

Biochemistry

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

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Generation of 3D Tumor Spheroids for Drug Evaluation Studies

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

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Learn to generate homogeneous tissue spheroids using a 3D printed stamp-like device for tissue engineering and drug development. This cost-effective method ensures high cell viability and consistent quality for biomedical research.

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

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