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>

<ol><li>Laschke, M., Menger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Life+is+3D%3A+Boosting+spheroid+function+for+tissue+engineering%5BTitle%5D)+AND+%22Trends+in+Biotechnology%22%5BJournal%5D)">Life is 3D: Boosting spheroid function for tissue engineering.</a> Trends in Biotechnology. 35 (2), 133-144 (2017).</li>
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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>
<li>Shakeri, A., Khan, S., Didar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conventional+and+emerging+strategies+for+the+fabrication+and+functionalization+of+PDMS-based+microfluidic+devices%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">Conventional and emerging strategies for the fabrication and functionalization of PDMS-based microfluidic devices.</a> Lab on a Chip. 21 (16), 3053-3075 (2021).</li>
<li>vander Valk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fetal+bovine+serum+%28FBS%29%3A+Past+-+present+-+future%5BTitle%5D)+AND+%22ALTEX%22%5BJournal%5D)">Fetal bovine serum (FBS): Past - present - future.</a> ALTEX. 35 (1), 99-118 (2018).</li>
<li>vander Valk, J., Brunner, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+chemically+defined+cell+culture+media+-+Replacing+fetal+bovine+serum+in+mammalian+in+vitro+methods%5BTitle%5D)+AND+%22Toxicology+in+Vitro%22%5BJournal%5D)">Optimization of chemically defined cell culture media - Replacing fetal bovine serum in mammalian in vitro methods.</a> Toxicology in Vitro. 24 (4), 1053-1063 (2010).</li>
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<li>McMillen, P., Holley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integration+of+cell-cell+and+cell-ECM+adhesion+in+vertebrate+morphogenesis%5BTitle%5D)+AND+%22Current+Opinion+in+Cell+Biology%22%5BJournal%5D)">Integration of cell-cell and cell-ECM adhesion in vertebrate morphogenesis.</a> Current Opinion in Cell Biology. 36, 48-53 (2015).</li>
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<li>Skardal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-tissue+interactions+in+an+integrated+three-tissue+organ-on-a-chip+platform%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform.</a> Scientific Reports. 7, 8837(2017).</li>
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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

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 &#181;m (Figure 2A). Its use as a master mold to produce non-adherent micro resections was achieved by...

Read this Scientific Article Protocol about Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device at JoVE.com

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

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

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

1.7K Views.  Federal University of Rio de Janeiro (UFRJ). The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

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

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

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

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

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Bioengineering

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

Cancer Research

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

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

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 &#956;m microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

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.

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

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.

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

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

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

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.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

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.

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 Tissue Spheroids via a 3D Printed Stamp-Like Device

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

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

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

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

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

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