We thank the National Institute of Metrology, Quality and Technology (INMETRO, RJ, Brazil) for the use of their facilities. This study was partially supported by the Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (Faperj) (finance Code: E26/202.682/2018 and E-26/010.001771/2019, the National Council for Scientific and Technological Development (CNPq) (finance code: 307460/2019-3), and the Office of Naval Research (ONR) (finance code: N62909-21-1-2091). This work was partially supported by the National Center of Science and Technology on Regenerative Medicine-INCT Regenera (http://www.inctregenera.org.br/).

The ASCs used in this study were previously isolated from healthy human donors and cryopreserved as described14 according to the Research Ethics Committee of Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Brazil (25818719.4.0000.5257). See Table of Materials for details regarding all the materials and equipment used in this study.
1. Trypsinization of ASC monolayer at passage three
<ol>
	<li>Open the tissue culture 175 cm2 flask containing the monolayer of ASCs at 80% confluence and discard the supernatant.</li>
	<li>Add 7 mL of phosphate-buffered saline (PBS, 0.01 M) and wash the monolayer twice. Next, discard the liquid.</li>
	<li>Add 5 mL of 0.125% trypsin with 0.78 mM ethylenediaminetetraacetic acid (EDTA). Next, incubate for 5 min at 37 &#176;C.</li>
	<li>Add 10 mL of low-glucose Dulbecco&#39;s Modified Eagle Medium (DMEM LOW) with 10% fetal bovine serum (FBS) to the tissue culture 175 cm2 flask and mix the cell suspension well with a 10 mL sorologic pipette.</li>
	<li>Harvest the cell suspension with a 10 mL serological pipette and transfer it to a 50 mL centrifuge tube. Next, centrifuge at 400 &#215; g for 5 min to obtain the pellet of ASCs. Resuspend the cell pellet using DMEM LOW containing 10% FBS.</li>
	<li>Perform a cell count by trypan exclusion.</li>
	<li>After counting, take a total of 1 &#215; 106 ASCs in a separate 15 mL centrifuge tube to seed the 81 recessions micromolded with non-adherent hydrogel (a total of 10,000 cells/mL seeded here). To seed 256 recessions of micromolded non-adherent hydrogel, take a total of 5 &#215; 105 ASCs into a centrifuge tube (a total of 2,500 cells/mL seeded here).</li>
</ol>
2. Micromolded non-adherent agarose hydrogel fabrication
<ol>
	<li>Prepare 50 mL of a sterile solution of 2% ultrapure agarose in 0.9% NaCl in ultrapure water. Autoclave the solution for 30 min at 121 &#176;C.</li>
	<li>Add 500 &#181;L of sterile 2% ultrapure agarose solution in the center of a silicone mold containing 81 or 256 circular recesses.</li>
	<li>After 40 min, unmold the ultrapure agarose from the silicone mold and place it in a well of a 12-well plastic plate.</li>
	<li>Add 2 mL of DMEM LOW in the well with the micromolded agarose and incubate in an incubator at 37 &#176;C in a 5% CO2 atmosphere for at least 12 h before seeding the ASCs.</li>
</ol>
3. Biofabrication of ASC spheroids using the automated pipetting system
<ol>
	<li>Check the following:
	<ol>
		<li>Check whether laminar flow is on, and the airflow of the cabinet is working properly.</li>
		<li>Check whether the equipment is connected to the correct voltage.</li>
		<li>Check whether the tablet is connected to the equipment.</li>
		<li>Check whether the height of the cabinet protection glass is at the same height as the sensor marking of the equipment.</li>
	</ol>
	</li>
	<li>Press the On/Off button on the left side of the equipment and wait for the tablet and the equipment to start.</li>
	<li>Position the tip boxes, the rack for centrifuge tubes, and the plate containing the micromolded agarose hydrogel in the workspace of the equipment.</li>
	<li>At the tablet with the software open, first click on Labware Editor.</li>
	<li>Set up a virtual worktable and choose the positions of the pipettes, tip boxes, the rack for the plastic tubes, and the plates. 
	NOTE: The virtual worktable must represent the physical positions of the accessories in the equipment.</li>
	<li>Click on the Switch to Produce button to include the parameters (see step 3.10) and commands that the equipment will carry out throughout the experiment. Wait for a toolbar and a Produce List to open in the software.</li>
	<li>Initially, to indicate the commands, include the number of samples to be pipetted and drag it to the Produce List. 
	NOTE: The number of samples are the number of plastic centrifuge tubes containing the ASC suspension to be seeded in the center of the micromolds.</li>
	<li>After entering the number of samples, click on the Sample Transfer command button on the software to transfer the ASC suspension to the wells of the 12-well plate, containing the micromolds, and drag it to the Produce List.</li>
	<li>Click on Properties to set up the start and end positions for the equipment to transfer the sample.</li>
	<li>Wait for the software to return to the Work Table page. Click on the Options button to set up the parameters for automated seeding of the ASCs: i) Aspiration Speed of 15.4 s-1; ii) Dispense Speed of 1 s-1; iii) Blow Delay of 0 ms; iv) Blow Speed of 15.4 s-1; v) Initial Stroke of 100%. Select Water as the Standard Liquid Type.</li>
	<li>Set up the parameters to mix the ASCs to obtain a homogeneous suspension: i) Number of Cycles of 5; ii) Speed of 6.5 s-1; iii) Volume of 300 &#181;L.</li>
	<li>After setting up the parameters, add the Time of 40 min to the Produce List. 
	NOTE: This time is needed to wait for the ASC suspension to decant at the bottom of the micromold.</li>
	<li>In the Produce List, include the Reagent Transfer option to transfer the spheroid culture medium to the corresponding wells of the plate.</li>
	<li>Repeat steps 3.9-3.11 to set up the position and parameter configurations to transfer the spheroid culture medium.</li>
	<li>Click on the button with a check symbol on the top bar of the software to ensure there is no programming error.</li>
	<li>Click on the Play button (with a triangle symbol) on the top bar of the software to start the program.</li>
	<li>Let the equipment start, as programmed, and wait for it to make a sound, indicating it has finished.</li>
	<li>Collect the 12-well plate and incubate it in an incubator at 37 &#176;C, 5% CO2 for at least 18 h for the spheroids to be completely formed and compact.</li>
	<li>Manually harvest the spheroids after 1, 3, and 7 days of culture for analysis. Manually flush the medium with a micropipette to liberate the spheroids from the micromolded non-adherent agarose hydrogel.</li>
	<li>After 5 min, check under the microscope to determine whether the spheroids are completely released from the micromolded non-adherent agarose hydrogel.</li>
	<li>Collect the spheroids manually using a micropipette and transfer them to a 15 mL centrifuge tube.</li>
	<li>Wash the spheroids at least two times with 0.01 M PBS. Assess the viability and perform biomechanical analyses on the fresh spheroid samples. For morphology analysis (histology), incubate the spheroid samples in 4% paraformaldehyde in PBS for at least 18 h at 2-8 &#176;C.</li>
</ol>

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The authors declare no conflicts of interest.

This paper presents the large-scale generation of ASC spheroids using an automated pipette system. The critical step of the protocol is to precisely set up the software to ensure the correct volume of cell suspension, speed, and distance for pipetting. The parameters described in the protocol were determined after a number of trials to optimize the dispensing of the ASC cell suspension into the wells of 12-well plates containing the micromolded, non-adherent hydrogels. The optimization was evaluated by measuring the diameter and sphericity of the spheroids. The area and height of the micromolded non-adherent hydrogels were used to calculate the ideal speed to dispense the cell suspension. If the cell suspension is seeded by the equipment at a different dispensing speed, this will directly affect the formation of the spheroids. Hence, these are crucial parameters to be optimized after trials with the equipment. This protocol has some limitations. Only seeding of the ASCs in the non-adherent hydrogel in the micromold can be automated, followed by the addition of cell culture medium. The features of the system are limited by the version of the equipment and the software. However, the main advantage of this reproducible approach is to produce up to 3,072 ASC spheroids-homogeneous in size and shape and viable-with minor risks of human errors or contamination. The large number of biofabricated ASC spheroids can be directly loaded into a 3D bioprinter to produce more complex tissue models.
In general, it is widely known that accurate and automated pipetting plays a central role in the automation of cell culture. Automated cell culture systems enable large-scale production and increase technical accuracy, reproducibility, and process efficiency15. Therefore, automated systems can facilitate large-scale production, which is common and beneficial for industrial environments16,17. However, there are few studies describing the development of protocols to automate 3D cell culture systems7,18,19,20,21.
Mehesz and collaborators7 and Meseguer-Ripolles and colleagues21 used a similar protocol to biofabricate ASC spheroids. In the study by Mehesz et al.7, the authors used the same automated pipetting system. However, their protocol produced only 96 spheroids, whereas the protocol developed in the present work was able to produce up to 3,072 ASC spheroids at once, homogeneous in size and shape. Meseguer-Ripolles et al.21 also used an automated pipetting system to produce up to 73 liver spheroids. Another approach being discussed to fabricate spheroids on a large scale nowadays is via microfluidics devices. However, the articles published so far have explored the technology to develop better devices that can produce the spheroids rather than increasing the scale for spheroid fabrication7,20.
Another method that can be used to fabricate a high number of spheroids is by using spinner flasks. However, one main issue regarding this technique is the difficulty in controlling the size and shape of the spheroids22,23. In addition, 3D bioprinting was also already applied to biofabricate spheroids on a large scale. In the study by Daly and Kelly19, the authors used an inkjet technique to produce a large number of spheroids in polycaprolactone microchambers. The spheroids were viable and homogeneous in size and shape.
The main application of the protocol described in this work is to biofabricate ASC spheroids on a large scale needed for 3D bioprinting. As discussed by Mironov and collaborators24, thousands of spheroids will be necessary to biofabricate complex tissues and organ models. The automated method described in this protocol will allow for the fabrication of a large number of ASC spheroids that can be loaded directly in the 3D bioprinter. It is also possible to differentiate these spheroids to a desired mesodermal pathway to engineer musculoskeletal tissues. De Moor and collaborators25 developed a high-throughput method to fabricate a large number of vascularized spheroids for future 3D bioprinting applications.
The equipment can be modified to improve the protocol. More recent versions of the equipment allow the use of a larger number of plates, which could enhance the number of ASC spheroids produced. This same recent version allows the accurate stacking of the plates, and this also would provide a better efficiency of the protocol. In conclusion, we successfully showed a protocol allowing the biofabrication up to 3,072 ASC spheroids homogeneous in size and shape in 15 min, and that this is considered the first step to build a biofabrication line for Tissue Engineering applications.

Spheroids are considered a scaffold-free approach in tissue engineering. ASCs are capable of forming spheroids by the self-assembly process. The spheroid&#39;s 3D microarchitecture increases the regenerative potential of ASCs, including the differentiation capacity into multiple lineages1,2,3. This research group has been working with ASC spheroids for cartilage and bone tissue engineering4,5,6. More importantly, spheroids are considered building-blocks in the biofabrication of tissues and organs, mainly due to their fusion capacity.
The use of spheroids for tissue formation depends on three main points: (1) the development of standardized and scalable robotic methods for their biofabrication7, (2) the systematic phenotyping of tissue spheroids8, (3) the development of methods for the assembly of 3D tissues9. These spheroids can be formed with different cell types and obtained through various methods, including hanging drop, reaggregation, microfluidics, and micromolds8,9,10. Each of these methods has advantages and disadvantages related to the homogeneity of size and shape of the spheroids, recovery of the spheroids after formation, the number of spheroids produced, process automation, labor intensity, and costs11.
In the micromold method, the cells are dispensed and deposited at the bottom of the micromold because of gravity. The non-adhesive hydrogel does not allow the cells to adhere to the bottom, and cell-to-cell interactions lead to the formation of a single spheroid per recession8,12. This biofabrication method generates spheroids of homogeneous and controlled size, can be robotized for large-scale production in a time-efficient manner with minimal effort, and has good cost-effectiveness-critical factors in the design of a biofabrication of tissue spheroid7,8. This method can be applied to form spheroids of any cell lineage to prepare a new tissue type with predictable, optimal, and controllable characteristics8.
Biofabrication is defined as &#34;the automated generation of biologically functional products with structural organization ...&#34;13. Therefore, the automated 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 bioprinted tissue by spheroid fusion. In this study, to improve the scalability of ASC spheroid biofabrication, we use an automated pipetting system to seed the cell suspension, thus ensuring the homogeneity of spheroid size and shape. This paper shows that it was possible to produce a large number (thousands) of spheroids needed for 3D bioprinting approaches to biofabricate more complex tissue models.

<table><tbody><tr><td>12-well plastic plate</td><td>Corning</td><td>3512</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Corning</td><td>CLS430828</td><td></td></tr><tr><td>EpMotion 5070</td><td>Eppendorf</td><td>5070000282</td><td></td></tr><tr><td>epT.I.P.S. Motion</td><td>Eppendorf</td><td>30015231</td><td></td></tr><tr><td>ethylenediaminetetraacetic acid (EDTA)</td><td>Invitrogen</td><td>15576028</td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>Gibco</td><td>10082147</td><td></td></tr><tr><td>&lt;em&gt;Low Glucose Dulbecco&#039;s Modified Eagle Medium (DMEM LOW)&lt;/em&gt;</td><td>Gibco</td><td>31600034</td><td></td></tr><tr><td>MicroTissues 3D Petri Dish micro-mold spheroids - 16 x 16 array</td><td>Sigma</td><td>Z764000</td><td></td></tr><tr><td>MicroTissues 3D Petri Dish micro-mold spheroids - 9 x 9 array</td><td>Sigma</td><td>Z764019</td><td></td></tr><tr><td>phosphate saline buffer (PBS)</td><td>Sigma</td><td>806552</td><td></td></tr><tr><td>sodium chloride (NaCl)</td><td>Sigma</td><td>S8776</td><td></td></tr><tr><td>tissue culture flask</td><td>Corning</td><td>430720U</td><td></td></tr><tr><td>trypan</td><td>Lonza</td><td>17-942E</td><td></td></tr><tr><td>trypsin</td><td>Gibco</td><td>27250018</td><td></td></tr><tr><td>ultrapure agarose</td><td>Invitrogen</td><td>16500100</td><td></td></tr></tbody></table>

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.

The automatic pipette system can seed the ASC cell suspension into 12 wells of one 12-well plate in 15 min. Using the 81 micromolded non-adherent hydrogel will produce 972 spheroids at the end of the protocol. Using the 256 micromolded non-adherent hydrogel will produce 3,072 spheroids at the end of the protocol. ASC spheroids were analyzed for the homogeneity of their size and shape. ASC spheroids from micromolds with 81 recessions showed homogeneous diameter during the culture period in contrast to ASC spheroids from micromolds with 256 recessions (Figure 1C,D). The ratio of the smallest and largest diameters (sphericity) was close to 1 in ASC spheroids from micromolds with 81 and 256 recessions (Figure 1E,F). The results from viability, morphology, and force (Figure 2) analyses provide evidence for the successful, large-scale production of ASC spheroids.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63430/63430fig01.jpg" /> 
Figure 1:&#160;ASC spheroids showed high diameter reproducibility. Representative optical micrographs of ASC spheroids from micromolded non-adhesive hydrogels with (A) 81 circular recessions and (B) 256 circular recessions. (C,D) Spheroid diameter at 1, 3, and 7 days from five micromolded non-adhesive hydrogels with 81 and 256 circular recessions, respectively. (E,F) Ratio of the smallest and largest diameters from micromolded non-adhesive hydrogels with 81 and 256 circular recessions, respectively. To measure the smallest and largest diameters of the spheroids, images were acquired weekly using an optical microscope equipped with a digital camera. Width and length were measured using ImageJ software. A diameter ratio of each spheroid was obtained by dividing width by length (spheroid sphericity). A total of 85 spheroids were measured from micromolded, non-adhesive hydrogels with 81 circular recessions, and a total of 160 spheroids were measured from micromolded, non-adhesive hydrogels with 256 circular recessions. The data are expressed as mean &#177; standard deviation. The asterisks indicate values of P obtained by ANOVA nonparametric and unpaired followed by multiple comparisons (**P &#60; 0.001; ****P &#60; 0.0001). Scale bars = 100 &#181;m. Abbreviation: ASC = adipose tissue stem/stromal cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63430/63430fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63430/63430fig02.jpg" /> 
Figure 2:&#160;ASC spheroids showed high cell viability. (A) Fluorescence micrographs of viability assay of ASC spheroids at day 7 from micromolded, non-adhesive hydrogels with 256 circular recessions. Live and dead cells are observed in green and red, respectively. Death control is shown in the inset. (B) Section of an ASC spheroid stained by hematoxylin and eosin from micromolded, non-adhesive hydrogels with 81 circular recessions. The fixed spheroids samples were embedded in paraffin, and samples were cut into 5 &#181;m thickness sections using a microtome. (C) Compressive resistance force measured in &#181;N of ASC spheroids at day 7. The data were collected from five spheroids and expressed as mean &#177; standard deviation, according to previously described methodology5. The asterisks indicate the value of P obtained by two-way ANOVA, nonparametric and unpaired, followed by multiple comparisons. Scale bars = 50 &#181;m. Abbreviation: ASC = adipose tissue stem/stromal cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63430/63430fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

large-scale-automated-production-adipose-derived-stem-cell-spheroids

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.5K Views.  Federal University of Rio de Janeiro. De betekenis van het protocol is het verminderen van menselijke fouten en microbiologische besmetting. Daarnaast verantwoordelijk voor het produceren van duizenden sferoïden voor 3D-bioprinting. Het belangrijkste voordeel van deze kwestie is echter dat het de arbeidsuren heeft geoptimaliseerd.De softwareparameters zijn cruciaal omdat ze de uiteindelijke kwaliteit van de sferoïden kunnen beïnvloeden. En het beste advies zou zijn om alleen tests uit te voeren met celkweekmedia. De vis...

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

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

Developmental Biology

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

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 ...

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 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.

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

Biochemistry

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 ...

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 ...

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...

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

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