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...

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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 diam...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>Low Glucose Dulbecco&#39;s Modified Eagle Medium (DMEM LOW)</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 m...

Watch this Scientific Journal Video about Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting at JoVE.com

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 ...

The significance of the protocol relies reducing human errors and microbiology contamination. In addition, responsible to produce thousands of spheroids for 3D bioprinting approaches. The main advantage of this matter, though, is that it's optimized the labor hours.The software parameters are crucial because they can influence the final quality of the spheroids. And the best advice would be to perform tests only with cell culture media. The visual demonstration of this method is critical because it is an old traditional 3D cell culture method based on Begin by harvesting the stem cell suspension with a 10 milliliter serological pipette and transferring it to a 50 milliliter centrifuge tube.Then, centrifuge at 400 RCF for 5 minutes to obtain the ASC pellet. Resuspend the cell pellet using DMEM-Low containing 10%FBS. After performing cell count, take ASCs in separate 15 milliliter centrifuge tubes to seed the 81 and 256 recessions micromolded with non-adherent hydrogel.Refer to the text manuscript for details of the number of cells used. Add 500 microliters of sterile 2%ultra pure agarose solution in the center of a silicone mold containing 81 or 256 circular recesses. After 40 minutes, unmold the ultra pure agarose from the silicone mold and place it in a well of a 12-well plastic plate.Add two milliliters of DMEM-Low in the well with the micromolded agarose and incubate at 37 degrees Celsius with 5%atmospheric carbon dioxide supply for at least 12 hours before seeding the ASCs. Ensure that the laminar flow is on and the cabinet's airflow is working properly. Confirm that the equipment is connected to the correct voltage and the tablet is connected to the equipment.Check whether the height of the cabinet protection glass is at the same level as the sensor marking of the equipment. Press the on or off button on the left side of the equipment. Then, wait for the tablet and the equipment to start.Position the tip boxes, the rack for centrifuge tubes, and the plate containing the micromolded agarose hydrogel in the workspace of the equipment. In the software opened in the tablet, click LabWare Editor. Set up a virtual work table and choose the positions of the pipettes, tip boxes, the rack for the plastic tubes, and the plates.Click on the switch to procedure"button to include all the parameters and commands carried out by the equipment throughout the experiment. Wait for a toolbar and a procedure list to open in the software. Initially, to indicate the commands, incorporate the number of samples to be pipetted and drag it to the procedure list.Then, click on the reagent transfer"command button on the software to transfer the ASC suspension to the wells of the 12-well plate containing the micro molds and drag it to the procedure list. Click on properties"to set up the start"and end"positions for the equipment to transfer the sample. Wait for the software to return to the Work Table page.Set up the parameters for automated seeding of the ASCs as described in the text manuscript. Select water as the standard liquid type. Click on the options"button to set up the parameters to create a homogeneous suspension of the ASCs as described in the text manuscript.Click on the button with a check"symbol on the top bar of the software to ensure there is no programming error. Click on the play"button on the top bar of the software to start the program. Let the equipment start as programmed.And wait for it to make a sound indicating it has finished. Collect the 12-well plate then incubate it at 37 degrees Celsius with a 5%atmospheric carbon dioxide supply for at least 18 hours, to allow complete and compact spheroids formation. Manually harvest the spheroids after one, three, and seven days of culture for analysis.Flush the medium with a micro pipette to release the spheroids from the micromolded non-adherent agarose hydrogel. Collect the spheroids manually using a micro pipette and transfer them to a 15 milliliter centrifuge tube. A total of 85 and 160 spheroids were measured from micromolded non-adhesive hydrogels with 81 and 256 circular recessions, respectively.The automatic pipette system seeded the ASC cell suspension into 12 wells of a single 12-well plate in 15 minutes. Using the 81 micromolded non-adherent hydrogel, produced 972 spheroids in this study. While the 256 micromolded non-adherent hydrogel, produced 3072 spheroids.ASC spheroids were analyzed for the homogeneity of their size and shape. ASC steroids from micro molds with 81 recessions showed homogeneous diameter during the culture period. In contrast to ASC steroids from micro molds with 256 recessions, the smallest and largest diameters ratio was close to one in ASC spheroids from micro molds with 81 and 256 recessions.The viability, morphology and force analyses provided evidence for the successful, large scale production of ASC spheroids. The most case of is to ensure that the problem was solved abruptly without errors.

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

Research

JoVE Journal

Bioengineering

2.4K Görüntüleme.  Federal University of Rio de Janeiro. Protokolün önemi, insan hatalarını ve mikrobiyoloji kontaminasyonunu azaltmaya dayanmaktadır. Buna ek olarak, 3D biyobaskı yaklaşımları için binlerce sferoid üretmekten sorumludur. Bununla birlikte, bu konunun en büyük avantajı, çalışma saatlerini optimize etmesidir.Yazılım parametreleri çok önemlidir çünkü sferoidlerin nihai kalitesini etkileyebilirler. Ve en iyi tavsiye, sadece hücre kültürü ortamı ile testler yapmak olacaktır. Bu yöntemin görsel göst...