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 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...

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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

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2.4K Views.  Federal University of Rio de Janeiro. 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.

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

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 ...

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

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 ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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 ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

Formulating and Characterizing an Exosome-based Dopamine Carrier System

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an active role in intercellular cargo and communication. Exosomes are mostly found in body fluids such as plasma, cerebrospinal fluid, urine, saliva, amniotic fluid, colostrum, breast milk, joint fluid, semen, and pleural acid. Considering the size of exosomes, it is thought that they may play an important role in central nervous system diseases because they can pass through the blood-brain barrier (BBB). Hence, this study aimed to develop an exosome-based nanocarrier system by encapsulating dopamine into exosomes isolated from Wharton's jelly mesenchymal stem cells (WJ-MSCs). Exosomes that passed the characterization process were incubated with dopamine. The dopamine-loaded exosomes were recharacterized at the end of incubation. Dopamine-loaded exosomes were investigated in drug release and cytotoxicity assays. The results showed that dopamine could be successfully encapsulated within the exosomes and that the dopamine-loaded exosomes did not affect fibroblast viability.

Here, we aimed to obtain a formulation by dopamine loading of the isolated exosomes of stem cells from Wharton's jelly mesenchymal stem cells. Exosome isolation and characterization, drug loading into the resulting exosomes, and the cytotoxic activity of the developed formulation are described in this protocol.

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an ...