The authors thank Shu Hashimoto, Aoi Yoshida, and Taeko Ohta at Doshisha University for their generous assistance with the experimental setup. This work was supported by KAKENHI (15H02121, 15K05400, 25103012, 50587441) and by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities. This study was also supported by a Polish grant from the KNOW (Leading National Research Centre) Scientific Consortium &#34;Healthy Animal - Safe Food&#34;, decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015..

1. Preparation of Cells
<ol>
	<li>Maintain NAMRU mouse mammary gland epithelial cells (NMuMG cells) with 5 mL of D-MEM containing 10% (v) fetal bovine serum (FBS) and 1% (v) Penicillin-Streptomycin (P/S) in a 25 cm3 flask. Remove Dulbecco&#39;s modified Eagle&#39;s medium (D-MEM), containing 10% FBS and 1% P/S.</li>
	<li>Add 3-5 mL of 37 &#176;C phosphate-buffered saline (PBS) (-) to the flask. Note that the pH of PBS is 7.1-7.3.</li>
	<li>Remove all the PBS from the flask using an aspirator.</li>
	<li>Add 1.5 mL of 37 &#176;C trypsin (0.25%, w/v) to the flask.</li>
	<li>Incubate the flask for approximately 1-2&#160;minutes at 37 &#176;C in a CO2 incubator.</li>
	<li>Add 3.5 mL of D-MEM containing 10% FBS and 1% P/S to the flask. Pipette to mix.</li>
	<li>Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge it for 3 min at room temperature. Note that the rotation radius and rotation speed of the centrifuge are 16.6 cm and 1500 rpm, respectively. Under this condition, the relative centrifugal force is 417 x g.</li>
	<li>Add 5 mL of fresh D-MEM (10% FBS and 1% P/S) to the flask after aspirating the medium (if required, cryopreserve the cells with cryopreservation solution, following the manufacturer&#39;s directions).</li>
</ol>
2. Preparation of Dextran (DEX)
<ol>
	<li>Prepare 80 mg/mL of DEX solution by mixing 10 mL of D-MEM (10% FBS, 1%P/S) and 0.8 g of DEX. Note that the DEX solution can be filtered with a syringe filter (0.22 &#181;m) after the cells have cultured for a sufficient time.</li>
	<li>Prepare a cell suspension containing 40 mg/mL DEX medium by mixing 200 &#181;L of DEX solution and 200 &#181;L of the cell suspension prepared in 2.1. Note that the number density of cells in the solution is approximately 2.3&#215;105 cells/mL.</li>
</ol>
3. Preparation for Laser and Microscopy
<ol>
	<li>Turn on the laser (continuous wave, 1,064 nm wavelength) (Figure 1a). Note that the use of a laser beam with a wavelength in the red to near-infrared region is most effective; this wavelength region is called the diagnostic and therapeutic window10 since it is minimally absorbed by cells.</li>
	<li>Double-click the software icon.</li>
	<li>Double-click the icons for the &#9312; camera, &#9313; light-emitting diode (LED), &#9314; focus adjust, and &#9315; moving stage. The displays corresponding to &#9312;-&#9315; will show up (Figure 1b).</li>
</ol>
4. Cell Manipulation using the Laser Trapping System
<ol>
	<li>Place 20 &#181;L of the sample prepared in step 2.2 on the bottom cover glass (0.17 mm thickness, size = 30 mm &#215; 40 mm), and cover it with the top cover glass (0.17 mm thickness, size = 18 mm &#215; 18 mm), with separation provided by two spacers (0.17 mm thickness, size = 10 mm &#215; 24 mm), as shown in Figure 2. Note that these glasses do not require a special coating.</li>
	<li>Place the sample cell prepared in step 4.1 on the lower objective Lens (water immersion with ca. 10 &#956;l, magnification = 60X, working distance = 0.28 mm, numerical aperture = 1.2)).</li>
	<li>Attach the upper objective lens (water immersion with ca. 10 &#956;l, magnification = 60X, working distance = 2 mm, numerical aperture = 1.0) at the top of the sample cell.</li>
	<li>Turn on the LED light by clicking icon 2 (Figure 1b).</li>
	<li>Adjust the distance between the sample and the lower objective lens by clicking the icons on panel 3 (Figure 1b) until it is in focus.</li>
	<li>Irradiate the laser beams at Position 1 and Position 2 (Figure 1B) of the sample by clicking icons I, II, and III (Figure 1b).</li>
	<li>Set the intensity of each laser beam to 1,500 mW by entering this value at icon IV (Figure 1b).</li>
	<li>Move the sample stage by clicking icon 4 indicating directions (Figure 1b) until a cell is trapped at Position 1 (Figure 1b).</li>
	<li>Drag the cursor indicating Position 2 (Figure 1b) until another cell is trapped at Position 2.</li>
</ol>
5. Construction of a 3-Dimensional (3D) Cell Structure
<ol>
	<li>Manipulate a single cell so that it is in contact with another cell. Maintain this condition for 300 s; i.e., each cell is exposed to a laser for 300 s.</li>
	<li>Record the X-Y axis shown in Figure 1b.</li>
	<li>Construct an arbitrary 2D cell assembly by trapping and transporting another cell to the cells.</li>
	<li>Construct a 3D cellular assembly by moving the stage up and down.</li>
	<li>Confirm that the assembly remains stable after the laser is switched off.</li>
</ol>

<ol>
	<li>Aloysious, N., Nair, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+survival+and+function+of+islet-like+clusters+differentiated+from+adipose+stem+cells+on+a+three-dimensional+natural+polymeric+scaffold:+an+in+vitro+study.">Enhanced survival and function of islet-like clusters differentiated from adipose stem cells on a three-dimensional natural polymeric scaffold: an in vitro study.</a> Tissue Engineering Part A. 20 (9-10), 1508-1522 (2014).</li><li>Fatehullah, A., Tan, S. H., Barker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+as+an+in+vitro+model+of+human+development+and+disease.">Organoids as an in vitro model of human development and disease.</a> Nature Cell Biology. 18 (3), 246-254 (2016).</li><li>Kirkham, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+assembly+of+complex+cellular+microenvironments+using+holographic+optical+tweezers.">Precision assembly of complex cellular microenvironments using holographic optical tweezers.</a> Scientific Reports. 5, 8577 (2015).</li><li>Liu, Y., Rayatpisheh, S., Chew, S. Y., Chan-Park, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+endothelial+cells+on+3D+cultured+smooth+muscle+cells+in+a+biomimetic+hydrogel.">Impact of endothelial cells on 3D cultured smooth muscle cells in a biomimetic hydrogel.</a> ACS Applied Materials &amp; Interfaces. 4 (3), 1378-1387 (2012).</li><li>Liu, Z. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dextran-based+hydrogel+formed+by+thiol-Michael+addition+reaction+for+3D+cell+encapsulation.">Dextran-based hydrogel formed by thiol-Michael addition reaction for 3D cell encapsulation.</a> Colloids and Surfaces B. 128, 140-148 (2015).</li><li>McKee, C., Perez-Cruet, M., Chavez, F., Chaudhry, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+three-dimensional+culture+system+for+long-term+expansion+of+embryonic+stem+cells.">Simplified three-dimensional culture system for long-term expansion of embryonic stem cells.</a> World Journal of Stem Cells. 7 (7), 1064-1077 (2015).</li><li>Sadovska, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+heterotypic+spheroid+model+for+studying+extracellular+vesicle-mediated+tumor+and+immune+cell+communication.">A novel 3D heterotypic spheroid model for studying extracellular vesicle-mediated tumor and immune cell communication.</a> Biochemical and Biophysical Research Communications. , (2017).</li><li>Srinivasan, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Salivary+Human+Stem/Progenitor+Cells+Undergo+Microenvironment-Driven+Acinar-Like+Differentiation+in+Hyaluronate+Hydrogel+Culture.">Primary Salivary Human Stem/Progenitor Cells Undergo Microenvironment-Driven Acinar-Like Differentiation in Hyaluronate Hydrogel Culture.</a> Stem Cells Translational Medicine. 6 (1), 110-120 (2017).</li><li>Yoshida, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+living+cells+to+construct+a+3D+single-cell+assembly+without+an+artificial+scaffold.">Manipulating living cells to construct a 3D single-cell assembly without an artificial scaffold.</a> Polymers. 9, 319 (2017).</li><li>Anderson, R. R., Parrish, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+photothermolysis:+precise+microsurgery+by+selective+absorption+of+pulsed+radiation.">Selective photothermolysis: precise microsurgery by selective absorption of pulsed radiation.</a> Science. 220, 524-527 (1983).</li><li>Hashimoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+Stable+Cell-Cell+Contact+without+a+Solid/Gel+Scaffold:+Non-invasive+Manipulation+by+Laser+under+Depletion+Interaction+with+a+Polymer.">Formation of Stable Cell-Cell Contact without a Solid/Gel Scaffold: Non-invasive Manipulation by Laser under Depletion Interaction with a Polymer.</a> Chemical Physics Letters. 655, 11-16 (2016).</li><li>Rauch, P., J&#228;hnke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Tweezers+for+Quantitative+Force+Measurements+and+Live+Cell+Experiments.">Optical Tweezers for Quantitative Force Measurements and Live Cell Experiments.</a> Microscopy Today. 22, 26-30 (2014).</li><li>Ichikawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tilt+control+in+optical+tweezers.">Tilt control in optical tweezers.</a> Journal of Biomedical Optics. 13, 010503 (2008).</li></ol>

The authors have nothing to disclose.

The present study shows a concrete application of our recent reports9,11 on the use of soluble polymers for the construction of 3D single-cell assemblies. Such assemblies are stably formed in the bulk solution when the number of cells is up to 10, and can be held by a single laser beam. Assemblies precipitate on the glass surface when there are more than 10 cells. Although the experiments are still in a primitive stage, we expect that the novel methodology could be a powerful tool for the construction of next-generation 3D single-cell assemblies, which are indispensable for progress in the fields of cell biology and regenerative medicine.
In a solution containing no polymer, cells repel each other due to the electrostatic repulsion arising from the surface charge, the hydration repulsion force, the glycocalyx repulsion effect, and membrane undulation. Our previous study showed that cell pairs can be stable for a long time when the cells are treated with PEG. More importantly, the successful transport of a cell pair to a region without PEG, after the cells had been held in contact for 5 minutes in PEG, suggests that cellular contact is maintained in a stable manner. This is well explained in terms of the depletion effect11, and essentially the same mechanism applies to the cellular assemblies generated using DEX9. Our current results suggest that other kinds of natural macromolecules could also be used to construct stable 3D cellular assemblies.
For the prompt transport of cells, the concentration of polymer is important. Generally, the viscosity of the solution drastically increases when the polymer is dissolved above the overlap concentration. Under this condition, it is difficult to manipulate cells using optical tweezers. Hence, the experiment should be performed below the overlap concentration. For a DEX solution, the overlap concentration is ca. 50 mg/mL (the kinetic viscosity is 5.5 mm2/s). As shown in Ref. 9, a stable cellular assembly was observed when the concentration of DEX was 10 mg/mL to 40 mg/mL. This result suggests that the depletion effect is sufficiently large to maintain stable cell-cell contact even when the DEX concentration is lower than the overlap concentration. It has been shown that the addition of DEX does not affect cell viability up to 40 mg/mL 9.
The establishment of a method for the construction of 3D cellular assemblies is important in the field of regenerative medicine, since mimicking an in vivo cellular microenvironment by structuring single cells may facilitate stem cell-derived tissue formation. So far, we have used the present protocol to construct cellular assemblies using Neuro2A cells9 in addition to NMuMG cells. We hope to establish an experimental methodology for constructing 3D cellular assemblies of a larger number of cells of various morphologies. The optical tweezers system developed by Ichikawa et al.13 would be applicable for this purpose since the orientation of the cells can be controlled. Further trials along these lines should be promising.

While human tissues are composed of several assemblies of cells and can help to maintain homeostasis of the body, single cells by themselves also play important roles via cell-to-cell&#12288;interaction. Therefore, it is important to elucidate how single cells can be stimulated by external signals and how they transfer such signals to other adherent cells. For this purpose, several methods have been established for the construction of single-cell-based 3-dimensional (3D) assemblies1,2,3,4,5,6,7,8. However, the materials that are used to construct cellular assemblies can still be improved. For example, synthetic gels and polymers including polyethylene glycol (PEG) possess certain chemical physicochemical properties and may affect target cells (e.g., toxicity).
We recently reported a novel system that could generate a single-cell-based 3D assembly of cells using dextran (DEX) by establishing stable cell-cell contact9. We considered that this technology could be useful in several research fields, including regenerative medicine and even cancer biology. In this report, we describe how we manipulate single cells and construct 3-dimensional (3D) cellular assemblies in the presence of various hydrophilic biomacromolecules including DEX without an artificial scaffold.

<table><tbody><tr><td>Microscope IX71</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>Dextran(200,000; molecular biology-grade)</td><td>Wako</td><td>CAS.NO&amp;nbsp; 9004-54-0</td><td></td></tr><tr><td>Laser Trapping System (NanoTracker 2)</td><td>JPK Instruments</td><td>S/N T-05-0200</td><td></td></tr><tr><td>Upper Objective Lens</td><td>Olympus</td><td>LUMPLFLN60XW</td><td></td></tr><tr><td>Lower Objective Lens</td><td>Olympus</td><td>UPLSAPO60XW</td><td></td></tr><tr><td>Top Cover Glass</td><td>MATUNAMI</td><td>C022401</td><td></td></tr><tr><td>Intermediate Cover Glass (Spacer)</td><td>MATUNAMI</td><td>-</td><td>custom-made (size = 10mm&amp;times;10mm, thickness = 0.17mm)</td></tr><tr><td>Bottom Cover Glass</td><td>MATUNAMI</td><td>C030401</td><td></td></tr><tr><td>Camera</td><td>The Imaging Source</td><td>DFK 31AF03</td><td></td></tr><tr><td>Software</td><td>JPK Instruments</td><td>NanoTracker2 PFM software</td><td></td></tr><tr><td>NMuMG cells&amp;nbsp;</td><td>RIKEN BRC</td><td>RCB2868</td><td></td></tr><tr><td>PBS</td><td>Wako</td><td>166-23555</td><td></td></tr><tr><td>Cell banker</td><td>Nippon Zenyaku Kogyo</td><td>ZR621</td><td></td></tr><tr><td>D-MEM</td><td>Wako Pure Chem. Ind., Japan</td><td>044-29765</td><td></td></tr><tr><td>FBS</td><td>Cell Culture Biosci., Nichirei Biosci. Inc., Japan</td><td>172012-500ML</td><td></td></tr><tr><td>Trypsin</td><td>Thermo Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Wako Pure Chem. Ind., Japan</td><td>161-23181</td><td></td></tr></tbody></table>

manipulating living cells to construct stable 3d cellular assembly without artificial scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

Figure 1 shows the microscope and software used in this study. Figure 2 is a schematic representation of the procedure for placing the sample solution containing cells. Figure 3 demonstrates the formation of a pyramid structure using double-beam optical tweezers. If the experiment is successful, these cellular assemblies remain stable even after the laser is switched off.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57815/57815fig1.jpg" /> 
Figure 1: (a) The control system for the Laser Trapping System (NanoTracker2 (11)). The system is activated by turning on the laser switch following steps &#9312;-&#9314;. (b) The software for controlling the Laser Trapping System. The camera, LED light, focus adjust, and moving stage are activated by clicking icons &#9312;, &#9313;, &#9314;, and &#9315;, respectively. The microscopic image is displayed in panel 1. The on/off control for the LED is in panel 2. The focus is controlled in panel 3. The laser beams are irradiated at Positions 1 and 2 by clicking icons I to IV. The details of this Laser Trapping Systemare provided in Ref. (12). <a href="https://www.jove.com/files/ftp_upload/57815/57815fig1large.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/57815/57815fig2.jpg" /> 
Figure 2: Representative schematic for placing the slide glass. 20 &#181;L of the sample (cell suspension containing dextran) is placed on the slide and used for laser manipulation. <a href="https://www.jove.com/files/ftp_upload/57815/57815fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57815/57815fig3.jpg" /> 
Figure 3: a) Assemblies of epithelial cells (NMuMG) of an intended shape in a medium with DEX (40 mg/mL): a pyramid is shown as an example of a 3D cluster. b) A schematic figure of the pyramid-shaped 3D cellular assembly is also shown. <a href="https://www.jove.com/files/ftp_upload/57815/57815fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold at JoVE.com

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

manipulating-living-cells-to-construct-stable-3d-cellular-assembly

Research

JoVE Journal

Biochemistry

6.1K Views.  Doshisha University. Deze nieuwe methodologie stelt ons in staat om driedimensionale samenstellingen te construeren, aangepast van de gewenste individuele cellen, in een waterige bufferoplossing die niet-specifiek hydrofiele polymeer bevat, door stabiel celcelcontact tot stand te brengen. In deze video laten we zien hoe we de gewenste enkele cellen manipuleren en 3D cellulaire assemblages bouwen zonder kunstmatige steiger. Hier tonen we de experimentele procedure in ons medium met Dextran als voorbeeld.Om ...

Video: Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

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

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

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

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

Bioengineering

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Biology

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

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 ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

Scalable Generation of Uniform Cell Spheroids Using a 3D Acoustic Assembly Device

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

Author Spotlight: Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture 

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

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