This research was supported by the Basic Science Research Program (2016R1D1A1B03934418) and the Bio &#38; Medical Technology Development Program (2018M3A9H1023141) of the NRF, and funded by the Korean government, MSIT.

1. Centrifugal microfluidic-based spheroid (CMS) culture chip fabrication
<ol>
	<li>Make PC molds for the top and bottom layers of the CMS culture chip by CNC machining. Detailed dimensions of the chip are given in Figure 1.</li>
	<li>Mix PDMS base and PDMS curing agent at a ratio of 10:1 (w/w) for 5 min and place in a desiccator for 1 h to remove air bubbles.</li>
	<li>After pouring the PDMS mixture into the molds of the CMS culture chip, remove air bubbles for 1 h more and cure in a heat chamber at 80 &#176;C for 2 h.</li>
	<li>Place them in the vacuumed plasma cleaner with the surfaces to be bonded facing up and expose them to air-assisted plasma at a power of 18 W for 30 s.</li>
	<li>Bond the two layers of the CMS culture chip and place it in the heat chamber at 80 &#176;C for 30 min to increase adhesion strength.</li>
	<li>Sterilize the CMS culture chip in an autoclave sterilizer at 121 &#176;C and 15 psi.</li>
</ol>
2. Cell preparation
<ol>
	<li>Thaw the 1 mL of the vial containing 5 &#215; 105&#8211;1 &#215; 106 hASCs or MRC-5s cells in a water bath at 36.5 &#176;C for 2 min.</li>
	<li>Add 1 mL of Dulbecco&#8217;s Modified Eagle Medium (DMEM) to a vial and gently mix with a 1,000 &#956;L pipette.</li>
	<li>Put 15 mL of the DMEM prewarmed to 36.5 &#176;C into a 150 mm diameter Petri dish using a pipette and add the cells from the vial.</li>
	<li>After 1 day, aspirate the DMEM and replace with 15 mL of fresh DMEM. Subsequently, change the media every 2 or 3 days.</li>
	<li>To detach the cells from the Petri dish, add 4 mL of trypsin to the Petri dishes and place them in an incubator at 36.5 &#176;C and 5% CO2 for 4 min.</li>
</ol>
3. Monoculture spheroid formation
<ol>
	<li>Put 2.5 mL of 4% (w/v) pluronic F-127 solution into the inlet hole of the CMS culture chip (Figure 2A) while rotating the chip at 500&#8211;1,000 rpm and then rotate the chip at 4,000 rpm for 3 min using the CMS system (Figure 2B). 
	NOTE: &#160;The pluronic coating prevents cell attachment to the inlet port while the chip rotates. Make sure air is not trapped in the microwells.</li>
	<li>Incubate the CMS culture chip filled with pluronic solution overnight at 36.5 &#176;C in 5% CO2.</li>
	<li>Remove the pluronic solution, wash out the remaining pluronic solution with DMEM, and dry the chip for 12 h on a clean bench.</li>
	<li>Add 2.5 mL of DMEM to the CMS culture chip and rotate the chip at ~4,000 rpm for 3 min for prewetting the inside of the chip.</li>
	<li>Stop the rotation and pull out 100 &#956;L of DMEM to make room for injecting the cell suspension.</li>
	<li>Add 100 &#956;L of cell suspensions that contain either 5&#160;&#215; 105 hASCs or 8&#215; 105 MRC-5s by pipetting Uniformly distribute the cells by pipetting 3&#8211;5x for resuspension.</li>
	<li>Rotate the chip at 3,000 rpm for 3 min to trap cells in each microwell by centrifugal force. 
	NOTE: &#160;Excessive rotational speed can cause cell escape through solution ejection holes.</li>
	<li>Culture the cells for 3 days in the incubator at 36.5 &#176;C, &#62;95% humidity, and 5% CO2, rotating at 1,000&#8211;2,000 rpm. Change culture medium every day.</li>
</ol>
4. Coculture spheroid formation
<ol>
	<li>Concentric spheroids formation
	<ol>
		<li>Add the first cells, 2.5 &#215; 105 hASCs, and rotate the chip at 3,000 rpm. After 3 min, add the second cells, 4 &#215; 105 MRC-5s, and rotate the chip at 3,000 rpm for 3 min. Inject a total of 100 &#956;L of cell suspensions by pipetting. When the cells are injected, shift the rotational speed to 500-1,000 rpm.</li>
		<li>Culture the cells in the incubator at 36.5 &#176;C, &#62;95% humidity%, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm. The concentric spheroids are created within 24 h. For long-term culture, change the culture medium every day.</li>
	</ol>
	</li>
	<li>Janus spheroid formation
	<ol>
		<li>Add 100 &#956;L of cell suspensions containing the first cells, 2.5 &#215; 105 hASCs, by pipetting while the chip rotates at 500&#8211;1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.</li>
		<li>Incubate the chip at 36.5 &#176;C, &#62;95% humidity, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm for 3 h.</li>
		<li>Add 100 &#956;L of the cell suspensions containing the second set of cells, 4 &#215; 105 MRC-5s, by pipetting while the chip rotates at 500&#8211;1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.</li>
		<li>Culture the cells in the incubator at 36.5 &#176;C, &#62;95% humidity, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm. The Janus spheroids are created within 24 h. For long-term culture, change the culture medium every day.</li>
	</ol>
	</li>
	<li>Sandwich spheroid formation
	<ol>
		<li>Add 100 &#956;L of cell suspensions containing the first cells, 1.5 &#215; 105 hASCs, by pipetting while the chip rotates at 500&#8211;1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.</li>
		<li>Incubate the chip at 36.5 &#176;C, &#62;95% humidity, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm for 3 h.</li>
		<li>Add 100 &#956;L of cell suspensions containing the second cells, 3 &#215; 105 MRC-5s, by pipetting while the chip rotates at 500&#8211;1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.</li>
		<li>Incubate the chip at 36.5 &#176;C, &#62;95% humidity%, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm for 3 h.</li>
		<li>Add 100 &#956;L of cell suspensions containing the third cells, 1.5 &#215; 105 hASCs, by pipetting while the chip rotates at 500&#8211;1,000 rpm. Then, rotate the chip at 3,000 rpm for 3 min.</li>
		<li>Culture the cells in the incubator at 36.5 &#176;C, &#62;95% humidity%, and 5% CO2 by rotating at 1,000&#8211;2,000 rpm. The sandwich spheroids are created within 12 h. For long-term culture, change the culture medium every day.</li>
	</ol>
	</li>
</ol>
5. Cell staining
<ol>
	<li>Warm the cell fluorescence dye to room temperature (20 &#176;C).</li>
	<li>Add 20 &#956;L of anhydrous dimethylsulfoxide (DMSO) per vial to make a 1 mM solution.</li>
	<li>Dilute the fluorescence to a final working concentration of 1 &#956;M using DMEM.</li>
	<li>Add the fluorescence to the cell suspension and gently resuspend using a pipette.</li>
	<li>Incubate 20 min at 36.5 &#176;C, humidity of &#62;95%, and 5% CO2.</li>
</ol>

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The authors have nothing to disclose.

The CMS is a closed system in which all injected cells enter the microwell without waste, making it more efficient and economical than conventional microwell-based spheroid generation methods. In the CMS system, the media is replaced every 12&#8211;24 h through a suction hole designed to remove the media in the chip (Figure 3A). During the media suction process, barely any media escapes from inside the microwell due to the surface tension between the media and the wall of the microwell. A us...

It is easier to simulate biological in vivo microenvironments with three-dimensional (3D) spheroid cell culture than with two-dimensional (2D) cell culture (e.g., conventional Petri dish cell culture) to produce more physiologically realistic experimental results1. Currently available spheroid formation methods include the hanging drop technique2, liquid-overlay technique3, carboxymethyl cellulose technique4, magnetic force-based microfluidic technique5, and the use of bioreactors6. Although each method has its own benefits, further improvement in reproducibility, productivity, and generating coculture spheroids is necessary. For example, while the magnetic force-based microfluidic technique5 is relatively inexpensive, the effects of strong magnetic fields on living cells must be carefully considered. The benefits of spheroid culture, particularly in the study of mesenchymal stem cell differentiation and proliferation, have been reported in several studies7,8,9.
The centrifugal microfluidic system, also known as lab-on-a-CD (compact disc), is useful for easily controlling the fluid inside and exploiting the rotation of the substrate and has thus been utilized in biomedical applications such as immunoassays10, colorimetric assays for detecting biochemical markers11, nucleic acid amplification (PCR) assays, automated blood analysis systems12, and all-in-one centrifugal microfluidic devices13. The driving force controlling the fluid is the centripetal force created by rotation. Additionally, multiple functions of mixing, valving, and sample splitting can be done simply in this single CD platform. However, compared to the above-mentioned biochemical analysis methods, there have been fewer trials applying CD platforms to culture cells, especially spheroids14.
In this study, we show the performance of the centrifugal microfluidic-based spheroid (CMS) system by monoculture or coculture of human adipose-derived stem cells (hASC) and human lung fibroblasts (MRC-5). This paper describes in detail our group&#39;s research methodology15. Thus, the spheroid culture lab-on-a-CD platform can be easily reproduced. A CMS generating system comprising a CMS culture chip, a chip holder, a DC motor, a motor mount, and a rotating platform, is presented. The motor mount is 3D printed with acrylonitrile butadiene styrene (ABS). The chip holder and rotating platform are CNC (computer numerical control) machined with the PC (polycarbonate). The rotational speed of the motor is controlled from 200 to 4,500 rpm by encoding a PID (proportional-integral-derivative) algorithm based on pulse-width modulation. Its dimensions are 100 mm x 100 mm x 150 mm and it weighs 860 g, making it easy to handle. Using the CMS system, spheroids can be generated under various gravity conditions from 1 x g to 521 x g, so the study of cell differentiation promotion under high gravity can be extended from 2D cells16,17 to 3D spheroid. Coculture of various types of cells is also a key technology for effectively mimicking the in vivo environment18. The CMS system can easily generate monoculture spheroids, as well as coculture spheroids of various structure types (e.g., concentric, Janus, and sandwich). The CMS system can be utilized not only in simple spheroid studies but also in 3D organoid studies, to consider human organ structures.

<table><tbody><tr><td>3D printer</td><td>Cubicon</td><td>3DP-210F</td><td></td></tr><tr><td>Adipose-derived mesenchymal stem cells (hASC)</td><td>ATCC</td><td>PCS-500-011</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Gibco</td><td>15240-062</td><td>Contained 1% of completed medium and buffer</td></tr><tr><td>CellTracker Green CMFDA</td><td>Thermo Fisher Scientific</td><td>C2925</td><td>10 mM</td></tr><tr><td>CellTracker Red CMTPX</td><td>Thermo Fisher Scientific</td><td>C34552</td><td>10 mM</td></tr><tr><td>Computer numerical control (CNC) rotary engraver</td><td>Roland DGA</td><td>EGX-350</td><td></td></tr><tr><td>DC motor</td><td>Nurielectricity Inc.</td><td>MB-4385E</td><td></td></tr><tr><td>Dimethylsulfoxide (DMSO)</td><td>Sigma Aldrich</td><td>D2650</td><td></td></tr><tr><td>Dulbecco&#039;s modified eaggle&#039;s medium (DMEM)</td><td>ATCC</td><td>30-2002</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (D-PBS)</td><td>ATCC</td><td>30-2200</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ATCC</td><td>30-2020</td><td>Contained 10% of completed medium</td></tr><tr><td>human lung fibroblasts (MRC-5)</td><td>ATCC</td><td>CCL-171</td><td></td></tr><tr><td>Inventor 2019</td><td>Autodesk</td><td></td><td>3D computer-aided design program</td></tr><tr><td>Petri dish &amp;Phi; 150 mm</td><td>JetBiofill</td><td>CAD010150</td><td>Surface Treated</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma Aldrich</td><td>11/6/9003</td><td>Dilute with phosphate buffered saline to 4% (w/v) solution</td></tr><tr><td>Polycarbonate (PC)</td><td>Acrylmall</td><td>AC15PC</td><td>200 x 200 x 15 mm</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dowcorning</td><td>Sylgard 184</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>12604021</td><td></td></tr></tbody></table>

lab-on-a-cd platform for generating multicellular three-dimensional spheroids

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

The 6 cm diameter CMS culture chip (Figure 2) was successfully made following the above protocol. First, the chip was made separately from a top layer and a bottom layer and then bonded together by plasma bonding. Resulting spheroids can be easily gathered by detaching the chip. The channel of the CMS culture chip comprises an inlet port and central, slide, and microwell regions (Figure 3). The cell, medium, and pluronic solutions are injected through an inlet h...

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

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

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

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6.2K Views.  Chung-Ang University. We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

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

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 ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

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

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

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

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

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

Biochemistry

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Cancer Research

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

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

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

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

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

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

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

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix.&#160;This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 &#176;C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization.&#160;The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) ...

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.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

Biology

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

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

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

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

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

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

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

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

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

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications