This work was supported by grants from the Czech Ministry of Education, Youth, and Sports (LO1304) and the Technological Agency of the Czech Republic (TE01020028). The authors would like to thank Dr. Lakshman Varanasi for taking the still images of environmental lids.

1. Preparation of Agarose-coated Plates
<ol>
	<li>Weigh 0.75 g of low-melting point agarose and add it to 100 mL of McCoy&#39;s 5A medium (with or without phenol red) without serum. Heat the solution in a microwave and swirl every 1-2 min to completely dissolve the agarose. Autoclave the solution to sterilize it.</li>
	<li>Cool the autoclaved agarose to about 70 &#176;C and filter it through a 500 mL, 0.22 &#181;m filter top by vacuum filtration in a laminar flow box. Aliquot the 0.75% filtered agarose solution (FAS) into smaller volumes if not using the entire solution at once. Store this ready-to-use agarose solution aseptically in a cold room or 4 &#176;C fridge for up to 4 weeks.</li>
	<li>Attach a small tube with a plastic- or metal-tipped dispensing cassette to a Combi reagent dispenser and prime the cassette with 70% ethanol (EtOH) and then with sterile phosphate-buffered saline (PBS) in a flow box.</li>
	<li>Prior to use, heat a stored aliquot of FAS in the microwave to melt it. Prime the dispensing cassette with agarose solution and coat 384-well, tissue culture (TC)-treated &#34;special&#34; microplates with 15 &#181;L of FAS. Allow the agarose in the plate to cool for 15-20 min before seeding the cells. Store the agarose-coated plates aseptically, wrapped in a polyethylene bag in a cold room or at 4 &#176;C and away from direct light. 
	NOTE: Avoid repeated heating of the 0.75% FAS to prevent a change in the concentration of agarose in the stock solution. Agarose-coated plates can be stored for up to 2 weeks when stored under the above-mentioned conditions. The FAS does not require heating during the coating of plates.</li>
	<li>Clean the dispensing cassette by priming it with 70-80 &#176;C sterile water to remove any remaining agarose in the cassette tips and tubes.</li>
</ol>
2. Cell Culture and MCTS Formation
<ol>
	<li>Culture human colorectal carcinoma HCT116 cells, as described previously8.</li>
	<li>Remove the required number of agarose-coated, 384-well, TC-treated microplates from cold storage and equilibrate them to room temperature (RT) for 15 min.</li>
	<li>Prepare the Combi reagent dispenser for cell seeding by priming a standard tube dispensing cassette with 70% EtOH and sterile PBS. Adjust the seeding volume to the required &#181;L and the dispensing speed to medium using the manual setting buttons on the dispenser. 
	NOTE: The entire cell seeding process is performed under sterile conditions and in a laminar flow box.</li>
	<li>Dissociate adherent cells from the tissue culture flask using a recombinant cell-dissociation enzyme. Make a cell suspension stock in a sterile beaker with seed cells at a density of 2.5 x 104 cells/mL per well in 50 &#181;L of complete growth medium. When seeding more than one 384-WP, stir the cells using a magnetic stirrer to prevent them from settling to the bottom of the beaker.</li>
	<li>Allow the plates to rest for 30 min at RT and then centrifuge for 15 min at 4 x g.</li>
	<li>Meanwhile, take an evaporation-reducing environmental lid and fill it with 8 mL of sterile H2O or 5% dimethyl sulfoxide (DMSO) in the short sides (left and right) using a 5 mL pipette (Figure 1A). First, dispense 4 mL of filling liquid into the left-side trough by sweeping the pipette tip slowly up and down. Repeat this step with the right-side trough. 
	NOTE: Ensure that the liquid added to the side troughs does not merge at the center of the lid, and leave a gap for gas exchange (Figure 1A). Adding an excess amount of H2O results in the seepage of H2O to the exterior of the lid and subsequently into the outer wells of the 384-well TC plates.</li>
	<li>Fill the liquid reservoir of the 384-well TC plate with sterile H2O and replace the regular plate lids with the liquid-filled environmental lids (Figure 2A).</li>
	<li>Place the plates in a 37 &#176;C rotary incubator with 95% humidity, 5% CO2, and 20% O2, and allow the cells to aggregate into MCTSes for 4 days. Avoid opening the incubator door for too long in the subsequent days in order to prevent the humidity level from dropping abruptly.</li>
</ol>
3. Medium Exchange Using a Robotic System
<ol>
	<li>On day 4 following MCTS formation, add 30 &#181;L of pre-warmed medium per well using an automated microplate washer dispenser. Allow the MCTSes to grow for additional 3 days. Replace the medium regularly every 3 days, until they attain the desired size for experimentation.</li>
	<li>To aspirate a defined volume of medium from each well, empirically adjust the z-height of the washer manifold. Aspirate 30 &#181;L of medium per well and replace it with 30 &#181;L of fresh, pre-warmed medium. 
	NOTE: Set the dispensing speed and the speed at which the washer manifold travels down into wells at the lowest rate to minimize turbulence in the wells.</li>
</ol>
4. High-content Imaging of MCTS and Semi-automated Image Analysis
<ol>
	<li>Image the MCTS in a high-content automated imaging system using a 4X air objective (N.A. 16).</li>
	<li>Set the exposure time to 11 ms and the binning to 4 x 4. Adjust the number and spacing of the z-stacks and the pixel binning as desired for the experiment to capture an entire MCTS per well.</li>
	<li>Process the images stepwise, as described in the &#34;readme,&#34; using an in-house routine written in programming language. 
	NOTE: The .m code and .txt readme files are available as supplementary code files (Figure 1B). The routine measures the MCTS area, major and minor axes, perimeter, and solidity from the 2D images.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55403/55403fig1.jpg" /> 
Figure 1: Preparation of environmental lids and a flowchart of the semi-automatic routine. (A) Image of an evaporation-reducing environmental lid filled with the correct (left) and excess (right) amount of filling liquid (here, 5% DMSO). The arrow indicates the short-side trough for adding liquid. The asterisk shows the gap in the middle of the lid following the addition of the correct volume of DMSO. (B) A workflow showing the steps involved in the semi-automated image analysis routine. <a href="http://cloudfront.jove.com/files/ftp_upload/55403/55403fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+need+for+complex+3D+culture+models+to+unravel+novel+pathways+and+identify+accurate+biomarkers+in+breast+cancer.">The need for complex 3D culture models to unravel novel pathways and identify accurate biomarkers in breast cancer.</a> Adv. Drug Deliv. Rev. , 69-70 (2014).</li><li>Fischbach, C., Kong, H. J., Hsiong, S. X., Evangelista, M. B., Yuen, W., Mooney, D. 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The authors have nothing to disclose.

Coating 384-Well TC Plates with Filtered Agarose
The standard practice in LOT is to use a 1-1.5% low-melting point agarose to coat the plates, which requires the agarose and/or dispensing unit to be kept heated to prevent the gelling of the agarose6. The gelling of the agarose is of potential concern while preparing multiple plates using liquid dispensing cassettes with small tubing apertures ranging between 0.2 and 0.4 mm in diameter. To overcome the potential issue of cl...

Cancer cells in tumors are physiologically arranged in a complex, 3-dimensional (3D) structure surrounded by extracellular matrix and interacting cells. As nearly all cells in tissues reside in a 3D environment, the need for more physiologically relevant in vitro tumor models that mimic tumor traits has resulted in the development of several 3D culture techniques1,2,3. These models are now becoming fundamental research tools for studying the role of the tumor microenvironment on metastasis and cell response to therapeutics in 3D2. Moreover, compared to 2-dimensional (2D) cell cultures4, 3D models allow for a better understanding of tumor-stroma interactions, which affect cell signaling pathways.
Multicellular tumor spheroids (MCTSes) of cancer cell lines are frequently used in 3D cell culture models due to their relative closeness to in vivo tumors. Out of the several techniques in use, the liquid-overlay technique (LOT) of MCTS generation on agarose-coated plates has gained significant interest for lead optimization and target validation5,6,7,8,9,10,11. This is evident from the recent studies that were successfully able to run pilot screens of compound libraries in MCTS cultures using LOT6,7. However, well-to-well variability in MCTS morphology and growth due to the evaporation-induced uneven loss of medium are common hurdles that accompany the LOT using microtiter plates (MPs). Consequently, the formation of non-uniform MCTSes compromises the significance and relevance of data from pharmacological assays8,12,13. In addition to the reproducibility issues, another practical problem that affects LOT-based high-throughput assays is the coating of the MPs with agarose when using automatic liquid dispensing units. Although the dispensing unit can be kept heated to prevent the gelling of agarose, the clogging of the dispensing cassette and tubing is a potential concern for robotic systems6.
To overcome some of these challenges, we have recently devised a few modifications in the LOT for MCTS culture8. These modifications are mainly based on possible ways to prevent uneven medium loss from the MPs using instruments that are commonly found in high-throughput screening laboratories. A detailed procedure of the modified LOT for the generation of uniformly sized and reproducible MCTSes across 384-well plates (WPs) is presented here. The manuscript also presents a semi-automated routine for the evaluation of MCTS size, particularly in partially disintegrated, drug-treated MCTSes that do not have a clearly defined boundary for the measurement of cross-sectional area.

<table><tbody><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9414</td><td>Low-melting</td></tr><tr><td>McCOY&#039;s 5A Medium</td><td>Sigma-Aldrich</td><td>M8403</td><td></td></tr><tr><td>&amp;ldquo;rapid&amp;rdquo; Filtermax filter</td><td>TPP</td><td>99505</td><td>0.22 &amp;mu;m, 500 mL</td></tr><tr><td>Multidrop&amp;trade; Combi Reagent Dispenser&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>5840300</td><td></td></tr><tr><td>Small Tube Dispensing cassette&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>24073295</td><td>Metal tip&amp;nbsp;</td></tr><tr><td>384-well TC plate&amp;nbsp;</td><td>PerkinElmer</td><td>6057308</td><td>Plate type- CellCarrier</td></tr><tr><td>Standard Tube Dispensing Cassette</td><td>Thermo Fisher Scientific</td><td>24072670</td><td></td></tr><tr><td>MicroClime Environmental Lid</td><td>Labcyte</td><td>LLS-0310</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D4540</td><td></td></tr><tr><td>Rotary Incubator (SteriStore )</td><td>HighRes Biosolutions</td><td>23641</td><td>Serial No.: D00384</td></tr><tr><td>Microplate Washer Dispenser&amp;nbsp;</td><td>BioTek</td><td>Unspecified</td><td>Model: EL406&amp;nbsp;</td></tr><tr><td>High-Content Imaging System (CellVoyager )</td><td>Yokogawa Electric Corporation</td><td>Unspecified</td><td>Model: CV7000</td></tr><tr><td>Orange G</td><td>New England Biolabs</td><td>B7022S</td><td></td></tr><tr><td>TrypLE&amp;trade; Express recombinant cell dissociation reagent</td><td>Thermo Fisher Scientific</td><td>12604021</td><td>Phenol red free</td></tr></tbody></table>

evaporation-reducing culture condition increases the reproducibility of multicellular spheroid formation in microtiter plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium, particularly from peripheral wells, is a frequently encountered issue in MPs with small culture volumes. Substantially improved culture conditions, such as incubators with well-controlled temperature/humidification systems and evaporation-reducing MPs and plate lids, reduce the significant loss of medium across wells8. To measure the relative evaporation, equal volumes of Orange G (OG) were added to each well, and the change in OG absorba...

Watch this Scientific Journal Video about Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates at JoVE.com

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

evaporation-reducing-culture-condition-increases-reproducibility

Research

JoVE Journal

Cancer Research

6.9K Views.  Palacky University in Olomouc. The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

Video: Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

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 ...

Bioengineering

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Developmental Biology

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 ...

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 ...

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

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

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

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

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

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

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

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

Precise, High-throughput Analysis of Bacterial Growth

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells