Name: generation of 3d tumor spheroids for drug evaluation studies
Uploaded: 2023-02-24T13:20:00
Description: Watch this Scientific Journal Video about Generation of 3D Tumor Spheroids for Drug Evaluation Studies at JoVE.com

We thank all the members of our laboratories for their critical input and suggestions. This research was supported by the Key Project of Jiangsu Commission of Health (K2019030). Conceptualization was conducted by C.W. and Z.C., the methodology was performed by W.H. and M.L., the investigation was performed by W.H. and M.L., the data curation was performed by W.H., Z.Z., S.X., and M.L., the original draft preparation was performed by Z.Z., J.Z., S.X., W.H., and X.L., the review and editing was performed by Z.C., project administration was performed by C.W. and Z.C., and funding acquisition was conducted by C.W. All the authors have read and agreed to the published version of the manuscript.

1. Spheroid construction
<ol>
	<li>Anti-adhesion treatment of the culture plate
	<ol>
		<li>Pipette 100 &#181;L of anti-adhesion reagent into each well of a 48-well plate with a U-shape well bottom, and keep for 10 min. After 10 min, aspirate the coating reagent, and wash twice with sterilized PBS.</li>
		<li>Put the culture plate in an incubator (37 &#176;C in humidified air with 5% CO2) until use.</li>
	</ol>
	</li>
	<li>Cell preparation, collection, and counting
	<ol>
		<li>Use the cultur...

<ol>
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I., Tannock, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+penetration+in+solid+tumours.">Drug penetration in solid tumours.</a> Nature Reviews Cancer. 6 (8), 583-592 (2006).</li><li>Baker, B. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+the+third+dimension:+How+3D+culture+microenvironments+alter+cellular+cues.">Deconstructing the third dimension: How 3D culture microenvironments alter cellular cues.</a> Journal of Cell Science. 125 (13), 3015-3024 (2012).</li><li>Br&#252;ningk, S. C., Rivens, I., Box, C., Oelfke, U., Ter Haar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tumour+spheroids+for+the+prediction+of+the+effects+of+radiation+and+hyperthermia+treatments.">3D tumour spheroids for the prediction of the effects of radiation and hyperthermia treatments.</a> Scientific Reports. 10, 1653 (2020).</li><li>Graves, E. E., Maity, A., Thu Le, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumor+microenvironment+in+non-small-cell+lung+cancer.">The tumor microenvironment in non-small-cell lung cancer.</a> Seminars in Radiation Oncology. 20 (3), 156-163 (2010).</li><li>Kunz-Schughart, L. A., Frreyer, J. 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(144), e59020 (2019).</li><li>Yazdanfar, 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=Simple+and+robust+image-baed+autofocusing+for+digital+microscopy.">Simple and robust image-baed autofocusing for digital microscopy.</a> Optics Express. 16 (12), 8670-8677 (2008).</li><li>Chen, Z., 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=Automated+evaluation+of+tumor+spheroid+behavior+in+3D+culture+using+deep+learning-based+recognition.">Automated evaluation of tumor spheroid behavior in 3D culture using deep learning-based recognition.</a> Biomaterials. 22 (272), 120770 (2021).</li><li>Boucherit, N., Gorvel, L., Olive, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tumor+models+and+their+use+for+the+testing+of+immunotherapies.">3D tumor models and their use for the testing of immunotherapies.</a> Frontiers in Immunology. 11, 603640 (2020).</li><li>Anastasiou, D., 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=Microenvironment+factors+shaping+the+cancer+metabolism+landscape.">Microenvironment factors shaping the cancer metabolism landscape.</a> British Journal of Cancer. 116 (3), 277-286 (2017).</li><li>Zhou, H., 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=Functions+and+clinical+significance+of+mechanical+tumor+microenvironment:+Cancer+cell+sensing,+mechanobiology+and+metastasis.">Functions and clinical significance of mechanical tumor microenvironment: Cancer cell sensing, mechanobiology and metastasis.</a> Cancer Communications. 43 (5), 374-400 (2022).</li><li>Zhu, G. G., 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=Targeting+KRAS+cancers:+From+druggable+therapy+to+druggable+resistance.">Targeting KRAS cancers: From druggable therapy to druggable resistance.</a> Molecular Cancer. 21 (1), 159 (2022).</li><li>Ando, Y., Mariano, C., Shen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+in+vitro+tumor+models+for+cell-based+immunotherapy.">Engineered in vitro tumor models for cell-based immunotherapy.</a> Acta Biomaterialia. 132, 345-359 (2021).</li><li>Timmins, N. E., Dietmair, S., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hanging-drop+multicellular+spheroids+as+a+model+of+tumor+angiogenesis.">Hanging-drop multicellular spheroids as a model of tumor angiogenesis.</a> Angiogenesis. 7 (2), 97-103 (2004).</li><li>Costa, E. C., 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=3D+tumor+spheroids:+An+overview+on+the+tools+and+techniques+used+for+their+analysis.">3D tumor spheroids: An overview on the tools and techniques used for their analysis.</a> Biotechnology Advances. 34 (8), 1427-1441 (2016).</li><li>Sant, S., Johnston, P. 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The microenvironment plays an important role in tumor growth. It may affect the provision of extracellular matrices, oxygen gradients, nutrition, and mechanical interaction and, thus, affect gene expression, signal pathways, and many functions of tumor cells19,20,21. In many cases, 2D cells do not produce such effects or even produce opposite effects, thus affecting the evaluation of drug treatments. However, the emergence of 3D...

Cancer is one of the diseases most feared by human beings, not least because of its high mortality rate1. In recent years, the possibility of treating cancer has increased as new therapies have been introduced2,3,4,5. Two-dimensional (2D) and three-dimensional (3D) in vitro models are used to study cancer in a laboratory setting. However, 2D models cannot immediately and accurately assess all of the important parameters that indicate antitumor sensitivity; therefore, they fail to fully represent in vivo interactions in drug therapy testing6.
Since 2020, the global three-dimensional (3D) culture market has been greatly boosted. According to one report from NASDAQ OMX, the global value of the 3D cell culture market will exceed USD 2.7 billion by the end of 2025. Compared with 2D culture methods, 3D cell culturing exhibits advantageous properties, which can be optimized not only for proliferation and differentiation but also for long-term survival7,8. By such means, in vivo cellular microenvironments can be simulated to obtain more accurate tumor characterization, as well as metabolic profiling, so that genomic and protein alterations can be better understood. Due to this, 3D test systems should now be included in mainstream drug development operations, especially those with a focus on screening and evaluating novel antitumor drugs. Three-dimensional growths of immortalized established cell lines or primary cell cultures in spheroid structures possess in vivo features of tumors such as hypoxia and drug penetration, as well as cell interaction, response, and resistance, and can be regarded as a stringent and representative model for performing in vitro drug screening9,10,11.
However, these 3D culture models also suffer from several problems that may take some time to solve. Cell spheroids can be formed using these protocols, but they differ in certain details, such as culture time or embedding gels12, so these constructed cell spheroids cannot be well controlled under a restricted size range. The size of the spheroids may influence the consistency of the viability test and imaging analysis. The growth microenvironments and growth factors also vary, which may lead to different morphologies due to differences in the differentiation among cells13. There is now an obvious need for a standard, simple, and cost-effective method for constructing all types of tumors with controlled sizes.
From another perspective, although homogeneous assays and high-content imaging approaches have been developed to evaluate morphology, viability, and growth rate, the high-throughput screening of 3D models remains a challenge for various reasons reported in the literature, such as the lack of uniformity in the position, size, and morphology of tumor spheroids14,15,16.
In the protocol presented here, we list each step in the construction of 3D tumor spheroids and describe a method for spheroid observation and analysis using a high-throughput, high-content imaging system that involves auto-focus, auto-imaging, and analysis, among other advantageous characteristics. We show how this method can produce 3D tumor spheroids of uniform size that are suitable for high-throughput imaging. These spheroids also demonstrate a high sensitivity to cancer drug treatment, and morphological changes in the spheroids can be monitored using high-content imaging. In summary, we demonstrate the robustness of this methodology as a means to generate 3D tumor constructs for drug evaluation purposes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5-10 &#956;L Pipette tips</td>
			<td>AXYGEN</td>
			<td>T-300</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5&#160;mL Boil proof microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>100-1000&#956;L Pipette tips</td>
			<td>KIRGEN</td>
			<td>KG1313</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tube</td>
			<td>Nest</td>
			<td>601052</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L Pipette tips</td>
			<td>AXYGEN</td>
			<td>T-200-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>3D gel</td>
			<td>Avatarget</td>
			<td>MA02</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well U bottom Plate</td>
			<td>Avatarget</td>
			<td>P02-48UWP</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>Nest</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>Alamar Blue</td>
			<td>Thermo</td>
			<td>&#160;DAL1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>STEMCELL</td>
			<td>#07010</td>
			<td></td>
		</tr>
		<tr>
			<td>Certified FBS</td>
			<td>BI</td>
			<td>04-001-1ACS</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>aladdin</td>
			<td>W433884-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>sigma</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel sofware&#160;</td>
			<td>Microsoft office</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad prism sofware&#160;</td>
			<td>GraphPad software&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High Content Microscope and SMART system</td>
			<td>Avatarget</td>
			<td>1-I01</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-A Supplement (100X)</td>
			<td>Gibco</td>
			<td>51300-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>P1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin Sol</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Scientific Fluoroskan Ascent</td>
			<td>Thermo</td>
			<td>Fluoroskan Ascent</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 Flask</td>
			<td>JET Biofil</td>
			<td>TCF012050</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, 0.25% (1X)</td>
			<td>Hyclone</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of 3d tumor spheroids for drug evaluation studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

Figure 1A,B shows the process used for constructing tumor spheroids in this study. We first seeded the cells in a 48-well U-bottom plate. This step is almost the same as that used in 2D cell culture. We kept the plate in a common incubator with water surrounding the wells so that the deposited cells started to form spheroids in a self-assembly process. Under normal operational conditions, most types of tumor spheroids were completely formed after 5 days when a targeted mediu...

Watch this Scientific Journal Video about Generation of 3D Tumor Spheroids for Drug Evaluation Studies at JoVE.com

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

This protocol demonstrated a standardized method of constructing three-dimensional tumors of spheroids. And this methodology provided a high circuit and high content analysis of 3D tumor constructs. By using a standard method of tumorous spheroid construction and a high throughput imaging and analysis system, the effectiveness and accuracy of drug tests formed on three-dimensional spheroids can be dramatically increased.In this protocol, we used AMG510 to treat NCI-H23 spheroid as an example. From the experiment, we could observe a significant effect of cancerous targeted drug on the tumor spheroids. To begin, pipette 100 microliters of anti adhesion reagent into each well of a 48 well plate with a U-shaped well bottom and keep for 10 minutes.After 10 minutes, aspirate the coating reagent and wash twice with sterilized PBS. Place the culture plate in an incubator at 37 degrees Celsius in humidified air with 5%carbon dioxide until use. Observe the cells under the microscope.Then, wash the cells cultured in a T25 flask twice with PBS to remove the culture medium and treat the expanded cells with one milliliter of 0.25%trypsin EDTA for one to two minutes in an incubator at 37 degrees Celsius, 5%carbon dioxide. Confirm the cell shape under the microscope. And then stop the trypsin treatment by aspirating the used trypsin EDTA suspension in the T25 flask and washing the cells with four milliliters of fresh medium.Transfer all of the suspension to a 15 milliliter tube and use one milliliter of fresh medium to wash down the residual cells and add it to the tube. Centrifuge the cells at 186.48 G for five minutes at room temperature and discard the supernatant. Add 10 milliliters of fresh medium to the cell pellet and gently pipette until the cells are in a homogeneous suspension.Aspirate 0.1 milliliters of cell suspension into a new centrifuge tube. Add 0.9 milliliters of fresh medium and then pipette the suspension well. Extract 10 microliters of the cell suspension for cell counting.Repeat this process once or twice and take an average value. Dilute the suspension to reach a final seeding density of 50, 000 cells per milliliter. Next, add 200 microliters of the cell suspension to each well of a 48 well U-bottom plate.Wrap the sealing film around the plate and centrifuge it at 119.35 G for five minutes at room temperature. After centrifugation, pull off the protective film and add five to eight milliliters of sterilized water into the water channel surrounding the wells. Incubate the plate at 37 degrees Celsius for five days.Do not change or supplement any water to the water channel during this period and observe the cell aggregation during the following five days. For the gel embedding, after taking out the frozen gel from the minus 20 degrees Celsius fridge, place it on an ice box for the whole time during the experiment. Observe the cell spheroids under the microscope.Before the gel embedding begins, the status of the spheroids should again be checked. Carefully remove 150 microliters of the medium and embed each spheroid in the gel by slowly adding the liquid gel from the wall side of the well while moving the pre-cooled pipette tip around and inside the well. Wait for five minutes and if the gel does not spread evenly, gently pipette the gel with a 10 microliter pipette tip.Each well contains a tumor spheroid, 25 microliters of 3.5 milligrams per milliliter gel, and 50 microliters of complete culture medium. Add 75 microliters of medium to the controls as well. Incubate the plate at 37 degrees Celsius for 30 minutes until the hydrogelation is fully completed.Confirm the gelation status under the microscope. Overlay 125 microliters of fresh medium on each sample and culture the spheroids for another 7 to 10 days. Prepare groups of spheroids with four to six wells each and choose at least three of them for analysis.Dissolve the drug according to the manufacturer's instructions and prepare 100 fold working solutions with DMSO. Use 0.1%DMSO as a positive control and add 125 microliters of drug-treated medium to each well. Place the plate back into the incubator at 37 degrees Celsius in humidified air with 5%carbon dioxide.At this stage, each well contains a tumor spheroid, 25 microliters of 3.5 milligrams per milliliter gel, and 175 microliters of the medium. The controls contain 200 microliters of the medium. Measure the spheroid viability using an alamarBlue assay kit according to the manufacturer's guidelines.Aspirate 100 microliters of the supernatant medium from each well to a new test plate. Then, measure the viability using a microplate photometer. Measure it on day one, day four, day seven, and day 10 after embedding the spheroids in the gel.Add 80 microliters of fresh medium to each well of the culture plate. Then replace another 100 microliters of the drug-treated medium. Ensure there are no remains of alamarBlue in the well.Aspirate 100 microliters of the medium out before imaging and place the plate on the stage. Obtain digital images of the spheroids using an automated microscope with a tenfold objective. The microscope can focus and centralize these spheroids automatically.Wait for the automatic imaging. Four images are acquired for each spheroid. An integrated image is formed and processed with the software connected to the high content imaging system.Click on the image patch process button and choose the integrated images in the software. Choose U net model and type in the conversion rate. Click at the bottom of the screen to start the image processing.Save the diameter, perimeter, and roughness data in the spreadsheet software. Finally, add 100 microliters of fresh medium with the drug and place the plate back into the incubator at 37 degrees Celsius in humidified air with 5%carbon dioxide. The brightfield images of NCI-H23 cell spheroids treated with different concentrations of AMG510 and automatically captured by a high content microscope are shown in this figure.The columns represent different days and the rows represent different drug concentrations. The results include three spheroids for each condition. Tumor spheroid viability of the AMG510 treated sample groups was measured on day one, day four, day seven, and day 10.The terminal cell viability of the samples with concentration gradients is shown in this figure. The tumor spheroid diameters were measured on day one, day four, day seven, and day 10. The spheroid growth ratio was defined as the terminal volume relative to the original volume and calculated using the spheroid diameters.The spheroid growth inhibition ratio was defined relative to the volume and calculated using the spheroid diameters. The tumor roughness was measured by the software on day one, day four, day seven, and day 10, indicating the invasiveness of the tumor spheroids. The spheroid's perimeter was located and drawn by the software using deep learning algorithms.The spheroid areas at the focal plane were then measured by image J and an excess perimeter index was calculated based on these data. Although this message is easy to follow, the samples still need to be handled carefully.

Research

JoVE Journal

Cancer Research

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

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 ...