The authors thank M. Gordon of the Queen's University Biomedical Imaging Centre for assistance. This work was supported by operating grants from the Cancer Research Society of Canada (19439) and the Canadian Institutes for Health Research (MOP-142303) (LMM), and by Ontario Graduate Scholarships and studentships from the Terry Fox Research Institute Training Program in Transdisciplinary Cancer Research (SMM, EYL), and by a Craig Jury Summer Studentship (SMM).

<ol>
	<li>Zimmermann, M., Box, C., Eccles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+vs.+three-dimensional+in+vitro+tumor+migration+and+invasion+assays.">Two-dimensional vs. three-dimensional in vitro tumor migration and invasion assays.</a> Methods Mol Biol. 986 (986), 227-252 (2013).</li><li>Albini, A., Noonan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'chemoinvasion'+assay,+25+years+and+still+going+strong:+the+use+of+reconstituted+basement+membranes+to+study+cell+invasion+and+angiogenesis.">The 'chemoinvasion' assay, 25 years and still going strong: the use of reconstituted basement membranes to study cell invasion and angiogenesis.</a> Curr Opin Cell Biol. 22 (5), 677-689 (2010).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat. Rev. Mol. Cell Biol. 8 (10), 839-845 (2007).</li><li>Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., de Boer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+as+a+tool+for+creating+3D+complex+tissues.">Spheroid culture as a tool for creating 3D complex tissues.</a> Trends Biotechnol. 31 (2), 108-115 (2013).</li><li>Carletti, E., Motta, A., Migliaresi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+for+tissue+engineering+and+3D+cell+culture.">Scaffolds for tissue engineering and 3D cell culture.</a> Methods Mol Biol. 695, 17-39 (2011).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> J Vis Exp. (51), (2011).</li><li>Handschel, J. 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=Prospects+of+micromass+culture+technology+in+tissue+engineering.">Prospects of micromass culture technology in tissue engineering.</a> Head Face Med. 3, 4 (2007).</li><li>Stuckhoff, A. P., DelValle, L., Amini, S., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuronal Cell Culture Methods and Protocols Vol. 1078 Methods in Molecular Biology. 1078, 119-132 (2013).</li><li>Stewart, G. J., Wang, Y., Niewiarowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylcellulose+protects+the+ability+of+anchorage-dependent+cells+to+adhere+following+isolation+and+holding+in+suspension.">Methylcellulose protects the ability of anchorage-dependent cells to adhere following isolation and holding in suspension.</a> Biotechniques. 19 (4), 598-604 (1995).</li><li>Vinci, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+establishment+and+analysis+of+three-dimensional+tumor+spheroid-based+functional+assays+for+target+validation+and+drug+evaluation.">Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation.</a> BMC Biol. 10, 29 (2012).</li><li>Nagelkerke, A., Bussink, J., Sweep, F. C. G. J., Span, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+of+breast+cancer+cells:+How+to+go+three-dimensional.">Generation of multicellular tumor spheroids of breast cancer cells: How to go three-dimensional.</a> Anal. Biochem. 437 (1), 17-19 (2013).</li></ol>

The authors have nothing to disclose.

نقدم طريقة سريعة ومرنة لإنتاج الأجسام الشبه الكروية خلية 3D في تصميم نموذج لهندسة الأنسجة في الجسم الحي باستخدام الكواشف مكلفة ومتاحة على نطاق واسع. بروتوكول لدينا يستغل الخصائص غير السامة للخلايا وتعزيز التصاق MC 8 و 9 للتوسط تجمي?...

تقييم بيولوجيا السلوك الخلايا السرطانية يتحدى باستخدام ثنائي الأبعاد (2D) منهجيات زراعة الخلايا التقليدية، في جزء منه لأن هذه لا تعكس بشكل كاف المكروية الخلايا الموجودة في الجسم الحي. النهج البديلة تتضمن مكونات المصفوفة خارج الخلية في الثقافة (على سبيل المثال، بويدن المقايسات غرفة) هي ممثلة أكثر من الناحية الفسيولوجية للفي الجسم الحي بيئة الأنسجة. ومع ذلك، فإنها يمكن أن تقتصر على تقييم سلوك الخلية الفردية، وليس تلخيص مجمع في مجموعات المجراة من خلية مصفوفة وخلية خلية التفاعلات التي تساهم في الأنسجة أو الورم النمو 1، 2، 3. استخدام الكروية المتعددة الخلايا هو نهج مؤخرا أن يستنسخ أكثر دقة الهندسة المعمارية المدمجة في الجسم الحي نمو الخلايا 1،4. الكروية يمكن استخدامها لتحقيق التفاعلات خلية مصفوفة من الخلايا الطبيعية، ولكن يمكن أن تعمل أيضا نظائرها ورم لنموذج خصائص تطور الورم، مثل النمو المتنقل أو المقاومة للأدوية (4). ويمكن تشكيل الأجسام الشبه الكروية من انتشار الخلايا واحد جزءا لا يتجزأ من مصفوفة 5 أو أكثر بسرعة، من خلال تشجيع تجميع خلايا متعددة لتشكيل كتلة خلية واحدة (على سبيل المثال، والشنق قطرة، وأساليب الطرد المركزي) 6 و 7. قد تتطلب تقنيات خلية تجميع القائمة مواد مكلفة أو المعدات المتخصصة. وبالإضافة إلى ذلك، فإن هذه الأجسام الشبه الكروية لديها مجموعة واسعة من الأحجام والأشكال التضاريسية وقد يكون من الصعب إنتاج بكميات كبيرة، مما يجعل المقارنات بين ظروف النمو أو العلاجات الصعبة. وأخيرا، يمكن الكروية التي تولدها هذه الأساليب يكون من الصعب عزل من extracel البروتينيةمصفوفة lular التي هي جزء لا يتجزأ من أجل الاستخدام في تطبيقات أخرى. هنا، نحن تصف منهجية خلية تجميع قوية وسهولة التعديل على الإسراع في تشكيل الأجسام الشبه الكروية خلية الحجم باستمرار باستخدام لوحات المتاحة تجاريا U-أسفل الخلية طارد ومصفوفة تعزيز التصاق الخاملة، ميثيل السليلوز. تشكلت مرة واحدة، يتم عزل هذه الأجسام الشبه الكروية المتعددة الخلايا بسهولة لاستخدامها في مجموعة واسعة من التطبيقات. كما تكييفها بروتوكول بسهولة لتوليد الأجسام الشبه الكروية من خلال تكاثر الخلايا، والتي يمكن استخدامها لتقييم عمليات الخلايا الأخرى. هنا، وتبين لنا المقايسات غزو الخلايا، كميا عن طريق تلطيخ المناعي، وفحص anoikis، كمثال تطبيقات هذين مختلف البروتوكولات تشكيل كروي.

<table><tbody><tr><td>&lt;strong&gt;Buffers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x Phosphate buffered saline</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AM9625</td><td></td></tr><tr><td>Calcium Chloride Solution</td><td>Sigma-Aldrich</td><td>21114</td><td>Used for PBS&lt;sup&gt;+&lt;/sup&gt; wash buffer; Do not autoclave PBS&lt;sup&gt;+&lt;/sup&gt; wash buffer upon addition of calcium chloride</td></tr><tr><td>Magnesium Chloride Solution</td><td>Sigma-Aldrich</td><td>M1028</td><td>Used for PBS&lt;sup&gt;+&lt;/sup&gt; wash buffer; Do not autoclave PBS&lt;sup&gt;+&lt;/sup&gt; wash buffer upon addition of magnesium chloride</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Spheroid Formation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96-well U-bottom Cell-Repellent Plate</td><td>Greiner Bio-One</td><td>650970</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Sigma-Aldrich</td><td>D5546</td><td>For culturing SH-SY5Y, PANC-1, TPC-1 cell lines</td></tr><tr><td>F12K Medium</td><td>Thermo Fisher Scientific</td><td>2112722</td><td>For culturing TT cell line</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F1051</td><td>Filter prior to use to remove particulate contaminants</td></tr><tr><td>Methyl cellulose</td><td>Sigma-Aldrich</td><td>M7027</td><td>Prepare in water to 100 mg/mL</td></tr><tr><td>Roswell Park Memorial Institute Medium</td><td>Sigma-Aldrich</td><td>R8758</td><td>For culturing HCT-116, BxPC-3 cell lines</td></tr><tr><td>TrypLE Express</td><td>Thermo Fisher Scientific</td><td>12605028</td><td>Dissociation buffer</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Invasion Assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bovine Type I Collagen</td><td>Corning Incorporated</td><td>354231</td><td>Stock 3.1 mg/mL; Maintain on ice when in use</td></tr><tr><td>DMEM Phenol Red Free Low Glucose&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11054-20</td><td>Less background fluorescence compared to Phenol Red supplemented medium</td></tr><tr><td>Glial Cell Line Derived Neurotrophic Factor</td><td>Peprotech</td><td>450-10</td><td>Chemoattractant</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Immunofluorescence Microscopy&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>#1.5 Coverglass</td><td>Electron Microscopy Sciences</td><td>72225-01</td><td>For mounting excised spheroids</td></tr><tr><td>Alexa-Fluor 488 Phalloidin</td><td>Thermo Fisher Scientific</td><td>A12379</td><td>Used to stain actin at 1:200</td></tr><tr><td>Bovine Serum Albumin</td><td>Bioshop Canada Incorporated</td><td>ALB001</td><td>Used in BSA blocking buffer</td></tr><tr><td>Dabco 33-LV</td><td>Sigma-Aldrich</td><td>290734</td><td>Antifade</td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Bioshop Canada Incorporated</td><td>GLY001</td><td>Used in MOWIOL mounting medium</td></tr><tr><td>ImageJ Software</td><td>Freeware, NIH</td><td>-</td><td>Used for image analysis&amp;nbsp;</td></tr><tr><td>Microslides</td><td>VWR International</td><td>48312-024</td><td>For mounting excised spheroids</td></tr><tr><td>MOWIOL 4-88</td><td>EMD-Millipore</td><td>475904</td><td>Used in MOWIOL mounting medium</td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>EMD-Millipore</td><td>PX0055-3</td><td>Used in fixation buffer</td></tr><tr><td>Triton X-100</td><td>Bioshop Canada Incorporated</td><td>TRX777</td><td>Used in permeabilization buffer</td></tr></tbody></table>

an efficient and flexible cell aggregation method for 3d spheroid production

ثقافة الخلية أحادي الطبقة لا نموذجا كاف السلوك في الجسم الحي من الأنسجة، الذي ينطوي على خلية خلية وخلية مصفوفة التفاعلات المعقدة. ثلاثي الأبعاد تقنيات زراعة (3D) الخلية هي ابتكار حديث وضعت لمعالجة أوجه القصور في زراعة الخلايا الملتصقة. بينما العديد من التقنيات لتوليد نظائرها الأنسجة في المختبر تم وضعها، وهذه الأساليب هي في كثير من الأحيان معقدة ومكلفة لتأسيس، تتطلب معدات متخصصة، وتقتصر عموما التوافق مع فقط أنواع الخلايا معينة. هنا، نحن تصف بروتوكول سريع ومرن لتجميع الخلايا في الأجسام الشبه الكروية 3D متعددة الخلايا من حجم ثابت متوافق مع نمو مجموعة متنوعة من الأورام وخطوط الخلايا العادية. نحن نستخدم تركيزات مختلفة من المصل وميثيل السليلوز (MC) لتشجيع جيل كروي مستقل مرسى ومنع تشكيل الطبقات الوحيدة الخلية بطريقة استنساخه للغاية. الظروف المثلى لالكلاسيكي فرديخطوط الخلايا idual لا يمكن أن يتحقق من خلال تعديل MC أو تركيزات مصل في المتوسط ​​تشكيل كروي. الأجسام الشبه الكروية 3D المتولدة يمكن جمعها لاستخدامها في مجموعة واسعة من التطبيقات، بما في ذلك إشارات الخلية أو دراسات التعبير الجيني، مرشح فحص المخدرات، أو في دراسة العمليات الخلوية مثل غزو الخلايا السرطانية والهجرة. كما تكييف بروتوكول بسهولة لتوليد الأجسام الشبه الكروية نسيلي من الخلايا واحدة، ويمكن تكييفها لتقييم النمو المستقل مرسى وanoikis المقاومة. وعموما، يوفر بروتوكول لدينا وسيلة للتعديل بسهولة لتوليد واستخدام الكروية خلية 3D من أجل تلخيص المكروية 3D الأنسجة ونموذج للنمو في الجسم الحي من الخلايا الطبيعية والسرطانية.

<html> نحن تصف طريقة مرنة وفعالة لتوليد الأجسام الشبه الكروية منفصلة باستخدام ألواح الخلايا طارد وسائل الإعلام تشكيل كروي تستكمل مع MC. في ظل الظروف المناسبة من مولودية ومصل الدم، والخلايا الفردية تسوية والالتزام معا في مركز البئر لتشكيل الأجسام الشبه...

Watch this Scientific Journal Video about An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production at JoVE.com

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

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.

على طريقة تجميع خلية فعالة ومرنة ل3D كروي الإنتاج

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

an-efficient-flexible-cell-aggregation-method-for-3d-spheroid

Research

JoVE Journal

Biology

23.7K Views.  Queen's University Cancer Research Institute. 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. 

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

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.

إسقاط خلية بسيطة الشنق بروتوكول الثقافة للجيل 3D الأجسام الشبه الكروية

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

Bioengineering

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.

<meta charset="utf8"></head><body>توليد الأنسجة الكروية عبر جهاز يشبه الطوابع المطبوعة ثلاثية الأبعاد

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

توليد الأنسجة الكروية عبر جهاز يشبه الطوابع المطبوعة ثلاثية الأبعاد

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.

تحليل النمط الظاهري شبه الآلي لثقافات الخلايا الكروية 3D الوظيفية

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

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.

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.

منصة مختبر علي القرص المضغوط لتوليد خزان ثلاثي الابعاد متعدد الخلايا

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

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

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

الإنتاج الآلي على نطاق واسع من الخلايا الجذعية المشتقة من الدهون كروية للطباعة الحيوية 3D

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

جيل قابل للتطوير من كرويات الخلايا الموحدة باستخدام جهاز تجميع صوتي 3D

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

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

تسليط الضوء على المؤلف: تطوير طريقة تجميع صوتية خالية من السقالات لثقافة كروية خلية 3D عالية الجودة 

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

جهاز تجميع صوتي ثلاثي الأبعاد للتصنيع الشامل للكرويات الخلوية

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

تشكيل كروي متعدد الخلايا لتحليل التمثيل الغذائي ونقص الأكسجة القائم على FLIM

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.

تسليط الضوء على المؤلف: توحيد طرق تكوين كروية لتحليل التمثيل الغذائي والأوكسجين باستخدام المجهر التصويري مدى الحياة الفلوري

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.

الإنتاج والفحص المجهري للتصوير مدى الحياة للخلايا الحية متعدد المعلمات (FLIM) للكرويات متعددة الخلايا

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

على طريقة تجميع خلية فعالة ومرنة ل3D كروي الإنتاج

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إسقاط خلية بسيطة الشنق بروتوكول الثقافة للجيل 3D الأجسام الشبه الكروية

توليد الأنسجة الكروية عبر جهاز يشبه الطوابع المطبوعة ثلاثية الأبعاد

توليد الأنسجة الكروية عبر جهاز يشبه الطوابع المطبوعة ثلاثية الأبعاد

تحليل النمط الظاهري شبه الآلي لثقافات الخلايا الكروية 3D الوظيفية

منصة مختبر علي القرص المضغوط لتوليد خزان ثلاثي الابعاد متعدد الخلايا

الإنتاج الآلي على نطاق واسع من الخلايا الجذعية المشتقة من الدهون كروية للطباعة الحيوية 3D

جهاز تجميع صوتي ثلاثي الأبعاد للتصنيع الشامل للكرويات الخلوية

جهاز تجميع صوتي ثلاثي الأبعاد للتصنيع الشامل للكرويات الخلوية

جهاز تجميع صوتي ثلاثي الأبعاد للتصنيع الشامل للكرويات الخلوية

الإنتاج والفحص المجهري للتصوير مدى الحياة للخلايا الحية متعدد المعلمات (FLIM) للكرويات متعددة الخلايا