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).

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	<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.

אנו מציגים שיטה מהירה וגמישה להפקת spheroids תאי 3D מודל הארכיטקטורה של רקמות in vivo באמצעות ריאגנטים זול וזמין באופן נרחב. הפרוטוקול שלנו מנצל את המאפיינים הלא-ציטוטוקסיות וקידום-הידבקות של 8 MC, 9 לתווך צבירת תא ולמזער היווצרות monolayer התא....

ביולוגית רלוונטית הערכת התנהגות תאים הסרטניים היא מאתגר באמצעות דו ממדים מסורתיות (2D) מתודולוגיות תרבית תאים, בין שאר משום אלה אינם משקפים באופן נאות את המיקרו-הסביבה הניידת שנמצאה in vivo. גישות חלופיות שילוב רכיבים תאים מטריקס לתוך התרבות (למשל, מבחנים קאמריים בוידן) מייצגות פיסיולוגי יותר של סביבת רקמות in vivo. עם זאת, הם יכולים להיות מוגבלים הערכת התנהגות תאים בודדת, ואינו לשחזר את המורכבות בצירופי vivo של אינטראקציות התא-מטריקס תאי תאים תורמים לצמיחת רקמות או גידול 1, 2, 3. שימוש spheroids הרב התאי הוא גישה אחרונה יותר משחזרת את הארכיטקטורה הקומפקטית של צמיחת תאי vivo ב 1,4. Spheroids יכול לשמש כדי לחקור אינטראקציות תא-מטריצה של תאים נורמלים, אבל יכול לשמש גם אנלוגים גידול מודל מאפיינים של התקדמות גידול, כגון צמיחה גרורתי או סמי התנגדות 4. Spheroids עלול להיוצר על ידי ההתפשטות של תאים בודדים משוקעים בתוך גוש 5, או יותר במהירות, על ידי קידום הצבירה של מספר תאים כדי ליצור אשכול תא בודד (למשל, ירידה תלויה, שיטות צנטריפוגה) 6, 7. טכניקות צבירת תא קיימות מסוימות עשויות לדרוש חומרים יקרים או ציוד מיוחד. בנוסף, יש spheroids אלה מגוון רחב של גדלי מורפולוגיות ועלול להיות קשה לייצר בכמויות גדולות, לערוך השוואות בין תנאי גידול או טיפולים קשים. לבסוף, spheroids המיוצרים בשיטות אלה יכול להיות קשה לבודד מן extracel חלבונייםמטריקס lular שבה הם משובצים לשימוש ביישומים אחרים. כאן אנו מתארים מתודולוגיה צבירת תא חזקה ובקלות לשינוי להיווצרות של spheroids תא בגודל העקבי באמצעות זמינים מסחריים U-תחתונות צלחות תא-דוחה מטריצת קידום הידבקות אינרטי, תאי מתיל. מרגע היווצרותם, spheroids רב-תאיים אלה מבודדים בקלות לשימוש במגוון רחב של יישומים. הפרוטוקול גם להתאים בקלות ליצור spheroids באמצעות שגשוג תאים, אשר ניתן להשתמש כדי להעריך תהליכי תאים אחרים. כאן, אנו מציגים מבחני פלישת תא, לכמת ידי מכתים immunofluorescence, וכן assay anoikis, כמו יישומים לדוגמא של שני אלה פרוטוקולי היווצרות אליפטית שונים.

<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

תרבית תאים חד שכבתי כראוי אינו מודל ההתנהגות in vivo של רקמות, אשר כרוך אינטראקציות תאים תאים ותא-מטריצה מורכבת. תלת ממדי (3D) תא טכניקות תרבות הם חידוש אחרון פותח כדי לענות על החסרונות של תרבית תאים חסידה. בעוד מספר טכניקות ליצירת אנלוגים רקמות במבחנה פותחו, שיטות אלה הן מורכבים לעתים קרובות, יקרים להקים, דורשות ציוד מיוחד, והם מוגבלים בדרך כלל על ידי תאימות עם סוגי תאים מסוימים בלבד. כאן אנו מתארים פרוטוקול מהיר וגמיש לאיסוף תאים לתוך spheroids 3D רב-תאיים של גודל עקבי תואמת צמיחה של מגוון רחב של גידולי שורות תאים נורמלים. אנו מנצלים ריכוזים שונים של תאית בסרום מתיל (MC) לקדם דור אליפטית-עצמאית למעגן ולמנוע היווצרות של monolayers התא באופן מאוד לשחזור. תנאים אופטימליים indivשורות תאים idual יכולה להיות מושגת על ידי התאמת MC או ריכוזים בסרום בטווח הבינוני היווצרות אליפטית. הספרואידים 3D שנוצרו ניתן לאסוף לשימוש במגוון רחב של יישומים, כולל איתות תא או ביטוי במחקרי גן, הקרנת סמים מועמדת, או בחקר תהליכים תאיים כגון פלישת תאים סרטניים והגירה. הפרוטוקול גם להתאים בקלות ליצור spheroids המשובט מתאים יחידים, והוא יכול להיות מותאם על מנת להעריך צמיחה למעגן-עצמאי-התנגדות anoikis. בסך הכל, הפרוטוקול שלנו מספק שיטה וניתנת לשינוי להפקה וניצול spheroids תאי 3D כדי לשחזר את המיקרו-סביבת 3D של רקמות מודל צמיחת vivo של תאים נורמלים גידולים.

<html> אנו מתארים שיטה גמישה ויעילה כדי ליצור spheroids הבדיד באמצעות צלחות תא דוחים ומדיה היווצרות אליפטית השלימה עם MC. תחת התנאים המתאימים של MC וסרום, תאים בודדים להתיישב לדבוק יחד במרכז הבאר כדי ליצור spheroids עם דבקות מזערית על קרקעית הבאר. שימוש בפרוטוקול ...

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.

שיטת Aggregation Cell יעילה וגמישה עבור הפקת 3D אליפטית

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

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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.

תא פשוט תלוי תרבות Drop פרוטוקול הדור של Spheroids 3D

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>יצירת כדורי רקמות באמצעות מכשיר דמוי בול מודפס בתלת מימד

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

יצירת כדורי רקמות באמצעות מכשיר דמוי בול מודפס בתלת מימד

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.

ניתוח פנוטיפי חצי אוטומטי של תרביות תאים ספרואידים תלת-ממדיים פונקציונליים

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.

פלטפורמת מעבדה-על-CD ליצירת רב-תאיים תלת מימדי

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.

ייצור אוטומטי בקנה מידה גדול של כדורי תאי גזע שמקורם בשומן להדפסה ביולוגית תלת-ממדית

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

ייצור מדרגי של כדורי תאים אחידים באמצעות התקן הרכבה אקוסטית תלת מימדית

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.

מחבר זרקור: פיתוח שיטת הרכבה אקוסטית ללא פיגומים לתרבית תאי תלת מימד באיכות גבוהה 

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.

התקן הרכבה אקוסטית תלת מימדי לייצור המוני של ספרואידים סלולריים

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

היווצרות ספרואידים רב-תאיים לניתוח מטבולי והיפוקסיה מבוסס 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.

מחבר זרקור: סטנדרטיזציה של שיטות היווצרות כדוריד לניתוח מטבולי וחמצון באמצעות מיקרוסקופ הדמיה פלואורסצנטית לכל החיים

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.

ייצור ומיקרוסקופ הדמיה לכל החיים של תאים חיים מרובי פרמטרים (FLIM) של ספרואידים רב-תאיים

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

שיטת Aggregation Cell יעילה וגמישה עבור הפקת 3D אליפטית

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תא פשוט תלוי תרבות Drop פרוטוקול הדור של Spheroids 3D

יצירת כדורי רקמות באמצעות מכשיר דמוי בול מודפס בתלת מימד

יצירת כדורי רקמות באמצעות מכשיר דמוי בול מודפס בתלת מימד

ניתוח פנוטיפי חצי אוטומטי של תרביות תאים ספרואידים תלת-ממדיים פונקציונליים

פלטפורמת מעבדה-על-CD ליצירת רב-תאיים תלת מימדי

ייצור אוטומטי בקנה מידה גדול של כדורי תאי גזע שמקורם בשומן להדפסה ביולוגית תלת-ממדית

התקן הרכבה אקוסטית תלת מימדי לייצור המוני של ספרואידים סלולריים

התקן הרכבה אקוסטית תלת מימדי לייצור המוני של ספרואידים סלולריים

התקן הרכבה אקוסטית תלת מימדי לייצור המוני של ספרואידים סלולריים

ייצור ומיקרוסקופ הדמיה לכל החיים של תאים חיים מרובי פרמטרים (FLIM) של ספרואידים רב-תאיים