Name: an efficient and flexible cell aggregation method for 3d spheroid production
Uploaded: 2017-03-27T15:00:00.000+00:00
Duration: 7 min 46 s
Description: Watch this Scientific Journal Video about An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production at JoVE.com

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.

우리는 저렴하고 광범위하게 이용할 시약을 이용하여 생체 조직의 구조를 모델링하는 3 차원 셀 구 상체를 제조하기위한 신속하고 융통성있는 방법을 제시한다. 우리 프로토콜 MC (8, 9)의 비 독성 및 접착 촉진 성질 세포 응집을 매개 세포 단층의 형성을 최소화하기를 이용한다. 동물 소스로부터 단리 된 단백질 - 기초 매트릭스는 달리, MC는 불활?...

종양 세포의 생물학적 행위 관련 사정이 적절히 생체 내에서 발견되는 세포 미세 환경을 반영하지 않기 때문에, 부분적으로는 기존의 2 차원 세포 배양 방법을 사용하여 도전하고있다. 배양 (예, 보이든 챔버 분석법)에 세포 외 기질 성분을 통합하는 대안 적 방법은 생체 조직 환경 이상의 생리 대표. 그러나, 개별 셀 동작의 평가에 한정 될 수 있으며, 조직 또는 종양 성장을 1, 2, 3에 기여하는 세포 기질 및 세포 - 세포 상호 작용의 생체 조합 복소 요점을 되풀이하지 않는다. 다세포 타원체의 사용은,보다 정확하게 생체 내 세포 성장 (1)의 콤팩트 한 구조를 재생하는 방법이다 최근4. 구 상체는 정상 세포의 세포 - 기질 상호 작용을 조사하기 위하여 사용될 수 있으며,뿐만 아니라, 전이성 성장 또는 약물 내성 4와 같은 종양 진행의 특성을 모델링하기 위해, 종양 유사체로서 작용할 수있다. 타원체가 매트릭스 5 또는 더 빠르게 내장 단일 세포의 증식에 의해 형성 될 수 있고, 다수의 세포의 응집을 촉진함으로써 단일 셀 클러스터 (예를 들어, 걸려 강하를 원심 분리 방법) (6, 7)을 형성한다. 기존의 세포 응집 기술은 비용이 많이 드는 재료 또는 특수 장비가 필요할 수 있습니다. 또한, 이러한 회전 타원체는 크기 및 모폴로지의 넓은 범위가 성장 조건이나 곤란 치료 간의 비교를, 대량 생산하기가 어려울 수있다. 마지막으로, 이러한 방법에 의해 생성 된 타원체는 단백질 extracel에서 분리하기가 어려울 수 있습니다적인 다공질 행렬을하는 그들은 다른 응용 프로그램에서 사용하기 위해 포함됩니다. 여기에서는 시판 U 바닥 셀 반발 판 불활성 접착 촉진 행렬 메틸 셀룰로오스를 이용하여 지속적으로 크기가 셀 타원체의 신속한 형성을위한 견고하고 용이하게 수정 세포 응집 방법을 설명한다. 형성 후에,이 다세포 타원체 용이하게 다양한 애플리케이션에서 사용하기 위해 분리된다. 프로토콜은 쉽게 다른 세포 프로세스를 평가하기 위해 사용될 수있다 세포 증식을 통해 구 상체를 생성하도록 구성된다. 여기, 우리는이 두 개의 서로 다른 회전 타원체 형성 프로토콜의 예 응용 프로그램과 같은 면역 형광 염색법에 의해 정량화 셀 침공 분석, 및 anoikis 분석은 보여줍니다.

<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

단층 세포 배양 충분히 복잡한 세포 - 세포 및 세포 - 기질 상호 작용을 포함한다 조직의 생체 내 행동을 모델링하지 않습니다. 3 차원 (3D) 세포 배양 기술을 접착 세포 배양의 단점을 해결하기 위해 개발 된 최근의 기술 혁신이다. 시험관 내에서 조직 유사체를 생성하기위한 몇 가지 기술이 개발되었지만,이 방법은 비용이 확립 자주 복잡 특수 장비를 필요로하고, 일반적으로 특정 유형의 세포와의 호환성에 의해 제한된다. 여기서는 종양 정상적인 다양한 세포주의 성장과 호환 일관된 크기 다세포 차원 타원체로 세포 응집을위한 신속하고 유연한 프로토콜을 설명한다. 우리 앵커리지 독립적 타원체 생성을 촉진하고, 높은 재현성으로 세포 단층의 형성을 방지하기 위해 혈청 및 메틸 셀룰로오스 (MC)의 다양한 농도를 사용한다. 개인 라지을위한 최적의 조건idual 세포주 회전 타원체 형성 매체 MC 또는 혈청 농도를 조정함으로써 달성 될 수있다. 생성 된 3D 타원체 셀 시그널링 또는 유전자 발현 연구 후보 약물 스크리닝, 또는 종양 세포의 침윤 및 이동과 같은 세포 과정의 연구 등의 다양한 애플리케이션에 사용하기 위해 수집 될 수있다. 프로토콜은 또한 용이하게 단일 세포 클론 타원체를 생성하도록 구성되고, 앵커리지 - 독립된 성장 anoikis 저항을 평가하도록 구성 될 수있다. 전체적으로, 우리는 프로토콜을 생성하고, 정상 및 종양 세포의 생체 내 성장을 조직의 3 차원 미세 요점을 되풀이하고 모델링하기 위해 3 차원 셀 타원체를 이용하는 쉽게 수정 방법을 제공한다.

우리는 세포 반발 판과 MC로 보충 회전 타원체 형성 미디어를 사용하여 개별 회전 타원체를 생성하는 유연하고 효율적인 방법을 설명합니다. MC 혈청의 적절한 조건 하에서, 각각의 세포가 정착 웰 바닥에 최소한으로 부착 구 상체를 형성하는 웰의 중심에 함께 부착. 이 프로토콜을 이용하여, 구 상체는 다양한 세포주 (도 2b)에서 발생 하였다. MC 혈청 농도 적정...

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.

3D 회전 타원체 생산을위한 효율적이고 유연한 셀 집계 방법

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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Biology

23.7K 조회수.  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. 

비디오: 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.

3D Spheroids의 생성을위한 간단한 한깅 드롭 세포 배양 프로토콜

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

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생체공학

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>3D 프린팅된 스탬프와 같은 장치를 통한 조직 스페로이드 생성

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

3D 프린팅된 스탬프와 같은 장치를 통한 조직 스페로이드 생성

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

생화학

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.

기능적 3D 스페로이드 세포 배양의 반자동 표현형 분석

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.

다세포 3차원 스페로이드 생성을 위한 랩 온 어 CD 플랫폼

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.

3D 바이오프린팅을 위한 지방유래 줄기세포 구상체의 대규모, 자동화 생산

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

3D Acoustic Assembly Device를 사용한 균일한 세포 스페로이드의 확장 가능한 생성

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.

저자 스포트라이트: 고품질 3D 세포 스페로이드 배양을 위한 스캐폴드가 없는 음향 조립 방법 개발 

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.

세포 스페로이드의 대량 생산을 위한 3차원 음향 조립 장치

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

FLIM 기반 대사 및 저산소증 분석을 위한 다세포 스페로이드 형성

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.

저자 스포트라이트: Fluorescence Lifetime Imaging Microscopy를 사용한 대사 및 산소화 분석을 위한 스페로이드 형성 방법 표준화

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.

다세포 스페로이드의 생산 및 다중 파라미터 Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM)

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

3D 회전 타원체 생산을위한 효율적이고 유연한 셀 집계 방법

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3D Spheroids의 생성을위한 간단한 한깅 드롭 세포 배양 프로토콜

3D 프린팅된 스탬프와 같은 장치를 통한 조직 스페로이드 생성

3D 프린팅된 스탬프와 같은 장치를 통한 조직 스페로이드 생성

기능적 3D 스페로이드 세포 배양의 반자동 표현형 분석

다세포 3차원 스페로이드 생성을 위한 랩 온 어 CD 플랫폼

3D 바이오프린팅을 위한 지방유래 줄기세포 구상체의 대규모, 자동화 생산

세포 스페로이드의 대량 생산을 위한 3차원 음향 조립 장치

세포 스페로이드의 대량 생산을 위한 3차원 음향 조립 장치

세포 스페로이드의 대량 생산을 위한 3차원 음향 조립 장치

다세포 스페로이드의 생산 및 다중 파라미터 Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM)