Name: nutrient regulation by continuous feeding for large-scale expansion of mammalian cells in spheroids
Uploaded: 2016-09-25T13:00:00.000+00:00
Duration: 11 min 1 s
Description: Watch this Scientific Journal Video about Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids at JoVE.com

The authors thank Michael Garwood and Sam Stein for their helpful comments, and Kristen M. Maynard for assistance with manuscript preparation.

The authors have nothing to disclose.

(58) - 생물학적 제제와 세포 치료에 대한 생산을위한 포유 동물 세포의 제품을 생성하는 대규모 (55)의 문화와 포유 동물 세포의 모니터링이 필요합니다. 또한,이 응용 프로그램은 정의되고 검증 된 배양 조건을 요구. 단순히 이러한 요건을 모두 만족하지 조사 기술을 사용하여 세포의 양을 증가시킨다. 영양분과 노폐물의 축적에 변동을 일으키는 설명서 매체의 변화는 세?...

이식 가능한 기능적 인간 세포의 다수를 생성하기 위해, 배양 조건의 조절이 필수적이다. 3 - 신진 대사 폐기물의 축적과 함께 영양분의 고갈은 셀 제품 1의 품질이 저하 노화 및 대사 변화에 중요한 기여한다. 이 절차는 배양 기간에 걸쳐 생리 학적 범위 4 글루코스를 조절하는 조절 된 속도로 관류 공급 시스템과 결합 교반 생물 반응기를 사용 타원체의 포유 동물 세포 배양하는 방법을 보여준다. 이 연구의 목적은 생리적 인 범위는 100 내지 200 밀리그램 / DL로 정의 하였다. 동일한 방법이 다른 영양소 및 락 테이트 대사 폐기물을 조절하기 위해 사용될 수있다. 작은 볼륨의 정적 문화 (1-30 ml)을 일반적으로 experime에 대한 세포 라인을 유지하고 차별화하는 실험실 환경에서 사용되는ntal 목적. 세포 계대는 정기적으로 필요한 완전 배지 변경과 함께 수행된다. 대부분의 &quot;기존의&quot;문화 매체는 영양 제한의 위험없이 덜 자주 매체 변경을 허용하는 높은 포도당 농도 (이 연구에 사용 된 DMEM 450 밀리그램 / DL)가 있습니다. 그러나,이 배치 급지에있어서 여전히 빈번한 조작을 필요로하는 세포의 환경에서 변화를 도입하고, 5 오염의 위험이 증가한다 - 9. 교반 정지 생물 반응기는 (SSB)는 더 나은 혼합을 제공하고 3,10 취급 감소 - 20 만, 정적 문화처럼, 영양 및 폐기물 제품 수준의 잠재적 인 손상 변동에 기여 설명서 매체의 변화가 필요합니다. SSB 문화의 관류 공급 연속 주입 및 매체의 제거,하지만 인해 세포 성장에 영양 수준에서 큰 변화로 이러한 문제가 문제가 남아 줄일 수 있습니다. 조정 된 공급 라의 사용예상 셀 요구 사항에 따라 영양소 사용량 계산에서 TE는 세포 생존을 최적화하는데 필요한 안정적인 셀 환경을 제공하고, 21 작용할 수 - 24. 25 구체적으로 만능 세포의 배양 및 확장을위한 포유 동물 세포의 확장 SSB 문화에 대한 방법을 설명하는 문학의 큰 몸이있다 - 다른 사람과 (32), 섬 (베타) 세포 17,33,34에 초점을 맞춘이, 또는 생물 의약품 24의 생산, 35-38. 이러한 연구 세포 종류의 많은 구형 배양에서 성장 될 수 있고, 사용되는 세포 유형에 대한 구체적인 절차는 연속 공급 시스템을 구현하기 이전에 최적화되어야한다. 43 -이 예제에서, 관류 공급 방법은 교반 생물 반응기 (39) 회전 타원체로 성장 베타 세포주를 확장 하였다. 본원에 기재된 방법을 제공한다타겟 배양 조건을 달성하기 위해 오프라인 혈당 측정에 기초하여 조정 레이트 공급의 간단한 구현. 이 방법으로 공급 속도를 조절하는 생리적 포도당 수준이 증가 세포 수율에 표시됩니다 유지합니다. 포유 동물 세포는 에너지 생산을 위해 중요한 영양소, 포도당에 의존하므로,이 세포주의 사용은 많은 포유류 배양 세포 (44)의 모델을 나타낸다. 또한,이 라인은 포도당 45 만성 높은 수준에 민감한 베타 세포의 추가의 복잡성을 예시한다. 본 연구를 위해, β-TC6 세포는 생체 내에서 랑게르한스섬의 평균 크기는 대략 배양 타원체를 형성시켰다. 관류 생물 반응기 시스템 (17) - 소비 포도당 조정 된 공급 속도와 19,21,46은, 생존의 변화없이 생리 학적 조건과 높은 셀 수율을 유지하는 결과.

<table><tbody><tr><td>&amp;#946; TC-6 Cells</td><td>ATCC, Manassas, VA</td><td>CRL-11506</td><td>Mouse Insulinoma cell line (adherent cell type)</td></tr><tr><td>DPBS No Ca, No Mg</td><td>Invitrogen, Carlsbad, CA</td><td>14190-144</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144&quot;&gt;https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144&lt;/a&gt;</td></tr><tr><td>Dulbecco&#039;s Modified Eagles Medium</td><td>Invitrogen, Carlsbad, CA</td><td></td><td>See below for product numbers&amp;nbsp;</td></tr><tr><td>DMEM High Glucose (500 mM)</td><td>Invitrogen, Carlsbad, CA</td><td>11965-092</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/11965092&quot;&gt;http://www.lifetechnologies.com/order/catalog/product/11965092&lt;/a&gt;</td></tr><tr><td>DMEM Low Glucose (100 mM)</td><td>Invitrogen, Carlsbad, CA</td><td>11885-084</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/11885084%20(note%20that%20this%20medium%20already%20contains%20pyruvate)&quot;&gt;http://www.lifetechnologies.com/order/catalog/product/11885084 (note that this medium already contains pyruvate)&lt;/a&gt;</td></tr><tr><td>L-glutamine</td><td>Invitrogen, Carlsbad, CA</td><td>25030081</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product&quot;&gt;http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product&lt;/a&gt;</td></tr><tr><td>Sodium Pyruvate</td><td>Invitrogen, Carlsbad, CA</td><td>11360070</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product&quot;&gt;https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product&lt;/a&gt;</td></tr><tr><td>Heat Inactivated Porcine Serum</td><td>Gibco - Life Technologies</td><td>10082147</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/10082147&quot;&gt;http://www.lifetechnologies.com/order/catalog/product/10082147&lt;/a&gt;</td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen, Carlsbad, CA</td><td>25200056</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product&quot;&gt;https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product&lt;/a&gt;</td></tr><tr><td>T-150 Tissue Culture Treated Flasks</td><td>Corning, Corning, NY</td><td>430825</td><td>http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430825(Lifesciences)&lt;br/&gt;&amp;amp;categoryname=</td></tr><tr><td>NuAire Cell culture incubator</td><td>Princeton, MN</td><td></td><td>US Autoflow. Any water-jacketed CO&lt;sub&gt;2&lt;/sub&gt; regulating cell culture incubator could be used.</td></tr><tr><td>Centrifuge</td><td></td><td></td><td>Sorvall RT 7 (Any similar benchtop centrifuge may be used)</td></tr><tr><td>Refrigerator</td><td></td><td></td><td>Any laboratory refrigerator could be used (a small table-top version was used for these studies)</td></tr><tr><td>1 L Glass Bottle</td><td>Corning, Corning, NY</td><td>1395-1L</td><td>Any vendor could be used. http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=1395-1L(Lifesciences)&lt;br/&gt; &amp;amp;categoryname=</td></tr><tr><td>2 L Glass Bottle</td><td>Corning, Corning, NY</td><td>1395-2L</td><td>Any vendor could be used</td></tr><tr><td>250 ml stirred bioreactors&amp;nbsp;</td><td>Corning, Corning, NY</td><td>4500-250</td><td>http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=4500-250(Lifesciences)&lt;br/&gt;&amp;amp;categoryname=</td></tr><tr><td>Stir Plate</td><td>Fisher Scientific</td><td>11-496-104A</td><td>Any incubator safe stir-plate can be used, any vendor</td></tr><tr><td>Tissue Culture Dishes 100 mm Diameter</td><td>Nunc, Rochester, NY (Fisher Scientific)</td><td>1256598&amp;nbsp;</td><td>Any vendor could be used (ordered through Fisher Sci)</td></tr><tr><td>FALCON 50 ml Conical Tubes</td><td>Falcon, San Jose, CA</td><td>1256598</td><td>Any vendor could be used</td></tr><tr><td>Delran Plastic Used for Custom Parts</td><td>McMaster Carr</td><td>Various</td><td>Any material of choice could be used, but Deran is chosen because it is autoclave safe, non-reactive, and easy to machine. http://www.mcmaster.com/#acetal-homopolymer-sheets/=rjrcac</td></tr><tr><td>Stainless Steel Pipe for custom lids</td><td>McMaster Carr</td><td>Various</td><td>Any vendor could be used. http://www.mcmaster.com/#standard-stainless-steel-tubing/=rjrd91</td></tr><tr><td>Custom Modified Delran Bioreactor Lids for Continuous Feeding</td><td>Custom made</td><td></td><td>Not aware of any vendors producing a similar product</td></tr><tr><td>Custom Modified Glass Bottle Lids for Continuous feeding</td><td>Custom made</td><td></td><td>Some vendors (&lt;em&gt;e.g.&lt;/em&gt;, Fischer Sci, Corning) make similar products in the links below.</td></tr><tr><td>Masterflex Digital Peristaltic Pump</td><td>Cole Parmer, Vernon Hills, IL</td><td>EW-77919-25</td><td>Any precision peristaltic pump could be used. http://www.coleparmer.com/Product/L_S_Eight_Channel_Four_Roller_&lt;br/&gt; Cartridge_Pump_System_115_230&lt;br/&gt; _VAC/EW-77919-25</td></tr><tr><td>PVDF Tubing Connectors (various)</td><td>Cole Parmer, Vernon Hills, IL</td><td>see link</td><td>Any vendor could be used. http://www.coleparmer.com/Category/Cole_Parmer_PVDF_Premium&lt;br/&gt; _Luer_Fittings/55889</td></tr><tr><td>Pharmed BPT Tubing L/S 16</td><td>Cole Parmer, Vernon Hills, IL</td><td>WU-06508-16</td><td>Any vendor could be used. http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing&lt;br/&gt; _L_S_13_25/WU-06508-16</td></tr><tr><td>Pharmed BPT Tubing L/S 14</td><td>Cole Parmer, Vernon Hills, IL</td><td>WU-06508-14</td><td>Any vendor could be used. http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing&lt;br/&gt; _L_S_13_25/WU-06508-14</td></tr><tr><td>Pharmed BPT Tubing L/S 13</td><td>Cole Parmer, Vernon Hills, IL</td><td>WU-06508-13</td><td>Any vendor could be used. http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing&lt;br/&gt; _L_S_13_25/WU-06508-13</td></tr><tr><td>Millipore Millex GP PES membrane 0.22 &amp;#181;l sterile syringe filter (used for venting, and medium filtration)</td><td>Fisher Scientific</td><td>SLGP033RS</td><td>Any vendor could be used</td></tr><tr><td>25 ml Graduated Pipette</td><td>Fisher Scientific</td><td>13-678-11</td><td>Any vendor could be used, and various sizes may be used</td></tr><tr><td>Pipetter</td><td>Fisher Scientific</td><td>13-681-15E</td><td>Any vendor, or similar product could be used</td></tr><tr><td>Hemocytometer</td><td>Fisher Scientific</td><td>02-671-6</td><td>Any vendor, or similar product could be used</td></tr><tr><td>Trypan Blue</td><td>Gibco - Life Technologies</td><td>15250-061</td><td>Any vendor, or similar product could be used. https://www.lifetechnologies.com/order/catalog/product/15250061</td></tr><tr><td>Inverted Light Microscope</td><td>Leica</td><td></td><td>Any vendor, or similar product could be used</td></tr><tr><td>One Touch Ultra Blood Glucose Meter</td><td>Fisher Scientific</td><td>22-029-293</td><td>Any vendor, or similar product could be used (&lt;i&gt;e.g.&lt;/i&gt;, Bayer)</td></tr><tr><td>One Touch Ultra-Strips</td><td>Fisher Scientific</td><td>22-029-292&amp;nbsp;</td><td>Any vendor, or similar product could be used (&lt;i&gt;e.g.&lt;/i&gt;, Bayer)</td></tr></tbody></table>

nutrient regulation by continuous feeding for large-scale expansion of mammalian cells in spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

중간 포도당 수준 및 변동 표준 SSB 문화의 세포 확장을 제한 포도당 수준은 배양 기간 3를 통해 정적 문화와 SSB 문화에 변동. 이러한 변동은 21 일의 배양 기간 동안 세포 수의 증가와 함께 강화하고 정적 배양 SSB 모두에서 거의 동일 하였다. 이러한 관찰은 이전 발행 3에 제시되어있다. 포도당 수준은 두 가지 방법의 배양 기간의 기간 동안 슈퍼 생...

Watch this Scientific Journal Video about Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids at JoVE.com

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

회전 타원체의 포유 동물 세포의 대규모 확장을위한 지속적인 공급함으로써 영양 규정

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

nutrient-regulation-continuous-feeding-for-large-scale-expansion

Research

JoVE Journal

Bioengineering

7.7K 조회수.  University of Minnesota. Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

비디오: Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

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

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

마이크로 웰 플레이트를 사용하여 크기 조절 종양 상체 많은 수의 생산

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

연구

생체공학

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

대장암 줄기세포를 연구하는 스페로이드의 3차원 모델

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

암 연구학

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

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

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

세포외 플럭스 분석을 이용한 단일 3D 미세조직 구상체의 미토콘드리아 에너지 대사 탐구

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

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 ...

생화학

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 ...

Assembloid Technology: Pioneering Liver Development Research

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver ...

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

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

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. ...

생물학

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

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

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

후성유전학적 변형과 생체역학적 단서를 결합하여 포유류 다능성 세포를 생성하는 두 단계 전략

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

회전 타원체의 포유 동물 세포의 대규모 확장을위한 지속적인 공급함으로써 영양 규정

요약

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이 비디오의 챕터

마이크로 웰 플레이트를 사용하여 크기 조절 종양 상체 많은 수의 생산

대장암 줄기세포를 연구하는 스페로이드의 3차원 모델

세포외 플럭스 분석을 이용한 단일 3D 미세조직 구상체의 미토콘드리아 에너지 대사 탐구

기능적 3D 스페로이드 세포 배양의 반자동 표현형 분석

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

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

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

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

3D 회전 타원체 생산을위한 효율적이고 유연한 셀 집계 방법

후성유전학적 변형과 생체역학적 단서를 결합하여 포유류 다능성 세포를 생성하는 두 단계 전략