Name: easy manipulation of architectures in protein-based hydrogels for cell culture applications
Uploaded: 2017-08-04T11:00:00
Description: Watch this Scientific Journal Video about Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications at JoVE.com

저자는 바 덴-뷔르템베르크 재단 "Bioinspired 소재 합성" 프레임 워크 (BioMatS-14)에 그들의 재정 지원에 대 한 감사 하 고 싶습니다.

1. 하이드로 겔 준비
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	<li>BSA의 200 mg 이온된 H2O를 20% (w/v) BSA 주식 (재고 솔루션 A)을 만드는의 1 mL와 함께 믹스.</li>
	<li>THPC 솔루션 (134 mg/mL)의 이온된 수를 만드는 THPC의 4.835 mL와 혼합 165 µ L 재고 솔루션 (재고 솔루션 B).</li>
	<li>KCSSGKSRGDS (1,111.1 g/mol) 펩 티 드 (또는 동등한 세포 접착 펩 티 드)의 1 밀리 그램의 무게 그리고 살 균 H2O 10 mg/mL 해결책 (재고 솔루션 C)를 100 µ L에 희석.<br...

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The production of macroporous matrices can be beneficial to many different fields. It has high technical and economic potential due to the defined structure of the hydrogel and the ability to control and tune specific material properties. However, the introduction of supramolecular structural elements, such as pores or channels, to a 3D template might influence the overall properties of a material, such as the swelling ratio or the stiffness. This can result in the undesired decomposition, degradation, or breakdown of th...

Hydrogels는 많은 양의 물 바인딩 수 불용 성 3 차원 네트워크를 형성 하는 물자. 이러한 자료는 살아있는 세포에 대 한 우수한 환경 조건을 제공할 수 있습니다. 현재, 3 차원 하이드로 겔 구조의 세대에 그들의 화학 및 물리적 특성에 맞게 프로세스 개발에 관심이 증가 하고있다. 이 달성 되 면 세포의 성장 및 세포 행동의 조작에 대 한 템플릿이 생성된1,2,,34될 수 있습니다. 이러한 3 차원 구조 뿐만 아니라 기존의 2 차원 방법, 하지만 그들은 보다 더 자연스럽 고 현실적인 환경 또한 공개 새로운 가능성을 만들 줄기 세포의 성장에 대 한 또는 종양 모델5. 다른 재료는 젤6의 기 공 크기에 주로 의존 하는 특성의 범위를 소유한 다. 모 공 세포 문화 응용 프로그램, 조직 공학 및 줄기 세포의 감독된 성장에 중요 한 역할을 한다. 예를 들어, 매트릭스를 통해 확산 하는 산소와 영양분 그리고 충분 한 양의 세포7에 도달 할 수 있어야 합니다. 다른 한편으로, 유해한 대사 산물, 최대한 빨리 제거 되어야 한다 고 세포 성장 위한 충분 한 공간이 사용 가능한7수 있어야 합니다. 따라서, 재료의 기 공 크기, 속성 심각 하 게 영향을 미칠 행렬의 잠재적인 혜택 및 가능한 응용 프로그램. 재료의 특성에 따라, 다른 세포-성장 과정; 신경 구조의 형성을 포함 하 여 3 차원 세포 배양에서 발생할 수 있습니다. 성장과 분화의 피부 나 뼈 세포; 그리고 hepatocytes 또는 섬유 아 세포2,3,,89,10,11같은 특별 한 줄기 세포 라인의 감독된 성장. 또 다른 중요 한 포인트는 물질의 가능한 응용 프로그램에 영향을 미치는 외부 자극12쪽으로 그것의 안정성입니다. 예를 들어는 히드로 세포 배양 또는 인체에 기계적 무결성을 유지 해야 합니다.
최근 몇 년 동안, 강화, 3D 셀 문화 hydrogels에 연구와 많은 연구 시스템13의 3 차원 구조를 해결 하기 위해 실시 했다. 화학적 합성된 구성 요소 구성 Hydrogels 가장 일반적으로 쉽게 합성 및 화학적 수정 수 있고 그들은 높은 안정성을 전시 하기 때문에 조사 (주 외., 2011 리뷰 참조)5. 그러나, 단백질에는 많은 유익한 속성이:으로 소위 "정밀 고분자," 그들은 생체; 그들은 정의 길이; 그들은 상대적으로 쉽게 수정; 그리고 그들은 많은 수의 대상 사이트14,15. 이와 관련, 많은 분야에서 응용 프로그램에 대 한 매우 구체적인, 혁신적인 구조를 생성할 수 있습니다. 본이 연구에서는 단백질 기반 하이드로 겔16 자료의 3 차원 구조에 영향을 잘 설립 방법의 능력을 보여 주기 위해 사용 되었다. 또한, 능력과 기 공 세대에 적용도 조사 했다.
많은 다른 기술이 간단한 방법 및 재료 과학의 다른 분야에서 정교 하 고, 고도로 전문화 된 기술 모두를 포함 하 여 3 차원 구조를 수정할 수 있습니다. 광범위 한 기술을 잘 정의 된 구조17를 생성 하는 전기의 사용 이다. 충전된 섬유는 전기 분야에 의해 해결책에서 가져온 고 산소에 노출 시 응고. 이 방법에서는, 몇몇 미크론까지 여러 나노미터의 범위에서 섬유를 생산 수 있습니다. 추가 기술을 크기, 구조, 그리고 매트릭스 내의 숨 구멍의 배포를 조정 하는 소프트 리소 그래피, 사진 평판, 유체역학 초점, 전기 살포 바이오 인쇄18,,1920. 이러한 기술의 중요 한 결점은 특정, 비싼 장비 및 특수 화학 제품 또는 자료에 대 한 의존도. 또한, 이러한 기술을 경험 종종 단백질 기반 재료를 직접 양도 이며 화학 물질 및 방법의 많은 셀 호환 되지 않습니다.
다른 한편으로, 많은 기술을 쉽고 저렴 적용 하 고 재현 하 그들을 만드는 특별 한 장비에 의존 하지 마십시오. 구조 조작에 대 한 광범위 한 방법은 용 매 캐스팅21,,2223입니다. 입자 중 합 반응 전에 추가 되 고 솔루션 포화 homogenously 배포 됩니다. 중 합 후 희석 또는 pH 변화, 같은 조건의 변화를 모는 재료 내에서 유지 하는 동안 입자의 solvation에 지도 한다. 소금, 설탕, 파라핀, 젤라틴, 분필, 등이 기술에 사용 되는 화학 물질 저렴 하 고 쉽게 사용할 수 있다 동결에 부 어 hydrogels 동결 됩니다. 진공에서 액체 단계의 후속 승화는23,,2425수행. 네트워크에서 물 승화 만큼 부드러운 재료의 특정 3D 구조를 유지 하는. 가스 거품, 동안에 중 합, 젤21내 숨 구멍을 떠나는 해결책 가스와 스트리밍됩니다. 크기와 숨 구멍의 분포는 가스 흐름에 따라 조정할 수 있습니다.
형성 단백질 히드로, BSA는 tetrakis (hydroxymethyl) phosphonium 염화 물 (THPC) 1 차 아민 4 무장 링커 분자26의 hydroxy 그룹 사이 공유 결합의 형성에 대 한 있도록 Mannich 형 반응에서으로 반작용 했다. 반응 발생 가능한 유해한 중간체 물질의 과도 한 씻기에 의해 제거 됩니다.
이 연구에서는 조작 하 고 숨 구멍의 크기에 맞게 다른 기술로 BSA 기반 소재를 치료의 가능성을 보여 줍니다. 각 기술을 사용할 수 있습니다 어떤 실험실에 전세계, 특별 한 장비는 필요 하다. 또한, 다른 매개 변수를 같은 붓기 비율, 효소 분해성, pH 안정성, 온도 감도, 검사 그리고 서로에 비해 특히 존중 다른 기술의 영향에 3D 아키텍처의 생성에. 마지막으로, 자료 했다 기능성 세포 배양 물질의 가능한 응용 프로그램을 조사 하기 위해 셀 접착제 펩 티 드와 함께. 두 개의 서로 다른 모델 셀 라인 사용 되었다: A549 및 MCF7.

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			<td height="20" style="height:20px;width:479px;">Phosphate Buffered Saline (PBS)</td>
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			<td>Thermo Fisher Scientific</td>
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			<td height="20" style="height:20px;">Ethanol 99.8 %, verg&#228;llt</td>
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			<td>Carl Roth&#160;</td>
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			<td>Sigma Aldrich</td>
			<td>404861-100ML</td>
			<td>Toxic</td>
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			<td>Sigma Aldrich</td>
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			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>R415</td>
			<td></td>
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			<td height="20" style="height:20px;">3.7 % Formaldehyde&#160;</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>F8775-25ML</td>
			<td>Toxic</td>
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			<td>Sigma Aldrich</td>
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			<td height="20" style="height:20px;">&#181; slide 8 well</td>
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			<td height="20" style="height:20px;">Ethanol 99.8 %, verg&#228;llt</td>
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			<td height="20" style="height:20px;">Falcon 5 ml Polysterene Round-Bottom Tube&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>62.547.254&#160; &#160;&#160;</td>
			<td></td>
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			<td height="20" style="height:20px;">Tubes 50 ml&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>62.547.254&#160; &#160;&#160;</td>
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			<td height="20" style="height:20px;">Tubes 1,5 ml&#160;&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>72,690,001</td>
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			<td height="20" style="height:20px;">Tubes 2 ml&#160;&#160;</td>
			<td>Sarsted Ag und Co.</td>
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			<td>GSA GmbH</td>
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easy manipulation of architectures in protein-based hydrogels for cell culture applications

Hydrogels 셀 문화 응용 프로그램 때문에 높은 수 화 셀 환경을 제공 하는 그들의 능력에 대 한 유망 자료로 인정 받고 있습니다. 3 차원 서식 파일의 필드는 자연적인 기질을 그 물질의 잠재적인 정체 때문 상승 이다. 단백질 기반 hydrogels가 있습니다 특히 유망한 functionalized 쉽게 수 있기 때문에 조정 가능한 물리 속성으로 정의 된 구조를 얻을 수 있습니다. 그러나, 천연 재료를 사용 하 여 셀 문화 응용 프로그램에 대 한 macroporous 3D 템플릿의 생산 종종 합성 물질의 그들과 비교 된 그들의 약한 기계적 성질에 의해 제한 됩니다. 여기, 다른 방법을 macroporous 소 혈 청 알 부 민 (BSA)를 생산 하기 위해 평가 했다-하이드로 겔 시스템, 반경에서 10 ~ 70 µ m의 범위에서 조정 가능한 기 공 크기를 기반으로. 또한, 몇 백 미크론 긴은이 단백질 기반 자료 채널을 생성 하는 방법 설치 되었다. 모 공, 기 공 크기의 영향 비율, pH, 온도 안정성, 그리고 효소 저하 행동, 붓기 등의 소재 속성에 생산 하는 여러 가지 방법은 분석 했다. 기 공 크기 hydrogels confocal 레이저 스캐닝 현미경 검사 법을 사용 하 여 네이티브, 부푼 상태에서 조사 되었다. 셀 문화 응용 프로그램에 대 한 타당성 단백질 시스템 및 두 개의 모델 셀 라인의 세포 접착제 RGD 펩타이드 수정을 사용 하 여 평가 했다: 인간 유방암 세포 (A549)와 adenocarcinomic 인간의 치경 기저 상피 세포 (MCF7).

Hydrogel development has become one of the most prominent fields in material research-related biological studies, with thousands of entries indexed in scientific research archives. Although the behavior of many systems is well studied, the manipulation of 3D networks, especially of sensitive protein-based materials, is often a major issue in material science. Another commonly underestimated challenge is the correct measurement of the native structure of a material using cryo electron micr...

Watch this Scientific Journal Video about Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications at JoVE.com

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

단백질 기반 hydrogels 3 차원 구조를 조작 하는 다른 메서드 여기 소재 속성에 대해 계산 됩니다. 세포 접착 펩 티 드와 함께 macroporous 네트워크는 공업화 하 고 두 개의 서로 다른 모델 셀 라인을 사용 하 여 세포 배양에서 그들의 타당성을 평가.

셀 문화 응용 프로그램에 대 한 단백질 기반 Hydrogels 아키텍처의 쉬운 조작

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

The overall goal of this methodology is to create macroporous hydrogels with varying pore sizes for cell culture applications. This method can help answer key questions in the field of 3D cell culture such as the principle feasibility of a novel hydrogel material for 3D cell culture applications. The main advantage of this methodology is that it offers a range of techniques to optimize and adjust a novel material to the requirements of different types of cells.To begin, dilute one milligram of the RGD peptide or an equivalent cell adhesive peptide in 100 microliters of sterile water to obtain the 10 milligram per milliliter solution. Next, remove the bottom of a 96 well plate and replace it with removal plastic wrap. Mix 100 microliters of BSA stock solution and 100 microliters of THPC stock solution in the 96 well plate to obtain 200 microliters of the hydrogel.Mix the components by pipetting up and down at least five times to guarantee a uniform hydrogel after polymerization. Place the 96 well plate at room temperature for about 10 minutes until all hydrogels are properly polymerized. Following polymerization, carefully and slowly remove the plastic wrap from the bottom of the plate.Press the hydrogels out of the 96 well plate using a small stab and transfer them to 1.5 milliliter tubes with sterile PBS pH 7.4. Store the hydrogels in phosphate buffered saline at four degrees Celsius for up to several months. To freeze-dry the hydrogels, first fill 1.5 milliliter tubes with 500 microliters of sterile deionized water and remove the caps.Transfer the hydrogels to the 1.5 milliliter reaction tubes using forceps. Wrap the 1.5 milliliter tubes tightly with paraffin film using at least three layers for each vial. Use a needle to pierce small holes in the film to enable gas release from the tubes.To guarantee that the water and hydrogel completely freeze, transfer the vial to liquid nitrogen solution for five minutes. Immediately after removal from the liquid nitrogen, transfer the vials to the freeze-dryer to prevent the material from thawing. As an alternative freezing step, keep the vials at minus 20 degrees Celsius overnight to slowly freeze the hydrogels.Immediately after removal from minus 20 degrees Celsius, transfer the vials to the freeze-dryer to prevent the material from thawing. After 24 hours and the complete evaporation of the water in and around the hydrogel, thaw the material by removing it from the freeze-dryer. For particle leaching, mix the components of the hydrogel as before.Following mixing, immediately add sodium chloride until saturation occurs. Add salt until salt crystals can be seen in the solution as a white precipitate. Pore sizes can be altered by grinding the salt crystals prior to use.Transfer the 96 well plate to a shaker and shake until polymerization takes place. After about 10 minutes, remove the hydrogels from the plate as before. Incubate the hydrogels for at least 24 hours at room temperature in sterile water on a rotator to elute all salt from the hydrogel template.Store the hydrogels at four degrees Celsius in PBS for up to several months. For gentle formation, prepare hydrogels as described. Then remove a hydrogel from solution using a pincer.Remove the excess water with highly absorbent paper and place it on top of a block of dry ice. Freeze the hydrogel for 30 seconds and carefully remove it from the block. Do not damage the hydrogel.Carefully use a spatula to scrape it off the block. Transfer the hydrogel to a 1.5 milliliter tube and dry it overnight at 37 degrees Celsius. Prepare a Rhodamine B stock solution as well as serial dilutions as described in the text protocol.Remove the hydrogel from the stored solution and transfer it to a 1.5 milliliter tube with one milliliter of 0.01 milligram per milliliter Rhodamine B solution. Stain the hydrogel overnight in Rhodamine B solution at room temperature. The next day, transfer the hydrogel that is to be visualized to 10 milliliters of PBS and wash for at least three hours.Cut small slices out of the hydrogel using a blade. Then transfer the hydrogel onto an eight well microscopic slide and cover it with PBS. Using a confocal laser scanning microscope, visualize the hydrogel at a wavelength of 514 nanometers.Sterile filter all stock solutions with a 0.45 micron filter. Begin preparation of the hydrogel and include the cell adhesive peptide prior to hydrogel polymerization. After mixing the components, immediately pipette the mixture into an eight well microscopic slide until the bottom is completely covered.Transfer the cells into pre-warmed sterile DMEM cell culture medium supplemented with fetal bovine serum, penicillin streptomycin, and nonessential amino acid solution. After counting the cells with a Neubauer counting chamber, carefully pipette 200 microliters of the desired number of cells onto the hydrogel surface. Cover the eight well microscopic slide with a lid and transfer it to an incubator.After at least four hours of cellular attachment, wash the cells twice with 200 microliters of sterile cell culture PBS. Next, fix the cells with 200 microliters of 3.7%formaldehyde for 10 minutes at room temperature and wash twice with PBS. Permeabilize the cells with 200 microliters of 0.1%Triton X for five minutes before washing twice more with PBS.Stain the cells with Toluidine Rhodamine by mixing five microliters of methanolic stock with 195 microliters of PBS and adding it to the cells at room temperature. Stain the cells for 20 minutes in the dark before washing twice with PBS. Investigate the cell adhesion properties using a confocal microscope at 514 nanometers.Analyze the hydrogel properties as described in the text protocol. As shown here, the polymerized and stained hydrogels have no distinctive architectures. After freeze-drying the hydrogels at different temperatures, the average size of the pores within the hydrogel can be extended up to 60 microns.Salt leaching results in uniform networks with pore sizes of about 10 microns. The size can be further adjusted by grinding salt crystals prior to use. The use of a gradient freezing approach on a block of dry ice leads to the formation of channels with a diameter of about 20 microns and a length of several hundreds of microns.Prior to functionalization with the cell adhesive moiety, cells cannot adhere to the surface of the hydrogel and show a round morphology. Only after providing the material with the cell adhesive peptide, the cells find focal adhesion points which enable a proper adhesion of the cell onto a material. This is crucial for use of novel materials in 3D cell culture applications.After watching this video, you should have a good understanding of how to manipulate a hydrogel's architecture with simple methods. This method can be applied for most novel hydrogel systems to learn about the use in cell culture and to optimize the hydrogel's architecture depending on the intended use.

Research

JoVE Journal

Biochemistry

Preparation of Homogeneous MALDI Samples for Quantitative Applications

양적 응용 프로그램에 대한 균일 MALDI 시료의 제조

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

연구

생화학

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

마그네틱 핀셋으로 G-quadruplexes의 단일 분자 조작

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

분해 활동에 대 한 화면에 Fluorogenic 펩 티 드 분열 분석 결과: 코로나 바이러스에 대 한 응용 프로그램 스파이크 단백질 활성화

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

차에서 파생된 새로 설립된 세포 현탁액 배양에서 6개의 전신 살충제의 물질대사에 관한 연구(동백나무시넨시스 L.) 잎

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

싱크로트론 X선 형광 나노 분석을 위한 실리콘 질화물 멤브레인의 세포 배양 및 저온 준비

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

탠덤 질량 분석과 결합 된 액체 크로마토그래피를 사용하여 사카로 미세화세세비시아의 세포 지질의 정량분석

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

성숙한 쌀의 횡단 단면(오리자 사티 라 L.) 파이펫 팁을 사용하여 전자 현미경 이미징을 스캐닝하기 위한 커널

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

탠덤 질량 분광과 결합 된 액체 크로마토그래피를 사용하여 사카로미세스 Cerevisiae의 정량적 Metabolomics

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA 메틸트랜스퍼라제 억제제를 스크리닝하기 위한 연속 형광 기반 엔도뉴클레아제 결합 DNA 메틸화 분석

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...