Name: tunable hydrogels from pulmonary extracellular matrix for 3d cell culture
Uploaded: 2017-01-17T13:00:00
Description: Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

우리는 그대로 돼지의 폐 조직 기증에 대한 스미스 필드 팜을 감사드립니다. 우리는 또한 우리 자신의 장비를 사용할 수 있도록 박사 후 양 박사 크리스티나 당나라와 VCU 성형 외과 부서에 감사의 말씀을 전합니다. 히드로 겔 및 조직 샘플은 자금 조달 형태 NIH-NCI 암 센터 지원 그랜트에 의해 부분적으로 NIH - NINDS 센터 코어 그랜트 (5) P30 NS047463에서 자금에 의해 부분적으로 지원되는 해부학 및 신경 생물학 현미경 시설의 VCU 부서에서 SEM을 준비했다 P30의 CA016059. VCU 나노 기술 핵심 특성화 시설 (NCC)에서 샘플의 SEM 영상. 이 작품은 국립 과학 재단 (National Science Foundation), CMMI 1351162에 의해 투자되었다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 해결책 </td><td> 살균 필터 </td><td> 지도 </td></tr><tr><td> DiH 2 O </td><td> 예 </td><td> DiH 2 O; 무균 여과 </td></tr><tr><td> 0.1 % 트리톤 X-100 솔루션 </td><td> 예 </td><td> 흄 후드에서 DiH 2 O 100 ㎖에 100 μL 트리톤-X 100 솔루션을 추가하고 용해 될 때까지 교반; 살균 필터. </td></tr><tr><td> 2 % ...

<ol>
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E., Wu, J., Shah, M., Hong, Y., Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+Material+Properties+of+Cardiac+Matrix+Hydrogels+to+Induce+Endothelial+Differentiation+of+Human+Mesenchymal+Stem+Cells.">Tailoring Material Properties of Cardiac Matrix Hydrogels to Induce Endothelial Differentiation of Human Mesenchymal Stem Cells.</a> ACS Appl. Mater. Interfaces. 7 (20), 11053-11061 (2015).</li><li>Suckow, M. A., Voytik-Harbin, S. L., Terril, L. A., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+bone+regeneration+using+porcine+small+intestinal+submucosa.">Enhanced bone regeneration using porcine small intestinal submucosa.</a> J. Invest. Surg. 12 (5), 277-287 (1998).</li><li>Brown, B. N., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+as+an+inductive+scaffold+for+functional+tissue+reconstruction.">Extracellular matrix as an inductive scaffold for functional tissue reconstruction.</a> Transl. Res. J. Lab. Clin. Med. 163 (4), 268-285 (2014).</li><li>Pouliot, R. A., 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=Development+and+characterization+of+a+naturally+derived+lung+extracellular+matrix+hydrogel.">Development and characterization of a naturally derived lung extracellular matrix hydrogel.</a> J.Biomed.Mater.Res.A. , (2016).</li><li>Price, A. P., England, K. A., Matson, A. M., Blazar, B. R., Panoskaltsis-Mortari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+decellularized+lung+bioreactor+system+for+bioengineering+the+lung:+the+matrix+reloaded.">Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.</a> Tissue Eng. Part A. 16 (8), 2581-2591 (2010).</li><li>Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+rheological+characterization+of+a+gel+form+of+the+porcine+urinary+bladder+matrix.">Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.</a> Biomaterials. 29 (11), 1630-1637 (2008).</li><li>Tilghman, R. W., 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=Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype.">Matrix rigidity regulates cancer cell growth and cellular phenotype.</a> PloS One. 5 (9), e12905 (2010).</li><li>Ratner, B. D., Bryant, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials:+where+we+have+been+and+where+we+are+going.">Biomaterials: where we have been and where we are going.</a> Annu. Rev. Biomed. Eng. 6, 41-75 (2004).</li><li>Fridman, R., Benton, G., Aranoutova, I., Kleinman, H. K., Bonfil, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+initiation+and+growth+of+tumor+cell+lines,+cancer+stem+cells+and+biopsy+material+in+mice+using+basement+membrane+matrix+protein+(Cultrex+or+Matrigel)+co-injection.">Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection.</a> Nat Protoc. 7 (6), 1138-1144 (2012).</li><li>Zhang, W. -. J., 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=The+reconstruction+of+lung+alveolus-like+structure+in+collagen-matrigel/microcapsules+scaffolds+in+vitro.">The reconstruction of lung alveolus-like structure in collagen-matrigel/microcapsules scaffolds in vitro.</a> J. Cell. Mol. Med. 15 (9), 1878-1886 (2011).</li><li>Booth, A. J., 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=Acellular+Normal+and+Fibrotic+Human+Lung+Matrices+as+a+Culture+System+for+In+Vitro+Investigation.">Acellular Normal and Fibrotic Human Lung Matrices as a Culture System for In Vitro Investigation.</a> Am. J. Respir. Crit. Care Med. 186 (9), 866-876 (2016).</li><li>Matsuda, A., 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=Evidence+for+human+lung+stem+cells.">Evidence for human lung stem cells.</a> N. Engl. J. Med. 364 (19), 1795-1806 (2011).</li><li>Zuo, W., 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=p63+Krt5++distal+airway+stem+cells+are+essential+for+lung+regeneration.">p63+Krt5+ distal airway stem cells are essential for lung regeneration.</a> Nature. 517 (7536), 616-620 (2015).</li><li>Keane, T. J., Londono, R., Turner, N. J., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+ineffective+decellularization+of+biologic+scaffolds+on+the+host+response.">Consequences of ineffective decellularization of biologic scaffolds on the host response.</a> Biomaterials. 33 (6), 1771-1781 (2012).</li><li>Neill, J. D., 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=Decellularization+of+human+and+porcine+lung+tissues+for+pulmonary+tissue+engineering.">Decellularization of human and porcine lung tissues for pulmonary tissue engineering.</a> Ann Thorac Surg. 96 (3), 1046-1055 (2013).</li></ol>

생물학 적분 측면 중 하나는 특정한 작업을 수행하는 계층 적 구조로 분자의 자기 조직이다. 연구실에서 (13)는, 자기 조립은 염 농도, 산도, 소화 기간 등 다양한 요인에 따라 달라집니다. 도시 된 바와 같이, 자기 조직화 하이드로 폼 가용화 된 단백질은 생리 학적 온도로 돌아 오면. 형성된 하이드로 겔은 생체 외에서 세포의 부착과 증식을 촉진 할 수있다. ECM?...

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6. 
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research. 
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

<table border="0" cellpadding="0" cellspacing="0" width="841">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100&#160;</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td style="width:371px;">Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Deoxycholate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6750-100g</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1016-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>D5025-150KU</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>258148-500ML</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepsin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>P6887-5G</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Hydroxide</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP359-500</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Genipin</td>
			<td style="width:135px;">Wako Chemicals</td>
			<td style="width:119px;">078-03021</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 10x</td>
			<td style="width:135px;">Quality Biological</td>
			<td>119-069-151</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:135px;">VWR</td>
			<td>45000-448</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter Paper</td>
			<td>Whatman</td>
			<td align="right">8519</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hand pump</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>10-239-1</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graduate Beaker</td>
			<td style="width:135px;">VitLab</td>
			<td align="right" style="width:119px;">445941</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomill</td>
			<td>SPEX</td>
			<td>6700</td>
			<td>Use cryogloves and eye protection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lyophilizer</td>
			<td>FTS FlexiDry</td>
			<td></td>
			<td>Use gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rheometer</td>
			<td>Discovery</td>
			<td>HR-2</td>
			<td>Use gloves and eye protection.</td>
		</tr>
	</tbody>
</table>

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

여기에서는 시험 관내 폐 세포 배양 여러 컴포넌트 세포 배양 하이드로 겔을 형성하기위한 방법을 제시한다. 돼지, 쥐, 또는 마우스에서 블록의 폐 조직 엔 건강을 시작으로, 조직은 관류 및 세포 파편을 제거하기 위해 후속 화학 세제에 잠겨있다. 처리 전후의 조직 학적 비교는 이중 가닥 DNA 및 알파 갈 락토시다 제 염색을 95 % 이상 제거하는 것을 확인 세포 파편의 대부분이 제거되어 나왔다. 탈세 포화 후, 조직을 동결 건조 후 분말로 cryomilled이다. 매트릭스 분말 산성 펩신 소화 용액에 48 시간 동안 절단 한 후, 프리 겔 용액을 형성하기 위해 중화된다. 프레 겔 용액의 겔화 37 ° C에서 배양하여 유도 할 수 있고, 곧바로 다음 중화 사용 또는 최대 2 주 동안 4 ℃에서 저장 될 수있다. 코팅은 C에 대한 비 처리 판의 프리 겔 용액을 사용하여 형성 될 수있다엘 첨부. 세포는 세포 골격을 통해 이전 또는 코팅에 도금 할 수있는 형성된 겔의 표면 상에 도금 된 3 차원 배양을 달성하기 위해 자기 조립 전에 프레 겔에 현탁 될 수있다. 전략에 변화가 겔화 온도, 강도, 또는 단백질 단편의 크기에 영향을 미칠 수 있습니다 발표했다. 하이드로 겔 형성 외에도 하이드로 겔 강성 genipin을 사용하여 증가 될 수있다.

이 방법을 사용하여, 우리는 정상 돼지, 래트, 마우스 폐 (도 1)로부터의 하이드로 겔을 제조 하였다. 폐 처리는 각각 5 ㎎, 40 ㎎, 및 ECM 분말 10g을 추정 제공한다. 공정의 개요는도 2에 도시되어있다. 과정에서 주요 시각화은 다음과 같습니다 : 데 옥시 콜레이트 세척 후 폐의 흰색 외관; 프레 겔을 형성 한 후에, 용액을 은폐되어야하고 4 ℃에서 저장...

Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

이 폐 세포 외 기질에서 3 차원 세포 배양 지지체를 제작하는 방법이다. 그대로 폐 세 차원 세포의 성장을 지원할 수있는 하이드로 겔로 처리된다.

3 차원 세포 배양을위한 폐 세포 외 매트릭스에서 조정 히드로 겔

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Research

JoVE Journal

Bioengineering

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

세포 배양 응용 프로그램에 대한 히드로 겔의 빛 매개 형성 및 패터닝

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

연구

생체공학

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

조직 특이 적 세포 배양 및 전달 플랫폼과 같은 세포 외 기질 유래 거품과 마이크로 담체의 제조

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

날조는 신장 피 질 세포 외 매트릭스 파생 하이드로 겔

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

희생 빈 섬유 막 세포 배양을 통해 기질 섬유의 생산

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

글로벌 세포 매트릭스 Metalloproteinase 및 3D Hydrogels 신진 대사 활동 측정

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

인쇄 3D 세포 -라덴 췌장 조직 구성을 인쇄를위한 췌장 조직 유래 세포 외 매트릭스 바이오 잉크

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

매트릭스 세포 대사 크로스토크 연구를 위한 인간 3D 세포외 세포외 매트릭스-세포세포 배양 모델

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

슈완 세포 표현형 사양을 평가하기 위한 튜블드 세포 외 세포 매트릭스 미세 환경의 준비

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

인간 신경 구조물의 임베디드 3D 프린팅을 위한 마이크로겔-세포외 기질 복합체 지원

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels (Video) | JoVE

섬유아세포 활성화를 연구하기 위한 3D 바이오프린팅 광조정 가능 하이드로겔

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...