이 연구는 국립 보건원에서 미국 재향 군인의 실무 계열 (워싱턴 DC), 보조금 0,852,048, 0,731,201 및 0,854,430 국립 과학 재단 (알링턴, VA)에서, 그리고 부여 RO1 DK080897 (베데스다, 메릴랜드)에 의해 지원되었다 . 씨는 Khanna는 빌 &amp; 멜린다 게이츠 재단 (시애틀, WA)에서 에드워드 로스 박사 모니카 모야하여 관대한 기부로부터 지원을 받았습니다.

여기서 프로토콜 multilayered 알지네이트요의 microbeads (그림 1)을 생성하는 3 단계 절차를 설명합니다. 첫째, 알지네이트요의 microbeads은 (그림 2A) 형성됩니다. 이 절차는 아래 섹션 1에 설명되어 있습니다. 세포 또는 단백질이 전달 시스템의 역할을하기 위해이 단계에서 microbeads에 추가할 수 있습니다. 다음 단계는 microbeads에 permselective 레이어의 형성을 포함하고 섹션 2에 설명되어 있습니다. 마지막 단계는 추가 알지네이트요 계층의 형성을 포함하며 제 3에 설명되어 있습니다. 구슬의 표면 (그림 2B)의 외부에이 레이어 형태는 시스템에 다음과 이식에 대한 세포 반응을 지시하는 치료 분자를 (그림 2C) 캡슐 및 전달하는 데 사용할 수 있습니다. 1. 알긴산의 Microbead 준비 <ol><li> 1 ML에서 LVM의 알지네이트요의 15 그램을 해소하여 1.5 % (W / V) LVM의 알지네이트요 솔루션을 준비내부 레이어 알지네이트요 솔루션 (산도 7.4로 조정 DI 워터에서 25 밀리미터 HEPES 버퍼, 118 MM NaCl, 5.6 MM KCl, 및 2.5 밀리미터 MgCl 2). 알지네이트요 전원이 완전히 투명, 점성 용액을 형성 해산 때까지 Vortexer에 섞는다. 참고 :이 프로토콜은 알지네이트요의 microcapsules 이내 섬 캡슐을위한 최적의 조건을 설명 한 농도와 알지네이트요 microspheres의 성분이 다른 응용 프로그램 (예, 약물 전달, 조직 공학 등)주의에 대한 속성을 조정 변경될 수 있습니다. 섬 캡슐 들어 islets 이전 microencapsulator에 로딩이 단계에서 알지네이트요 솔루션에 추가할 수 있습니다. </li><li> 100 MM CaCl 2 및 DI 워터에서 10 밀리미터 HEPES 버퍼를 용해하고 pH를 7.4로 조정하여 crosslinking 솔루션 (DI 워터 22 MM CaCl 2)를 준비합니다. 참고 사항 : BR이 같은 다른 이가의 양이온은 +, 시니어 2 + 등 + 알지네이트요 겔화 D의 특성에 따라 대신 칼슘 2 사용될 수esired. </li><li> 주사기에 25 게이지 바늘을 추가하고, 바늘이 공기 재킷의 중심에 있는지 확인하기 위해 양옆의 밸브를 조절하여 2 채널 에어 자켓 알지네이트요의 microencapsulator를 설정합니다. 다른 게이지 바늘이 대상 알지네이트요의 microbeads의 크기에 따라이 단계에 사용될 수 있습니다. 이 단계도 주사기로 수행할 수 microencapsulator를 사용할 수 없습니다. 주사기로 알지네이트요 솔루션을 추가하고 원하는 microbeads의 크기에 따라 바늘 게이지를 선택합니다. </li><li> 직접 바늘 아래 crosslinking 솔루션의 10 ML이 들어있는 플라스크를 놓습니다. 솔루션 볶음 막대를 놓습니다. </li><li> microbeads를 형성 알지네이트요를 crosslink하기 위해 CaCl 2 용액에 직접 방울을 주입. 계속 저어으로, 적어도 15 분간 치료 crosslinking 솔루션의 구슬을 품어. </li><li> 15 ML의 원심 관에 비즈를 전송합니다. , 잔류 용액을 제거두 분마다 정상 생리 식염수에 0.2 % CaCl의이 세 가지 세차장을 수행합니다. </li></ol> 2. 폴리-L-ornithine과 코팅 Microbeads를 <ol><li> 정상 식염수에있는 폴리-L-ornithine (PLO)의 0.1 % (W / V) 솔루션 3 ML을 준비합니다. 그것이 완전히 명확한 솔루션을 형성 해산되기 전까지 와동에 솔루션을 놓습니다. 폴리-L-라이신 (PLL)는 permselectivity 비슷한 수준의 결과를이 단계를 위해 대신 PLO에 사용될 수 있습니다. </li><li> PLO 솔루션으로 알지네이트요의 microbeads를 전송합니다. PLO 충분한 시간이 polycation-polyanion 복합을 형성 알지네이트요과 상호 작용할 수 있도록 30 분 소용돌이에 장소. 30 분 말에, 알지네이트요 microbeads의 주위에 분명히 보이는 흰색 코팅도 있어야합니다. </li><li> PLO 솔루션을 제거 두 분마다 정상 식염수에 0.2 % CaCl의이 세 가지 세차장을 수행합니다. </li></ol> 3. 바깥 알긴산을 만드는 것은 때려치어 <ol><li> 외부 레이어를 만드는 데 사용되어야하는 원하는 농도 알지네이트요 솔루션을 준비합니다. (1.1) 상기에 설명된대로 솔루션은 준비가되어 있습니다. 외부 레이어의 크기는 (그림 3) 사용 알지네이트요의 조성과 농도에 의해 영향을받습니다. </li><li> 셀 - 스트레이너로 알지네이트요의 microbeads를 전송합니다. microbeads 가능한 건조하기 위해 여분의 용액을 흡수할 수 있도록 Kimwipe을 사용합니다. </li><li> Parafilm 표면에 microbeads를 전송합니다. 원하는 경우 이러한 유리 또는 플라스틱 배양 접시와 같은 다른 부드러운 표면은 Parafilm에 대한 대체하실 수 있습니다. </li><li> 알지네이트요의 microbeads에 (3.1)에서 준비 알지네이트요 솔루션을 전송합니다. 알지네이트요 솔루션의 볼륨이 완전히 알지네이트요의 microbeads를 충당해야한다. microbeads 45 분 알지네이트요 솔루션 유지합시다. </li><li> microbeads에 바인딩되지 과잉 알지네이트요 솔루션을 제거하는 피펫을 사용합니다. </li><li> 에 microbeads를 전송22mM CaCl 2 용액 있습니다. 이것은 별개의 외부 알지네이트요 계층의 형성 그 결과 microbeads 주위 알지네이트요 솔루션을 crosslinks. </li><li> 이분 각각 정상 식염수에 CaCl 2 22 밀리미터 든 세 세차장을 수행합니다. 현미경 microbeads을 관찰 하는게 핵심 microbead 주위에 형성된 볼 수있는 뚜렷한 외부 알지네이트요 층 수 있도록해야합니다. </li></ol> 4. 대표 결과 <img src="/files/ftp_upload/3388/3388fig1.jpg" alt="그림 1" /> 그림 1. multilayered 알지네이트요의 microbeads를 만드는 절차의 개략도. Khanna 외로부터 허가를 재현. J Biomed Mater 해상도 A. 11월, 95 (2), 632-40 (2010). <img src="/files/ftp_upload/3388/3388fig2.jpg" alt="그림 2" /> 그림 2. ()와 (B)의 알지네이트요 microbeads의 위상 콘트라스트 이미지입니다. () microbead을 보여줍니다이후 합성 단계 (1.7), (B) 단계 (3.7)을 마친 후 현재 뚜렷한 외부 알지네이트요 레이어를 표시하면서. (C) 외부 계층에 캡슐 fluorescently-라벨 BSA 단백질의 FITC 이미지입니다. Khanna 외로부터 허가를 재현. J Biomed Mater 해상도 A. 11월, 95 (2), 632-40 (2010). <img src="/files/ftp_upload/3388/3388fig3.jpg" alt="그림 3" /> 그림 3. 외부 알지네이트요 레이어의 크기는 사용 알지네이트요의 조성과 농도를 기반으로 다양한 수 있습니다. 우리의 결과는 LVM과 LVG의 알지네이트요 모두를 위해 증가 알지네이트요 농도와 외부 레이어의 크기 증가, 그 LVG 알지네이트요가 동일 농도에서 LVM의 알지네이트요보다 두꺼운 아우터 레이어를 얻을, 것을 보여줍니다. Khanna 외로부터 허가를 재현. J Biomed Mater 해상도 A. 11월, 95 (2), 632-40 (2010). <img src="/files/ftp_upload/3388/3388fig4.jpg" alt="그림 4"/&gt; 그림 4. FGF-1의 출시, LVM 및 LVG의 알지네이트요 사용의 농도를 기반으로 다양한 외부 알지네이트요 계층에서 angiogenic 성장 인자 단백질. (A)와 (B) 쇼 퍼센트 릴리스, 그리고 (C)와 (D) 다른 외부 레이어 공법 시간 대 FGF-1의 해당 대량 릴리스를 나타냅니다. 버스트 초기 5 H (A와 C) 이내에 모든 조건을 위해 전시 발표하고, 최대 30 일 대한 낮은 선량 연속 릴리스 (B와 D)이 있습니다. Khanna 외로부터 허가를 재현. J Biomed Mater 해상도 A. 11월;. 95 (2), 632-40 (2010) <a href="http://www.jove.com/files/ftp_upload/3388/3388fig4large.jpg" target="_blank">큰 그림을 보려면 여기를 누르십시오</a> .

<ol>
	<li>Darrabie, M. D., Kendall, W. F., Opara, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+Poly-L-+Ornithine-coated+alginate+microcapsules.">Characteristics of Poly-L- Ornithine-coated alginate microcapsules.</a> Biomaterials. 26, 6846-6852 (2005).</li><li>Yamagiwa, K., Kozawa, T., Ohkawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+alginate+composition+and+gelling+conditions+on+diffusional+and+mechanical+properties+of+calcium-alginate+gel+beads.">Effects of alginate composition and gelling conditions on diffusional and mechanical properties of calcium-alginate gel beads.</a> J. Chem. Eng. Jpn. 28, 462-467 (1995).</li><li>Amsden, B., Turner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+characteristics+of+calcium+alginate+gels.">Diffusion characteristics of calcium alginate gels.</a> Biotechnol. Bioeng. 65, 605-610 (1999).</li><li>Uriel, S., Brey, E. M., Greisler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+low+levels+of+fibroblast+growth+factor-1+promote+persistent+microvascular+network+formation.">Sustained low levels of fibroblast growth factor-1 promote persistent microvascular network formation.</a> Am. J. Surg. 192, 604-609 (2006).</li><li>Moya, M. L., Lucas, S., Francis-Sedlak, M., Liu, X., Garfinkel, M. R., Huang, J. J., Cheng, M. H., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+delivery+of+FGF-1+increases+vascular+density+in+comparison+to+bolus+administration.">Sustained delivery of FGF-1 increases vascular density in comparison to bolus administration.</a> Microvasc. Res. 78, 142-147 (2009).</li><li>Moya, M. L., Cheng, M. H., Huang, J. J., Francis-Sedlak, M. E., Kao, S. W., Opara, E. C., Brey, E. 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+effect+of+FGF-1+loaded+alginate+microbeads+on+neovascularization+and+adipogenesis+in+a+vascular+pedicle+model+of+adipose+tissue+engineering.">The effect of FGF-1 loaded alginate microbeads on neovascularization and adipogenesis in a vascular pedicle model of adipose tissue engineering.</a> Biomaterials. 31, 2816-2826 (2010).</li><li>Khanna, O., Moya, M. L., Greisler, H. P., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayered+Microcapsules+for+the+Sustained-Release+of+Angiogenic+Proteins+From+Encapsulated+Cells.">Multilayered Microcapsules for the Sustained-Release of Angiogenic Proteins From Encapsulated Cells.</a> Am. J. Surg. 200, 655-658 (2010).</li><li>Khanna, O., Moya, M. L., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+multilayered+alginate+microcapsules+for+the+sustained-release+of+fibroblast+growth+factor-1.">Synthesis of multilayered alginate microcapsules for the sustained-release of fibroblast growth factor-1.</a> J. Biomed. Mater. Res. A. 95, 632-640 (2010).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

알긴산은 해조류에서 추출 및 1,4 &#39;-β-D-mannuronic 산 (M)와 α-L-guluronic 산성 (G) 2,3 단위로 구성되어 자연, 산성 다당류이다. 단순 겔화가 발생했을 때 같은 칼슘이 같은 이가의 양이온, +, 시니어 2 +, 또는 바 2 + 인접 알지네이트요 체인 사이의 이온 가교를 형성 G-단량체와 상호 작용. 알지네이트요의 Microbeads은 chondrocytes, hepatocytes를 포함한 fibroblast의 성장 인...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> Pronova Ultrapure LVG의 알지네이트요 </td><td> 노바 - 매트릭스 </td><td> 4,200,006 </td><td> 알지네이트요 공법의 종류가 있습니다. 알지네이트요의 선택은 크기를 포함하여 microbeads 끝 특성, 기계적 특성 및 전송에 영향을 미칩니다. 사용된 구성은 주어진 응용에 최적화된해야합니다. </td></tr><tr><td> Pronova Ultrapure LVM의 알지네이트요 </td><td> 노바 - 매트릭스 </td><td> 4,200,206 </td><td> 알지네이트요 공법의 종류가 있습니다. 알지네이트요의 선택은 크기를 포함하여 microbeads 끝 특성, 기계적 특성 및 전송에 영향을 미칩니다. 사용된 구성은 주어진 응용에 최적화된해야합니다. </td></tr><tr><td> 폴리-L-ornithine 하이드로 클로라이드 </td><td> 시그마 - 올드 리치 </td><td> P2533 </td><td></td></tr></tbody></table>

generation of alginate microspheres for biomedical applications

알긴산 기반의 자료가 있기 때문에 그들의 친수성 ​​자연, biocompatibility, 물리적 아키텍처 생명 의학 애플 리케이션에 상당한 주목을 받았습니다. 응용 프로그램은 세포 캡슐, 약물 전달, 줄기 세포 배양 및 조직 엔지니어링 공사장 공중 발판을 포함합니다. 실제로 임상 시험은 현재 islets는 유형 I 당뇨병의 치료로서 PLO 코팅 알지네이트요의 microbeads에 캡슐되는 수행되고있다. 그러나, islets 많은 수의는 가난한 생존 다음의 이식에 의한 효능 필요합니다. 로컬 캡슐 세포 주위 microvascular 네트워크 형성을 자극하는 능력은 산소, 포도당 및 기타 필수 영양소 개선된 교통 수단을 통해 그들의 생존을 증가시킬 수 있습니다. Fibroblast 성장 인자-1 (FGF-1)은 혈관 형성을 자극하고 허혈성 조직의 산소 수준을 향상시킬 수 자연스럽게 발생하는 성장 인자이다. 그것이 sust으로 전달되었을 때 FGF-1의 효능이 향상됩니다오히려 하나의 대형 bolus 행정부보다 ained 패션. 섬 캡슐 시스템의 성장 요인의 지역 장기 릴리스는 잠재적으로 기능성 그래프트 결과를 개선, 이식된 세포를 향해 직접적으로 혈관의 성장을 자극 수 있습니다. 이 문서에서 우리는 생명 의학 애플 리케이션에서 사용할 수 알지네이트요 microspheres의 준비 절차를 설명합니다. 또한, 우리는 multilayered 알지네이트요의 microbeads를 생성용으로 개발된 방법을 설명합니다. 세포는 외부 알지네이트요 계층의 내부 알지네이트요 코어, 그리고 angiogenic 단백질에 캡슐 수 있습니다. 이 외부 레이어에서 단백질의 릴리스는 이식된 islets 향해 직접 현지 microvascular 네트워크의 형성을 자극합니다.

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Generation of Alginate Microspheres for Biomedical Applications

다음 섹션에서 우리는 생명 의학 애플 리케이션에서 사용할 수 알지네이트요 microspheres의 준비 절차를 설명합니다. 우리는 특히 1 형 당뇨병에 대한 잠재적인 치료로서 세포 및 단백질 캡슐의 이중 목적 multilayered 알지네이트요 microspheres를 만들기위한 기술을 설명합니다.

바이오 메디컬 응용 프로그램을위한 알긴산 Microspheres의 생성

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

generation-of-alginate-microspheres-for-biomedical-applications

Research

JoVE Journal

Medicine

20.6K 조회수.  Illinois Institute of Technology. 다음 섹션에서 우리는 생명 의학 애플 리케이션에서 사용할 수 알지네이트요 microspheres의 준비 절차를 설명합니다. 우리는 특히 1 형 당뇨병에 대한 잠재적인 치료로서 세포 및 단백질 캡슐의 이중 목적 multilayered 알지네이트요 microspheres를 만들기위한 기술을 설명합니다.

비디오: Generation of Alginate Microspheres for Biomedical Applications

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

종합 흉부 종양학 데이터베이스의 생성 - Translational 연구 도구

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

연구

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Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

임상 및 전임상 연구 응용 프로그램에 대한 종양 세포 (CTC) 분석 실험을 순환 반자동의 적응

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

차세대 시퀀싱에 대한 수집 및 타액 DNA의 추출

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

분리, 냉동 보존 및 문화 임상 응용 프로그램에 대한 인간 양막 상피 세포

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

실험 녹내장은 안구하는 미소 자석을 투여 한

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

인간의 뇌하수체 선종 세포의 3 차원 알긴산 비드 문화

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Kinomic 프로파일 의약품 응답 테스트를위한 3D 인간의를 Biogel 문화 시스템을 사용 Microtumors 및 환자 유래 아교 모세포종 세포의 생성

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

유방암 전이성 행동에 장기 별 영향 유학을위한 장기 에어컨 미디어 및 응용 프로그램의 생성

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Drug Repurposing Hypothesis Generation Using the &quot;RE:fine Drugs&quot; System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

은 &quot;RE를 : 좋은 약&quot;을 사용하여 의약품 용도 변경 가설 발전 시스템

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

샘플링 전략 및 돼지 생물 의학 모델에서 Biobank 조직 샘플의 처리

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...