웰컴 트러스트의 자금께서는 (부여 번호 085245/Z/08/Z) 인정됩니다. 노팅엄 (Nottingham)의 나노 기술과 Nanoscience 센터께서는이 장비를 자금에 대한 라만 시스템에 대한 액세스를 제공와 이스트 미들랜드 진흥원에 인정된다.

<ol>
	<li>Hook, A. L., Anderson, D. G., Langer, R., Williams, P., Davies, M. C., Alexander, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+methods+applied+in+biomaterial+development+and+discovery.">High throughput methods applied in biomaterial development and discovery.</a> Biomaterials. 31, 187-198 (2010).</li><li>Anderson, D. G., Levenberg, S., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoliter-scale+synthesis+of+arrayed+biomaterials+and+application+to+human+embryonic+stem+cells.">Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells.</a> Nature Biotechnology. 22, 863-866 (2004).</li><li>Mei, Y., Saha, K., Bogatyrev, S. R., Yang, J., Hook, A. L., Kalcioglu, Z. I., Cho, S. W., Mitalipova, M., Pyzocha, N., Rojas, F., Van Vliet, K. J., Davies, M. C., Alexander, M. R., Langer, R., Jaenisch, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+development+of+biomaterials+for+clonal+growth+of+human+pluripotent+stem+cells.">Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells.</a> Nature Materials. 9, 768-778 (2010).</li><li>Pernagallo, S., Wu, M., Gallagher, M. P., Bradley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonising+new+frontiers-microarrays+reveal+biofilm+modulating+polymers.">Colonising new frontiers-microarrays reveal biofilm modulating polymers.</a> Journal of Materials Chemistry. 21, 96-101 (2011).</li><li>Yang, J., Mei, Y., Hook, A. L., Taylor, M., Urquhart, A. J., Bogatyrev, S. R., Langer, R., Anderson, D. G., Davies, M. C., Alexander, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+surface+functionalities+that+control+human+embryoid+body+cell+adhesion+revealed+by+high+throughput+surface+characterization+of+combinatorial+material+microarrays.">Polymer surface functionalities that control human embryoid body cell adhesion revealed by high throughput surface characterization of combinatorial material microarrays.</a> Biomaterials. 31, 8827-8838 (2010).</li><li>Urquhart, A. J., Anderson, D. G., Taylor, M., Alexander, M. R., Langer, R., Davies, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+surface+characterisation+of+a+combinatorial+material+library.">High throughput surface characterisation of a combinatorial material library.</a> Advanced Materials. 19, 2486-2491 (2007).</li><li>Taylor, M., Urquhart, A. J., Zelzer, M., Davies, M. C., Alexander, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picoliter+water+contact+angle+measurement+on+polymers.">Picoliter water contact angle measurement on polymers.</a> Langmuir. 23, 6875-6878 (2007).</li><li>Urquhart, A. J., Taylor, M., Anderson, D. G., Langer, R., Davies, M. C., Alexander, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TOF-SIMS+analysis+of+a+576+micropatterned+copolymer+array+to+reveal+surface+moieties+that+control+wettability.">TOF-SIMS analysis of a 576 micropatterned copolymer array to reveal surface moieties that control wettability.</a> Analytical Chemistry. 80, 135-142 (2008).</li><li>Hook, A. L., Yang, J., Chen, X., Roberts, C. J., Mei, Y., Anderson, D. G., Langer, R., Alexander, M. R., Davies, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrylate+polymers+with+hydro-responsive+topography.">Acrylate polymers with hydro-responsive topography.</a> Soft Matter. 7, 7194-9197 (2011).</li><li>Pernagallo, S., Unciti-Broceta, A., Diaz-Mochon, J. J., Bradley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+cellular+morphology+and+biocompatibility+using+polymer+microarrays.">Deciphering cellular morphology and biocompatibility using polymer microarrays.</a> Biomedical Materials. 3, (2008).</li></ol>

우리는 공개 할 아무 것도 없습니다.

폴리머 microarrays이 성공적으로 생물 검정의 소설 폴리머의 심사 수백명의 새로운 자료의 발견에 사용되며 이후 유용한 장치를 확장 할 수 있습니다 &#39;히트&#39;자료를 식별되었습니다. 이 경우 설명 된 표면 특성은 생물학적 분석에 후속 고용 될 수 있으며 전용 &#39;히트&#39;자료에 자세히 그 자료를 공부합니다. HTSC이 방법을 채용 experimentalist을 사용할 수없는 경우이 전략은 관심이있을 것으로 생각됩니다. 그러나, 완전히 생물 - 물질 상호 작용을 할 이전 이후 일반적으로 구조 기능 동향을 관찰하는 데 사용할 수있는 HTSC 방법을 사용하여 생물 assays에 분석해야 재료의 수백의 전체 배열을 공부 폴리머 microarrays를 활용합니다. 연락처 인쇄 핀 홀더 내에서 자유롭게 등을 아래로 슬라이딩 금속 핀에 의존하고 있습니다. 핀과 핀 홀더 청결은 파라마운트가 P를 보장합니다 것입니다rinting 성공적으로 발생하고 엄격하게 실시해야합니다. 인쇄 핀 홀더 내에서 핀의 적절한 운동을 실행 시작하기 전에 현재의 노 단량체와, 드라이 실행을 수행하여 테스트 할 수 있습니다. 핀의 움직임이 reproducibly 달성 될 때까지 청소 단계는 계속해야합니다. 상당한 생각은 단량체 혼합물의 설계로 이동합니다. 쉽게 폴리머의 조합 라이브러리를 생성하기 위해, 공중 합체의 수백은 다른 비율에서 몇 단량체를 혼합하여 형성된다. 이 유리 슬라이드의 구조에 적합한 24 X 24 배열을 형성으로 일반적으로 우리는 576 회원 라이브러리를 생산하고 있습니다. 가장 쉬운 방법은 2시 1분 비율 pairwise 24 단량체를 혼합하는 것입니다 가장 조합 공간을 탐구 조합 라이브러리를 생성하기 위해. 또한, 배열 내의 작곡 그라디언트의 포함은 최적의 단량체 조성은 절망 할 수 동향 관찰을 가능하게에 유용합니다etermined. 이 22 단량체의 예는 순차적으로 1 6의 제 2 구성 요소로 희석 된 공동 단량체 혼합물의 첫 번째 구성 요소로 사용할 수 있습니다. 5 dilutions를 사용하는 경우, 예를 들어 90:10, 75:25, 50:50, 25:75 및 10:90의 비율에서 첫 번째와 두 번째 구성 요소를 혼합이 488 독특한 공중 합체 솔루션에 나타날 수 있습니다. 576에 총을 가져, 종종 중요한 표본이 도입 될 수있다 사용되는 단량체의 homopolymers의 복제합니다. 576 단량체 솔루션은 2 개의 384도 접시에 투여해야합니다. 로봇을 프로그래밍은 단량체의 위치를​​ 기준으로 두 개의 동일한 번호판을 가지고 쉽게이며, 따라서 단량체 솔루션은 두 판 사이에 균등하게 분할해야합니다. 시간의 상당한 금액이 채널 피펫을 사용하여 소스 플레이트의 준비에 저장 될 수 있으며, 소스 플레이트의 디자인은 멀티 채널 피펫의 사용을 악용하기 위해 결정해야합니다. &lt;/ P&gt; 배열의 자동화 HTSC을 달성하기 위해 현장 위치가 성공적으로 특성화 장치와 정렬되어야합니다. 일반적으로 아크릴 레이트 배열의 피치 500-1000 μm이며, 폴리머 장소 직경은 300 μm입니다. 대부분의 XY 단계는 10 μm 이하 해상도를 가지고, 따라서 올바른 크기의 샘플 위치 소프트웨어에 입력 한 후 배열의 위치를​​ 안정적으로 액세스 할 수있는 표면 특성화 장치에 대한 충분한 내성이 있습니다. 자동 위치에 대한 제한은 실제로 배열의 정확한 인쇄합니다. 이 인쇄 무대에서 기판의 움직임을 방지하는 것이 중요합니다 정확하게 인쇄되도록하려면 어느 적절한 슬라이드 치수 (미국 및 EU 표준 슬라이드 크기가 모두 존재합니다)와 함께 진공 흡입 또는 스프링 클램프를 사용합니다. ToF - 심스는 샘플에 대한 오염을 준수합니다 매우 민감한 표면 기술입니다. 따라서, 맨 먼저주의가되어야합니다표면과의 접촉을 피합니다. 샘플은 처리하지만, 관심의 표면은 깨끗 장갑 (바람직 폴리에틸렌)와 갓 청소 핀셋과와 접촉하지해야합니다. 우리는 일반적으로 클로로포름과 헥산으로 씻어. 측정하기 전에 샘플 보관소가 가장 차별화 슬라이드를 보유하고 샘플 홀더에서 수행, 예를 들어 5 슬라이드 홀더 또는 20 슬라이드 홀더 (Holder).입니다 배열은 즉, 많은 생물 검정 형식과 판독과 호환되도록 특별히 설계되어 사용 된 기판은 이상적으로 형광 스캐너 가벼운 현미경에 적합한 현미경 슬라이드입니다. 이 형식이 잘 많은 소재 생물학적 상호 작용을 여행에 적합한 의미합니다. 또한, 형식은 소재의 수백이 병렬로 검사를 할 수 있습니다. 이 더 많은 자료를 각각의 새 재료 화학은 개별적으로 상영되어 상기 기존의 방법보다 검사를 할 수 있습니다. 생물 - 물질 상호 작용에 대한 증가 범위는 허용생물학적 표면의 상호 작용뿐만 아니라 특정 응용 프로그램에 대한 최적의 자료를 찾는 메커니즘의 해설을위한 s입니다.

<table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 / 모델 번호 </td></tr><tr><td> 에폭시 슬라이드 </td><td> Genetix </td><td> K2652 </td></tr><tr><td> 프린터에 문의 </td><td> Biodot </td><td> XYZ3060 플랫폼 </td></tr><tr><td> 금속 핀 </td><td> ArrayIt </td><td> 946MP6B </td></tr><tr><td> ToF - 심스 장비 </td><td> ION-TOF </td><td></td></tr><tr><td> XPS 악기 </td><td> Kratos </td><td></td></tr><tr><td> WCA 장치 </td><td> Krüss </td><td> DSA 100 </td></tr><tr><td> AFM </td><td> Bruker </td><td> 치수 아이콘 </td></tr><tr><td> RPMI-1640 세포 배양 미디어 </td><td> 시그마 - 알드리치 </td><td> R0883 </td></tr><tr><td> SYTO17 </td><td> Invitrogen </td><td> S-7579 </td></tr></tbody></table>

polymer microarrays for high throughput discovery of biomaterials

혼합이 균일 한 혼합물에 두 개 이상의 구성 요소를 결합 단위 조작입니다. 이 작품은 인라인 정적 믹서를 사용하여 두 점성 액체 스트림을 혼합하는 것입니다. 믹서는 구성 요소 사이의 계면 접촉을 증가 전단과 extensional 흐름을 채용 분할 및 재조합 디자인입니다. 프로토 타입 분할 - 및 - 재조합 (SAR) 믹서는 접시는 PVC 파이프에서 개최에서 개최 얇은 레이저 컷 폴리 (메틸 메타 크릴 레이트) (PMMA)의 일련의 정렬에 의해 건설되었습니다. 이 장치에 혼합하는 것은 그림에있는 사진에 설명되어 있습니다. 1. 레드 염색은 시험 유체의 일부에 추가되고 주요 (undyed) 구성 요소에 혼합되는 작은 구성 요소로 사용되었다. 이 혼합 섹션을 통해 유동하는 믹서의 입구에서 추적 유체의 주입 층은 두 개의 레이어로 나뉩니다. 이후의 각 혼합 섹션에서, 수평 레이어의 번호가 중복됩니다. 궁극적으로, 염료의 단일 스트림이 균일하게 분산되어 thr장치의 단면 oughout. 장치에 혼합 0.2 % Carbopol와 유사한 구성의 도핑 추적 유체의 비 뉴턴 시험 유체를 사용하면 자기 공명 영상 (MRI)를 사용하여 시각화합니다. MRI는 분자 화학 및 물리적 환경뿐만 아니라 마이크론에서 센티미터에 길이 저울에 샘플 구조의 매우 강력한 실험 프로브입니다. 이 감도는 물리적, 화학적 및 / 또는 인간의 다공성 매체, 1, 2로 음식에 이르기까지 물질의 생물학적 특성을 특징 이러한 기술의 광범위한 응용 프로그램에서 발생했습니다. 여기에 사용되는 장비와 조건은 오일 등의 일반 생수와 유기농 액체 등의 NMR 모바일 1 H의 상당한 양의를 포함하는 영상 액체에 적합합니다. 전통적으로 MRI는 산업 환경에 적합하고 실험실 (그림 2)에서 휴대하지하지 않은 슈퍼 수행 자석을 활용하고 있습니다. 최근 발전 전N 자석 기술은 대량의 건설 이미징 프로세스 흐름에 적합한 산업 상 호환 자석을 허용하고 있습니다. 여기 MRI는 혼합 과정에서 다른 축 위치에 spatially 해결 구성 요소 농도를 제공합니다. 이 작품 문서는 실시간 개인 케어 제품에 응용 프로그램과 함께 혼합 분배를 통해 고점도 유체의 혼합.

Watch this Scientific Journal Video about Polymer Microarrays for High Throughput Discovery of Biomaterials at JoVE.com

Polymer Microarrays for High Throughput Discovery of Biomaterials

온 - 칩 photopolymerization 기술을 사용하여 폴리머 microarray의 형성에 대한 설명입니다. 원자 힘 현미경, 물 접촉 각도 측정, X-선 광전자 분광법 및 비행 차 이온 질량 분광법과 세포 부착 분석 시간을 사용하여 높은 처리량 표면 특성도 설명되어 있습니다.

Biomaterials 높은 처리량 발견을위한 폴리머 Microarrays

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

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Bioengineering

14.4K 조회수.  University of Nottingham. 온 - 칩 photopolymerization 기술을 사용하여 폴리머 microarray의 형성에 대한 설명입니다. 원자 힘 현미경, 물 접촉 각도 측정, X-선 광전자 분광법 및 비행 차 이온 질량 분광법과 세포 부착 분석 시간을 사용하여 높은 처리량 표면 특성도 설명되어 있습니다.

비디오: Polymer Microarrays for High Throughput Discovery of Biomaterials

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

높은 처리량 Antifungal 약물 검사를위한 칸디다 albicans Biofilm 칩 (CA BChip)

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

연구

생체공학

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

조합의 합성과 고분자 필름과 Nanoparticle 도서관에서 높은 처리량 단백질 출시

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Polyanhydride의 Nanoparticles의 높은 처리량 탄수화물의 합성과 작용화

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

각종 설치류 모델에 Cystometric 분석을 사용하여 방광 증원을위한 Biomaterials 평가

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

전체 피부 결함 모델은 생체의 혈관 신생을 평가하기<em&gt; 생체</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

A Coupled Experiment-finite Element Modeling Methodology for Assessing High Strain Rate Mechanical Response of Soft Biomaterials

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. brain, liver, tendon, fat, etc.) when exposed to high strain rates. This study utilized a Split-Hopkinson Pressure Bar (SHPB) to generate strain rates of 100-1,500 sec-1. The SHPB employed a striker bar consisting of a viscoelastic material (polycarbonate). A sample of the biomaterial was obtained shortly postmortem and prepared for SHPB testing. The specimen was interposed between the incident and transmitted bars, and the pneumatic components of the SHPB were activated to drive the striker bar toward the incident bar. The resulting impact generated a compressive stress wave (i.e. incident wave) that traveled through the incident bar. When the compressive stress wave reached the end of the incident bar, a portion continued forward through the sample and transmitted bar (i.e. transmitted wave) while another portion reversed through the incident bar as a tensile wave (i.e. reflected wave). These waves were measured using strain gages mounted on the incident and transmitted bars. The true stress-strain behavior of the sample was determined from equations based on wave propagation and dynamic force equilibrium. The experimental stress-strain response was three dimensional in nature because the specimen bulged. As such, the hydrostatic stress (first invariant) was used to generate the stress-strain response. In order to extract the uniaxial (one-dimensional) mechanical response of the tissue, an iterative coupled optimization was performed using experimental results and Finite Element Analysis (FEA), which contained an Internal State Variable (ISV) material model used for the tissue. The ISV material model used in the FE simulations of the experimental setup was iteratively calibrated (i.e. optimized) to the experimental data such that the experiment and FEA strain gage values and first invariant of stresses were in good agreement.

The current study prescribes a coupled experiment-finite element simulation methodology to obtain the uniaxial dynamic mechanical response of soft biomaterials (brain, liver, tendon, fat, etc.). The multiaxial experimental results that arose because of specimen bulging obtained from Split-Hopkinson Pressure Bar testing were rendered to a uniaxial true stress-strain behavior when simulated through iterative optimization of the finite element analysis of the biomaterial.

소프트 생체 재료의 높은 변형 속도 기계 응답을 평가하기위한 결합 실험 - 유한 요소 모델링 방법론

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Tomoauto 사용 : 프로토콜 높은 처리량 자동화 된 곳을 알아내는 - 전자 단층 촬영을 위해

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

높은 처리량 단일 세포의 분리 및 문화를위한 미세 유체 플랫폼

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

의료 기기에 대한 박테리아의 높은 처리량 식별 구충제 폴리머

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.