국립 심장 폐 혈액 연구소 (수상 # R00HL093358에 LDB) -이 작품은 국립 보건원에 의해 지원되었다.

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical,+biochemical,+and+extracellular+matrix+effects+on+vascular+smooth+muscle+cell+phenotype.">Mechanical, biochemical, and extracellular matrix effects on vascular smooth muscle cell phenotype.</a> Journal of applied physiology. 98, 2321-2327 (2005).</li><li>Guarnieri, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalently+immobilized+RGD+gradient+on+PEG+hydrogel+scaffold+influences+cell+migration+parameters.">Covalently immobilized RGD gradient on PEG hydrogel scaffold influences cell migration parameters.</a> Acta. 6, 2532-2539 (2010).</li><li>Krebs, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+poly(lactic-co-glycolic)+acid+scaffolds+with+in+situ+pore+formation+for+tissue+engineering.">Injectable poly(lactic-co-glycolic) acid scaffolds with in situ pore formation for tissue engineering.</a> Acta. 5, 2847-2859 (2009).</li><li>Zimmermann, W. -. 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관심 없음 충돌 선언하지 않습니다.

심근 시스템의 체외 모델에서 일관성 있고 가능한 삼차원의 섬유소 젤 결과에 신생아 쥐 cardiomyocytes의 캡슐. 세포 속은 때, 그들은 metabolically 활성화하고, 압축 개조 및 원시 심장 조직을 12 일치 세포외 매트릭스를 재현 할 수 있기 때문에 섬유소가 선호 biomaterial입니다. 우리가 cardiomyocytes이 환경에 자신을 맞출 수 있기 때문에, 그들의 기능은 등방성 조직 6 비해 큰 수축 강제의 결과로 심장 근육에 더 많은 특징입니다. 잠재적인 치료 어플 리케이션을 위해, 그것은 생존과 기능을 모두 촉진 물질 내에서 세포를 캡슐이 필요합니다. 프로토콜은 3 차원 microenvironment에서 심장 세포의 동작을 제어하는​​ 섬유소 네트워크를 만들기위한 효율적이고 정확한 방법을 보여 여기 소개. 몇 가지 잠재적인 문제는 이러한 구조의 생성과 문화 중에 발생할 수 있습니다. 한 잠재적인 문제는 크게 구조의 기능에 영향을 미칩니다 이전 캡슐화하기 위해 세포 생존 능력을 유지하고 있습니다. 노력은 격리와 섬유소 젤 내의 세포 캡슐 사이의 시간을 줄임으로써 격리 다음과 세포 죽음을 제한하여야한다. 구조는 엄격한 일정에 따라 매일 미디어를 제공하여야한다. 또한, 사용되는 솔루션의 모든 균등을 보장하기 위해 중요합니다. 피브리노겐, 트롬빈​​과 세포의 혼합물은 이기종 환경을 생성하는 경우, ECM을 리모델링하는 세포의 능력은 기계적 부부와 계약이 잠재적으로 방해합니다. 설계 조직의 연속성에 장애를 방지하기 위해 구성의 주입시 공기 방울의 형성을 방지하는 것도 중요하다. 이 문제를 완화하는 한 가지 방법은 주사기로 구성을 천천히 주입하는 데 필요한 것보다 더 많은 겔을 그릴 수 있습니다. 마지막으로, 한번 섬유소 매트릭스가 설정하고 24 시간 동안 문화 매체되었습니다, 그것은 섬유소 젤의 셀 기반의 압축을 촉진하기 위해 링 몰드의 측면에서 구조를 분리하는 것이 필수적입니다. 금형의 중간에있는 구조를 유지할 수있다는 것은 가스와 영양소 교환을 용이하게합니다. 반지의 금형의 측면에 준수는 원하는 세포 정렬을 방해 수 있습니다. 그 의식이 잘린가 건강 및 미국 수의학 협회의 의학 국립 연구소 모두의 지침에 따라 적절한 방법으로이 프로토콜의 euthanization의 방법으로 사용됩니다하는 것이 중요합니다. 그러나, 일부 기관은 신생아 쥐에 대한 잘린 다음 마취제 사용을 권장합니다. 그것은 excised 심장 조직 / 세포 hypoxic 조건에서 시간의 최소한을 보장하기 때문에 우리는 의식 잘린을 선택했습니다. hypoxia의 비교적 적은 금액은 국소 빈혈과 크게이 프로토콜의 결과에 영향을 미칠 수있는 잠재적 myocyte 죽음을, myocyte 발생할 수 있습니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 나트륨 염화물 </td><td> 시그마 </td><td> S7653 </td><td> PBS </td></tr><tr><td> 염화칼륨 </td><td> 시그마 </td><td> P9333 </td><td> PBS </td></tr><tr><td> 나트륨 인산염 이염 </td><td> 시그마 </td><td> S7907 </td><td> PBS </td></tr><tr><td> 인산 칼륨 일염기의 </td><td> 시그마 </td><td> P5655 </td><td> PBS </td></tr><tr><td> 포도당 </td><td> 시그마 </td><td> G5400 </td><td> 절연 </td></tr><tr><td> hemostat </td><td> 파인 과학 도구 </td><td> 91308-12 </td><td> 절연 </td></tr><tr><td> 고급 핀셋 </td><td> 파인 과학 도구 </td><td> 11251-20 </td><td> 절연 </td></tr><tr><td> 큰 가위 </td><td> 파인 과학 도구 </td><td> 91401-14 </td><td> 절연 </td></tr><tr><td> 마이크로 가위 </td><td> 파인 과학 도구 </td><td> 91501-09 </td><td> 절연 </td></tr><tr><td> 메스 핸들 </td><td> 파인 과학 도구 </td><td> 10008-13 </td><td> 절연 </td></tr><tr><td> 메스 블레이드 </td><td> 피셔 과학 </td><td> 08-918 - 5A </td><td> 절연 </td></tr><tr><td> 흡수 벤치 underpad </td><td> VWR </td><td> 56617-014 </td><td> 절연 </td></tr><tr><td> 무균 드레이프 </td><td> 피셔 과학 </td><td> GM42526 </td><td> 절연 </td></tr><tr><td> 압력솥 가방 </td><td> 어부 </td><td> 01-812-54 </td><td> 절연 </td></tr><tr><td> 거즈 패드 </td><td> 피셔 과학 </td><td> 13-761-52 </td><td> 절연 </td></tr><tr><td> betadine </td><td> Purdue 제품 </td><td> 67618-150-01 </td><td> 절연 </td></tr><tr><td> 멸균 장갑 </td><td> 피셔 과학 </td><td> 19-020 </td><td> 절연 </td></tr><tr><td> 무균 전송 피펫 </td><td> 피셔 과학 </td><td> 9962 </td><td> 절연 </td></tr><tr><td> collagenase </td><td> 워싱턴 </td><td> CLS2 </td><td> 절연 </td></tr><tr><td> 테플론로드 4분의 1 인치 직경 </td><td> McMaster - 카 </td><td> 8546K11 </td><td> 금형 부품 </td></tr><tr><td> 테플론 튜브 4분의 1 인치 ID, 2분의 1 인치 OD </td><td> McMaster - 카 </td><td> 8547K31 </td><td> 금형 부품 </td></tr><tr><td> 실리콘 O - 링 4분의 1 인치 ID, 2분의 1 인치 OD </td><td> McMaster - 카 </td><td> 9396K204 </td><td> 금형 부품 </td></tr><tr><td> 테플론 튜브 4분의 1 인치 ID, 16분의 5 인치 OD </td><td> McMaster - 카 </td><td> 52355K14 </td><td> 금형 부품 </td></tr><tr><td> 켄달 monoject의 주사기 6cc </td><td> 피셔 과학 </td><td> 05-561-41 </td><td> 금형 부품 </td></tr><tr><td> BD 주사기 3cc </td><td> 피셔 과학 </td><td> 309585 </td><td> 금형 부품 </td></tr><tr><td> 소 피브리노겐 </td><td> 시그마 </td><td> F8630 </td><td> 구축 </td></tr><tr><td> 소 트롬빈 </td><td> 시그마 </td><td> T7513 </td><td> 구축 </td></tr><tr><td> 1 M HEPES </td><td> 시그마 </td><td> H0887 </td><td> 구축 </td></tr><tr><td> 나트륨 염화물 </td><td> 시그마 </td><td> S7653 </td><td> 구축 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 10,569 </td><td> 구축 </td></tr><tr><td> Pluronic F - 127 </td><td> 시그마 </td><td> P2443 </td><td> 구축 </td></tr><tr><td> 염화칼슘 </td><td> 시그마 </td><td> 383147 </td><td> 구축 </td></tr><tr><td> 0.2 미크론 필터 </td><td> 피셔 과학 </td><td> SCGVT05RE </td><td> 구축 </td></tr><tr><td> 40 마이크론 셀 strainers </td><td> 피셔 과학 </td><td> 22-363-547 </td><td> 구축 </td></tr><tr><td> 0.45 미크론 병 톱 필터 </td><td> 코닝 </td><td> 430627 </td><td> 구축 </td></tr><tr><td> 유리 프리 필터 </td><td> Millipore </td><td> AP2007500 </td><td> 구축 </td></tr><tr><td> 18G 1 2분의 1 인치 긴 바늘 </td><td> 피셔 과학 </td><td> 14-826 - 5D </td><td> 구축 </td></tr><tr><td> 21G 1 인치 바늘 </td><td> 피셔 과학 </td><td> 14 - 826C </td><td> 구축 </td></tr><tr><td> 단지를 구축 </td><td> 피셔 과학 </td><td> 2116 </td><td> 구축 </td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> Invitrogen </td><td> 15,140 </td><td> 미디어 </td></tr><tr><td> 말 혈청 </td><td> 시그마 </td><td> H1138 </td><td> 미디어 </td></tr><tr><td> 태아 소 혈청 </td><td> Invitrogen </td><td> 16,000 </td><td> 미디어 </td></tr><tr><td> aminocaproic 산 </td><td> Acros의 생명체 </td><td> 103305000 </td><td> 미디어 </td></tr><tr><td> 아스코르비 산 </td><td> 시그마 </td><td> A5960 </td><td> 미디어 </td></tr><tr><td> 인슐린 </td><td> 시그마 </td><td> I9278 </td><td> 미디어 </td></tr><tr><td> Paraformaldehyde, 16% </td><td> 전자 현미경 과학 </td><td> 15,710 </td><td> 조직학 </td></tr><tr><td> 최적의 절삭 온도 (-10 월) </td><td> 테드 펠라 </td><td> 27,050 </td><td> 조직학 </td></tr><tr><td> 2 methylbutane </td><td> 어부 </td><td> 03551-4 </td><td> 조직학 </td></tr><tr><td> 마우스 MYH1/2/4/6 일차 항체 </td><td> 산타 크루즈 생명 공학 </td><td> SC - 32732 </td><td> 조직학 </td></tr><tr><td> 토끼 Connexin 43 일차 항체 </td><td> 세포 신호 기술 </td><td> 3512 </td><td> 조직학 </td></tr><tr><td> Dylight 549 - 복합 당나귀 안티 마우스 차 항체 </td><td> 잭슨 ImmunoResearch 연구소 </td><td> 715-505-151 </td><td> 조직학 </td></tr><tr><td> Dylight 488 - 복합 Donke 안티 토끼 이차 항체 </td><td> 잭슨 ImmunoResearch 연구소 </td><td> 711-485-152 </td><td> 조직학 </td></tr><tr><td> 죽은 / 라이브 분석 </td><td> Invitrogen </td><td> L - 3224 </td><td> 분석 </td></tr></tbody></table>

encapsulation of cardiomyocytes in a fibrin hydrogel for cardiac tissue engineering

입체 히드로겔 환경에서 Culturing 세포는 조직 공학에 대한 구조를 개발뿐만 아니라 체외에서 다양한 문화 조건 하에서 세포 반응을 연구를위한 중요한 기술이다. 입체 환경은 더 밀접하게 세포로 인해 모든 크기 1 기계적 및 화학적 자극의 응용 프로그램에 생체내에서 관찰 무엇 모방한 것이었 지요. 입체 hydrogels는 중 같은 PEG - DA 2와 3 또는 PLGA 등 콜라겐 4, hyaluronic 산성 5 섬유소 6,7와 같은 자연 발생 단백질의 숫자로 합성 고분자로부터 만들 수 있습니다. 섬유소, 자연스럽게 발생 혈액 응고 단백질에서 생성된 Hydrogels는 신체의 자연 상처 치유 8 프로세스의 일부로 메쉬를 형성 중합 수 있습니다. 섬유소는 조직 공학을위한 이상적인 임시 발판 만들기, 셀 분해 및 9 잠재적 autologous입니다. 여기 자세히 삼일 늙은 쥐의 새끼와 조직 공학을위한 섬유소의 히드로겔 구조의 캡슐에 대한 세포의 준비에서 신생아 cardiomyocytes의 절연을 설명합니다. 신생아 myocytes은 심장 조직 형성 및 엔지니어링 4 체외 연구에서 사용되는 일반적인 셀 소스입니다. 섬유소 젤이 효소 트롬빈과 혼합 섬유소에 의해 생성됩니다. 트롬빈은 다른 단량체 10과 상호 작용하는 구속력이 사이트를 공개, 피브리노겐에서 fibrinopeptides FpA 및 FpB을 클리브. 이러한 상호 작용은 단량체가 히드로겔 메쉬를 형성 섬유로 자체 조립 원인. 이 효소 반응의 타이밍이 섬유소로 트롬빈의 비율을 변경하거나, 트롬빈에 칼슘의 비율로 조정할 수 있기 때문에, 하나는 주사 다른 형상 11,12의 번호와 구조를 주형 수 있습니다. 더 나아가 우리 문화 13시 겔을 제한하는 방법으로 인해 조직의 정렬을 생성할 수 있습니다. culturing 정적 조건에서 두 주 동안 설계 심장 조직 구성 후 심장 세포의 구조를 개조하기 시작하고 전기 서성 거려 조건 6에 따라 수축 힘을 생성할 수 있습니다. 이 프로토콜의 일환으로, 우리는 기능 심장 근육에 의해 생성된 활성 힘을 분석 최종 세포 생존을 결정하기위한 전기 자극을뿐만 아니라, 방법에 따라 구축 (라이브 죽었을 포함한 문화 시대 이후 myocardium을 설계 조직을 분석하는 방법을 설명합니다 분석)과 수축 (마이 오신 중쇄 또는 MHC)와 myocytes 사이 세포 커플링 (Connexin 43 또는 Cx43)에 대한 중요한 전형적인 단백질의 표현과 형태를 검토 immunohistological 염색법.

Watch this Scientific Journal Video about Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering at JoVE.com

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

우리는 신생아 cardiomyocytes 및 조직 공학을위한 섬유소의 히드로겔 구조의 캡슐에 대한 세포의 준비의 절연을 설명합니다. 우리는 전기 자극 및 세포 생존과 immunohistological 얼룩에 따라 생성되는 활성 강제를 포함하여 문화 시대 이후 조직 공학 m​​yocardium을 분석하는 방법을 설명합니다.

심장 조직 공학을위한 섬유소 히드로겔에 Cardiomyocytes의 캡슐

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

encapsulation-cardiomyocytes-fibrin-hydrogel-for-cardiac-tissue

Research

JoVE Journal

Bioengineering

24.0K 조회수.  Tufts University. 우리는 신생아 cardiomyocytes 및 조직 공학을위한 섬유소의 히드로겔 구조의 캡슐에 대한 세포의 준비의 절연을 설명합니다. 우리는 전기 자극 및 세포 생존과 immunohistological 얼룩에 따라 생성되는 활성 강제를 포함하여 문화 시대 이후 조직 공학 m​​yocardium을 분석하는 방법을 설명합니다.

비디오: Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

탄성 PGS는 아티 조직 공학 공사장 공중 발판

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

연구

생체공학

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

Proteolytically 분해 3 차원 시스템에서 난소 대과의 캡슐화 및 문화에 대한 방법

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

ASC의 분화를 제어하는​​ Bilayered 히드로겔 꾸민

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

조직 공학을위한 Electrospun의 Fibres의 Postproduction 처리

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

손쥐 모델에서 창자의 조직 공학

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

심장 조직 공학을위한 모세관 력 리소그래피

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

인간의 3 차원의 조직 공학<em&gt; 시험관</em&gt; 종양 테스트 시스템

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting Cellularized는 조직 고유의 하이드로 겔 Bioink를 사용하여 구축

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

에 내장 혈관 신생에 의하여 조직 공학<em&gt; 생체</em&gt; 조직 공학 상공 회의소

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...