트레이시 C. Grikscheit, 에릭 R. Barthel, 그리고 글쓴이 G. 살라은 캘리포니아 재생 의료 연구소 (CIRM), 교부금 번호 RN2 - 00946-1 (TCG)과 TG2-01168 (ERB, FGS)에 의해 지원됩니다. 앨리슨 L. Speer은 대학 외과 전문의 Ethicon 학자의 사회입니다. Yashuhiro Torashima는 어린이 병원 로스 앤젤레스 Saban 연구소 연구 경력 개발 친목으로 운용되고 있습니다.

1. Organoid 단위 준비 <ol><li> 마우스 해부 (가위와 집게)에 적합한 악기 압력솥의 소독을해야합니다. </li><li> 편안하게 지역 IACUC 프로토콜에 따라 기증자 마우스를 안락사시켜야. 동물이하기 전에 죽은 있는지 확인합니다. </li><li> 복막 캐비티에 액세스 할 수있는 중간 선 절개를합니다. 노출을 개선하기 위해 필요에 따라 피부 플랩이 반영 될 수 있습니다. </li><li> 작은 창자를 창자를 빼내다와 Treitz의 인대에 단지 말초을 나눕니다. 샤프하고 부드러운 무딘 절개를 사용하여 장간막에서 작은 창자를 분리합니다. ileocecal 접합을 파악하고 이에 대한 근위 작은 창자 5mm를 나눕니다. </li><li> 가위를 사용하면 (Invitrogen, 안티 - 안티) 10 ML 4 ° C, 멸균 행크스 &#39;버퍼 생리 (HBSS, Invitrogen, Carlsbad CA) / 1X 항생제 - 항 곰팡이를 배양 접시에있는 antimesenteric 국경을 따라 길이 솔루션을 장을 엽니 다. 로 연 장에서 대변을 해제합니다부드러운 교반 한 다음 10 4의 ML ° C, 멸균 HBSS / 1X 안티 - 안티을 15ml 원심 분리기 튜브로 열린 장을 전송하기 만하면됩니다. </li><li> 4 10 ML에 열려있는 장을 세 번 씻어 ° C, 테스트 튜브에 안티 - 안티 살균 HBSS / 1X. 각 세척은 30 초에 15 ML 튜브의 약한 흔들림을 수행 할 수 있습니다. 진동 후, 장 조직은 관의 바닥에 가라. 중간 엽 파편이 부동 물질을 폐기하십시오. pipet을주의 깊게 세척 솔루션을 제거합니다. </li><li> 4 10 ML과 배양 접시에있는 세탁 장을 이기다 ° C, 멸균 HBSS / 가위를 사용하여보다 1mm 제곱 조각에 1X 안티 - 안티. 자동 pipet으로 다진 자료를 수집하고 테스트 튜브에 넣습니다. </li><li> 8 분 500 rpm으로 튜브를 원심 분리기. 뚱뚱하고 mesenchyme 포함되어있는 표면에 뜨는을 폐기하십시오. </li><li> 멸균 HBSS / 1X 안티 - 안티 플러스 0.125 MG / ML dispase (Invitrogen) 및 800 단위 / m의 10 ML과 다진, 씻은 자료를 소화리터 collagenase 타입 1 (워싱턴, 레이크 우드 NJ). 소화 솔루션의 40 ML를 준비하려면 dispase, collagenase의 142 밀리그램의 5 밀리그램을 무게, 40 ML의 볼륨으로 살균 HBSS를 추가 할 수 있습니다. 갓 때마다 organoid 단위 준비가되어이 솔루션을 준비하고, 사용하기 4에 ° C까지 준비해 놓아요. 단계 1.8에서 직접 펠릿으로 소화 솔루션을 추가합니다. </li><li> 20 분에 37 ° C에서 소화 솔루션과 다진, 씻은 재료를 포함하는 테스트 튜브를 품다. </li><li> 테스트 튜브를 검색하여 추가로 10 ML의 pipet으로 분쇄하여 소화 조직을 중단. 균일 한 모양을 얻을 때까지 20-50 시간 사이에 반복합니다. </li><li> 800 rpm으로 5 분 동안 테스트 튜브를 원심 분리기. 하나의 셀을 포함하는 표면에 뜨는을 폐기하십시오. </li><li> 4 ° C, 멸균 Dulbecco의 수정 된 이글 중간 (DMEM, Invitrogen) 플러스 10% 브이 / v 열 inactivated 태아 소 혈청 (HI-FBS, Invitrogen)의 10 ML과 소화 반응을 중지합니다. pelle을 Resuspendt와 튜브를 흔들. </li><li> 800 rpm으로 5 분 동안 테스트 튜브를 원심 분리기. 마지막 몇 방울까지 자동 pipet으로 조심스럽게 표면에 뜨는을 제거합니다. 펠렛을 resuspending 방지하기 위해 지난 몇 방울을위한 일회용 플라스틱 pipet을 사용합니다. </li></ol> 2. Polyglycolic 산성 발판으로로드 <ol><li> 로 참조에 설명, nonwoven polyglycolic 산 (다공성&gt; 95 %, Concordia는 섬유, 코번 트리의 RI 2 mm 시트의 두께, 60 MG cm-3 벌크 밀도)에서 양식 2 mm 길이 5 mm 외경 원통 공사장 공중 발판. 4. </li><li> 증류수에 70 % 에탄올로 만든 가위로 팁을 일회용 1000 마이크로 리터의 pipet의 말초 2mm를 잘라. </li><li> 4 잘 문화 판에 비계를 놓으십시오. 처음 루멘으로하고 바깥 쪽 표면에, 1000 마이크로 리터의 pipet으로 발판 위에 organoid 단위를로드합니다. 루멘의 코팅을 보장하기 위해 집게를 사용하십시오. 중단 또는 폴리머의 원통형 모양을 깨뜨하지 마십시오. </리&gt; </ol> 3. 호스트 마우스에 주입 <ol><li> 기증자 가능한 경우와 같은 배경 syngeneic 호스트 마우스를 사용합니다. 그렇지 않으면, 면역 저하 nonobese의 당뇨병 환자 (잭슨 연구소, 새크라멘토 CA) 동물 / 심한 결합 immunodeficient 또는 NOD / SCID를 사용합니다. </li><li> isoflurane과 전신 마취를 유도. , 사립를 면도하고 마우스의 복부를 내리면 t. </li><li> 복막 캐비티에 입학을 얻기 위해 5mm 중간 선 절개를합니다. 확인하고 조심스럽게 큰 omentum을 골자를 빼 버리다. omentum로로드 된 폴리머를 배치하고 조직과 함께 포장. omentum을 찢어하지 마십시오. </li><li> 5-0 monocryl의 봉합과 함께 omentum에 폴리머를 고정합니다. 부드럽게의 해부학 위치로 포장 폴리머로 omentum를 교체하십시오. </li><li> 4-0 vicryl 봉합을 사용하여 레이어의 복부 절개를 닫습니다. 근육 폐쇄를 실행하고 절개 아래 복부 내장을 손상하지 돌. 피부 중단 봉합을 사용하여. </li><li> 절개에 인접 피하 wheal으로 멸균 물에 2 밀리그램 / kg 케토 프로 펜 (Ketofen, 포트 닷지 동물 건강)와 수술 후 진통제를 관리 할 수​​ 있습니다. 동물은 매일 평가해야하고 동물이 통증이나 고통의 흔적을 보여주는 경우 케토 프로 펜의 추가 투여는 수술 후 2 일에 실시 될 수 있습니다. 세 번째 수술 일까지, 동물 완전히 통증이나 고통의 증거도없이 복구 할 수 있어야합니다. 통증이나 고통이 포스트 수술 일 3 지속되면, 이것은 비정상적인 것으로 간주되고 IACUC과 동물 보호 시설 프로토콜에 따라 해결해야합니다. </li><li> 회복 할 수있는 마우스와 조직 - 엔지니어링 소장은 4 주 동안 성장 할 수 있습니다. 쥐 수유 (랩 다이어트 5001, PMI 영양, 세인트 루이스 MO) 5 ML (하이테크 Pharmacal, Amityville NY)에 1:100 희석 당 Septra 200 밀리그램 / 40 MG와 물 동물 광고 libitum 액세스를 제공합니다. </li></ol> 4. 수확 <ol><li> Humanely 사주 주입 한 후 호스트 동물을 안락사시켜야. </li><li> 원래 절개을 열고 피부를 반영는 복막 캐비티에 대한 액세스를 용이하게 cephalad. </li><li> 근육 레이어를 열고 조직의 세계로 조직 - 엔지니어링 구조를 확인합니다. </li><li> 날카로운 해부를 사용하여 intraabdominal 내장의 구조에 adhesions을보십시오. </li><li> 설치하기 파라핀을위한 포르말린에 구조를 수정, 또는 실시간 PCR 또는 단백질 분리와 같은 생화학 assays에 대한 새로운 조직을 사용합니다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

우리는 organoid 단위 온 발판 접근 방식을 사용하여 마우스에 조직 - 엔지니어링 소장을 생성하기위한 프로토콜을 제시한다. 가장 중요한 단계는 organoid 단위의 준비가 일반적입니다. 주의 적절하게 청소하고 기계적으로 조직을 처리하는 이동해야하지만 소화를 수행 한 후에 동일한주의 (단계 1.11) organoid 단위를 overdigest 또는 overtriturate하지 않도록해야합니다. 이 작업을 수행하는 경우, organoid 단위...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> HBSS </td><td> Gibco </td><td> 114170-112 </td><td></td></tr><tr><td> 항생제 - Antimycotic 100X </td><td> Invitrogen </td><td> 15240-062 </td><td></td></tr><tr><td> Dispase </td><td> Gibco </td><td> 17105-041 </td><td></td></tr><tr><td> Collagenase의 타입 1 </td><td> 워싱턴 </td><td> LS004194 </td><td></td></tr><tr><td> DMEM 높은 포도당 1X </td><td> Gibco </td><td> 11995-065 </td><td></td></tr><tr><td> 열 inactivated FBS </td><td> Invitrogen </td><td> 16140-071 </td><td></td></tr><tr><td> Biofelt 백퍼센트 PGA </td><td> Concordia는 의료 </td><td> FELT01-1005 </td><td> 참조에서와 같이 폴리머 준비하십시오.4 </td></tr><tr><td> 폴리-L-락트산 </td><td> Durect </td><td> B6002-1 </td><td> 참조에서와 같이 폴리머 준비하십시오. 4 </td></tr><tr><td> I 콜라겐, 쥐 꼬리 입력 </td><td> 시그마 - 알드리치 </td><td> C3867-1VL </td><td> 참조에서와 같이 폴리머 준비하십시오. 4 </td></tr><tr><td> 케토 프로 펜 100 MG / ML </td><td> 요새 닷지 동물 건강 </td><td> 71 - KETOI - 100-50 </td><td></td></tr><tr><td> LabDiet 5,001 쥐 급식 </td><td> LabDiet </td><td> 5001 </td><td></td></tr><tr><td> 5 ML, USP 당 Septra 200 밀리그램 / 40 밀리그램 </td><td> 하이테크 Pharmacal </td><td> 50383-824-16 </td><td></td></tr><tr><td> Isoflurane, USP </td><td> 피닉스 제약 </td><td> 57319-507-06 </td><td></td></tr></tbody></table>

tissue engineering of the intestine in a murine model

조직 - 엔지니어링 소장 (TESI)가 성공적으로 40일 이내에 수술 전 근력로 돌아 발생, 거대한 작은 창자 절제술 후 루이스의 쥐를 구출하는 데 사용되었습니다. 인간 1, 대규모 작은 창자 절제술 짧은 창자 증후군, 기능 malabsorptive이 발생할 수 있습니다 중요한 병적 상태, 사망률, 그리고 비경 영양 의존도, 간 장애 및 간경변, 그리고 multivisceral 장기 이식의 필요성 2.이 문서에 포함 의료 비용을 수여 국가는, 우리는 마우스 모델에서 조직 - 엔지니어링 소장을 만들기위한 우리의 프로토콜을 설명하고 문서화 다세포 organoid 단위 온 발판 방식으로. Organoid 단위는 점막과 중간 엽 모두 요소를 포함하는 장에서 파생 다세포 집계, 3 장 줄기 세포 틈새 시장을 유지하는 사이의 관계입니다. 지속적인와 미래의 연구에서 4로 우리 기술의 전환마우스는이 종에서 사용할 수있는 유전자 변형 도구를 이용하여 TESI 형성하는 동안 관련된 프로세스의 조사를 허용합니다. 면역 저하 마우스 종자의 5 가용성도 우리 인간의 장 조직에 기술을 적용하고 같은 인간 TESI의 형성을 최적화 할 수 있도록 허용합니다 인간에의 전환하기 전에 마우스 이종 이식. 우리의 방법은 좋은 제조 연습 (GMP) 시약 및 이미 인간의 환자에 사용하기 위해 승인, 따라서 decellularized 동물의 조직에 의존 접근 방식에 비해 상당한 장점을 제공 한 자료를 사용하고 있습니다. 이 방법의 궁극적 인 목표는 짧은 창자 증후군의 재생 의료 치료 전략으로 인간의 번역입니다.

그림 1은 여기에 설명 된 프로토콜에 대한 전반적인 스키마를 보여줍니다. 이 프로토콜의 최종 결과는 동일한 구조를 표시하는 루멘, 점막, submucosa, 그리고 muscularis. 그림 2A를 둘러싼 것은 시작 폴리머 비계. 그림 2B에 비해 전형적인 세계를 보여줍니다과 조직 - 엔지니어링 손쥐 소장의 세계 또는 구형 구조입니다 급의 루멘을 나타 내기 위해 bivalved. 그?...

Watch this Scientific Journal Video about Tissue Engineering of the Intestine in a Murine Model at JoVE.com

Tissue Engineering of the Intestine in a Murine Model

이 문서와 함께 비디오 organoid 단위 온 발판 접근법을 사용하여 마우스에 조직 - 엔지니어링 소장을 생성하기 위해 우리의 프로토콜을 제시한다.

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 ...

tissue-engineering-of-the-intestine-in-a-murine-model

Research

JoVE Journal

Bioengineering

14.8K 조회수.  Keck School of Medicine of the University of Southern California. 이 문서와 함께 비디오 organoid 단위 온 발판 접근법을 사용하여 마우스에 조직 - 엔지니어링 소장을 생성하기 위해 우리의 프로토콜을 제시한다.

비디오: Tissue Engineering of the Intestine in a Murine Model

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

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 ...

연구

생체공학

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 ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

전기 자극의 손쥐 Myoblast의 전구 세포 및 응용에서 공학 골격근 조직도

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

조직 공학을위한 이축 기계 로딩 생물 반응기의 설계

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

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. ...

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 ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

소형 동물 모델에서의 동정맥 (AV) 루프는 혈관 신생과 혈관을 조성한 조직 공학을 공부하기

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

운동 조건의 혈청을 이용한 인대 구조의 치료 : Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...