This work was funded by a New Faculty Award II from the California Institute of Regenerative Medicine (CIRM; RN2-00921-1), NIH-funded National Research Award (F32-HL104924), and CIRM Training Grant (TG21163). Materials were provided by: Glycosan Biosystems Inc / BioTime and Dr. Stephen Badylak (University of Pittsburgh)

pNIPAAM 코팅 된 플레이트의 1 창조 <ol><li> 60 % 톨루엔 / 40 % 헥산 용액 2 ㎖에 pNIPAAM의 2.6 g을 녹인다. </li><li> pNIPAAM이 용해 될 때까지 교반 10 분 동안 60 ° C로 가열하여 혼합합니다. </li><li> 뷰 흐너 깔때기에 60mm 직경의 원과 장소 종이에 종이 필터를 잘라. </li><li> 미리 칭량 유리 비이커 흐너 깔때기를 통해 용액 (헥산 플라스틱을 용융하는 바와 같이, 플라스틱을 사용하지 않음) 필터. </li><li> 벨 진공 (24 PSI) O / N (16 시간)로 비커와 내용을 놓습니다. 참고 : 잔류 이소 프로필과 반응 할 때까지 그렇게는 산소와 접촉하지 않는지 확인 산화됩니다. </li><li> pNIPAAM의 무게를 설정하는 비커의 무게를 측정. </li><li> 솔루션 w / w 50 대 50을 만들어 pNIPAAM에 이소 프로필 알코올을 추가합니다. </li><li> UV 빛 아래 5 분의 조직 배양 판의 표면에 솔루션 및 코트 2 ㎖를 놓습니다. </li><li> 두 번 따뜻한 PBS의 2 ㎖의 접시를 씻으세포 배양에 사용하기 전에. </li></ol> 세포 시트의 2 창조 주 : 표적 장기에 대한 일차 세포의 세포 시트를 서로 다른 방법들을 사용하여 생성 될 수 있고, 또는 여기에서 설명한 바와 같이 열 응답 성 폴리머로 조직 배양 표면을 코팅하여. 사전 코팅 온도에 민감한 판은 공급 업체들에 의해 제공됩니다. 참고 :이 프로토콜은 35mm 접시를 사용하여 문화입니다. 간단히, 세포는 제 1 인접 셀 사이의 수평 연결을 설정할 수 합류에 24 시간의 최소 37 ° C에서 배양된다. 세포 시트를 해제하려면 판은 32 ° C 이하의 온도로 실시한다. 세포 시트는 후 혈관 내피 세포와 혈관 신생 허용 하이드로 겔을 포함하는 강염기 섬유상 매트릭스에 전송된다. <ol><li> 세포 집단을 분리합니다. 주 :이 방법은 개개의 도출 과정과 세포 유형에 의존한다. 쥐 대동맥 부드러운 무scle 세포 (RASMC)이 예에서 사용됩니다. 이들은 쥐의 복부 대동맥으로부터 분리 일차 평활근 세포이다. </li><li> 따뜻한 PBS의 2 ㎖의 세포를 씻으십시오. </li><li> 5 분 동안 세포에 트립신 (또는 다른 클 리빙 / 연결 해제 용액) 3 ㎖를 추가합니다. </li><li> 10 % 태아 소 혈청 (FBS)을 함유하는 배지, 또는 인산 완충 용액 (PBS) 3 ㎖을 첨가함으로써 트립신을 억제한다. </li><li> 원뿔 튜브에 세포를 수집하고 나누어지는 계산합니다. </li><li> 5 분 동안 1,000 rpm으로 (228 XG)에서 세포를 스핀. </li><li> 뜨는을 기음과 성장 배지에서 세포를 재현 탁 (SmGM2 플러스 총알 키트 배양액 RASMC에 사용됩니다). </li><li> 백퍼센트 합류를 달성하기위한 농도 pNIPAAM 코팅 플레이트 - 35mm의 온도에 민감한 접시에 세포를 포함하는 미디어를 넣습니다. 주 : RASMCs 들어 그 숫자 / cm 2 100,000 세포로 측정되었다. 그 버지니아의 그러나, 통과하는 동안 세포의 손실, 120 %<html> 루이 사용됩니다. </li><li> 37 ° CO / N에서 인큐베이터에 놓습니다. 참고 : 접시에 세포 부착을 유지하기 위해 37 ° C에서 세포를 유지하는 것이 중요합니다. </li></ol> 파운데이션 매트릭스의 3 준비 참고 : 다양한 3D 섬유 행렬이 섬세한 세포 시트 사이에 강한 섬유 매트릭스를 레이어 할 수 있습니다. 예를 들면 다음과 같습니다 : gelfoam을, 바이오 글라스, 자연 acellularized 소재 26 또는 nanospun 재료 (27, 28)을이 연구에 사용 된 돼지 방광 매트릭스 (UBM)가 관대하게 우리의 협력자, 박사 Badylak에서 제공 한 29. <ol><li> decellularized 행렬이 사용되는 경우, 셀룰러 함량의 부족을 포함 매트릭스의 특성을 결정 27,28 특정 세포 생존 능력, 및 보이드 공간을 사용하기 전에. 22 </li><li> 원하는 크기와 형상으로 예비 살균 행렬을 잘라. 주 : 여기에서, 홀 펀치 4mm 직경의 원을 절단하는 데 사용된다. </li></ol>ve_title &quot;&gt; 4. 신생 혈관 허용 히드로 겔로 시드 내피 세포 주 : 내피 줄기 세포 또는 전구 세포로부터 분화 포함한 다양한 소스로부터 획득 될 수있다. 여기에, HUVECs를 사용한다. <ol><li> 가교 시간만큼 긴 허용 하이드로 겔 (피브린, 콜라겐 젤)를 사용하면 세포가 가능한 머물 수 있도록 충분히 짧다. 주 : 여기에서, 히알루 론산 (HA)을 기반 겔 디설파이드 브릿지가 사용으로 가교. </li><li> 회사 프로토콜에 따라 HA 하이드로 젤을 준비한다. </li><li> 내피 세포를 수집하고 1X 트립신을 사용하여 단일 세포로 분산 용액. 참고 : Accutase 또는 세포 분열 버퍼는 단일 세포 분산에 사용될 수 있습니다. </li><li> 또는 PBS에 10 % FBS 용액 / 15 ㎖ 중에 세포 원추형 튜브를 수집 (이것은 세포가 혈청과 접촉하지 않는 것이 중요한 경우) 대두 트립신 억제제의 동등한 양을 사용하여 트립신 효소를 비활성화. </li><li> C세포 ount과 패치 사이즈에 필요한 양을 계산한다 (이전 정량화). 참고 : 4mm 패치, 여기 2,000,000 내피 세포가 사용된다. </li><li> 2,000,000 세포를 추출, 새로운 15 ML 원뿔 튜브에 배치합니다. </li><li> 5 분 (228 XG)에 스핀. </li><li> 원뿔 튜브에서 펠릿으로 세포를 떠나, 뜨는을 대기음. </li><li> 한 배급 : 1의 HA 및 젤라틴 액체 재료를 섞는다. 이어서 펠렛을 함유하는 원뿔형 튜브에 전체 부피의 80 %를 추가한다. </li><li> 한 HA / 젤라틴 혼합물 : 1의 내피 세포를 재현 탁 </li><li> 2 단계에서 기본 섬유 매트릭스로 HA / 젤라틴 혼합물에서 일시 중단 된 세포를 놓습니다. </li><li> 가교제의 원하는 총 부피 1/5 (20 %)를 추가 </li><li> 37 ° C에서 1 시간 동안 품어. </li></ol> 세포 시트의 5 분리 <ol><li> 세포 배양 후드에서의 인큐베이터 및 장소에서 세포를 함유하는 35mm pNIPAAM 처리 판을 제거RT. </li><li> 신속하게 세포에서 미디어를 대기음, 37 ° C로 가열 된 6 % 정상 젤라틴 2 ㎖를 추가합니다. </li><li> 젤라틴이 남아있는 동안, 일반 젤라틴 (동영상 1)의 표면 아래에 침수, 젤라틴으로 금속 격자를 배치합니다. </li><li> 젤라틴은 강화 할 수 있도록 5-7 분 동안 얼음에 전체 판을 놓습니다. </li><li> 7 분 후, 조심스럽게 플레이트 측으로부터 젤라틴 에지를 분리하는 주걱을 사용하고 플레이트 주부터 금속 격자 리프트 집게를 사용 : 6 % 젤라틴, 및 세포 시트는 격자 리프트한다. </li><li> 접시에 세포 시트를 이동하고 조심스럽게 구조의 상단에있는 격자를 설정, 기본 섬유 매트릭스 하이드로 겔 조합의 상단에 배치합니다. 참고 : 셀 시트의 꼭대기 쪽은 여전히​​ 최고 위치에있을 것입니다. </li><li> 따뜻한 미디어 (37 ° C) 2 ㎖를 추가합니다. </li><li> O / N은 ​​하이드로 겔 표면에 부착하기 위해 세포 시트를 허용 부화. </li><li> t 제거용액 (약 1 시간), 또는 그 다음날 따뜻해 후 그는 금속 격자. </li></ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+extracellular+matrix+as+an+inductive+scaffold+for+the+partial+replacement+of+functional+myocardium.">The use of extracellular matrix as an inductive scaffold for the partial replacement of functional myocardium.</a> Cell Transplant. 15, S29-S40 (2006).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restriction+of+human+hepatic+stem+cells+to+mature+fates+is+made+efficient+by+tissue-specific+biomatrix+scaffolds.">Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.</a> Hepatology. 53, 293-305 (2011).</li><li>Gilbert, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fiber+alignment+and+biaxial+mechanical+behavior+of+porcine+urinary+bladder+derived+extracellular+matrix.">Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix.</a> Biomaterials. 29, 4775-4782 (2008).</li><li>Luna, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+biomimetic+topography+for+the+alignment+of+neonatal+and+embryonic+stem+cell-derived+heart+cells.">Multiscale biomimetic topography for the alignment of neonatal and embryonic stem cell-derived heart cells.</a> Tissue Eng Part C Methods. 17, 579-588 (2011).</li></ol>

We have nothing to disclose.

프로토콜의 중요한 단계는 다음과 같습니다 thermoresponsive 폴리머 플레이트 표면을 코팅하고 판을 냉각 한 후 세포 시트를 조작. 상이한 셀들이 서로 다른 물리적 특성을 보일 수 있기 때문에, 점착성 같이, 승강 시간은 서로 다른 세포 유형에 대해 최적화되어야한다. 이 프로토콜의 제 2 및 가장 현저 도전 성분은, 세포 시트, 티슈 조립 방법의 중요한 양태의 조작을 중심. 세포 시트의 단일 셀 층은 ...

Injection of cells and/or single materials alone has shown variable success in other organ systems and limited success in cardiac regeneration.5,7-12 Currently, stem cell-derived cells are delivered to damaged tissue using a variety of delivery methods including: direct cell injection into tissue and perfusion into the blood supply.13-17 Others have implanted cells alone, materials alone and/or in combination with material carriers to help regenerate damaged organs.18-21 This design combines multiple strategies that provide material strength, patterning in multiple materials and multiple cell types. 
Specifically, the base acellularized fibrous matrix provides the foundational physical strength to the construct, making it suitable for suturing in into the patient, if necessary. The void spaces in the base matrix are filled with endothelial cells in a neovascular permissive hydrogel22 for rapidly establishing vascularization of the implanted construct. This composite is then integrated with pre-patterned cell sheets that allow enhanced cell-to-cell communication, more closely mimic the native tissue.1,23-25 The overall production process for the layered cellular patch is outlined by the flowchart in Figure 1.

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tissue engineering: construction of a multicellular 3d scaffold for the delivery of layered cell sheets

이러한 성인의 마음으로 많은 조직은 적절하게 손상 후 재생 할 수 없습니다. 조직 공학에서 2,3 전략 복구 및 수리에 몸을 지원하기 위해 혁신을 제안한다. 예를 들어, TE 방식은 심근 경색 (MI) 후 심장 리모델링을 약화 가능성이 거의 정상 사전 MI 수준으로 전체 심장 기능을 향상시킬 수 있습니다. 4, 심장 조직을 성공적으로 재생이의 적절한 전달이 포함 기능적 조직과 마찬가지로 이식 된 세포 / 조직 이식의 통합과 생존에 유리한 환경 단서 여러 세포 유형. 배달 차량, 세포 생존에 미치는 영향, 재료 강도 및 세포 간 조직의 조직의 촉진으로 평가 용해 신호, 세포 간 상호 작용 및 매트릭스 재료 : 엔지니어링 조직은 다음과 같은 여러 매개 변수를 해결해야합니다. 그래프트 세포의 직접 분사 만이 필수 요소를 무시를 이용하는 연구. 2,5,6이 성분을 결합 조직 설계는 아직 개발되어야한다. 여기서, 우리는 &quot;조직&quot;에서 새로운 혈관의 형성을 향상시키기위한 표적 장기 세포 유형 및 내피 세포를 포함하는 생물학적 유래 물질의 두 가지 유형의 패턴 화 된 세포 시트의 레이어를 이용하여 통합 된 디자인의 예를 제시한다. 이러한 연구는 심장과 같은 조직의 생성에 집중하더라도,이 조직 설계는 최소한의 디자인 및 재질 변경 심장 이외의 여러 장기에 적용 할 수 있고, 회생 치료를위한 상용 제품이 될 것을 의미한다. 이 프로토콜은 다섯 자세한 단계가 포함되어 있습니다. 온도에 민감한 폴리 (N의 -isopropylacrylamide) (pNIPAAM은) 코트 조직 문화 요리에 사용됩니다. 이어서, 조직 특이 세포는 강한 횡 유착 세포 시트를 형성하기 위해 코팅 된 플레이트 / 미세 패턴 표면의 표면 상에 배양한다. 셋째, 기본 매트릭스는 신생 혈관 permissi 다공성 매트릭스와 결합하여 조직을 위해 생성 된하이드로 겔과 내피 세포를했습니다. 마지막으로, 세포 시트 pNIPAAM 코팅 접시로부터 해제되어 완전한 구조를 만드는 기본 요소로 전송.

흐름도 (도 1)는 다층 패치를 제조하는 전체적인 방법을 도시한다. 세포 시트를 32 ° C 이하의 온도를 떨어 뜨려 pNIPAAM 처리 플레이트에서 분리된다. 이어서 세포 시트는 기본 섬유 매트릭스 (도 1)로 파종 혈관 내피 세포를 포함하는 가교 된 하이드로 겔의 상단에 배치된다. 전처리 감열 플레이트는 또한 세포 시트를 만드는 데 사용할 수있다. 특별 위상 표면은 특별히 패턴...

Watch this Scientific Journal Video about Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets at JoVE.com

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

조직 공학 : 계층화 된 세포 시트의 전달을위한 다세포 3D 비계 건설

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

tissue-engineering-construction-multicellular-3d-scaffold-for

Research

JoVE Journal

Bioengineering

14.3K 조회수.  University of California, Merced. For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

비디오: Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

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  ...

연구

생체공학

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3 차원 세포 배양을위한 자기보고 발판

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

전기 방사를 적응하는 것은 트라이 계층에 대한 세 가지 독특한 환경을 제공하기 위해<em&gt; 체외</em&gt;기도 벽의 모델

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

미세 패턴 매크로 스케일 3D 셀룰러 아키텍처 모듈 형 하이드로 겔 시트의 건설

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

반전 콜로이드 결정체 폴리 (에틸렌 글리콜) 비계의 제작 : 간 조직 공학을위한 삼차원 세포 배양 플랫폼

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

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

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

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

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

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

셀 시드를 위한 사용자 지정 Agarose 우물 및 자기 조립 금형 3D 인쇄를 사용 하 여 조직 반지 제작

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

신경과 같은 조직을 위한 Murine 피질 성상 세포의 3D 생물 인쇄

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...