我々は、バイオリアクターの開発を支援するためMaegan B. Colehourに感謝。これらの研究は、麻酔のエール大学学科によっておよびNIHの助成金HL 098220（LENまで）によって賄われた。 THPは、NIH T32 GM007171によってサポートされていました。

<ol>
	<li>American Lung Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lung Disease Data. , (2008).</li><li>Petersen, T. H., Calle, E. A., Colehour, M. B., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreactor+for+the+Long+Term+Culture+of+Lung+Tissue.">Bioreactor for the Long Term Culture of Lung Tissue.</a> Cell Transplant. , (2010).</li><li>Bilek, A. M., Dee, K. C., Gaver, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+surface+tension+induced+epithelial+cell+damage+in+a+model+of+pulmonary+airway+reopening.">Mechanisms of surface tension induced epithelial cell damage in a model of pulmonary airway reopening.</a> J Appl Physiol. 94, 770-783 (2003).</li><li>Petersen, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-Engineerined+Lungs+for+in+Vivo+Implantation.">Tissue-Engineerined Lungs for in Vivo Implantation.</a> Science. 329, 538-5341 (2010).</li><li>Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung.">Regeneration and orthotopic transplantation of a bioartificial lung.</a> Nat Med. 16, 927-9233 (2010).</li><li>Cortiella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+acellular+natural+lung+matrix+on+murine+embryonic+stem+cell+differentiation+and+tissue+formation.">Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation.</a> Tissue Eng. Part A. 16, 2565-2580 (2010).</li><li>Sugihara, H., Toda, S., Miyabara, S., Fujiuyama, C., Yonemitsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+alveolus-like+structure+from+alveolar+type+II+epithelial+cells+in+three-dimensional+collagen+gel+matrix+culture.">Reconstruction of alveolus-like structure from alveolar type II epithelial cells in three-dimensional collagen gel matrix culture.</a> Am J Pathol. 142, 783-7892 (1993).</li><li>Choe, M. M., Sporn, P. H., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+remodeling+by+dynamic+strain+in+a+three-dimensional+tissue-engineered+human+airway+wall+model.">Extracellular matrix remodeling by dynamic strain in a three-dimensional tissue-engineered human airway wall model.</a> American Journal Respiratory Cell Molecular Biology. 35, 306-306 (2006).</li><li>Cortiella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-enginered+lung:+an+in+vivo+and+in+vitro+comparison+of+polyglycolic+acid+and+pluronic+F-127+hydrogel/somatic+lung+progenitor+cell+constructs+to+support+tissue+growth.">Tissue-enginered lung: an in vivo and in vitro comparison of polyglycolic acid and pluronic F-127 hydrogel/somatic lung progenitor cell constructs to support tissue growth.</a> Tissue Engineering. 12, 1213-1213 (2006).</li><li>Price, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Decellularized+Lung+Bioreactor+System+for+Bioengineering+the+Lung:+The+Matrix+Reloaded.">Development of a Decellularized Lung Bioreactor System for Bioengineering the Lung: The Matrix Reloaded.</a> Tissue Eng Part A. , (2010).</li><li>Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat. Med. 14, 213-221 (2008).</li><li>Uygun, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat. Med. 16, 814-820 (2010).</li><li>Shannon, J. M., Mason, R. J., Jennings, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differentiation+of+alveolar+type+II+epithelial+cells+in+vitro:+effects+of+cell+shape,+cell-matrix+interactions+and+cell-cell+interactions.">Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions.</a> Biochim. Biophys. Acta. 931, 143-156 (1987).</li><li>Cortiella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Acellular+Natural+Lung+Matrix+on+Murine+Embryonic+Stem+Cell+Differentiation+and+Tissue+Formation.">Influence of Acellular Natural Lung Matrix on Murine Embryonic Stem Cell Differentiation and Tissue Formation.</a> Tissue Eng Part A. , (2010).</li></ol>

LENはHumacyte、再生医療の会社の株式を取得しています。 Humacyteは、これらの研究に資金を供給していない、と記載される実験や方法のいずれかの設計、解釈、またはレポートには影響しなかった。著者ら（LEN、THP、EAC）とエール大学は、肺の組織工学に関連する特許出願を提出した。

システムの最も重要な側面は、足場と培養再移入の肺を準備し、播種の過程で血管床に適用される圧力の無菌性の維持、および綿密なモニタリングを含むここに紹介。無菌性は最高の、使用前にすべての材料をオートクレーブで、間もなく植後の閉鎖系で肺をマウントし、この障壁のその後の違反を回避することによって維持されます。脱細胞マトリクスを徹底的に洗浄し、培養用の滅菌バイオリアクターに転送された後に、シリコンキャップや他のシールとの接続が妨げまたは削除しないでください。可能な限りチェックで圧力を保つために、重力によって駆動される流れが望ましい。ポンプは、流体の循環のために必要なときは、PBSで洗浄し、培養中に継続して後に始まる、我々が直接流体を圧トランスデューサーと肺動脈に入る直前に圧力を測定することをお勧めします。適用される圧力の大きさは、15mmのHgを超えないようにしてください。 Recellularizedの肺は、通常、4日から最大3週間までに至るまでの期間を変化させるための培養することができる。換気は、通常、上皮培養中に1呼吸/分の速度で印加した状態で血管灌流は一般的に、内皮培養中1〜3ミリリットル/分で行われます。複合文化の期間中、同時換気と血流が適切です。換気は、液体培地または空気のいずれかで行うことができます。 過去数十年にわたり、いくつかのグループは、in vitroで肺上皮の分化と肺微小解剖学7,8,9、10のいくつかの側面を複製の可能性を示す重要な組織工学の作業を行っている。しかし、最近まで、肺組織を設計するためにこれらの試みの4,5いずれも、血液および気道のコンパートメント間の分離を維持し、ガス交換に参加できることができたインプラント臓器をもたらしたなかった。説明した方法は機能的な肺組織を生成するという長期目標に​​向かってのみ最初のステップであるけれどもそのため、この作業は、移植のための利用可能な肺組織の量を増やす可能性に向けて有望なステップです。また、この作業は、組織工学、複雑な三次元構造の足場としての脱細胞外マトリックスの有効性を実証し、細胞の様々な種類の成長と生存を支える、オットらとUygunと同僚11,12によって行われた仕事を詳しく説明。この作品は、呼吸器細胞および分子生物学の装備一式への貢献のためにも重要です。また、適切な機械的刺激を提供できるユニークな、三次元環境を提供することにより、それが急速な脱分化のアテンダントの危険を共有しないものは、より伝統的な方法13の実験室で培養するの齧歯類上皮時に発生する可能性がある、科学者が使用することができます細胞分化と機能の役割を果たす細胞間および細胞 - マトリックス相互作用の新たな洞察を得るために私たちのシステム。 Cortiellaのグループは初期の研究14に示されているように、様々な幹細胞集団の運命を導くために活用するとして使用する場合は、この知識は、特に強力な可能性があります。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> 安楽死 </td><td></td><td></td><td></td></tr><tr><td>ヘパリン</td><td>シグマアルドリッチ</td><td> H4784 </td><td></td></tr><tr><td>ニトロプルシドナトリウム</td><td>フルカ</td><td> 71778 </td><td></td></tr><tr><td> Euthasolの安楽死のソリューション</td><td> Virbac AH </td><td> 710101 </td><td> 390 mg / mlのペントバルビタールナトリウムの原液は、IP投与のために適切に希釈してください</td></tr><tr><td colspan="4"></td></tr><tr><td> Decellソリューション </td><td></td><td></td><td></td></tr><tr><td> CHAPS </td><td>シグマアルドリッチ</td><td> C3023 </td><td></td></tr><tr><td> NaClの</td><td>バイオ分析アメリカン</td><td> AB01915 </td><td></td></tr><tr><td> EDTA </td><td>シグマ</td><td> E5134 </td><td></td></tr><tr><td> NaOHで</td><td> JTベイカー</td><td> 3722〜01 </td><td></td></tr><tr><td> 1X PBS </td><td>ギブコ</td><td> 14190 </td><td></td></tr><tr><td colspan="4"></td></tr><tr><td> バイオリアクターのコンポーネント </td><td></td><td></td><td></td></tr><tr><td> 480ミリリットルの瓶</td><td>コー​​ルパーマー</td><td> EW - 3460560 </td><td></td></tr><tr><td>シリコンストッパー、サイズ14 </td><td>コー​​ルパーマー</td><td> EW - 06298〜26 </td><td></td></tr><tr><td> Y -コネクター</td><td>コー​​ルパーマー</td><td> ED - 30614〜08 </td><td>動脈と気管カニューレのために使用</td></tr><tr><td>シリコンチューブ</td><td> Masterflex </td><td> 96420〜14、16 </td><td> L / S 14、16 </td></tr><tr><td>圧力トランスデューサ</td><td>エドワーズライフサイエンス</td><td> PX - 212 </td><td></td></tr><tr><td>バルブをチェックする</td><td>コー​​ルパーマー</td><td> EW - 98553〜20 </td><td>ワンウェイバルブ</td></tr><tr><td> 4ウェイストップコック</td><td>エドワーズライフサイエンス</td><td> 594WSC </td><td></td></tr><tr><td>シリンジフィルター</td><td>コー​​ルパーマー</td><td> 2915〜08 </td><td> PTFE、0.2μmの</td></tr><tr><td colspan="4"></td></tr><tr><td> Decellとすすぎapparati（バイオリアクターへの追加） </td><td></td><td></td><td></td></tr><tr><td> 500および1000 mlのガラス瓶</td><td>コー​​ニング</td><td> 1395-500; - 1L </td><td> decellのために使用され、重力によるすすぎ</td></tr><tr><td colspan="4"></td></tr><tr><td> ベンゾナーゼ処理 </td><td></td><td></td><td></td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td>ギブコ</td><td> 15140122 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30071.03 </td><td></td></tr><tr><td>トリス塩酸</td><td>バイオ分析アメリカン</td><td> AB14043 </td><td> 1M、pH8.0の</td></tr><tr><td> MgCl 2の </td><td> JTベイカー</td><td> 2444〜01 </td><td></td></tr><tr><td> BSA </td><td>シグマ</td><td> A9647 </td><td></td></tr><tr><td>ベンゾナーゼヌクレアーゼ</td><td>シグマ</td><td> E1014 </td><td>行列から残留DNAを除去するために使用されるエンドヌクレアーゼ</td></tr></tbody></table>

procedure for lung engineering

肺がんや慢性閉塞性肺疾患などの慢性肺疾患、毎年いくつか28万死亡の累積的にアカウントを含む肺組織、慢性閉塞性肺疾患は、現在、米国1の死因の第4位です。この死亡率に寄与すると肺が一般的に顕微鏡、細胞レベルを超えて修理または再生成されないという事実です。したがって、変性または感染、または外科的に切除された肺組織が ​​損傷している肺組織は、機能的に生体内で置き換えられません。肺組織をin vitroで生成することができるかどうかを調べるために、我々は、無細胞肺癌細胞外マトリックス足場を生成するために細胞成分を除去する手順を用いて成体ラットから肺を治療。この足場は、気道と血管系の階層的な分岐構造だけでなく、コラーゲンIV、ラミニン、およびフィブロネクチンを含有する主に無傷の基底膜を、保持します。足場は、負圧換気と拍動性血管灌流などの肺の生理機能の重要な側面を、模倣するように設計されたバイオリアクターに取り付けられている。培養肺上皮とバイオリアクターを取り付けられた足場内の血管内皮によって、我々は、ネイティブの肺組織の表現型と同等であり、それが短い時間間隔（45〜120分）のためのガスの交換に参加することができる肺組織を生成することができます。これらの結果は有望、および肺行列のその再増殖が肺の再生のための実行可能な戦略であることが示唆されています。この可能性だけでなく、移植用の肺組織の供給を増やす努力するだけでなく、より長い期間のためのin vitroで 、以前可能であったよりも正確な微小環境での呼吸器細胞および分子生物学を研究する機会を提供します。

Watch this Scientific Journal Video about Procedure for Lung Engineering at JoVE.com

Procedure for Lung Engineering

私たちは、脱細胞肺癌細胞外マトリックスと機能的な肺組織を生成するために使用することができる新規バイオミメティックバイオリアクターを開発している。バイオリアクターにおけるマトリックスと培養に細胞を播種することにより、我々は短期間のためのin vivoで移植すると効果的なガス交換を示す組織を生成する。

肺のエンジニアリングのための手順

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Research

JoVE Journal

Bioengineering

46.5K ビュー.  Yale University. 私たちは、脱細胞肺癌細胞外マトリックスと機能的な肺組織を生成するために使用することができる新規バイオミメティックバイオリアクターを開発している。バイオリアクターにおけるマトリックスと培養に細胞を播種することにより、我々は短期間のためのin vivoで移植すると効果的なガス交換を示す組織を生成する。

ビデオ: Procedure for Lung 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.

動脈組織工学におけるエラストマーのPGSの足場

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

生物工学

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.

組織工学のためのエレクトロ繊維のポスト処理

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

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.

心臓組織工学のためのキャピラリーフォースリソグラフィ

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

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.

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

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

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

医工学のためのケラチンベースのナノファイバーの合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

マイクロ流体工学バイオプリンティング血管組織や Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

組織工学用植物組織の Decellularization の 2 つの方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

浸透哺乳動物の心臓のトランスムラル吸光分光法を用いた細胞代謝モニタリングのための心臓内側発射光カテーテル

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

脱細胞化肺スライスから調製された人工肺組織

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

ウシ肺ECMハイドロゲルの脱細胞化技術の比較研究

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypic<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: black; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&#160;alternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

著者スポットライト:器官型ハイドロゲルのためのウシ肺脱細胞化の最適化

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

脱細胞化肺細胞外マトリックスハイドロゲルの開発と特性評価

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...