この研究は、LSH 2Treatプログラム(436001003)とオランダ腎臓財団(14a2d507)の一環としてZonMwによって財政的に支援されています。N.A.K.は、欧州研究評議会(851960)からの支援を認めています。オランダ科学研究機構(024.003.013)が出資する重力プログラム「物質主導再生」を感謝します。

著者らは開示するものは何もない。

本明細書に記載されたバイオリアクターは、尿細管付動性足場における炎症および組織再生に対する剪断応力および環状ストレッチの個々および結合された影響の系統的評価を可能にする。このアプローチはまた、代表的な結果セクションで例示されるように、血管構造上で行われる多種多様な分析を可能にする。これらの結果は、TEVG構造の成長とリモデリングの両方に異なる血行力負荷レ...

心臓血管組織工学(TE)は、患者1、2、3、4の大コホートに最適でない現在使用されている永久的な心臓血管補修(例えば、血管移植片、心臓弁置換術)に対する代替治療選択肢として追求されている。多くの求められているアプリケーションは、組織工学的に設計された血管移植片(TEVGs)5、6および心臓弁(TEHV)7、8を含む。ほとんどの場合、心臓血管TEの方法論は、新しい組織が形成される有益な足場として機能する再ソーブルバイオマテリアル(天然または合成のいずれか)を利用する。新しい組織の形成は、細胞で足場を播種し、移植前にバイオリアクターで培養することによって完全にインビトロで設計することができる(in vitroTE)9、10、11、または合成足場が体内に新しい組織の形成を直接誘導するために前培養せずに移植されるその中で直接(situ TEにおいて)12,12, 13.in vitroとininin心臓血管TEアプローチの両方について、機能的再生の成功は、移植された構築物に対する宿主免疫応答および適切な生体力学的負荷の両方に主に依存する。
心血管TEのための生体力学的負荷の重要性は、十分に認められている15.心臓血管インプラントの場合、足場に入る細胞は、血行力学的環境の結果として生じる周期的なストレッチおよびせん断ストレスにさらされる。多くの研究は、様々な細胞タイプによる、例えばコラーゲン16、17、18、19、グリコサミノグリカン(GAGs)20、およびエラスチン21、22、などのマトリックス成分の形成に対する(環状)ストレッチの刺激効果を報告している。例えば、Huang et al. は、二軸ストレッチが血管バイオリアクター23を用いてインビトロTEVGsにおけるコラーゲンおよびエラスチンの堆積および組織を上昇させたことを実証した。主な負荷として強調される一方で、これらの研究は、多くの場合、サンプルがせん断流に曝露されるフロー駆動のバイオリアクターを利用しています。シアストレスが3Dの組織形成や炎症に及ぼす孤立した影響については比較的ほとんど知られていないが、いくつかのデータが利用可能である。例えば、ヒンダーらおよびEohら.は、剪断流が、3D足場微細構造に加えて、インビトロモデルシステム24、25におけるヒト血管平滑筋細胞による成熟エラスチンの形成にとって重要であることを実証した。全体として、これらの知見は、心血管TEの環状ストレッチとせん断ストレスの両方の関連性を示している。
TEインプラントの成功または失敗に対するもう一つの重要な決定要因は、移植された移植片26に対する宿主の免疫応答である。これは、細胞流入および内因性組織形成および再モデリング27のその後のプロセスを開始するために足場に対する急性炎症反応に実際に依存する、その中での物質駆動のTE戦略にとって特に重要である。マクロファージは、機能的組織再生の重要なイニシエータであり、複数の研究28、29、30によって示されている。創傷治癒に類似して、組織の再生は、マクロファージと線維芽細胞と筋線維芽細胞31、32、33などの組織産生細胞との間のパラクリンシグナル伝達によって支配される。新しい組織堆積の調整に加えて、マクロファージは、外来足場材料34、35の活性再吸収に関与している。このように、生体材料に対するインビトロマクロファージ応答は、インプラント36、37、38の生体内成功の予測パラメータとして同定されている。
埋め込まれた足場に対するマクロファージ応答は、材料組成および微細構造35、39、40などの足場設計特徴に依存する。足場の特性に加えて、足場に対するマクロファージ応答および筋線維芽細胞とのクロストークも血行力学的負荷の影響を受ける。例えば、環状ストレッチは、マクロファージ表現型41、42、43、44およびサイトカイン43、44、45、46の分泌の重要なモジュレーターであることが示された。マクロファージと血管平滑筋細胞の共培養システムを用いて、Battistonらは、マクロファージの存在がエラスチンおよびGAGのレベルの増加につながり、適度なレベルの環状ストレッチ(1.07-1.10)がコラーゲンIおよびエラスチン47の沈着を刺激することを実証した。これまでの研究では、剪断応力が3Dエレクトロスパン足場48,49への単球採用にとって重要な決定要因であり、剪断応力と環状ストレッチの両方がヒト単球と間葉間質細胞50との間のパラクリンシグナル伝達に影響を及ぼすことを実証した。Fahy et al. は、剪断流がヒト単球51による炎症性サイトカインの分泌を増加することを実証した。
まとめると、上記の証拠は、血行力学的負荷の十分な理解と制御が心血管TEにとって重要であり、これを達成するために炎症反応を考慮することが重要であることを示しています。多数のバイオリアクターが、心血管組織のinvitro 52、53、54、55、56、57、58またはex vivo 59、60、61培養物について以前に説明されている。しかし、これらのシステムはすべて、可能な限り生理学的な血行負荷条件を模倣するように設計されています。これは、体外で心血管組織を作成したり、エキビボ培養を維持する目的で非常に貴重であるが,そのようなシステムは、個々の手がかりの個々の効果に体系的な研究を可能にしません.これは、これらのバイオリアクターにおける環状ストレッチとせん断応力の両方の適用は、本質的にそれらをリンクする同じ加圧流によって駆動されるためです。正確なマルチキュー機械操作を可能にするマイクロシステムは、2D基板62または3Dハイドロゲルセットアップ63、64のために記述されているが、そのようなセットアップは、エラストマー3D生体材料足場の組み込みを可能にしていない。
ここでは、せん断応力と環状ストレッチの分離を独自に可能にし、個々の効果と組み合わせた効果を機械論的に調査するのに役立つ管状バイオリアクターシステムの適用を提示する。このシステムは、多種多様な組織工学的に設計された血管移植片(例えば、合成または天然起源、異なるマイクロアーキテクチャ、様々な多孔質)の試験を可能にする。せん断応力とストレッチの適用を効果的に切り離すために、バイオリアクターが使用する重要な概念は、(1)異なるポンプシステムを使用したせん断応力とストレッチの制御の分離と(2)計算駆動寸法を持つ「インサイドアウト」方式で足場の刺激です。流れは流れポンプの使用によって管状足場の外面に適用され、足場の周伸張は、足場が別のひずみポンプを使用して取り付けられるシリコーン管を拡大することによって誘発される。シリコンチューブと構成体を含むガラス管の寸法は、スキャフォールド(流れによる)と周伸縮(チューブ膨張による)のせん断応力が互いに大きな影響を与えないようにするために、計算流体力学シミュレーションを使用して慎重に選択され、検証されます。この裏返しの設計には、いくつかの実用的な根拠があります。ストレッチが(生理学的負荷に似た)発光流体圧力によって適用される場合、サンプル設計は本質的に漏れのないものでなければなりません。さらに、サンプルの伸張に必要な圧力は、サンプルの剛性によって完全に決定され、サンプル間およびサンプル内で時間の経過とずれによって変化し、ストレッチの制御が困難になります。このバイオリアクターは、シリコーンチューブの周りに組織工学的に設計された移植片を取り付け、移植片の外壁に壁せん断応力(WSS)の適用を可能にし、内部から移植片を加圧する。このようにして、サンプルとサンプル内の間の等しい負荷条件を時間をかけて確保することができ、また、多孔性血管足場19に対して一般的であるように、サンプルが漏れやすい状態が許される。この内部出力バイオリアクターは、従来の血管バイオリアクターのセットアップがより適しているインビトロのネイティブのような血管のエンジニアリングではなく、せん断および/またはストレッチの効果に関する体系的な研究のために特に意図されています。バイオリアクター設計図面の 図1A-B およびそれに対応する 表1 を参照して、バイオリアクターの主要成分の背後にある機能の説明と根拠を確認してください。
バイオリアクターの使用は、我々は、その現場の心血管組織19、43、44における再ソーブルエレクトロスパン足場における炎症および組織形成に関する剪断ストレスおよび環状ストレッチの個々および組み合わせた影響を調査した我々のグループによる一連の最近の研究に基づいて実証される。そのために、ヒトマクロファージと筋線維芽細胞をモノ培養または共培養で使用して、イン・シチュの再生カスケードの様々な相をシミュレートしました。ヒトマクロファージによるサイトカイン分泌は、周期的な伸縮とせん断応力の両方によって明らかに影響を受け、これらの足場におけるヒト筋線維芽細胞によるマトリックス沈着および組織に影響を及ぼすことを実証した。特に、これらの研究は、せん断応力とストレッチの併用の場合、組織形成および炎症への影響が2つの負荷のいずれかによって支配されるか、または両方の負荷の相乗効果があることを明らかにした。これらの知見は、両方の負荷を分離して、TE プロセスにおける機械的環境の寄与をより深く理解する関連性を示している。この理解は、関連する血行負荷のレジームでスキャフォールド設計パラメータを体系的に最適化するために適用できます。さらに、このような十分に制御された環境からの機械化データは、TEV65またはTEHV66に対して最近報告されているように、その場での組織改修の経過を予測するために開発されている数値モデルの入力として役立つ可能性があり、予測能力をさらに向上させる。

<table><tbody><tr><td>advanced Dulbecco&amp;rsquo;s modified EagleMedium (aDMEM)</td><td>Gibco</td><td>12491-015</td><td>cell culture medium for fibroblasts</td></tr><tr><td>Aqua Stabil</td><td>Julabo</td><td>8940012</td><td>prevent microorganism growth in bioreactor-hydraulic reservoir</td></tr><tr><td>Bovine fibrinogen</td><td>Sigma</td><td>F8630</td><td>to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td></tr><tr><td>Bovine thrombin</td><td>Sigma</td><td>T4648</td><td>to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804</td><td>to spin down cells and conditioned medium</td></tr><tr><td>Clamp scissor - &quot;kelly forceps&quot;</td><td>Almedic</td><td>P-422</td><td>clamp the silicone tubing and apply pre-stretch to the scaffold so the scaffold can be sutured into the engraved groove (autoclave at step 1, step 7)</td></tr><tr><td>CO2 cell culture incubators</td><td>Sanyo</td><td>MCO-170AIC-PE</td><td>for cell culturing</td></tr><tr><td>Compressed air reservoir</td><td>Festo</td><td>CRVZS-5</td><td>smoothing air pressure fluctuations and create time delays for pressure build-up</td></tr><tr><td>Custom Matlab script to calculate the maximum stretches</td><td>Matlab</td><td>R2017. The Mathworks, Natick, MA</td><td>calculate the minimum and maximum outer diameter of the electrospun scaffold</td></tr><tr><td>Data acquisition board</td><td>National Instruments</td><td>BNC-2090</td><td>data processing in between amplifier system and computer</td></tr><tr><td>Ethanol</td><td>VWR</td><td>VWRK4096-9005</td><td>to keep sterile working conditions</td></tr><tr><td>Fetal bovine calf serum (FBS)</td><td>Greiner</td><td>758087</td><td>cell culture medium supplement; serum-supplement</td></tr><tr><td>Flow culture chamber compartments, consisting of a pressure conduit with engraved grooves and small holes to apply pressure on silicone tubing, a screw thread, nose cone, top compartment with flow inlet and bottom compartment flow outlet, adapter bushing</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>flow culture chamber compartments (autoclave at step 1, step 7)</td></tr><tr><td>Glass Pasteur pipet</td><td>Assistant</td><td>HE40567002</td><td>apply vacuum on electrospun scaffold (autoclave at step 1)</td></tr><tr><td>Glass tubes of the flow culture chamber</td><td>Custon made, Equipment &amp;amp; Prototype Center, Eindhoven University of Technology</td><td>n.a.</td><td>part of the flow culture chamber (clean and store in 70% ethanol, at step 1 and 7)</td></tr><tr><td>GlutaMax</td><td>Gibco</td><td>35050061</td><td>cell culture medium amino acid supplement, minimizes ammonia build-up</td></tr><tr><td>High speed camera</td><td>MotionScope</td><td>M-5</td><td>to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td></tr><tr><td>High speed camera lens - Micro-NIKKOR 55mm f/2.8 - lens</td><td>Nikon</td><td>JAA616AB</td><td>to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td></tr><tr><td>Hose clip</td><td>ibidi GmbH</td><td>10821</td><td>block medium flow (autoclave at step 1, step 7)</td></tr><tr><td>Hydraulic reservoir with 8 screw threads for 8 flow culture chambers</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to apply pressure to the silicone mounted constructs (clean outside with a paper tissue with 70% ethanol, rinse reservoir with 70% ethanol followed by demi water, at step 1 and 7)</td></tr><tr><td>Ibidi pump system (8x) including ibidi pump, PumpControl software, fluidic unit, perfusion set (medium tubing), air pressure tubing, drying bottles with orange silica beads</td><td>ibidi GmbH</td><td>10902</td><td>set up used to control the flow in the flow culture chambers. Note 1: the ibidi pumps were modified by the manufacturer to enable 200 mbar capacity. Note 2: can be replaced by pump system of other manufacturer, as long as same flow regimes can be applied.</td></tr><tr><td>Knives (no.10 sterile blades, individual foil pack) and scalpel handle (stainless steel, individually wrapped)</td><td>Swann Morton</td><td>0301; 0933</td><td>to cut the silicone tubing in the correct size for the scaffold and to cut the suture material</td></tr><tr><td>LabVIEW Software</td><td>National Instruments</td><td>version 2018</td><td>to control the stretch applied to the scaffolds</td></tr><tr><td>Laminar flow biosafety cabinet with UV light</td><td>Labconco</td><td>302310001</td><td>to ensure sterile working conditions. The UV is used to decontaminate everything that cannot be autoclaved, or touched after autoclaving</td></tr><tr><td>Large and small petri dishes</td><td>Greiner</td><td>664-160</td><td>for sterile working conditions</td></tr><tr><td>L-ascorbic acid 2-phosphate (vitamin C)</td><td>Sigma</td><td>A8960</td><td>cell culture medium supplement, important for collagen production</td></tr><tr><td>LED light cold source KL2500</td><td>Zeiss</td><td>Schott AG</td><td>to aid in visualization for the time lapse of the scaffolds during monitoring of the stretch</td></tr><tr><td>Luer (female and male) locks and connectors, white luer caps</td><td>ibidi GmbH</td><td>various, see (https://ibidi.com/26-flow-accessories)</td><td>to close or connect parts of the bioreactor and the ibidi pump (autoclave at step 1, step 7)</td></tr><tr><td>Measuring amplifier (PICAS)</td><td>PEEKEL instruments B.V.</td><td>n.a.</td><td>to amplify the signal from the pressure sensor and feedback to LabView</td></tr><tr><td>Medium reservoir (large syringes 60 mL) and reservoir holders</td><td>ibidi GmbH</td><td>10974</td><td>medium reservoir (autoclave at step 1, step 7)</td></tr><tr><td>Medium tubing with 4.25 mm outer diameter and 1 mm inner diameter</td><td>Rubber BV</td><td>1805</td><td>to allow for a larger flow rate, the ibidi medium tubing with larger diameter is used. Note: the part of medium tubing guided through the fluidic unit valves are the same as the default ibidi medium tubing</td></tr><tr><td>Motion Studio Software</td><td>Idtvision</td><td>2.15.00</td><td>to make the high speed time lapse images for stretch monitoring</td></tr><tr><td>Needle (19G)</td><td>BD Microlance</td><td>301700</td><td>together with thin flexible tubing used to fill the hydraulic reservoir with ultrapure water without adding air bubbles</td></tr><tr><td>Needle driver</td><td>Adson</td><td>2429218</td><td>to handle the needle of the nylon suture through the silicone tube (autoclave at step 1, step 7)</td></tr><tr><td>Paper tissues</td><td>Kleenex</td><td>38044001</td><td>for cleaning of the equipment with 70% ethanol</td></tr><tr><td>Parafilm</td><td>Sigma</td><td>P7793-1EA</td><td>quick fix if leakage occurs</td></tr><tr><td>Penicillin/streptomycin (P/S)</td><td>Lonza</td><td>DE17-602E</td><td>cell culture medium supplement; prevent bacterial contamination</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma</td><td>P4417-100TAB</td><td>for storage and washing steps (autoclave at step 1)</td></tr><tr><td>Plastic containers (60 mL) with red screw caps</td><td>Greiner</td><td>206202</td><td>to prepare the fibrinogen solution</td></tr><tr><td>Pneumatic cylinder</td><td>Festo</td><td>AEVC-20-10-I-P</td><td>to actuate the Teflon bellow (clean with a paper tissue with 70% ethanol at step 1 and 7)</td></tr><tr><td>Polycaprolactone bisurea (PCL-BU) tubular scaffolds (3 mm inner diameter, 200 &amp;micro;m wall thickness, 20 mm length)</td><td>SyMO-Chem, Eindhoven, The Netherlands</td><td>n.a.</td><td>produced using electrospinning from 15% (w/w) chloroform (Sigma; 372978) polymer solutions. See Van Haaften et al Tissue Engineering Part C (2018) for more details</td></tr><tr><td>Pressure conduit without holes (for static control)</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to mount electrospun tubes on silicon tubing (autoclave at step 1, step 7)</td></tr><tr><td>Pressure sensor and transducer</td><td>BD</td><td>TC-XX and P 10 EZ</td><td>the air pressure going to the pneumatic actuated pump is raised until it reaches the set pressure</td></tr><tr><td>Proportional air pressure control valve and pressure sensor</td><td>Festo</td><td>MPPES-3-1/8-2-010, 159596</td><td>provides compressed air to the pneumatic actuated pump</td></tr><tr><td>Roswell Park Memorial Institute 1640 (RPMI-1640)</td><td>Gibco</td><td>A1049101</td><td>cell culture medium for monocyte/macrophage</td></tr><tr><td>Safe lock Eppendorf tubes (1.5 mL)</td><td>Eppendorf</td><td>30120086</td><td>multiple applications (autoclave at step 1)</td></tr><tr><td>Sodium dodecyl sulfate solution 20%</td><td>Sigma</td><td>5030</td><td>Used to clean materials, at a concentration of 0.1%.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Silicone O-rings</td><td>Technirub</td><td>1250S</td><td>to prevent leakage (autoclave at step 1, step 7)</td></tr><tr><td>Silicone tubing (2.8 mm outer diameter, 400 um wall thickness)</td><td>Rubber BV</td><td>1805</td><td>to mount the electrospun tubes on the pressure conduits (autoclave at step 1)</td></tr><tr><td>Sterile tube (15 mL)</td><td>Falcon</td><td>352095</td><td>multiple applications</td></tr><tr><td>Suture, 5-0 prolene with pre-attached taper point needle</td><td>Ethicon, Johnson&amp;amp;Johnson</td><td>EH7404H</td><td>Prolene suture wire 5-0 (75cm length, TF taper point needle, 1/2 circle, 13 mm needle length)</td></tr><tr><td>Syringe (24 mL)</td><td>B. Braun Melsungen AG</td><td>2057932</td><td>to add the ultrapure water or medium to the hydraulic reservoir or flow culture chamber</td></tr><tr><td>Syringe filter (0.2 &amp;micro;m)</td><td>Satorius</td><td>17597-K</td><td>to filter the fibrinogen solution</td></tr><tr><td>T150 cell culture flask with filter cap</td><td>Nunc</td><td>178983</td><td>to degas culture medium</td></tr><tr><td>T75 Cell culture flask with filter cap</td><td>Nunc</td><td>156499</td><td>to culture static control samples</td></tr><tr><td>Teflon bellow</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to load the hydraulic reservoir (clean outside with a paper tissue with 70% ethanol at step 1 and 7)</td></tr><tr><td>Tray (stainless steel)</td><td>PolarWare</td><td>15-248</td><td>for easy transport of the fluidic culture chambers and the bioreactor from incubator to laminar flow cabinet and back (clean with a paper tissue with 70% ethanol before and after use)</td></tr><tr><td>Tweezers</td><td>Wironit</td><td>4910</td><td>sterile handling of individual parts (autoclave at step 1 and 7)</td></tr><tr><td>Ultrapure water</td><td>Stakpure</td><td>Omniapure UV 18200002</td><td>to correct for medium evaporation, mixed with aqua stabil mixed and used as hydraulic fluid. (autoclave ultrapure water at step 1)</td></tr><tr><td>UV light</td><td>Philips</td><td>TUV 30W/G30 T8</td><td>for decontamination of grafts and bioreactor parts before seeding</td></tr></tbody></table>

a multi-cue bioreactor to evaluate the inflammatory and regenerative capacity of biomaterials under flow and stretch

体内で直接再生を誘導する再ソーブルバイオマテリアルの使用は、翻訳の観点から魅力的な戦略です。このような物質は、移植時に炎症反応を誘発し、これは材料のその後の再吸収および新しい組織の再生の原動力である。この戦略は、現場組織工学としても知られており、組織工学的に設計された血管移植片などの心血管置換を得るために追求される。炎症と再生プロセスの両方は、足場上の局所的な生体力学的手がかり(すなわち、ストレッチおよび剪断応力)によって決定される。ここでは、管状足場上のストレッチとせん断応力の分離を独自に可能にするカスタム開発バイオリアクターの使用について詳しく説明する。これにより、よく制御された機械的負荷の影響下での管状足場の炎症および再生能力の体系的かつ標準化された評価が可能となり、ヒトマクロファージおよびミオ線維芽細胞を用いた動的共培養実験に基づいて実証する。このアプローチの主要な実践的なステップ(バイオリアクターの構築とセットアップ、足場と細胞の播種の準備、ストレッチとせん断の流れの適用とメンテナンス、分析用のサンプル採取)について詳しく説明します。

このバイオリアクターは、3D生体材料足場における血管組織の成長とリモデリングに対するせん断応力と環状ストレッチの個々および組み合わせ効果を研究するために開発されました。バイオリアクターの設計により、様々な負荷条件下で最大8個の血管構造を培養することができます(図1A)。血管構築物は、周延伸とWSSの両方を独立して制御できるフロー培養チャンバー...

Watch this Scientific Journal Video about A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch at JoVE.com

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

このプロトコルの目的は、剪断応力と環状ストレッチの分離を可能にするバイオリアクターを使用して、材料駆動組織再生を調査するために、管状の電気スピン足場でヒトマクロファージと筋線維芽細胞の動的共培養を実行することです。

フローとストレッチの下でバイオ材料の炎症と再生能力を評価するマルチキューバイオリアクター

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

a-multi-cue-bioreactor-to-evaluate-inflammatory-regenerative-capacity

Research

JoVE Journal

Engineering

5.5K ビュー.  Eindhoven University of Technology. このプロトコルの目的は、剪断応力と環状ストレッチの分離を可能にするバイオリアクターを使用して、材料駆動組織再生を調査するために、管状の電気スピン足場でヒトマクロファージと筋線維芽細胞の動的共培養を実行することです。

ビデオ: A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

拍動バイオリアクターを用いた工学生物学ベースの人工血管

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

生物工学

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

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 ...

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.

組織工学のための二軸メカニカルローディングバイオリアクターの設計

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

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

大動脈リング共培養アッセイ:体外間葉系間質細胞の血管新生の可能性を評価するために便利なツール

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

発生生物学

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多臓器チップ - 長期マルチ組織の共培養のためのマイクロ流体プラットフォーム

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

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. ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

骨再生のための生体材料の生物学的互換性プロファイル

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

人工心臓組織のデータ取得とモデルスループットを向上させるバイオリアクターの設計

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

灌流中の血管のex vivo培養のための新しい3Dプリントバイオリアクター

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

著者スポットライト:EasyFlow - 大血管培養のための経済的で適応性の高い灌流バイオリアクター 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Ex Vivo(エクス・ビボ )3Dプリントされたバイオリアクターでの大血管の灌流培養

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...