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1。キトサン貼付製剤<ol><li>キトサン粉末が酢酸溶液に可溶であり、酢酸（2％v / v）のストック溶液を調製するために、490ミリリットルの脱イオン水（DI-H 2 O）に10 mlの氷酢酸を追加します。 </li><li>酢酸中ローズベンガル（RB）（0.01％w / v）のストック溶液の調製のために、退色を避けるためにアルミホイルで包まれたバイアル中でRBの5mgを量る。酢酸原液（1.1項で説明）の49.5ミリリットルを加えて粉末を溶解するためのDI-H 2 Oの0.5 mlを加える。 </li><li>キトサン溶液（1.7％w / v）の調製のために、キトサン粉末（ミディアムMW、アセチル化の85％程度）の0.85グラムの重量を量るとRB酢酸溶液50mlに追加します。コントロールは、RBなし酢酸溶液にキトサンを溶解することによって調製した。 </li><li>キトサン混合物にマグネチックスターラーを追加し、室温で撹拌。コンポーネントの完全な溶解を確実にするために、キトサンをかき混ぜる2週間と5日間酢酸中のキトサン（コントロール）用酢酸中+ RB。キトサンが解散されたときは、溶液のpHは約2.6から〜3.9に増加します。 RBは酸性pHに難溶性であり、それは溶液のpHと溶解する拡張撹拌時間の増加が必要であることに注意してください。 </li><li> 1時間3270 xgで遠心し、ソリューションを使用して上清を回収し、例えば、無菌の10mlシリンジ。不純物の沈殿を乱さないようにしてください。このステップの間、溶液は肉眼で見える不純物、不溶物から洗浄される。 </li><li>無菌の注射器を使用してフラットperpexスラブ上（遠心分離）キトサン溶液を注入します。乾燥したフィルムの厚さは、溶液の調剤量と溶液が広がっているその上に表面積との比に依存する。ソリューションは気泡がないと均等にスラブの上に広げていることを確認します。 〜13μmの厚さの薄膜を得るために、12 cm 2の表面の上に〜1mlの溶液を注入面積（寸法は〜3×4センチ、 図1A）。 </li><li>溶液の退色を避けるために、例えばアルミ箔製のスクリーンを持つ風防スラブを、カバーしています。十分な換気が画面の下に溶液を乾燥することが保証されていることを確認します。 </li><li>得られたフィルムは水に不溶であり、肉眼的に膨潤しないまで室温（25℃）及び圧力（1気圧）で乾燥するソリューションを残す。これは一般的に2週間で達成される。 </li><li>慎重に薄くて平らなへらを使用してフィルムをはずす：これらのフィルムは、簡単にそれらを損傷することなく、風防スラブに剥離することができる。 </li><li>マイクロメーターを用いてフィルムの厚さを測定します。 </li><li>きれいな鋭いはさみで短冊で接着剤をカットし、その平坦な形状を維持するために滅菌したガラススライドの間に配置します。遮光のためにアルミホイルでスライドを包み、室温で保管してください。または &quot;キトサンadhes&quot;接着バラ &quot;として、RBの有無にかかわらず、接着剤のラベルを付けますそれぞれ &quot;IVE。 </li><li>バラの接着フィルムをエタノール（90％）またはガンマ線（24時間0.1 KGy / h）で4,5とを用いて滅菌することができる。 </li></ol> 2。接着剤光減衰<ol><li>石英キュベット内のバラ接着剤を固定します。 </li><li>デュアルビーム分光光度計4を使用して 800nm ​​の- 400の範囲で、その減衰スペクトルを記録します。 </li><li>次のように接着剤の減衰長を計算し、ベールの法則の妥当性を仮定すると、I = I 0のe-AXここで私は0は入射ビーム強度は、1 /減衰長であり、xは膜厚である。 </li><li>キトサン接着剤の2.1から2.3にコントロールとして機能するように手順を繰り返します。 </li></ol> 3 体外アプリケーションで次の操作を行います 。小腸におけるレーザーティッシュボンディング<ol><li>収穫ウシ腸直後は-80℃で、動物安楽死と℃で保存</li><li>使用前に、霜と水和物TI10分間のDI-H 2 O中に浸漬してssue。 </li><li>手術用顕微鏡（×10）の下で＃10ブレードを使用してセクション（2×1cm）をに組織を二分。 DI-H 2 Oを使用してセクションの潤いを保つ</li><li> （エンドツーエンド）近くに一緒に切開の切り株を持参してください。綿のヒントを使用して表面から余分な水分を拭き取ります。 </li><li>腸（ 図2）と完全に接触を確保microforcepsと漿膜層に切開を横切っバラの粘着ストリップ（10×6 mm）を配置します。 </li><li>バラの接着剤の組織の癒着は、マルチモード光ファイバ（コア径200μm）を4に結合されているダイオード励起固体レーザによって活性化される。 </li><li> 180 mWにレーザーのパワーレベルを設定し、 表1に記載されたパラメータを使用して〜5mmのビームスポットサイズで〜6分、のための接着剤を照射します。各スポットは、隣接するスポットにビームを移動する前に、約5秒間照射されていることを確実に接着剤をスポット照射します。 T彼の手順では、レーザービームが接着剤を数回の全体の表面積をスキャンすることを保証します。 </li><li>組織接着強度を評価するために、機械的なグリップを使用して校正された単一の列テンシオメータ（インストロン、MA、USA）に試料をクランプします。 4つの独立した二つの組織の切り株まで22ミリメートル/分でグリップを移動します。 </li><li>ステップ3.5で説明されるように組織上のバラの接着剤を置きます。試料に照射し、手順3.8で説明したように、組織の接着強度を測定しないでください。このサンプルでは、​​コントロールとして機能します。 </li></ol> 4 in vivo用途で次の操作を行います 。脛骨神経の吻合<ol><li> O /イソフルラン2混合物（以後誘導中に4％、2％）5と落ち着いたWistarラット。皮下術中鎮痛作用を提供し、できるだけ痛みや不快感を軽減するためにBuprenexを（0.03 mg / kg）を注入します。 </li><li>以下の外科的処置は、手術用顕微鏡（×10）の下で行われるべきである。斜めの肌を作る右大腿部の背外側部で長く臀筋2を介して筋肉分割アプローチで脛骨神経を露出さ約3cm切開。 </li><li>部分的に分析し、ストレートマイクロ剪刀を用いて脛骨神経の外膜をトリミングして、滅菌ガーゼや綿のヒントと余分な水分を吸収する。 </li><li>マイクロはさみで脛骨神経を切断。 </li><li>マイクロ鉗子を用いて脛骨神経の下に位置無菌キトサンストリップ（寸法は5×4 mm）です。 </li><li>バラ粘着ストリップ上のマイクロピンセットで近似神経切り株エンドツーエンド。 </li><li>ストリップが完全にストリップのレーザー照射時に神経の回転に伴って、支援することができます襟を形成する神経にも準拠しています。 </li><li> ）ステップ3.6で説明されるようにレーザーでバラの接着剤を活性化）と3.7。 </li><li>作動神経の襟から冗長バラ接着剤をトリムします。 </li><li> 5 3から0縫合糸5と筋肉と皮膚を閉じます。 </lI&gt; <li>必要に応じて標準的な術後の回復手順は、裂開や膿形成や鎮痛剤投与のための日常点検を含む従うべきである。 </li></ol> 5。代表的な結果得られたフィルムは明るいカラーでバラ薄く、滑らかな表面（ 図1）を持っている。彼らはまた、柔軟であり、涙や他の明らかな損傷（ 図1B）を発生させることなく小径のチューブにロールバックすることができます。バラの接着剤は、530および562 nmの2つの吸収ピークを有し、緑のレーザーはこのように強力に接着剤で吸収し、532nmで対応する減衰長は〜12μmで（ 図3）である。これとは対照的に、RBなしキトサンフィルムはおそらく、弱く散乱によるレーザー（1 /〜162μm）を減衰させる。それはRBの有意な凝集が映画の中で発生していないことをスペクトルプロットから表示されます。 しっかりと接着結合をバラ0.9障害発生時の典型的な最大負荷を達成するため、レーザー照射（ 図2）±0.4（N = 30）の時に腸へ。接着強度は、接着表面積、すなわち、15で割った最大荷重±6キロパスカル（​​n = 30）のように推定された。非照射バラ接着剤は組織に結合して有意に少ない（2±2kPaと、n = 30の、対応のないt-検定、p &lt;10 6）。脛骨神経の生体内吻合は正常に緑色のレーザーと一緒にバラの接着剤の塗布及び接合後に達成された。術後一週間、運営神経が継続していたと修理の強度は17歳±9キロパスカル（​​n = 10）であった。 <img alt="図1" src="/files/ftp_upload/4158/4158fig1.jpg" /> 図1。）RB-キトサン溶液は、フラットパースペックススラブ（スラブ厚さ約5 mm）の上にキャストされています。方眼紙は、乾燥キャストステップを容易にするスラブの下に配置されています。水不溶性FILMは2週間鋳造後に得られる。 B）はバラ接着剤は柔軟性があり、小さなシリンダーにロールバックすることができます。 <img alt="図2" src="/files/ftp_upload/4158/4158fig2.jpg" /> 図2：バラの接着剤は、レーザー照射後の子牛の腸に接着されている。均一照射は組織ボンディング時のRB接着剤に適用されますが選択されたスポットは、接着剤にレーザーの光漂白効果を説明するために、この絵に示されている。 <img alt="図3" src="/files/ftp_upload/4158/4158fig3.jpg" /> 図3：バラの接着剤の吸収スペクトルは、（任意の単位）530および562 nmの2つのピークを示す。緑色レーザー（λ= 532 nm）をこのように強くレーザー露光時の接着剤によって吸収される。 RBなしキトサン接着が不十分なレーザー光を吸収。 <table border="1"><tbody><tr><td></td><td> N </td><td><s&gt;面積（mm 2）チョン· </td><td> 消費電力（W） </td><td> 時間（s） </td><td> フルエンス（J / cm 2）を </td><td> I（W / cm 2）を </td><td> 最大負荷/エリア（KPA） </td></tr><tr><td> 接着剤+レーザー </td><td> 30 </td><td> 60±10 </td><td> 0.18±0.03 </td><td> 365±5 </td><td> 〜110 </td><td> 〜0.9 </td><td> 15±1 </td></tr><tr><td> 接着剤 </td><td> 30 </td><td> 60±10 </td><td> NA </td><td> NA </td><td> NA </td><td> NA </td><td> 2±2 </td></tr></tbody></table> 表1。伝説。 nはサンプル番号、エリア、バラの接着剤の表面積（±最大誤差を意味する）、パワー、レーザパワー（±最大誤差を意味する）、時間、照射時間（±最大誤差を意味する）、フルエンス、平均レーザフルエンス、私、推定IRRadiance、最大負荷/エリア、接着表面積で割った修復組織の破壊時の最大荷重（平均±SE）。

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酸性pHにおけるRBの溶解が延長キトサン溶液の攪拌を要求している接着剤の製造は、シンプルな乾式キャスト法に基づいていました。それは水に不溶性の膜となるまで溶液が乾燥させることが重要です。重量含水率は乾燥膜6〜10％であるとき、これは起こります。不溶性のフィルムは、通常、温度と圧力の標準条件（〜25℃、約1気圧）で2週間乾燥キャスティング後に得られる。それは...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>ローズベンガル</td><td>シグマアルドリッチ</td><td> 632-69-9 </td><td></td></tr><tr><td>キトサン（ミディアムMW） </td><td>シグマアルドリッチ</td><td> 10318AJ </td><td></td></tr><tr><td>氷酢酸</td><td>シグマアルドリッチ</td><td> 08050051 </td><td> DI水で2％v / vの</td></tr><tr><td>マグネチックスターラー</td><td> Heidolph </td><td> MR特ミックスS </td><td></td></tr><tr><td>遠心</td><td>ベックマン·コールター</td><td>アレグラX-12R </td><td></td></tr><tr><td>分光光度計</td><td>バリアン</td><td>ケーリー4000紫外可視</td><td></td></tr><tr><td>レーザー</td><td> CNIレーザー</td><td> MGL-532 </td><td></td></tr><tr><td>マイクロメータ</td><td>ミツトヨ</td><td>シリーズ227 </td><td></td></tr><tr><td>外科用顕微鏡</td><td>ツァイス</td><td> OPMI </td><td></td></tr></tbody></table>

fabrication and application of rose bengal-chitosan films in laser tissue repair

光化学組織結合（PTB）は、2つの組織の縁部1,2の間にローズベンガル（RB）のソリューションを適用することによって達成される組織修復のための縫合テクニックです。次いで、これらは、選択的にRBによって吸収されるレーザーが照射される。その結果、光化学反応は、おそらく最小限の熱産生3で、組織内のコラーゲン繊維を架橋する。本報告では、RBはレーザーで活性化される新規の組織接着剤を製造するために薄いキトサン膜に組み込まれています。キトサンおよび〜0.1重量％RBを含むに基づく接着剤フィルムは、固体レーザ（λ= 532nmで、フルエンス〜110 J / cm 2であり 、スポットサイズは約0.5cm）により製造され、子牛腸およびラット脛骨神経に接合されている。パソコンとのインタフェースの単一列テンシオメータは、接合強度をテストするために使用されます。 RB-キトサン接着ボンドしっかりと15の強度を持つ腸±6 kPaで、（N = 30）。接着強度は、2＆に低下plusmn;レーザーを接着剤に適用されていない2キロパスカル（​​N = 30）。脛骨神経の吻合も縫合糸を使用せずに完了することができます。新規なキトサン接着剤が糸を光化学的に組織に結合し、必要としないことを試作した。

Watch this Scientific Journal Video about Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair at JoVE.com

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

縫合糸は通常、外科手術の際に組織を修復するために必要とされている。彼らは侵襲的であり、組織に損傷を与える可能性があるしかし、そのアプリケーションが問題になることがあります。小説組織接着剤の製造、およびアプリケーションの方法はここで報告されます。この接着フィルムは、レーザー活性化され、縫合糸の使用を必要としません。

レーザー組織修復にローズベンガル-キトサン膜の作製と応用

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

fabrication-application-rose-bengal-chitosan-films-laser-tissue

Research

JoVE Journal

Bioengineering

12.1K ビュー.  University of Western Sydney, NSW Australia. 縫合糸は通常、外科手術の際に組織を修復するために必要とされている。彼らは侵襲的であり、組織に損傷を与える可能性があるしかし、そのアプリケーションが問題になることがあります。小説組織接着剤の製造、およびアプリケーションの方法はここで報告されます。この接着フィルムは、レーザー活性化され、縫合糸の使用を必要としません。

ビデオ: Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心筋組織工学のための生物由来注射用材料の作製

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

生物工学

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2Dと3D環境での神経前駆細胞の電界制御監督移行

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

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

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

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

電気刺激のマウス筋芽細胞前駆細胞およびアプリケーションからの工学骨格筋組織

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

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次元細胞培養のための自己申告足場

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

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

三次元インクジェット印刷技術を用いて、ヒト軟骨組織の作製

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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.

三層のための3つのユニークな環境を提供するために、エレクトロスピニング法を適応させます<em&gt;インビトロ</em&gt;気道壁のモデル

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

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

圧縮されたコラーゲンで細分化した組織：シングル段再建修復のための細胞含有Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

血管由来のマイクロ流体ネットワークの画像誘導、レーザーベースの試作

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

マクロを含む光の組織ファントムの作製と評価

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

血管可視化のための二重ラスター走査光音響小動物イメージャー

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...