Name: construction of modular hydrogel sheets for micropatterned macro-scaled 3d cellular architecture
Uploaded: 2016-01-11T10:30:00.000+00:00
Duration: 10 min 55 s
Description: Watch this Scientific Journal Video about Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture at JoVE.com

This research was supported by a National Leading Research Laboratory Program (Grant NRF-2013R1A2A1A05006378) through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning. The authors also acknowledge a KAIST Systems Healthcare Program.

マイクロパターン金型及びヒドロゲルの作製<ol><li> PDMSモールドを鋳造するための標準的な2段階のフォトリソグラフィ技術15,17を介して、シリコンウェハの表面上のSU-8フォトレジストを用いて所望のマイクロスケールのパターンを生成します。示された例は、肝小葉様メッシュパターン（ 図1）を使用します 。 </li><li> 1の比でPDMSと硬化剤溶液を秤量：5（ すなわち 、PDMS 12.5gのと硬化剤2.5 g）を得ました。 </li><li>完全に溶液の15グラムを混合し、真空チャンバ内に気泡を脱ガスし、次に均一にフォイル鋳造皿内のシリコンウェーハのマイクロパターン化表面上に混合溶液を広げます。 </li><li> PDMSを硬化させるために、平らな面に90分間65℃で加熱したプレート上にシリコンウェーハを配置します。 </li><li>鋳造皿やシリコンウエハから硬化PDMSを削除します。 </li><li> PDMSの端をカットし、100 mmの直径のペトリ皿の上に置きますマイクロパターンサイドアップ。 </li><li>主な滅菌のために70％エタノールと蒸留水を用いてシャーレに微細化硬化PDMSを洗ってください。その後、65℃のオーブンで10分間完全に乾燥します。 </li><li>コー​​ティング溶液（w / v）の3％を作成し、蒸留水100ml中で粉末化非イオン性界面活性剤のO / N 3グラムを溶解し、混合します。 </li><li>プラズマクリーナーにマイクロパターンPDMS金型を置き、1分（85 W、0.73ミリバール）水性液体の追加を容易にするために、親水性表面を作成するためにそれらをきれいにします。その後、実験室コートロッカーを使用して、少なくとも3時間（ORO / N）を100 mlの界面活性剤溶液とPDMSの表面。 </li><li> PDMS型から界面活性剤溶液を洗浄し、10分間65℃のオーブンで完全に乾燥させます。その後、30分かけて紫外線（UV）放射線に曝露することによって、各微細金型を滅菌します。 </li></ol> 2.ヒドロゲル前駆体中の細胞懸濁液を準備<ol><li>に1％（w / v）のアルギン酸塩前駆体を生成する、ヒドロゲル前駆体を調製リン酸緩衝生理食塩水（PBS）10ml中のアルギン酸ナトリウム粉末を0.1gを溶解します。完全に粉末を溶解するために、O / Nそれらをインキュベートし、混合します。 </li><li> 1ml注射器に接続された0.22μmのフィルターを通して溶液をフィルタリングします。 </li><li>培養10％ウシ胎児血清および1％ペニシリン-ストレプトマイシン、従来の組織培養皿で70％までを補充したダルベッコ改変イーグル培地（DMEM）中でHepG2細胞- 37℃、5％CO 2の加湿インキュベーター中で80％コンフルエンス。 </li><li>一度PBSを用いて細胞を洗浄し、次いで37℃で5％CO 2加湿インキュベーター中で4分間、0.05％トリプシン-EDTAを1ml添加することによって、それらをトリプシン処理。遠心上清を除去した後、1mlのPBSを用いて3分間再懸濁し、250×gで培養皿から細胞を採取しました。 </li><li>自動化された細胞を用いてPBS中で単分散細胞の数を数えますカウンタ。 </li><li> 3分およびPBSを除去するために250×gで遠心分離した後、（w / v）の1％の1 mLを加え、残りの細胞ペレットのアルギン酸ナトリウム溶液を、穏やかにピペッティングを使用して混合します。 10 7細胞/ ml -細胞の最終播種密度は5×10 6でなければなりません。 37℃で5％CO 2の加湿インキュベーター中で、細胞/ヒドロゲルサスペンションをインキュベートします。 </li></ol> 3.ロードとセル/ハイドロサスペンションの架橋<ol><li>塩化カルシウム1.47 gを架橋試薬を生成するためのddH 2 O 100ml中に溶解する脱水（ すなわち 、100ミリモルのCaCl 2・2H 2 O中のddH 2 O）。 </li><li> 70％エタノールを用いた超音波トランスデューサと加湿器の内部をすすぎ、および架橋試薬200mlでそれを埋めます。加湿器は、110ミリメートル幅の広い300ミリメートル、長さ170ミリメートルの深さ（ すなわち 、110ミリメートル×300ミリメートル×170ミリメートル（幅×高さ×奥行））。 </li><li>マイクロパターンPDMSを配置プラズマクリーナーで金型や親水性表面を作成するために、85 Wで1分間のためにそれらをきれいにします。 </li><li>着実に金型内の微細パターンのエッジでよく混合細胞/ヒドロゲル懸濁液の7.2μLをロードします。示された例は、肝小葉様メッシュパターン（ 図1）を使用します 。懸濁液の容量は、地形のパターンに依存します。 </li><li>細胞/ヒドロゲル懸濁液のゲル化を達成するためには、加湿器をオンにし、加湿器は、架橋試薬のミストを生成することを確認します。 5cmの範囲内のPDMSモールドの地形表面を覆う、5分間のヒドロゲル前駆体上に架橋試薬を噴霧。 距離より長く、より短い5センチメートルはそれぞれ、不完全で、不均一なゲル化が生じる可能性があります。注意して​​ください。加湿器は、5分で架橋試薬のミスト20mlのを生産、250ミリリットル/時間の噴霧速度を持っていることを確認してください。 </li><li>架橋工程の後、加湿器の電源をオフにし、PDMS MOを埋めますPBSでLDS。 </li></ol>シングルモジュラーヒドロゲルシートの4取り扱い<ol><li> 200μlのピペットチップを使用して、ハイドロゲルシートの周りに優しくPBSをピペットを介しマイクロパターンを型から各硬化ヒドロゲルシートを外します。 </li><li>エンドカット千μlのピペット先端を使用して、各浮動ハイドロゲルシートをピックアップ。各肝小葉様メッシュハイドロゲルシートを8ミリメートル×8.7ミ​​リメートルの寸法を有し、かつ100である - 厚さ200μm。 </li><li>ハイドロゲルを使用して浮いた状態でのin vitroでの細胞は5週で12ウェルプレート中のユニット部品として構成する12ウェルプレートに1mlのDMEMにハイドロゲルシートの単層を移し、文化37℃での％のCO 2の加湿インキュベーター。一日おきに培地を交換します。 </li></ol> 5の組立多層ヒドロゲルシート<ol><li>繰り返しが18ミリメートル×18ミリメートル×4mmの寸法のPDMSフレームを生成するために1.5から1.1ステップ（幅x下面には170μmの高い柱状構造を含むのHのx D）、および。ステップ1.2と同じ比率で、PDMSと硬化剤の混合物の42グラムを使用してください。 ×9ミリメートル×2ミリメートル（幅×高さ×奥行）8mmの寸法の内部フレームを作成するために、PDMSフレーム用シリコンウェーハ上に特殊なポリカーボネートの金型を置きます。 </li><li> 121℃で15分間オートクレーブに蒸留水とピンセットに沈め、PDMSフレームと180μmの細孔ナイロン濾紙を滅菌します。 </li><li> 60 mmの直径のペトリ皿中（直径5cmの）ナイロンフィルター紙片の四分の一に滅菌PDMSフレームを配置します。 </li><li>エンドカット千μlのピペットチップを使用して、PDMSフレームの内部にモジュラーハイドロゲルシートを転送します。組み立てに使用されるハイドロゲルシートは、それらが作製した後、少なくとも一日のために培養されるべきです。 </li><li>空の200μlのピペットチップを使用して、PDMSフレームと各モジュール式のハイドロゲルシートの端を合わせます。 </li><li> 6層 - 繰り返し4のスタックを形成するために、モジュール式のヒドロゲルシートを使用して、5.4と5.5を繰り返します。 </li><li> PDMSフレームの一番下にある柱構造を介して流出することにより、培養液を除去します。そして、多層構造物の隅にアルギン酸塩溶液2μL（w / vの2％）を追加します。 </li><li>別のものとそれぞれの層の端部を取り付けるために、多層構造体の上に30秒間架橋試薬のミストを噴霧します。 （250ミリリットル/時間の噴霧速度で）架橋剤のミストの2ミリリットルを使用してください。 </li><li> 400μlのDMEMで穏やかに多層構造物をすすぎ、ピンセットを使用して、PDMSのフレームを削除します。 </li><li>優しくDMEMの4ミリリットルを添加した後、細胞リフターで拭いてろ紙から多層構造体を取り外します。 </li><li>ヒドロゲルと同様に構成する、3濾紙を使用して、DMEM mlの培養浮遊様式でインビトロで細胞を含む6ウェルプレートに、多層構築物を転写37℃で5％CO 2の加湿インキュベーター中で一週間以上、6ウェルプレート中のマルチスケールの細胞の足場。一日おきに培地を交換します。 </li></ol>

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The authors have nothing to disclose.

このプロトコルは、モジュラーヒドロゲルシートを作製し、3次元細胞の足場を形成するためにそれらを組み立てる単純な方法を提供します。 短時間で明確なパターン化アルギン酸塩構造を構築するために、我々は金型から複雑な微細パターンを維持するだけでなく、細胞の生存率および代謝を維持するのに十分な剛性構造を作成することができ、架橋プロセスを特定す?...

ヒドロゲルは、特に、生体材料を約束しており、基本的な生物学、薬理学的ア ​​ッセイおよび医学において重要であることが期待される。ヒドロゲル系の細胞構築物の1 Biofabrication動物実験の使用を低減するために提案されている、2,3移植組織4を交換し、改善細胞ベースのアッセイ。5,6-含水（ハイドロ）粘弾性物質（ゲル）は、多数のセルが3D細胞の微小環境を制御するために、骨格構造中にカプセル化され、維持されることを可能にします。微小流体または微細技術のガイダンスと組み合わせて、ヒドロゲル構築物の形状は正確に細胞のスケールで制御することができます。現在までに、粒子、7を含むヒドロゲルの様々な形状、 - 9繊維、10から12とシートは、13から15 は、ボトムアップアプロに構築単位として使用されていますマクロスケール多細胞アーキテクチャの製作に痛みます。 ヒドロゲルをベースとする粒子および繊維の両方は、マイクロ流体デバイスを用いて流体を制御して、マイクロスケール細胞環境のような用途のために容易にかつ迅速に製造されています。しかし、操作された組織の基本単位として、それはそれらを再配置し、マクロスケール構造物としての容積を拡大するために複雑になる。16マイクロメートルサイズの基本モジュールを製造することよりも、これは、マクロスケールの構造体を達成することはより困難です。ヒドロゲルに基づく構築物のシート状のユニットは、簡単な組立工程を介して足場の容積を増加させることができます。結果的に、ヒドロゲルシートの積層は、体積の増加だけでなく、3D空間の幾何学的な拡張だけでなく、を提供します。 マルチレイにそのアセンブリと一緒に15 -我々は以前、13マイクロパターンハイドロゲルシートを製造する方法を報告していますセルラーアーキテクチャをered。技術は、多層構造の積層工程を経て、複雑な微細と細胞構造物のモジュラー設計を可能にします。微細化されている積層型モジュラーヒドロゲルシートの製造を介して、制御マクロスケールの細胞微小環境と3D細胞培養系を実現することができます。このビデオプロトコルは、ヒト肝癌細胞株（HepG2細胞）に基づくモジュラーヒドロゲルシートを構築するために使用することができるシンプルで強力な製造方法について説明します。我々は、多層構造の中に、本明細書にこれらの単純なパターニングモジュラーヒドロゲルシートの操作、及びそれらの組み立てを実証します。

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning Corporation</td><td>000000000001064291</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td>Powdered nonionic surfactant&amp;nbsp;</td></tr><tr><td>Alginic acid sodium salt, low viscosity</td><td>Alfa Aesar</td><td>B25266</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>C7902</td><td></td></tr><tr><td>Ultrasonic humidifier</td><td>MediHeim</td><td>MH-2800</td><td>Modified equipment, Maximum sprayed rate: 250 ml/hr</td></tr><tr><td>Nylon net filter hydrofilic, 180 &amp;mu;m</td><td>EMD Millipore</td><td>NY8H04700</td><td></td></tr><tr><td>Polycarbonate mold</td><td></td><td></td><td>Customized mold for fabrication of a PDMS frame pattern</td></tr></tbody></table>

construction of modular hydrogel sheets for micropatterned macro-scaled 3d cellular architecture

ヒドロゲルは 、インビボ様の三次元（3D）組織形状を提供するために、マイクロ流体や微細化技術を使用してマイクロスケールでパターン化することができます。結果の3Dハイドロゲルベースの​​細胞構築物は、高度な生物学的研究、薬理学的アッセイおよび臓器移植アプリケーションのための動物実験の代替として導入されています。ヒドロゲルをベースとする粒子および繊維は、容易に製造することができるが、組織の再構築のためにそれらを操作することは困難です。この動画では、制御された細胞の微小環境とマクロスケール3D細胞培養系を形成するために、それらのアセンブリと共に、微細アルギネートヒドロゲルシートの製造方法を説明します。 200μmで、かつ正確な微細パターンを有する - カルシウムゲル化剤のミストの形を用いて、薄いハイドロゲルシートは容易に100の範囲の厚さで生成されます。次いで、細胞をヒドロゲルシートの幾何学的な指導を培養することができます自立型条件。また、ヒドロゲルシートは、容易にエンドカットチップでマイクロピペットを使用して操作することができ、パターン化ポリジメチルシロキサン（PDMS）フレームを使用してそれらを積層した多層構造に組み立てることができます。容易なプロセスを用いて製造することができ、これらのモジュラーヒドロゲルシートは、ミクロおよびマクロ構造および組織再構築の機能試験を含むインビトロ薬物アッセイおよび生物学的研究の潜在的な用途を有します。

我々は、製造や携帯ヒドロゲルシートを自立の操作を説明しています。 図1に示すように 、我々は、マイクロパターンPDMS金型を製作し、細胞を含むヒドロゲルは、ゲル化剤のエアロゾル化ミストを生成するための加湿器を使用して、これらの型の親水性表面上にロードされ、架橋されました。金型からの放出後に、HepG2細胞を様々なパターン（ 図2?...

Watch this Scientific Journal Video about Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture at JoVE.com

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

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

マイクロパターンマクロスケール3D細胞構造のためのモジュラーヒドロゲルシートの構築

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

construction-modular-hydrogel-sheets-for-micropatterned-macro-scaled

Research

JoVE Journal

Bioengineering

10.2K ビュー.  Korea Advanced Institute of Science and Technology (KAIST). We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment. 

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

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

動的なマスク投影リソグラフィーを用いたニューラル培養システムのためのマイクロパターンハイドロゲルの作製

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

生物工学

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

細胞培養用ヒドロゲルの蛋白質ベースのアーキテクチャの簡単な操作

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

生化学

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

模様の作成3D細胞培養のための2つの成分​​のために温度応答性ハイドロゲルリバース金型の印刷

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

免疫・感染学

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

細胞を含んだハイドロゲルの刺激細胞挙動のひずみ勾配チップ

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

細胞培養アプリケーションのためのヒドロゲルの光媒介形成とパターニング

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

パターニングタンパク質および細胞の多彩な方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

ハイドロゲルを用いたマイクロ磁気応答性の高いコンポーネントとの容易な迅速な製造のための製造技術の添加剤

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

反復可能なパターン形成のためのハイブリッド ゲル キューブ デバイスで初期の 3 D 細胞クラスター制御

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

ゼラチンメタクリロイルヒドロゲルベースバイオインクの3Dバイオプリンティングのプロトコル

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

ミクロゲルロッドからの連結マクロポーラス3D足場

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

マイクロパターンマクロスケール3D細胞構造のためのモジュラーヒドロゲルシートの構築

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動的なマスク投影リソグラフィーを用いたニューラル培養システムのためのマイクロパターンハイドロゲルの作製

細胞培養用ヒドロゲルの蛋白質ベースのアーキテクチャの簡単な操作

模様の作成3D細胞培養のための2つの成分のために温度応答性ハイドロゲルリバース金型の印刷

細胞を含んだハイドロゲルの刺激細胞挙動のひずみ勾配チップ

細胞培養アプリケーションのためのヒドロゲルの光媒介形成とパターニング

パターニングタンパク質および細胞の多彩な方法

ハイドロゲルを用いたマイクロ磁気応答性の高いコンポーネントとの容易な迅速な製造のための製造技術の添加剤

反復可能なパターン形成のためのハイブリッドゲルキューブデバイスで初期の 3 D 細胞クラスター制御

ゼラチンメタクリロイルヒドロゲルベースバイオインクの3Dバイオプリンティングのプロトコル

ミクロゲルロッドからの連結マクロポーラス3D足場

マイクロパターンマクロスケール3D細胞構造のためのモジュラーヒドロゲルシートの構築

要約

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動的なマスク投影リソグラフィーを用いたニューラル培養システムのためのマイクロパターンハイドロゲルの作製

細胞培養用ヒドロゲルの蛋白質ベースのアーキテクチャの簡単な操作

模様の作成3D細胞培養のための2つの成分​​のために温度応答性ハイドロゲルリバース金型の印刷

細胞を含んだハイドロゲルの刺激細胞挙動のひずみ勾配チップ

細胞培養アプリケーションのためのヒドロゲルの光媒介形成とパターニング

パターニングタンパク質および細胞の多彩な方法

ハイドロゲルを用いたマイクロ磁気応答性の高いコンポーネントとの容易な迅速な製造のための製造技術の添加剤

反復可能なパターン形成のためのハイブリッド ゲル キューブ デバイスで初期の 3 D 細胞クラスター制御

ゼラチンメタクリロイルヒドロゲルベースバイオインクの3Dバイオプリンティングのプロトコル

ミクロゲルロッドからの連結マクロポーラス3D足場

模様の作成3D細胞培養のための2つの成分のために温度応答性ハイドロゲルリバース金型の印刷

反復可能なパターン形成のためのハイブリッドゲルキューブデバイスで初期の 3 D 細胞クラスター制御