私たちは、無傷のブタの肺組織を供与するためのスミスフィールドファームを感謝したいと思います。我々はまた、私たちは、自社の機器を使用できるようにするための博士胡ヤン博士クリスティーナ唐とVCU形成外科部門に感謝したいと思います。ヒドロゲルおよび組織サンプルは、資金調達のフォームNIH-NCIがんセンターサポート助成金により、一部には、NIH-NINDSセンターコアグラント5 P30 NS047463からの資金提供により、部分的には、サポートされている解剖学と神経生物学顕微鏡施設のVCU科でSEMのために調製しましたP30のCA016059。 VCUナノテクノロジーコアキャラ施設（NCC）でのサンプルのSEM画像。この作品は、全米科学財団、CMMI 1351162によって賄われていました。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 溶液 </td><td> 無菌フィルター </td><td> 行き方 </td></tr><tr><td> DIH 2 O </td><td>はい</td><td> DIH 2 O;滅菌濾過</td></tr><tr><td> 0.1％トリトンX-100溶液</td><td>はい</td><td>ヒュームフードの下でDIH 2 O 100 mlに100μlのトリトンX 100ソリューションを追加し、溶解するまで撹拌します。 滅菌フィルター。 </td></tr><tr><td> 2％デオキシコール酸溶液</td><td>はい</td><td>ヒュームフードの下で2グラムデオキシコール酸ナトリウム溶液を加え 100ミリリットルDIH 2 Oあたり、溶解するまで撹拌します。無菌フィルター</td></tr><tr><td> 1MのNaCl </td><td>はい</td><td> DIH 2 Oの1 Lに58.44グラムのNaClを追加します。溶解するまで撹拌します。 無菌フィルター</td></tr><tr><td> DNase溶液</td><td>はい</td><td> 12,000を追加1 L DIH 2 O単位のDNアーゼ; 0.156グラムのMgSO 4を追加します。 （無水）、および0.222グラムのCaCl 2;溶解するまで撹拌します。無菌フィルター</td></tr><tr><td> PBS </td><td>はい</td><td> 、27.2グラムのNa 2 HPO 4・7H 2 O（二塩基性七水和物）を組み合わせ 80グラムのNaCl、および10 L DIH 2 Oで2グラムのKCl;溶解するまで撹拌します。 pHを7.4に調整します。無菌フィルター</td></tr></tbody></table> 表1： 組織の脱細胞化のために必要なソリューション。脱細胞化プロセスのための上記の解決策を行います。 4℃で保存。約2.5 Lは1ブタの肺のために必要とされるであろう。 1.ヒドロゲル形成<ol><li> ブタ肺脱細胞化（参考文献9,10から適応） <ol><li>心とVASCで、屠殺場から、 一括通常のブタの肺を入手そのままulature。 </li><li>心臓にできるだけ近い血管系を切断し、心を離れて分析する組織ハサミやメスを使用し、残りの血管系は、灌流のために、後の工程で使用されます。 </li><li>慎重にメスやハサミを使用して、気管、気管支、および血管系を周囲の結合組織を離れて解剖。 注：脱細胞化プロセスの有効性を妨げることが胸膜および無気肺または血管閉塞、大量の大きな切り傷や刺し傷なしで肺を選択することで、残りの手順のために使用するのに最適な肺を決定します。 </li><li> （廃棄することができる心、結合組織および削除肺葉）できるだけ気管に取り付けられた同じくらい気管支を残し次善の肺を切り取ります。気管や血管系に付着したまま1肺があるはずです。 注：必要であれば、組織学のためのセクションを保持します。 9 </li><li> Pにクランプまたは縫合糸で切断された気管支を閉じますrevent過剰逆流。 </li></ol><ol start="6"><li> （ 表1）を 、必要になるまで4℃で脱細胞化ソリューションをして冷蔵庫を準備します。 </li><li>ハンドポンプを使用して、肺動脈および気管の両方を介してDI水で肺組織を3回灌流、肺動脈に適合するようにカニューレ。最初のたびに血管系を灌流。血管系に約1 Lで始まり、1.5 L各灌流のための気管内に、しかし、細胞破片を除去されるより多くの液体は、システムに灌流することができます。 </li><li>トリトンX-100溶液で血管系および気管の両方を灌流。 </li><li> 4℃で24時間、トリトンX-100溶液中に肺組織を浸します。 </li><li>リンスするためにDI水で血管系および気管3回灌流。 </li><li>デオキシコール酸溶液を用いて血管系および気管の両方を灌流。 </li><li> 4℃で24時間、デオキシコール酸で組織切片を水没。 </li><li>リンスするためにDI水で血管系および気管3回灌流。 </li><li>NaCl溶液で血管系および気管の両方を灌流。 </li><li> 4℃で1時間濾過NaCl溶液中で組織を浸します。 </li><li>リンスするためにDI水で血管系および気管3回灌流。 </li><li> DNase溶液で血管系および気管の両方を灌流。 </li><li> 4°Cで1時間フィルタリングDNase溶液中で組織を水没。 </li><li>血管系および気管PBSで5回の両方を灌流。 </li><li>唯一の呼吸ゾーン（主に周辺領域）を残して、気道を行ってから顕著な軟骨組織、気管および（主にへそと肺の内側部分の周りに見られる）、直径が全て尿細管2ミリメートル以上を離れて解剖。 </li><li> 1インチのセクション以下に組織を解剖。組織の向きは、このステップのために重要ではありません。 </li><li>過剰の液体を捨て、50mlのコニカルチューブに組織を配置します。 -80℃で組織を凍結します。 注：細胞の除去を確実にするために、組織学のためのセクションを保持し、携帯debrisの、所望の場合。我々は、H＆E、αガラクトシダーゼ、ピコグリーン、ヒドロキシプロリン、ニンヒドリン、アルシアンブルー、SDS-PAGE、および特徴付けする質量分析法を使用しています。 9 </li></ol></li><li> 肺処理 <ol><li>脱細胞化肺組織を含む凍結したチューブから蓋を外します。 </li><li>チューブ開口部の上にろ紙を置き、ゴムバンドで固定します。 注：チューブおよび内容はまだ凍結するまでそれ以外の場合は-80℃に置く凍結保存してください。 </li><li>すべての過剰な液体は、メーカーの指示に従って凍結乾燥機を使用して、なくなるまで組織を凍結乾燥。ミルへの準備ができるまで-80℃で保管してください。 </li><li>労働条件に絶縁し、内部コンポーネントを冷却するためにフリーザーミルに液体窒素を追加し始める前に。 </li><li>ミルチューブから磁気ミルバーを取り外し、底部をカバーするために組織を追加します。 </li><li>ミルバーを元に戻し、ゆるく充填された組織を追加します。ミルバーはまだ自由に移動する必要があります。 </li><li>ミルチューブを閉じ、ナンプラーフリーザーミルでCE。 </li><li>最大充填ラインに液体窒素を充填します。 </li><li>フリーザーミル微粉末に全ての組織（約5分〜600分の衝突速度-1）、ステンレス鋼のインパクターを有するポリカーボネートシリンダーだけでなく、製造業者の指示に従ってフリーザーミルを用いてステンレス鋼製の端栓インチ使用直前まで-80℃で保管してください。 </li></ol></li><li> ミクロ多孔質ゲルの形成（8 mg / mlで）（参考文献から適応9,11） <ol><li> （w / v）のために、室温で、一定の攪拌（液体のトップレベルでの流れを見ることができるはずです）の下で、M HClを0.01にペプシンの（w / v）の脱細胞化粉末と0.1％の1％を追加します。 48時間。 注：粉末は静的に帯電したので、粉末がチューブ壁から過剰を洗浄するための機会を提供した後、塩酸を加えています。 </li><li> 48時間消化した後、氷上で5分間溶液および試薬を配置します。 </li><li>冷蔵10％を使用して（v / v）の0.1 MのNaOH、D 11.11パーセント（v / v）の10倍PBS（1倍濃度の溶液全体をもたらすために）、7.4の生理学的pHに消化されたタンパク質溶液をもたらします。 注：溶液が今までに1週間4℃で保存することができます。必要であればレオ9を使用してゲル化速度を実行します。 </li></ol></li></ol> 2.機械的強度を改善するためにヒドロゲルを架橋<ol><li> 1％、0.1％、および0.01％W / Vゲニピン架橋溶液の溶液を、10％DMSO中にゲニピン粉末の必要量を溶解することによって調製しました。 </li><li> 1時間ごとに溶液を15分間ボルテックス。 </li><li>以前部1.3.4に組み立てられたECMヒドロゲル（96ウェルプレートの各ウェルに100μL）を覆うゲニピン溶液を加えます。 </li><li> 37℃で24時間架橋するために、各ECMハイドロゲルのままにしておきます。 </li><li>洗浄液がもはや青になるまで、ECMハイドロゲルをPBSで3回リンスしません。架橋ヒドロゲルは、さらなる特性評価およびCEの準備ができているでしょうLL培養研究。 </li></ol>微多孔ジェル3.細胞培養<ol><li> 二次元のコーティング <ol><li> 1.3.3からの溶液を用いて、96ウェル非組織培養処理プレートの各ウェルにプレゲル溶液20μlを加えます。 </li><li>タンパク質吸着板できるように一晩4℃で冷蔵します。 </li><li>吸引しプレゲル溶液を、PBSですすいでください。 </li><li>継代細胞9。 </li><li>ウェルに10,000細胞/ cm 2で追加します。 </li><li>ウェルあたり100μlにメディアを増やして、37℃でインキュベートします。 </li></ol></li><li> 三次元細胞培養9 <ol><li> 1.3.3からの溶液を用いて、プレゲル溶液1,000,000細胞/ mlの濃度になるようにペレット化した細胞を再懸濁します。 </li><li>迅速な3D細胞培養〜500μmの厚さのヒドロゲルを形成するために、96ウェルプレートの各ウェルにプレゲル細胞懸濁液の16μLを分注します。 </li><li> 30、37℃でインキュベートゲル分間が形成されます。 </li><li>形成されたヒドロゲルの上部にメディアの100μlを添加して、37℃でインキュベートします。 </li></ol></li><li> 可変剛性ゲル上で三次元細胞培養 <ol><li> 96ウェルプレートの各ウェルにピペット、1.3.3からプレゲル溶液100μlの溶液を用いて。 </li><li>ゲルを形成するために37℃で30分間インキュベートします。 注：パート2からの架橋の手順は、ここで使用することができます。 </li><li>継代細胞9。 </li><li>ウェルに10,000細胞/ cm 2で追加します。 </li><li>ウェルあたり100μlにメディアを増やして、37℃でインキュベートします。 </li></ol></li></ol>

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著者らは、開示することは何もありません。

生物学の不可欠な側面の一つは、特定のタスクを実行する階層構造への分子の自己組織です。ラボで13は 、自己組織化は、このような塩濃度、pH、および消化時間など多くの要因に依存します。示されるように、自己組織化ハイドロゲルの形態で可溶化タンパク質は、生理的温度に戻るとき。形成されたヒドロゲルは、in vitroでの細胞付着及び増殖を促進することができます。 ...

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6. 
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research. 
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

<table><tbody><tr><td>Triton X-100&amp;nbsp;</td><td>Fisher Scientific</td><td>BP151-100</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma-Aldrich</td><td>D6750-100g</td><td>Use in hood with eye protection and gloves.</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506-500g</td><td>None</td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C1016-500g</td><td>None</td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-150KU</td><td>None</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>258148-500ML</td><td>Use with eye protection and gloves.</td></tr><tr><td>Pepsin</td><td>Sigma-Aldrich</td><td>P6887-5G</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>BP359-500</td><td>Use with eye protection and gloves.</td></tr><tr><td>Genipin</td><td>Wako Chemicals</td><td>078-03021</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>PBS 10x</td><td>Quality Biological</td><td>119-069-151</td><td>None</td></tr><tr><td>PBS</td><td>VWR</td><td>45000-448</td><td>None</td></tr><tr><td>Filter Paper</td><td>Whatman</td><td>8519</td><td>N/A</td></tr><tr><td>Hand pump</td><td>Fisher Scientific</td><td>10-239-1</td><td>N/A</td></tr><tr><td>Graduate Beaker</td><td>VitLab</td><td>445941</td><td>N/A</td></tr><tr><td>Cryomill</td><td>SPEX</td><td>6700</td><td>Use cryogloves and eye protection.</td></tr><tr><td>Lyophilizer</td><td>FTS FlexiDry</td><td></td><td>Use gloves.</td></tr><tr><td>Rheometer</td><td>Discovery</td><td>HR-2</td><td>Use gloves and eye protection.</td></tr></tbody></table>

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

ここでは、in vitroでの肺細胞培養のための複数の成分の細胞培養ヒドロゲルを確立するための方法を提示します。ブタ、ラット、またはマウスからのブロックの肺組織エン健康に始まり、組織は、灌流し、細胞破片を除去するために、後続の化学洗剤に浸漬されます。処理前と後の組織の組織学的比較は、二本鎖DNAとアルファガラクトシダーゼ染色の95％以上の除去を確認する細胞破片の大部分が除去される示唆しています。脱細胞化した後、組織を凍結乾燥し、次いで粉末にcryomilled。マトリックス粉末は、酸性ペプシン消化液中で48時間消化し、次いでプレゲル溶液を形成するために中和されます。プレゲル溶液のゲル化は、37℃でインキュベートすることによって誘導することができ、すぐに次の中和を使用するか、または2週間まで4℃で保存することができます。コーティングは、cのための非処理プレートにプレゲル溶液を用いて形成することができますエルアタッチメント。細胞は、細胞が足場を通って移動する、又はコーティング上に播種することができ、そこから形成されたゲルの表面にメッキ3D培養を達成するために自己集合の前プレゲルに懸濁することができます。提示戦略の変更は、ゲル化温度、強度、またはタンパク質断片の大きさに影響を与えることができます。ヒドロゲル形成超え、ヒドロゲルの剛性がゲニピンを用いて増加させることができます。

この方法を用いて、通常のブタ、ラット、およびマウスの肺（ 図1）からヒドロゲルを生産しています。処理された肺はそれぞれ5ミリグラム、40ミリグラム、およびECM粉末10gを推定与えます。プロセスの概要を図2に示されています。プロセス中の主な視覚化が含まれます：デオキシコール酸をすすいだ後、肺の白い外観を。プレゲル形成した...

Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

これは、肺の細胞外マトリックスからの3次元細胞培養の足場を作成するための方法です。無傷の肺は、三次元での細胞の増殖を支持することができるヒドロゲルに加工されます。

3D細胞培養のための肺細胞外マトリックスから調整可能なヒドロゲル

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

tunable-hydrogels-from-pulmonary-extracellular-matrix-for-3d-cell

Research

JoVE Journal

Bioengineering

11.6K ビュー.  Virginia Commonwealth University. これは、肺の細胞外マトリックスからの3次元細胞培養の足場を作成するための方法です。無傷の肺は、三次元での細胞の増殖を支持することができるヒドロゲルに加工されます。 

ビデオ: Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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

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

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

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

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 ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting細胞化は、組織特異的ヒドロゲルBioinkを使用して構築します

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

注射の再生療法と高スループットの薬剤のスクリーニングのための 3 D への放射能

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

デュアルのバイオミメティック基底膜として極薄 Porated 弾性ゲルの細胞培養

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

カプセル化と 3 D の細胞の免疫染色のエラスチン様タンパク質ゲルの生産

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

ライブ顕微鏡イメージング下での3Dヒドロゲルの制御株

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

ハイドロゲルアレイにより、3D腫瘍モデルにおけるマトリックス成分および治療薬の効果をスクリーニングするためのスループットの向上が可能

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

ECM硬化に対する細胞応答をプローブするためのフォトチューナブルバイオプリントハイドロゲル

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

著者スポットライト:3Dプリントされたフォトチューナブルハイドロゲルを使用した慢性肺疾患の理解 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

線維芽細胞の活性化を研究するための3Dバイオプリンティング用フォトチューナブルハイドロゲル

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...