著者に感謝 t. パーマーと H. バブー (スタンフォード大学脳神経外科)図4 マウスの Npc。 ベクター アートを提供するために使用され、クリエイティブ ・ コモンズ表示 3.0 Unported ライセンス (https://creativecommons.org/ の下で仏医術から適応licenses/by/3.0/legalcode)。この作業の一環で、スタンフォード大学ナノ共有施設 (SNSF)、賞 ECCS 1542152 下の国立科学財団によってサポートを行った。N.A.S. は、サポートから国立科学研究所の一般医療の健康の国民の協会 (32 GM 008412) を認めています。C.M.M. は、博士フェローシップ (F31 EB020502) と Siebel 学者プログラムの NIH NRSA からサポートを認めています。S.C.H. では、再生医療 (RT3-07948)、国立衛生研究所 (U19 AI116484 と R21 EB018407)、全米科学財団 (DMR 1508006)、およびカリフォルニア工科大学からサポートを認めています。この研究は、ユーニス · ケネディ · シュライバー国立衛生研究所の子と人間の開発 (NICHD)、国立研究所によってサポートされている再生のリハビリテーション研究・ トレーニング (AR3T)、連合から資金を受けてください。神経疾患と脳卒中 (NINDS)、医用イメージング、生体 (NIBIB) 賞を受賞番号 P2CHD086843 の下で健康の国立研究所の国立研究所。内容は著者の責任と健康の国民の協会の見解を必ずしも表さない。

<ol>
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著者が明らかに何もありません。

組換え蛋白質の表現および浄化は、再現性の高い生体材料を合成する強力なツールです。商品化された分子クローニングの出現を主因カスタム プラスミド購入できますいくつかのサプライヤーから ELPs のような材料で作業時間を大幅に削減します。同様に、元の仕事は、連邦契約によって支えられた非営利使用するため今後の作業になりますとプラスミドを元の研究室から直接要求できます...

過去世紀にわたって基礎的な細胞生物学の研究のための不可欠なツールセットに二次元 (2 D) 組織文化を開発しました。さらに、2 D の細胞培養のため比較的低コストでシンプルなプロトコルは、多くの生物的および医学の分野での採用につながっています。しかし、過去の研究を示しています伝統的な 2 D のプラットフォームは、それら収集した体内から著しく逸脱、原因の貴重な時間と資金を無駄に臨床的に方向づけられた研究1,2のための結果につながること 3。我々 と他のこの不一致は最適な増殖と種々 の細胞の成熟に必要なことができる 2D サーフェス上で培養した細胞に提供されるネイティブ生化学的および生物物理学的手がかりの不足に帰因するかもしれないことを仮定します。
これらの制限および助け橋 2 D の間のギャップに対処するための in vitroとin vivoの研究、研究者がある電池封止材1,4,5 の開発した三次元 (3 D) ゲル プラットフォーム ,6。ヒドロゲルは細胞外マトリックス (ECM) の生体内での組織のような機械的性質と栄養素の急速な輸送を可能にする構造の水膨潤により内因性の微小環境を要約する理想的な材料と7,8の要因シグナリングします。さらに、足場の力学的および生化学的特性を独立に制御する 3 D ゲルを設計できます。行列力学9,10、11,12と細胞接着配位子13,14,15の両方がよくセルに影響を与える知られています。生体外で動作し、体内。したがって、可変プロパティと 3 D ゲルは、セルとその微小環境の因果関係を研究するためのプラットフォームを提供しています。理想的な 3 D ゲルのマトリックスのための基準は、単純な非細胞電池封止材、生理学的に関連する機械的性質の独立した可変性およびネイティブな細胞接着モチーフの模倣者に含まれます。
両方合成 (e.g、ポリエチレング リコール、ポリ乳酸、ポリ (グリコール酸)) および自然由来 (e.g。、アルギン酸、コラーゲン、マトリゲル) ヒドロゲル文化プラットフォーム 2D体外に利点があります。しかし、また適用性を限る重大な欠点があります。まず、合成と天然物由来の多くのプラットフォームが必要な哺乳類細胞に潜在的に有毒することができます過酷な架橋条件につながる減少した細胞の生存率の7。また、多くの合成プラットフォームはネイティブ活性を欠いている、増加コストと複雑さの16を追加することができますセカンダリの化学反応によって修飾する必要があります。最後に、天然由来原料は通常本質的な生体活性ドメインを含みますが、しばしば高いバッチごとに変動に悩まされているため、比較的弱いゲル7,17を形成に制限が多い。
組換えタンパク質工学タンパク質シーケンスを明示的に制御により材料設計のためのユニークなツールセットを提示し、延長によって、最終的なハイドロゲルの潜在的な力学的および生化学的特性足場18。また、エシェリヒア属大腸菌(E. 大腸菌) 蛋白質を表現するためのよく知られている生物学的機械を活用し、材料生産できる低コストで一貫して限られたインター-内-バッチ可変。ここで紹介するエラスチン様タンパク質 (ELP) は 3 つのエンジニア リング ドメイン: 抗体のタグを介して蛍光ラベリングを可能に (1) の T7 および His6 タグ、弾性力学特性を付与し、化学のエラスチンのような地域 (2)架橋、(3) 細胞接着モチーフのエンコード 'バイオ アクティブ' 地域。
エラスチンのような地域は、'効きます' アミノ酸サイト イソロイシン (イル) がある可能性がありますの 4 つはプロリンを除くすべてのアミノ酸に設計されている標準 (ヴァル Pro Gly 効きます-ペプチド-)5エラスチン シーケンスに基づいています。このシーケンスは、熱循環19,20を介して簡単な浄化後式の利用できる下の臨界溶液温度 (LCST) 行動と遺伝子組換え ELPs を与えます。この LCST プロパティは、異なる温度で熱骨材へゲスト '効きます' 残渣21,22を変更することでチューニングできます。
ここでは、5 つのエラスチンのような繰り返しの 1 つに「効きます」位置はリシン (Lys) アミノ酸のアミン提示酸架橋ハイドロゲルを利用されるに置き換えられていますいます。我々 の以前の仕事は、アミン反応性架橋剤テトラキス (ヒドロキシメチル) ホスホニウム塩 (THPC)23の反応特異性と堅牢な架橋を示しています。さまざまな全体蛋白質内容と架橋剤濃度による、生理学的に関連する剛性の範囲 (~0.5-50 kPa)9,23,24にまたがることチューニングできるゲルを生成することができるがおります。機械的特性をチューニングに加えて ELP 蛋白質のバックボーン内の標準的な細胞接着ドメインの統合から、ハイドロゲル内細胞接着の結果します。たとえば、非バインド 'RDGS' バリアント拡張フィブロネクチン由来 'RGDS' アミノ酸シーケンスの組み込むことで柔軟な形態、スクランブル中の細胞接着と細胞-マトリックス接着24が制限されます。総蛋白濃度と同様に、非接着タンパク質に細胞接着の比率を調節することによって効果的に配位子濃度の広い範囲でゲルを生成することができるがおります。その結果、種々 の細胞の最適な 3 D 文化のため個別にチューニングできる分離された生化学的および生物物理プロパティを持つハイドロゲル プラットフォームを開発しました。
剛性マトリックスと接着性リガンドの可変性に加え組換えヒドロゲルは細胞拡散、増殖、および4の 3 D のコンテキスト内での移行に必要なデザイン特定の材料劣化プロファイル機能を提供します。,9. この劣化は拡張 'RGDS'9またはエラスチンのようなシーケンス25を標的プロテアーゼの細胞分泌によって与えられます。ELP ヒドロゲルは、細胞生存率及び量的な逆の DNA ・ RNA ・ タンパク質抽出と同様に、免疫細胞化学を含む関数を勉強するために必要なその後の生化学的アッセイをサポートに示されています。トランスクリプション ポリメラーゼの連鎖反応 (qRT PCR) と西9 をしみ。ELP の亜種も、生体内でのモデルの数で使用されているし、免疫システム26忍容性が知られています。
一緒に取られて、ELP 電池封止材研究材料プラットフォームを誇るさまざまな生化学的および生物物理可変性のと同じ程度に欠ける、合成や天然物由来の素材プラットフォームと比較しての利点として、再現性。さらに、ELP のシンプルで非細胞毒性を使用さまざまな種類の細胞 (e.gひよこ後根神経節14,24、マウス神経前駆細胞9、ヒト間葉系幹細胞27、牛。新生児軟骨細胞28、ひと血管内皮細胞細胞29,30) 2 D 細胞培養と比較して内因性 3 D ECM のもっと生理学的に関連するモデルのことができます。ここで、recombinantly 由来の表現のためのプロトコルを提案する、3 d 可変ハイドロゲルのプラットフォームとして使用するための非細胞カプセル化。さらにダウン ストリームの蛍光標識とカプセル化された細胞の共焦点顕微鏡観察の方法論を提案します。

<table><tbody><tr><td>&lt;strong&gt;Elastin-Like Protein Expression and Purification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 cm Petri Dishes</td><td>Thermo Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>70% Ethanol</td><td>RICCA Chemical</td><td>2546.70-1</td><td></td></tr><tr><td>Ammonium Sulfate</td><td>Sigma-Aldrich</td><td>A3920-500G</td><td></td></tr><tr><td>Ampicillin</td><td>Thermo Fisher Scientific</td><td>BP1760-25G</td><td></td></tr><tr><td>Bacto Agar</td><td>Thermo Fisher Scientific</td><td>9002-18-0&amp;nbsp;</td><td></td></tr><tr><td>BL21(DE3)pLysS Competent Cells</td><td>Invitrogen</td><td>C606003</td><td></td></tr><tr><td>Chloramphenicol</td><td>Amresco</td><td>0230-100G&amp;nbsp;</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>EDTA disodium salt, dihydrate</td><td>Thermo Fisher Scientific</td><td>O2793-500</td><td></td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific</td><td>BP229-4</td><td></td></tr><tr><td>Isopropanol</td><td>Thermo Fisher Scientific</td><td>A451-4</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>BP1755-10G</td><td></td></tr><tr><td>Luria Broth</td><td>EMD Millipore</td><td>1.10285.5007</td><td></td></tr><tr><td>Parafilm</td><td>VWR</td><td>52858-000</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>MP Biomedicals</td><td>195381</td><td></td></tr><tr><td>Sodium Chloride</td><td>Thermo Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S 8045-1KG</td><td></td></tr><tr><td>Syringe Filter Unit (0.22 &amp;mu;m)</td><td>Millipore</td><td>SLGP033RB</td><td></td></tr><tr><td>Terrific Broth</td><td>Millipore</td><td>71754-4</td><td></td></tr><tr><td>Tris Base</td><td>Thermo Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>&lt;strong&gt;Cell Encapsulation in 3D ELP Hydrogels&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.22 &amp;mu;m syringe filters</td><td>Millipore</td><td>SLGV004SL</td><td></td></tr><tr><td>0.5 mm thick silicone sheet</td><td>Electron Microscopy Science</td><td>70338-05</td><td></td></tr><tr><td>24-well tissue culture plates&amp;nbsp;</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>Disposable Biopsy Punch (2 mm)</td><td>Integra Miltex</td><td>33-31</td><td></td></tr><tr><td>Disposable Biopsy Punch (4 mm)</td><td>Integra Miltex</td><td>33-34</td><td></td></tr><tr><td>Disposable Biopsy Punch (5 mm)</td><td>Integra Miltex</td><td>33-35</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (DPBS)&amp;nbsp;</td><td>Corning</td><td>21-031-CM</td><td></td></tr><tr><td>No. 1 12 mm glass coverslips</td><td>Thermo Fisher Scientific</td><td>12-545-80</td><td></td></tr><tr><td>Tetrakis(hydroxymethyl)phosphonium chloride (THPC)</td><td>Sigma-Aldrich</td><td>404861-100ML</td><td></td></tr><tr><td>0.5% Tryspin/EDTA</td><td>Thermo Fisher&amp;nbsp;</td><td>15400054</td><td></td></tr><tr><td>&lt;strong&gt;Immunocytochemistry of Cells in 3D ELP Hydrogels&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>16% (w/v) Paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15701</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Roche</td><td>3116956001</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Molecular Probes</td><td>D1306&amp;nbsp;</td><td></td></tr><tr><td>Donkey Serum</td><td>Lampire Biological Labs</td><td>7332100</td><td></td></tr><tr><td>Goat anti-mouse Secondary Antibody (AF488)</td><td>Molecular Probes</td><td>A-11017</td><td></td></tr><tr><td>Goat anti-rabbit Secondary Antibody (AF546)</td><td>Molecular Probes</td><td>A-11071</td><td></td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>16210-072</td><td></td></tr><tr><td>Mouse Nestin Primary Antibody</td><td>BD Pharmingen</td><td>556309</td><td></td></tr><tr><td>Mouse Sox2 Primary Antibody</td><td>Cell Signaling Technology</td><td>23064S</td><td></td></tr><tr><td>Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-100ML</td><td></td></tr><tr><td>Vectashield Hardset Mounting Medium&amp;nbsp;</td><td>Vector Labs</td><td>H-1400&amp;nbsp;</td><td></td></tr></tbody></table>

production of elastin-like protein hydrogels for encapsulation and immunostaining of cells in 3d

二次元 (2 D) 組織培養技術は、基礎的な細胞生物学の私達の理解に不可欠されています。ただし、従来の 2次元培養システムに欠けて結果の間で重要な結果として、三次元 (3 D) マトリックス切断収集の in vitroおよびin vivo。この制限に対処するため、研究者は体内細胞微小環境の生化学的および生物物理学的特性を模倣することができます 3 D のハイドロゲル培養プラットフォームを設計しました。この研究、3 D の細胞のカプセル化と下流の生化学的アッセイをサポートする材料のプラットフォームを開発する必要が動機となった。組換えタンパク質工学 3 D ゲル材料の設計と開発のユニークなツールセットを提供は、蛋白質シーケンスの特定のコントロールを可能にするあり、拡張子、合力の潜在的な力学的および生化学的特性行列。ここでは、recombinantly 由来エラスチン様タンパク質 (ELP) 独立可変の機械的性質と細胞接着リガンド濃度フォーム ヒドロゲルを使用ことができますの表現のためのプロトコルを提案する.さらに ELP ヒドロゲル内細胞のカプセル化の方法論を提案して、下流の分析と定量化のための細胞を埋め込まれたの後蛍光抗体染色します。

このプロトコルで使われる ELPs は 5 つの地域から成る: T7 タグ、His6 タグ、enterokinase (EK) 胸の谷間サイト、バイオ アクティブ領域およびエラスチンのような地域 (図 1)。T7 と His6 タグは、標準的な西部のしみの技術によって簡単に識別できます。EK の胸の谷間のサイトの紹介はタグ領域の酵素の除去のため必要な場合ことができます。バイオ ア?...

Watch this Scientific Journal Video about Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D at JoVE.com

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

組換え蛋白質設計ヒドロゲルは、ポリマー主鎖としたがって、細胞の微小環境の完全な可変性を可能にする彼ら 3 D 細胞培養のために有利です。ここでは、組換えのエラスチンのような蛋白質の浄化のプロセスと 3 D ゲル電池封止材への応用について述べる。

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 ...

production-elastin-like-protein-hydrogels-for-encapsulation

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

12.3K ビュー.  Stanford University. 組換え蛋白質設計ヒドロゲルは、ポリマー主鎖としたがって、細胞の微小環境の完全な可変性を可能にする彼ら 3 D 細胞培養のために有利です。ここでは、組換えのエラスチンのような蛋白質の浄化のプロセスと 3 D ゲル電池封止材への応用について述べる。

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

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

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

ステップ成長PEG-ペプチドハイドロゲルからβ細胞のスフェロイドの発生と復旧

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

生物工学

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

キトサンを用いた注射ゲルおよび 3 D 細胞培養への応用の準備

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

化学

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

組織工学のための3Dハイドロゲルの細胞のカプセル化

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

生物学

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D相互作用の研究のための3Dコラーゲンゲルおよびマイクロチャネルの作製<em&gt;インビボ</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

細胞外マトリックス内に埋め込まれたエンジニアリング三次元上皮組織

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

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 ...

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 ...

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

懸濁3Dバイオプリンティングのためのゲル化中のせん断処理によって形成されたアガロース流体ゲル

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...

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 ...