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
Summary
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。
Abstract
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。
Introduction
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.
Protocol
Representative Results
Discussion
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。 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Triton X-100 | Fisher Scientific | BP151-100 | Use in fume hood with eye protection and gloves. |
| Sodium Deoxycholate | Sigma-Aldrich | D6750-100g | Use with eye protection and gloves. |
| Magnesium Sulfate | Sigma-Aldrich | M7506-500g | None |
| Calcium Chloride | Sigma-Aldrich | C1016-500g | None |
| DNase | Sigma-Aldrich | D5025-150KU | None |
| HCl | Sigma-Aldrich | 258148-500ML | Use with eye protection and gloves. |
| Pepsin | Sigma-Aldrich | P6887-5G | Use in fume hood with eye protection and gloves. |
| Sodium Hydroxide | Fisher Scientific | BP359-500 | Use with eye protection and gloves. |
| Genipin | Wako Chemicals | 078-03021 | Use in fume hood with eye protection and gloves. |
| PBS 10x | Quality Biological | 119-069-151 | None |
| PBS | VWR | 45000-448 | None |
| Filter Paper | Whatman | 8519 | N/A |
| Hand pump | Fisher Scientific | 10-239-1 | N/A |
| Graduate Beaker | VitLab | 445941 | N/A |
| Cryomill | SPEX | 6700 | Use cryogloves and eye protection. |
| Lyophilizer | FTS FlexiDry | Use gloves. | |
| Rheometer | Discovery | HR-2 | Use gloves and eye protection. |
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