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Bioengineering

長距離繊維配向によるマイクロエンジニアリング3Dコラーゲンヒドロゲル

Published: September 7th, 2022

DOI:

10.3791/64457

1Department of Biomedical Engineering, Kate Gleason College of Engineering, Rochester Institute of Technology

このプロトコルは、流体の流れ方向に沿って形状を変化させるマイクロ流体チャネルを使用して、3Dコラーゲンヒドロゲル(厚さ<250 μm)内の繊維を整列させるための伸長ひずみ(伸張)を生成する方法を示します。結果として得られるアライメントは数ミリメートルにまたがり、伸張ひずみ速度の影響を受けます。

整列コラーゲンI(COL1)繊維は、腫瘍細胞の運動性を導き、内皮細胞の形態に影響を与え、幹細胞の分化を制御し、心臓および筋骨格組織の特徴です。 in vitroで整列した微小環境に対する細胞応答を研究するために、磁気的、機械的、細胞ベース、およびマイクロ流体法を含む、定義されたファイバー整列を有するCOL1マトリックスを生成するためのいくつかのプロトコルが開発されている。これらのうち、マイクロ流体アプローチは、流体の流れや細胞の微小環境を正確に制御するなどの高度な機能を提供します。しかし、高度な in vitro 培養プラットフォーム用に整列したCOL1マトリックスを生成するためのマイクロ流体アプローチは、500 μm未満の距離に伸びるCOL1繊維の薄い「マット」(厚さ<40 μm)に限定されており、3D細胞培養アプリケーションに役立ちません。ここでは、マイクロ流体デバイス内で定義されたファイバーアライメントのミリメートルスケール領域を持つ3D COL1マトリックス(厚さ130〜250 μm)を作製するためのプロトコルを紹介します。このプラットフォームは、細胞培養用のマイクロエンジニアリングマトリックスへの直接アクセスを提供することにより、構造化された組織の微小環境をモデル化するための高度な細胞培養機能を提供します。

細胞は細胞外マトリックス(ECM)と呼ばれる複雑な3D線維ネットワークに存在し、その大部分は構造タンパク質コラーゲンI型(COL1)1,2で構成されています。ECMの生物物理学的特性は細胞にガイダンスの手がかりを提供し、それに応じて、細胞はECMマイクロアーキテクチャを再構築します3、4、5。これらの相互細胞-マトリックス相互作用は、腫瘍環境7,8,9における血管新生および細胞浸潤を促進し、細胞形態10,11,12、分極13、および分化14に影響を与える整列したCOL1ファイバ....

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1. 2ピースチャンネルとモジュラープラットフォームベースの製作

注:マイクロ流体チャネルは、定義された厚さのポリジメチルシロキサン(PDMS)シートからかみそりでカットされたマイクロ流体チャネル「カットアウト」と、カットアウトに可逆的に結合してチャネルを形成するチャネルカバーの2つの部分で構成されています。チャネルは、媒体リザーバーと?.......

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自己組織化COL1溶液が断面積の減少を伴うチャネルを流れると、COL1溶液の流れ方向の速度(v x)は、2つのセグメント間のくびれの長さ(∂x)に沿って局所的に大きさ∂v x増加し、ε̇=∂v x/∂xの伸長ひずみ速度(ε̇)になります。伸張ひずみ速度は、図2に示すように、粒子画像速度測定(PIV)を使用して測定される流体速度から計算できます。

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整列したファイバーでCOL1マトリックスを生成するためのプロトコルは、磁気的方法、機械的ひずみの直接適用、およびマイクロ流体技術を使用して説明されています47。マイクロ流体アプローチは、生化学的微小環境を正確に制御できる、明確に定義された流れと輸送特性により、マイクロ生理学的システムを作成するために一般的に使用されます。整列したCOL1ファイバー.......

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この研究の一部は、国立衛生研究所(授与番号R21GM143658)および国立科学財団(助成金番号2150798)によって支援されました。内容は著者の責任であり、必ずしも資金提供機関の公式見解を表すものではありません。

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NameCompanyCatalog NumberComments
(3-Aminopropyl)triethoxysilane, 99% (APTES)Sigma Aldrich440140-100ML
20 Gauge IT Series Angled Dispensing TipJensen GlobalJG-20-1.0-90
3/16" dia. x 1/16" thick Nickel Plated MagnetKJ MagneticsD31
3M (TC) 12X12-6-467MPDigiKey3M9726-ND
ACETONE ACS REAGENT ≥99.5%Signa Aldrich179124-4L
BD-20AC LABORATORY CORONA TREATERElectro-Technic Products12051A
Bovine Serum Albumin (BSA), Fraction V, 98%, Reagent Grade, Alfa AesarVWRAAJ64100-09
Clear cast acrylic sheetMcMaster-Carr8560K181
Corning 100 mL Trypsin 10x, 2.5% Trypsin in HBSS [-] calcium, magnesium, phenol red, Porcine Parvovirus TestedVWR45000-666
Countess II Automated Cell CounterThermo Fisher ScientificAMQAX1000
CT-FIRE softwareLOCI - University of Wisconsin
EGM-2 Endothelial Cell Growth Medium-2 BulletKit, (CC-3156 & CC-4176), Lonza CC-3162, 500 mLLonzaCC-3162
Glutaraldehyde 50% in aqueous solution, Reagent Grade, Packaging=HDPE Bottle, Size=100 mLVWRVWRV0875-100ML
Graphtec CELITE-50GraphtecCE LITE-50
HEPES (1 M)Thermo Fisher Scientific15-630-080
High-Purity Silicone Rubber .010" Thick, 6" X 8" Sheet, 55A DurometerMcMaster-Carr87315K62
Human Umbilical Vein Endothelial cellsThermo Fisher ScientificC0035C
Invitrogen Trypan Blue Stain (0.4%)Thermo Fisher ScientificT10282
IsopropanolFisher ScientificA4154
Laser cutterFull Spectrum20x12 H-series
Microfluidics Syringe pumpNew Era Syringe PumpsNE-1002X
Microman E Single Channel Pipettor, Gilson, Model M1000EGilsonFD10006
Molecular Probes Alexa Fluor 488 PhalloidinThermo Fisher ScientificA12379
Molecular Probes Hoechst 33342, Trihydrochloride, TrihydrateThermo Fisher ScientificH3570
Nutragen Bovine Atelo CollagenAdvanced BioMatrix5010-50ML
Pbs (10x), pH 7.4VWR70011044.00
PBS pH 7.4Thermo Fisher Scientific10010049.00
Phosphate-buffered saline (PBS, 10x), with Triton X-100Alfa AesarJ63521
Replacement carrier sheet for graphtec craft ROBO CC330L-20USCUTTERGRPCARSHTN
Restek Norm-Ject Plastic Syringe 1 mL Luer SlipRestek22766.00
Silicon waferUniversity wafer452
Sodium Hydroxide, ACS, Packaging=Poly Bottle, Size=500 gVWRBDH9292-500G
Sylgard 184VWR102092-312
Thermo Scientific Pierce 20x PBS Tween 20Thermo Fisher Scientific28352.00

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