This work was funded by a New Faculty Award II from the California Institute of Regenerative Medicine (CIRM; RN2-00921-1), NIH-funded National Research Award (F32-HL104924), and CIRM Training Grant (TG21163). Materials were provided by: Glycosan Biosystems Inc / BioTime and Dr. Stephen Badylak (University of Pittsburgh)

PNIPAAMコーティングされたプレートの1。作成<ol><li> 60％トルエン/ 40％ヘキサン溶液2ml中のPNIPAAmを2.6g溶解する。 </li><li> PNIPAAMが溶解するまで、撹拌を10分間60℃に混合物を加熱する。 </li><li>ブフナー漏斗中の直径60mmの円と場所紙にろ紙をカット。 </li><li>予め秤量したガラスビーカーにブフナー漏斗を通して溶液を濾過（ヘキサンは、プラスチックを溶かすように、プラスチックを使用しないでください）​​。 </li><li>ベル真空（24 psi）でO / N（16時間）にビーカーと内容を配置します。注：残留物をイソプロピルと反応させるまでは、それが酸素と接触しないことを確認しますので、酸化する。 </li><li> PNIPAAMの重量を確立するためにビーカーを秤量する。 </li><li> w / w溶液50:50の作成、のPNIPAAmにイソプロピルアルコールを追加します。 </li><li> UV光の下で組織培養プレートの表面に溶液2mlを置き、5分間コート。 </li><li>二回温PBS 2mlでプレートを洗浄し細胞培養に使用する前に。 </li></ol>細胞シートの2。作成注：標的器官の一次細胞の細胞シートは、多数の異なる方法を使用して作成することができ、または、ここで説明したように、熱応答性ポリマーを用いて組織培養表面をコーティングすることにより。プリコーティングされた感熱プレートはまた、多くのベンダーによって提供されています。 注：このプロトコルは、35mmディッシュを用いて培養するためのものである。簡単に述べると、細胞をまず、隣接セル間の横方向の接続を確立するためにコンフルエンスで24時間、最低37℃でインキュベートする。細胞シートを解放するために、プレートは32°C以下の温度にさらされる。細胞シートは、その後、血管内皮細胞と血管新生許容ヒドロゲルを含む強塩基繊維マトリックスに転送される。 <ol><li>細胞集団を分離します。注：この方法は、個別の導出手順や細胞の種類に依存している。ラットの大動脈平滑ムーSCLE細胞（RASMC）は、この例で使用されている。これらは、ラットの腹部大動脈から単離した初代平滑筋細胞である。 </li><li>温かい2mlのPBSで細胞を洗浄する。 </li><li> 5分間、細胞にトリプシン（または他の切断/解離溶液）3mlのを追加します。 </li><li> 10％ウシ胎児血清（FBS）を含む培地、またはリン酸緩衝溶液（PBS）を3mlの添加によりトリプシンを阻害する。 </li><li>円錐管中の細胞を収集し、アリコートを数える。 </li><li> 5分間1000rpmで（228×g）で細胞をスピン。 </li><li>上清を吸引し、その増殖培地中で細胞を再懸濁（SmGM2プラス弾丸キット培養液をRASMCに使用されます）。 </li><li> 100％コンフルエンスを達成する濃度でPNIPAAM塗装板 - 35mmの感熱プレート上の細胞を含む培地を配置します。注：のRASMCの場合その数は/ cm 2の 100,000個の細胞であると決定された。しかし、通過中の細胞の喪失に起因し、そのバージニア州の120％<html> LUEが使用されます。 </li><li> 37℃のCO / Nにおけるインキュベーターに置きます。注意：プレートへの細胞接着を維持するために、37℃で細胞を維持することが重要である。 </li></ol>ファウンマトリックスの調製注：さまざまな3D繊維状マトリックスは、繊細な細胞シートとの間に強い繊維状マトリックスの層に使用することができる。いくつかの例には、これらの研究に使用ゲルフォーム、バイオガラス、天然の無細胞化材料26またはnanospun材料27,28ブタ膀胱マトリックス（UBM）は寛大に私たちの共同研究者、博士Badylakから提供された29。 <ol><li>使用前に、脱細胞化マトリックスが使用される場合、細胞内容物の欠如を含むマトリックスの特性を決定し、27,28、細胞特異的生存率、および空隙22 </li><li>所望のサイズおよび形状に予め滅菌行列をカット。注：ここでは、ホールパンチが4mmの直径の円を切断するために使用される。 </li></ol>ve_title &quot;&gt; 4。新生血管許容的ヒドロゲルに内皮細胞を播種注：内皮細胞は幹細胞または前駆細胞からの分化を含む多様な供給源から得ることができる。ここで、HUVECを使用している。 <ol><li>架橋時間がある限り、任意の許容性ヒドロゲル（フィブリン、コラーゲンゲル）を使用して、細胞が生存留まるように十分に短い。注：ここでは、ジスルフィド架橋で架橋されたヒアルロン酸（HA）ベースのゲルが使用される。 </li><li>会社のプロトコルに従って、HAハイドロゲルを準備します。 </li><li>内皮細胞を回収し、1×トリプシンを用いて、単一細胞溶液中に分散。注：のAccutaseまたは細胞解離緩衝液はまた、単一の細胞分散のために使用することができる。 </li><li> 15ミリリットルの円錐チューブに溶液/細胞を回収、PBSでFBS（これは、細胞が血清に接触しないことが重要である場合）、大豆トリプシン阻害剤の等量を用いて、トリプシン酵素を失活または10％。 </li><li> C言語細胞をount、パッチ寸法（以前に定量化）するために必要な体積を計算する。注：4mmのパッチについては、こちらを200万内皮細胞が使用される。 </li><li> 200万細胞を抽出し、新たな15ミリリットルコニカルチューブに入れる。 </li><li> 5分間（228×g）でスピン。 </li><li>コニカルチューブ中のペレットとして細胞を残し、上澄み液を吸引。 </li><li> 1比：1でHAとゼラチン液体材料を混ぜる。その後、ペレットを含むコニカルチューブに全体積の80％を追加します。 </li><li> 1 HA /ゼラチン混合物：1の内皮細胞を再懸濁</li><li>ステップ2からベースの繊維状マトリックスにHA /ゼラチン混合物中に懸濁した細胞を配置します。 </li><li>架橋剤の所望の総体積の5分の1（20％）を追加</li><li> 37℃で1時間インキュベートする。 </li></ol>細胞シートの5の単離<ol><li>で細胞培養フード内インキュベーターと場所からの細胞を含む35ミリメートルPNIPAAM処理プレートを外しRT。 </li><li>すぐに細胞から培地を吸引し、37℃に加熱された6％の通常のゼラチンの2ミリリットルを追加します。 </li><li>ゼラチンはまだ暖かいですが、通常のゼラチン（ムービー1）の表面下に沈め、ゼラチン中に金属格子を配置します。 </li><li>ゼラチンを硬化させること、5〜7分間氷の上にプレート全体を置きます。 </li><li> 7分後、慎重にプレートの側からゼラチンエッジを分離するために、スパチュラを使用し、その後、プレート（注）から金属格子持ち上げるために鉗子を使用します。6％のゼラチンを、そして細胞シートは、格子を持ち上げる必要があります。 </li><li>皿に細胞シートを移動し、慎重に構造物の上に格子を設定し、ベース繊維マトリックスヒドロゲルの組み合わせの上に置きます。注：細胞シートの頂端側は、まだトップの位置になります。 </li><li>温かいメディア（37℃）の2ミリリットルを追加します。 </li><li> O / Nがヒドロゲル表面に付着した細胞のシートを可能にインキュベートする。 </li><li> Tを削除する解決策は（約1時間）、または翌日を温めた後、彼は金属格子。 </li></ol>

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We have nothing to disclose.

プロトコルにおける重要なステップは次のとおりです。温度応答性高分子の板表面を被覆し、プレートを冷却した後、細胞シートを操作する。異なるセルは、異なる物理的特性を示すので、接着性のような、昇降時間は、各異なる細胞型について最適化されるべきである。このプロトコルの第2、及び最も重要な挑戦的な成分は、細胞シート、組織アセンブリのための方法の重要な態様での操...

Injection of cells and/or single materials alone has shown variable success in other organ systems and limited success in cardiac regeneration.5,7-12 Currently, stem cell-derived cells are delivered to damaged tissue using a variety of delivery methods including: direct cell injection into tissue and perfusion into the blood supply.13-17 Others have implanted cells alone, materials alone and/or in combination with material carriers to help regenerate damaged organs.18-21 This design combines multiple strategies that provide material strength, patterning in multiple materials and multiple cell types. 
Specifically, the base acellularized fibrous matrix provides the foundational physical strength to the construct, making it suitable for suturing in into the patient, if necessary. The void spaces in the base matrix are filled with endothelial cells in a neovascular permissive hydrogel22 for rapidly establishing vascularization of the implanted construct. This composite is then integrated with pre-patterned cell sheets that allow enhanced cell-to-cell communication, more closely mimic the native tissue.1,23-25 The overall production process for the layered cellular patch is outlined by the flowchart in Figure 1.

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:207px;">Table of Reagents:</td>
			<td style="width:128px;"></td>
			<td style="width:140px;"></td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Reagent</td>
			<td style="width:128px;">Company</td>
			<td style="width:140px;">Catalogue number</td>
			<td style="width:221px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Calcein-AM</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:140px;">C3099</td>
			<td style="width:221px;">Cell tracker / live dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Lysotracker Red</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:140px;">L7528</td>
			<td style="width:221px;">Cell tracker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Neutral Red</td>
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tissue engineering: construction of a multicellular 3d scaffold for the delivery of layered cell sheets

そのような大人の人間の心のような多くの組織は、十分に損傷後に再生することができません。組織工学における2,3の戦略は、回復と修復に体を支援するために技術革新を提案する。例えば、TEアプローチは、心筋梗塞（MI）後の心臓リモデリングを減弱、おそらくほぼ正常プレMIレベルに総心臓機能を高めることができるかもしれません。4、心臓組織の正常な再生がの適切な送達を含む任意の機能的な組織と同様に移植された細胞/組織移植片の統合と生存に有利な環境合図で複数の細胞タイプ。送達ビヒクル、細胞の生存に及ぼす影響、材料強度、及び細胞 - 組織組織の促進として評価可溶性シグナル、細胞間相互作用、およびマトリックス材料：操作された組織が含む複数のパラメータに対処すべきである。のみこれらの必須要素を無視し、移植片細胞の直接注入を利用する研究。2,5,6これらの成分を組み合わせた組織の設計は、まだ開発されなければならない。ここでは、「組織」における新たな血管の形成を増強するための標的臓器の細胞型および内皮細胞を含む生物学的由来の材料の2つの異なる種類のパターン化された細胞シートの積層を使用して統合された設計の例を提示する。これらの研究は、心臓のような組織の発生に焦点を当てているが、この組織の設計は、最小限の設計および材料の変更と心臓以外の多くの臓器に適用することができ、再生治療のための既製品であることを意味する。プロトコルは5詳細な手順が含まれています。温度感受性ポリ（N -isopropylacrylamide）（PNIPAAM）が被覆組織培養皿に使用される。その後、組織特異的細胞は強い横癒着による細胞シートを形成するためにコーティングされたプレート/マイクロパターン表面の表面上で培養される。第三に、ベースマトリックスは、血管新生permissiを多孔性マトリックスを組み合わせることにより、組織のために作成されヒドロゲルおよび内皮ve細胞。最後に、細胞シートは、完全な構築物を作るPNIPAAM被覆皿から持ち上げ、ベース要素に転送される。

フロー図（ 図1）は 、多層パッチを製造する全体的な方法を示している。細胞シートは、32℃以下の温度をドロップすることによってPNIPAAM処理プレートから剥がれた。その後、細胞シートは、下層繊維性マトリックス（ 図1）に播種した内皮細胞を含有する架橋されたヒドロゲルの上に配置される。前処理された感熱プレートは、細胞シートを作成するために使用す?...

Watch this Scientific Journal Video about Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets at JoVE.com

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

組織工学：層状細胞シートの送達のための多細胞3D足場の構築

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

tissue-engineering-construction-multicellular-3d-scaffold-for

Research

JoVE Journal

Bioengineering

14.3K ビュー.  University of California, Merced. For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

ビデオ: Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

組織工学のための二軸メカニカルローディングバイオリアクターの設計

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

生物工学

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

人間の3次元の組織工学<em&gt; in vitroで</em&gt;腫瘍テストシステム

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3次元細胞培養のための自己申告足場

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

三層のための3つのユニークな環境を提供するために、エレクトロスピニング法を適応させます<em&gt;インビトロ</em&gt;気道壁のモデル

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

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 ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

肝組織工学のための三次元細胞培養プラットフォーム：逆コロイド結晶ポリ（エチレングリコール）足場の作製

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

組織特異的細胞培養および配信プラットフォームとして、細胞外マトリックス由来のフォームおよびマイクロキャリアの作製

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

細胞播種用カスタム Agarose 井戸とティッシュ リング自己組織型の 3 D プリントを使用しての作製

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

ニューラル様組織の工学的なマウス皮質アストロサイトの3Dバイオプリンティング

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...