この作品は、国立衛生研究所（DBCにHL068915）、（CAP）のスラッシャー研究基金からの新しい研究者賞、補助金、米国心臓協会（DBCへ12GRNT11910008）からの研究助成金によってサポートされていました（DBC）は、ボストン小児病院の心臓伝導基金、ライアンファミリー基金への寄付、そしてデビッド·プルマンによる子供の心臓財団からの研究助成金。

ここでは、子羊の皮からCOL1の分離のデモンストレーションを行います。書かれたように、このプロトコルはまた、正常ウサギおよびヒトの皮膚からCOL1を単離するために使用することができる。 1。皮膚のサンプルを準備します<ol><li>使用前に4℃にすべての試薬を平衡化させます。 </li><li>氷のように冷たいのdH 2 O中で真皮サンプル（25〜g）をすすぎ、脱毛クリームを持つウール、毛皮、または髪を削除します。 </li><li>結合組織および脂肪のサンプルクリーンをこすりするために単一のエッジかみそりの刃を使用してください。 </li><li>氷のように冷たいのdH 2 Oのサンプルをすすぐ</li><li>シングルエッジかみそりの刃で1センチメートル×1センチの部分に皮膚サンプルをスライス。 </li></ol> 2。非コラーゲン可溶化物質を除去<ol><li>サンプル50ミリリットルあたりのコニカルチューブの5グラムを秤量し、氷冷した0.5 M酢酸ナトリウム30ミリリットルを追加します。 </li><li>ベンチトップホモジナイザー中で50mlのチューブアダプタを使用して6m /秒の設定で1分間チューブを混合する。 </li><li> SUPを破棄7酢酸ナトリウムの洗浄サイクルの合計ernatant繰り返し。 </li><li>氷のように冷たいのdH 2 Oの中に試料をすすぎ、残留酢酸ナトリウムを除去するために、一度ミックス。 </li><li>過剰な液体を除去した後、新しい50mlコニカルチューブに転送するためにチューブの側面に対してサンプルを圧縮するヘラを使用しています。 </li></ol> 3。私コラーゲンの抽出を入力<ol><li>試料を圧縮し、各洗浄後に上清を捨てベンチトップホモジナイザーで6m /秒で1分間ミリリットル/ gの2倍のサンプルを洗浄0.075 Mクエン酸ナトリウム緩衝液。 </li><li>サンプルに0.075 Mクエン酸ナトリウム緩衝液の新鮮な2ミリリットル/ gのアリコートを追加します。 </li><li>各サイクルの間にバッファを削除せずに6連続した1分、攪拌の6メートル/秒のベンチトップホモジナイザーミックスサイクルを実行します。 </li><li>コレクションチューブに厚く、透明な上澄みを転送します。 </li><li>サンプルにミリリットル/ g、さらに1 0.075 Mクエン酸ナトリウム緩衝液を加え、1の最終ベンチトップホモジナイザー胸焼けを行うクル。 </li><li>コレクションチューブにこの最終上清を追加します。 </li><li>遠心機で4℃で10分間、3,200×gで回収チューブ</li><li>分子量100,000の最上部のコンパートメントに上清を移し、遠心フィルター装置を遮断。 </li><li> 4℃で30分間3200×gで遠心分離</li><li> 4℃のクリーンコニカルチューブおよび格納するために上部コンパートメントから精製されたCOL1を転送</li></ol>

<ol>
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著者らは、開示することなく、競合する金融利害関係はありません。

この方法の重要な要素は、過剰な液体を除去し、皮膚のサンプルを繰り返し効率的な圧縮です。これは、試料を圧縮することによって、ステップ2.5においてできるだけ酢酸ナトリウムを除去することが重要である。我々は、チューブの側面に対して皮膚を圧縮するためにスパチュラを使用しているが、チーズクロスを使用することもできる、これがチューブからのサンプルの除去を必要とし?...

何十年もの間、研究者や商業ベンダーが使用することができる組織化されたCOL1原線維のマトリックスの再懸濁、その結果中​​和し、単純な酸抽出プロトコルのいくつかのバリエーションを使用して皮膚や腱などの組織源の品揃えから可溶化COL1を単離した生物医学的応用1-4多数のため。 col1の臨床応用の多くの例がありますが、このタンパク質の調製は5-7を実行するために数日から数週間を取って長々抽出を必要とするので、これらのいくつかは自家由来COL1を採用しています。その結果、研究調査官や医師は、一般的には、ラット、ウシ、またはブタ真皮などの非ヒト組織を用いたか、死体から摘出皮膚を使用したり、男性の割礼に従って製造されたCOL1の高価な、市販製剤を使用しています。再生アプリケーション、固体形成する能力に関して、これらの製品の多くの展示可変性マトリックスまたは細胞の付着および成長を支持することが可能な組織構築物。そのため、すぐにアクセス可能で、豊富な自家組織源からCOL1を抽出するために設計された新しい標準化された方法のための明確な必要性がある。 そのような方法は、COL1は、関心のある動物モデルから抽出したコラーゲンベースの足場上で組み立て設計組織の前臨床試験を使用することができる研究環境で、時間とコストの両方を節約することになる。同時に、診療​​所で急速に分離された、自家由来COL1を使用するオプションを有する他の方法で同種または異種の準備を受け取ることになる患者のための全体的な安全性を増加させるであろう。自家調製を受けている患者の場合、この方法は非常に合理化された生検収集およびコラーゲンアプリケーションとの間の間隔を減少させるであろう。これらの理由から、我々は、高速AGを使用して、長い確立COL1の単離および精製方法を改善しようとしたitationおよびサイズ​​排除遠心分離。この方法は、多くの研究室で見られる標準バッファーと装置を使用して実行するのは簡単です。高速攪拌を用いることにより、我々のプロトコルは3未満〜8時間、約10日から可溶性、皮膚COL1を単離するために必要な時間を短縮する。重要なことに、この方法は、容易な手順の単一のセット中の患者に使用するための自​​家由来するCOL1を調製するために臨床設定で行うことができる。

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			<td height="43" style="height:43px;width:123px;">FastPrep-24 System</td>
			<td style="width:136px;">MP Biomedicals</td>
			<td style="width:147px;">116004500</td>
			<td style="width:139px;">-</td>
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			<td height="190" style="height:190px;width:123px;">Centrifuge</td>
			<td style="width:136px;">Beckman</td>
			<td style="width:147px;">J6-MI</td>
			<td style="width:139px;">Swing bucket rotor to accommodate 50 mL conical tubes at 3200 g.</td>
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an improved method for the preparation of type i collagen from skin

水溶性タイプ1コラーゲン（COL1）は、細胞培養用の接着基質として、再生アプリケーションのための携帯足場として広く使用されています。臨床的に、このタンパク質は、広く美容外科、皮膚注射、骨移植および再建手術のために使用される。これらの手順のcol1のソースは、炎症や拒絶反応の可能性だけでなく、生体異物疾病伝播を増加させる、一般的にヒト以外である。そこで、効率的かつ迅速に自家由来組織の限られた量からCOL1を精製する方法は、これらの問題の多くを回避するであろうが、標準の単離プロトコルが長いであり、多くの場合、コラーゲン様組織を大量に必要とする。ここでは、約10日から約3時間後に、このタンパク質を単離および精製するために必要な時間を短縮し、効率的なCOL1抽出方法を示す。我々は、多くの外科的処置の際に、その利用可能性の我々の組織源として真皮を選びました。 T彼の方法は、真皮、少量から高純度、溶解性のCOL1を得るための強力な撹拌及び遠心濾過と組み合わせた伝統的な抽出バッファを使用しています。簡潔には、皮膚生検、脂肪、結合組織、毛髪を除去した後、氷冷のdH 2 Oで十分に洗浄する。皮膚サンプルを0.5 M酢酸ナトリウムおよび高速ベンチトップホモジナイザーを用いて非コラーゲン性タンパク質及び多糖類から取り除かれる。残留固形物由来のコラーゲンは、その後、ホモジナイザーを用いて0.075 Mクエン酸ナトリウム緩衝液で抽出する。これらの抽出物は、100,000 MWカットオフ市販品または従来の手順を用いて得られたものと同等以上の品質のCOL1製剤を得る遠心フィルタを使用して精製される。我々は、この方法では、研究と臨床応用の多数のための自家由来COL1の利用を容易にするであろうと予想している。

このCOL1単離プロトコールは、完了するのに約2時間と15分が必要です。図1は、この手順の主なステップを概説する概略図を示す。試料を調製し、高速攪拌回転の7サイクルを実行し、酢酸ナトリウムでリンスすると、約35分（図1A-C）をとる。 1の攪拌を実施するとDH 2 Oですすぎサイクルは約5分（図1Dおよび1E）を取ります。クエン酸ナトリウムを加?...

Watch this Scientific Journal Video about An Improved Method for the Preparation of Type I Collagen From Skin at JoVE.com

An Improved Method for the Preparation of Type I Collagen From Skin

水溶性タイプ1コラーゲン（COL1）を単離するための伝統的な手続きが原因に長いバッファインキュベーションおよびフィブリルの面倒な再懸濁の開始から終了まで約10日を要する。ここでは、3時間未満の小さな皮膚生検からCOL1を精製するための手段を記載する。

皮膚からのI型コラーゲンの調製のための改良された方法

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

an-improved-method-for-the-preparation-of-type-i-collagen-from-skin

Research

JoVE Journal

Bioengineering

22.7K ビュー.  Boston Children's Hospital. 水溶性タイプ1コラーゲン（COL1）を単離するための伝統的な手続きが原因に長いバッファインキュベーションおよびフィブリルの面倒な再懸濁の開始から終了まで約10日を要する。ここでは、3時間未満の小さな皮膚生検からCOL1を精製するための手段を記載する。 

ビデオ: An Improved Method for the Preparation of Type I Collagen From Skin

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

三次元ヒト皮膚は、モデルを再構築：ノーマルスキンとメラノーマ進行を勉強するツールを

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

生物工学

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官型コラーゲンIアッセイ：3次元コンテキストでのセルの挙動を評価するための可鍛性プラットフォーム

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

幹細胞にロードされたキトサンミクロスフェアの配信のためにコラーゲンハイドロゲルの構築

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

ナノサイズの脂質小胞の調製のための一定の圧力制御押出法

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

人間の細胞に基づく、血液脳関門の接触モデルの作製のための改善された方法

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

変性コラーゲンストランドを画像化する<em&gt;インビボ</em&gt;と<em&gt;生体外</em&gt;リテーナ入りコラーゲン模倣ペプチド写真·トリガブリダイゼーション経由

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

骨 - インプラントアンカレッジ評価するために改良された機械的試験方法

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

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. ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

圧縮されたコラーゲンで細分化した組織：シングル段再建修復のための細胞含有Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

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 ...