This work was supported by NIH-NIDCR IRO1DE019355 (MJ Yost, PI), and NSF-EPSCoR (EPS-0903795).

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著者らは、開示することは何もありません。

Biofabricationは、生物学と工学の原則は、天然組織を模倣する複雑な材料を生成するためにマージする高度に学際的分野です。これを達成するために、in vivoでの組織から収集した情報を使用して、 インビトロ足場に翻訳技術を開発する必要があります。この方法では、プラットフォームは、密接にin vivoでの組織の、建築の機能的、および機械的特性に似ていることを操作?...

組織工学の分野では、組織スキャフォールドを製造する能力が不可欠です。適切な組織足場は、3次元構造を有する、生体適合性材料から構成されており、 生体内組織アーキテクチャの模倣は、細胞および組織の増殖及びリモデリングを容易にします。この足場は、栄養素の輸送や廃棄物1-4の除去を可能にしなければなりません。これらの足場の製造における主な障害の一つは、生体適合性材料に特有の幾何学的特徴を再現する能力です。いくつかのbiofabrication技術がこれらの足場の幾何学的特徴を制御するために報告されており、例としては、溶媒キャスティング9、造形10 5-8エレクトロスピニングされ、とりわけ、11 3Dは、印刷します。これらの技術は、制御可能な内部および外部の建築の特徴を比較的容易に転送を提供するには及ばない高価であり、（彼らの解像度と印刷適性によって制限されています<em&gt;例えば、ノズルゲージ、材料の制限）、または実行可能な足場12を生成するために長時間を必要後の製造技術を必要とします。 多くの商業的な製造システムでは、内部空隙、チャネル、および機能の作成は、砂または他の適切な取り外し可能または犠牲材料を使用して達成されます。金属又はプラスチック部品は、砂型の周囲に形成され、そしてそれが固化した後、砂を除去します。ほぼ同じ方法で、生体材料の次世代biosand相当を必要とします。したがって、BSAゴムはbiosandの代替品として開発されました。 BSAゴムはグルタルアルデヒドで架橋されたウシ血清アルブミンから成る新たに処方材料です。究極の目標は、生分解性コラーゲン足場に固有のアーキテクチャ機能を再作成することです。元の組織の型と次元の忠実度を維持し、犠牲biorubberの特性が記載されています。 <p clお尻= &quot;jove_content&quot;&gt; BSA及びグルタルアルデヒド濃度のいくつかの組み合わせは、様々な溶媒を用いて試験しました。この材料は、BSAとグルタルアルデヒドとの反応によって作成されました。 BSAゴムは、組織の型の複雑な幾何学的形状に注入反応することができます。架橋されたBSAは、不安定なトリプシンと容易に穏やかなpHおよび温度条件で酵素によって消化されます。逆に、Iコラーゲン無傷のタイプは、トリプシン消化に非常に耐性です。これらの機能は、選択の背後にあるコラーゲンを残すBSAゴムを除去するために資産計上されました。本研究は、生体適合性足場に固有のアーキテクチャ機能を提供することができます不安定な金型を得るために必要な理想的なパラメータを決定から成っていました。評価された特定の特徴は、混合性、酵素消化、耐荷重、及び負の型に注入反応される能力が含まれていました。 30％BSA及び3％のグルタルアルデヒドとの組み合わせは、これらの要件を満たします。このプロトコルは、necessを提供aryのガイドラインは、これらの3次元足場を作成します。プロトタイプは1流入し、それぞれ4および3-ミリメートルの直径を持つ2つの流出路を有する分岐アーキテクチャを表すコラーゲン足場から構成されています。この技術は、対象の組織のマクロおよびミクロ環境を模倣する可能性を有します。この技術は 、生体内の組織の弾力性と関心の組織の他の特性を模倣するように調整することができる高忠実度、比較的容易かつタイムリーな問題で生分解性材料に固有の幾何学的な有益を提供するために実行可能な技術を提供します。

<table border="0" cellpadding="0" cellspacing="0" width="770">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Collagen type I</td>
			<td style="width:156px;"></td>
			<td style="width:116px;"></td>
			<td style="width:217px;">Collagen extracted from calf hide</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrocloric Acid (HCl)</td>
			<td>Sigma-Aldrich</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffer Solution (PBS Tablets)</td>
			<td>MP Biomedical</td>
			<td>U5378</td>
			<td>1 tablet per 100 mL makes 1XPBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Albumium from bovine serum (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glutaraldehyde</td>
			<td>Sigma -Aldrich</td>
			<td>G5882</td>
			<td>Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lard</td>
			<td>Fields</td>
			<td>3090</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless Steel Molds</td>
			<td></td>
			<td></td>
			<td>Milled using Microlution Machine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Air Brush Kit</td>
			<td>Central Pneumatic</td>
			<td>47791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mixing Tip for double syringe</td>
			<td>Medmix</td>
			<td>ML2.5-16-LLM</td>
			<td>Mixer, DN2,5X16, 4:1 brown, med</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Small O ring for double syringe</td>
			<td>Medmix</td>
			<td>PPB-X05-04-02SM</td>
			<td>Piston B, 5mL, 4:1, PE natural</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double Syringe cap&#160;</td>
			<td>Medmix</td>
			<td>VLX002-SM</td>
			<td>Cap, 4:1/10:1, PE brown, med</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Big O ring for double syringe</td>
			<td>Medmix</td>
			<td>PPA-X05-04-02SM</td>
			<td>Piston A, 5 mL, 4:1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double Syringe&#160;</td>
			<td>Medmix</td>
			<td>SDL X05-04-50M</td>
			<td>Double syringe, 5 mL, 4:1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double Syringe Dispenser</td>
			<td>Medmix</td>
			<td>DL05-0400M</td>
			<td>Dispenser, 5 mL, 4:1, med , plain</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminim</td>
			<td></td>
			<td></td>
			<td>3.6 mg/mL- extracted USC lab</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20 mL Syringe Luer Lock Tip</td>
			<td>BD</td>
			<td>302830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luer Lock Caps</td>
			<td>Fisher</td>
			<td>JGTCBLLX</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma -Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gibco Minimum Essential Media 10X (MEM)</td>
			<td>&#160;Life Technologies</td>
			<td>1143-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Life Technologies</td>
			<td>27250-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV Crosslinker&#160;</td>
			<td>Spectroline UV</td>
			<td>XLE1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Cloride (NaCl)</td>
			<td>Fisher</td>
			<td>S271-10</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride (KCl)</td>
			<td>Sigma -Aldrich</td>
			<td>P5405-250</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Bicarbonate (NaHCO3)</td>
			<td>Fisher</td>
			<td>BP328-500</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma -Aldrich</td>
			<td>G-8270</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Phosphate didasic (NaH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S-7907</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Filter for syringes</td>
			<td>Corning</td>
			<td>431224</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional biomimetic technology: novel biorubber creates defined micro- and macro-scale architectures in collagen hydrogels

組織足場は、組織再生の過程において重要な役割を果たしています。理想的な足場は、そのような適切な組成を有するなど、いくつかの要件、対象と弾性率、および明確に定義されたアーキテクチャ機能を満たす必要があります。 in vivoでの組織の本質的なアーキテクチャを再現生体材料は、疾患を研究するだけでなく、失われ、不正な形式の軟組織の再生を促進するために不可欠です。新規biofabrication技術は、研究および臨床用途のための生体材料の新世代を作成する技術の画像、3次元（3D）印刷法、選択的酵素活性の状態を組み合わせた開発されました。開発した材料、ウシ血清アルブミンゴムは、特定の幾何学的特徴を掲げ型に注入反応です。この犠牲材料は、天然の足場材料への建築の特徴を十分に転送できます。プロトタイプは4と3ミリメートルチャンネルのreprで3Dコラーゲン足場で構成されてい分岐構造をESENT。本論文では、自然の構築物の生成のためのこのbiofabrication技術の使用を強調しています。このプロトコルは、足場材料内にその建築の特徴を残して、ゴムの酵素消化に続いて、BSAゴムを注入し、反応になる固体の金型を製造するためのコンピュータ支援ソフトウェア（CAD）を利用します。

結果は、このbiofabrication技術は、インビボで組織に見られる空間的配置を模倣することができる三次元足場を生成するのに効率的であることを示しています。建築の特徴は、組織の in vivoでの細胞の相互作用と機能性において重要な役割を果たしている組織工学アプリケーションのための重要なパラメータです。 <p class="jove_content" fo:keep-together.within-page...

Watch this Scientific Journal Video about Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels at JoVE.com

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三次元バイオミメティック・テクノロジー：小説Biorubberはコラーゲンハイドロゲルで定義されたマイクロ・マクロスケールのアーキテクチャを作成します。

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

three-dimensional-biomimetic-technology-novel-biorubber-creates

Research

JoVE Journal

Bioengineering

9.1K ビュー.  Medical University of South Carolina. An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

ビデオ: Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

コラーゲンゲル含有する細胞のモジュールを使用してマイクロ組織の作製

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

生物工学

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三次元光学分解能光音響顕微鏡

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巣と卵管の三次元器官培養用アルギン酸ゲル

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

導電性インクの平面および三次元印刷

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

はSiO：フォトリソグラフィによって画定されたパリレン-C上の細胞のパターニング<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

三次元インクジェット印刷技術を用いて、ヒト軟骨組織の作製

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

三次元コラーゲンマトリックス内の細胞移動の解析

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三次元の全層皮膚等価物と自動傷つけ処理の発生

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

組織工学的応用の制御可能な形態の三次元 Poly(ε-caprolactone) 足場のエレクトロスピニング記述を溶かす

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

デュアルのバイオミメティック基底膜として極薄 Porated 弾性ゲルの細胞培養

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...