This work was supported by the Biomedical Research Council, Singapore through core support to the Institute of Medical Biology and grant SPF2013/005. M.R. was supported by a NUS Faculty Research Committee Grant (Engineering in Medicine) (M.R.) R-397-000-081-112, and the NUS Tissue Engineering Program (NUSTEP). The authors would like to thank Professor Irene Leigh for providing the collagen VII antibody. Electron microscopy work was carried out at the National University of Singapore Electron Microscopy Unit.

<ol>
	<li>Breitkreutz, D., Mirancea, N., Nischt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membranes+in+skin:+unique+structures+with+diverse+functions.">Basement membranes in skin: unique structures with diverse functions.</a> Histochem Cell Biol. 132 (1), 1-10 (2009).</li><li>Lane, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mutation+in+the+conserved+helix+termination+peptide+of+keratin+5+in+hereditary+skin+blistering.">mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering.</a> Nature. 356 (6366), 244-246 (1992).</li><li>Fuchs, E., Raghavan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+under+the+skin+of+epidermal+morphogenesis.">Getting under the skin of epidermal morphogenesis.</a> Nat Rev Genet. 3 (3), 199-209 (2002).</li><li>Niessen, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junctions/adherens+junctions:+basic+structure+and+function.">Tight junctions/adherens junctions: basic structure and function.</a> J Invest Dermatol. 127 (11), 2525-2532 (2007).</li><li>Jarvelainen, H., Sainio, A., Koulu, M., Wight, T. N., Penttinen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+molecules:+potential+targets+in+pharmacotherapy.">Extracellular matrix molecules: potential targets in pharmacotherapy.</a> Pharmacol Rev. 61 (2), 198-223 (2009).</li><li>Schaefer, L., Schaefer, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteoglycans:+from+structural+compounds+to+signaling+molecules.">Proteoglycans: from structural compounds to signaling molecules.</a> Cell Tissue Res. 339 (1), 237-246 (2010).</li><li>Frantz, C., Stewart, K. M., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+at+a+glance.">The extracellular matrix at a glance.</a> J Cell Sci. 123 (Pt 24), 4195-4200 (2010).</li><li>Ghalbzouri, A., Jonkman, M., Dijkman, R., Ponec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membrane+reconstruction+in+human+skin+equivalents+is+regulated+by+fibroblasts+and/or+exogenously+activated+keratinocytes.">Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes.</a> J Invest Dermatol. 124 (1), 79-86 (2005).</li><li>Lareu, R. R., Arsianti, I., Harve, K. S., Peng, Y. X., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Enhancement+of+Collagen+Matrix+Formation+and+Crosslinking+for+Applications+in+Tissue+Engineering--a+Preliminary+Study.">In Vitro Enhancement of Collagen Matrix Formation and Crosslinking for Applications in Tissue Engineering--a Preliminary Study.</a> Tiss Eng. 13 (2), 385-391 (2007).</li><li>Lareu, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+matrix+deposition+is+dramatically+enhanced+in+vitro+when+crowded+with+charged+macromolecules:+the+biological+relevance+of+the+excluded+volume+effect.">Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect.</a> FEBS Lett. 581 (14), 2709-2714 (2007).</li><li>Lareu, R. R., Harve, K. S., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emulating+a+crowded+intracellular+environment+in+vitro.+dramatically+improves+RT-PCR+performance.">Emulating a crowded intracellular environment in vitro. dramatically improves RT-PCR performance.</a> Biophys Biochem Res Comm. 363 (1), 171-177 (2007).</li><li>Harve, K. S., Ramakrishnan, V., Rajagopalan, R., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding+in+vitro+as+means+of+emulating+cellular+interiors:+when+less+might+be+more.">Macromolecular crowding in vitro as means of emulating cellular interiors: when less might be more.</a> Proc Natl Acad USA Sci. 105 (51), E119 (2008).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Scar-in-a-Jar:+Studying+antifibrotic+lead+compounds+from+the+epigenetic+to+extracellular+level+in+a+single+well.">The Scar-in-a-Jar: Studying antifibrotic lead compounds from the epigenetic to extracellular level in a single well.</a> Br J Pharmacol. 158 (5), 1196-1209 (2009).</li><li>Harve, K. S., Lareu, R., Rajagopalan, R., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+how+the+crowded+interior+of+cells+stabilizes+DNA/DNA+and+DNA/RNA+hybrids-in+silico+predictions+and+in+vitro+evidence.">Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids-in silico predictions and in vitro evidence.</a> Nucleic Acids Res. 38 (1), 172-181 (2009).</li><li>Satyam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding+meets+tissue+engineering+by+self-assembly:+A+paradigm+shift+in+regenerative+medicine.">Macromolecular crowding meets tissue engineering by self-assembly: A paradigm shift in regenerative medicine.</a> Adv Mat. 26 (19), 3024-3034 (2014).</li><li>Chen, C. Z. C., Loe, F., Blocki, A., Peng, Y., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+macromolecular+crowding+to+enhance+extracellular+matrix+deposition+and+its+remodeling+in+vitro+for+tissue+engineering+and+cell-based+therapies.">Applying macromolecular crowding to enhance extracellular matrix deposition and its remodeling in vitro for tissue engineering and cell-based therapies.</a> Adv Drug Deliv Rev. 63 (4-5), 277-290 (2011).</li><li>Rashid, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+use+for+Polyvinylpyrrolidone+as+a+Macromolecular+Crowder+for+Enhanced+Extracellular+Matrix+Deposition+and+Cell+Proliferation.">Novel use for Polyvinylpyrrolidone as a Macromolecular Crowder for Enhanced Extracellular Matrix Deposition and Cell Proliferation.</a> Tiss Eng C. , (2014).</li><li>Dewavrin, J. Y., Hamzavi, N., Shim, V. P. W., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+the+Architecture+of+3D+Collagen+Hydrogels+by+Physiological+Macromolecular+Crowding.">Tuning the Architecture of 3D Collagen Hydrogels by Physiological Macromolecular Crowding.</a> Acta Biomaterialia. 10 (10), 4351-4359 (2014).</li><li>Dewavrin, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+Rate+Boosting+of+Collagen+Fibrillogenesis+in+Heterogeneous+Mixtures+of+Crowding+Agents.">Synergistic Rate Boosting of Collagen Fibrillogenesis in Heterogeneous Mixtures of Crowding Agents.</a> J Phys Chem B. 119 (12), 4350-4358 (2015).</li><li>Auger, F. A., Berthod, F., Moulin, V., Pouliot, R., Germain, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineered+skin+substitutes:+from+in+vitro+constructs+to+in+vivo+applications.">Tissue engineered skin substitutes: from in vitro constructs to in vivo applications.</a> Biotechnol Appl Biochem. 39, 263 (2004).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding+effect+on+cartilaginous+matrix+production:+a+comparison+of+two-dimensional+and+three-dimensional+models.">Macromolecular crowding effect on cartilaginous matrix production: a comparison of two-dimensional and three-dimensional models.</a> Tiss Eng Part C Methods. 19 (8), 586-595 (2013).</li><li>Zeiger, A. S., Loe, F. C., Li, R., Raghunath, M., van Vliet, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding+directs+extracellular+matrix+organization+and+mesenchymal+stem+cell+behavior.">Macromolecular crowding directs extracellular matrix organization and mesenchymal stem cell behavior.</a> PLOS One. 7 (5), e37904 (2012).</li><li>Kumar, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecularly+crowded+in+vitro+microenvironments+accelerate+the+production+of+extracellular+matrix-rich+supramolecular+assemblies.">Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.</a> Scientific Reports. 5, 8729 (2015).</li><li>Ang, X. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding+amplifies+adipogenesis+of+human+bone+marrow-derived+MSCs+by+enhancing+the+pro-adipogenic+microenvironment.">Macromolecular crowding amplifies adipogenesis of human bone marrow-derived MSCs by enhancing the pro-adipogenic microenvironment.</a> Tiss Eng Part A. 20 (5-6), 966-981 (2014).</li><li>Peng, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Fibroblast+Matrices+Bioassembled+Under+Macromolecular+Crowding+Support+Stable+Propagation+Of+Human+Embryonic+Stem+Cells.">Human Fibroblast Matrices Bioassembled Under Macromolecular Crowding Support Stable Propagation Of Human Embryonic Stem Cells.</a> J Tiss Eng Regen Med. 6 (10), e74-e84 (2012).</li><li>Benny, P., Badowski, C., Lane, E. B., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+More+Matrix:+Enhancing+the+deposition+of+dermal-epidermal+junction+components+in+vitro+and+accelerating+organotypic+skin+culture+development,+using+macromolecular+crowding.">Making More Matrix: Enhancing the deposition of dermal-epidermal junction components in vitro and accelerating organotypic skin culture development, using macromolecular crowding.</a> Tiss Eng A. 21 (1-2), 182-192 (2015).</li></ol>

The authors have no conflicting interests to disclose.

Enhanced extracellular matrix is obtained upon introduction of macromolecular crowders to cell culture, owing to the excluded volume effect which increases the propeptide cleavage by proteinases. This results in extracellular matrix, in particular collagen, to be processed faster and deposited on the culture surface. While other groups have obtained thick fibroblast cell layers, it involved a culture time of several weeks20. In contrast, MMC described in this Protocol, dramatically shortens the culture time while increasing the amount of ECM. The increase in reaction rates in crowded cell cultures has also been reported in chondrocyte matrix deposition21, extracellular matrix organization22,23 and adipogenesis24. 
After decellularization of crowded cell layers, cell-derived matrices are harvested and used for various tissue engineering applications, one of which is the seeding of secondary cell cultures on top of the cell-derived matrix25,26. As the matrices are cell-derived and natural products, degradation might occur if kept at non-ideal conditions for extended periods of time. A significant benefit of the cell-derived matrix, obtained under MMC, is that it is cell-derived and cell-specific. This could be a potential alternative to commercially available coatings which often are expensive and from animal sources. The skin equivalents must be handled with care, especially prior to fixation.
The use of interference reflection microscopy (IRM) in this Protocol shows, in much greater detail, the cell-derived matrix obtained with MMC. As compared with conventional fluorescent immunocytochemistry, only two or three proteins can be viewed at a time. With IRM, the total matrix can be visualized, with minimal manipulation to the sample. When immunocytochemistry was merged with the IRM field, it provided important information as to the ECM protein deposited in relation to the total amount of ECM. For example, it was observed that fibroblasts produced most ECM components, but a fibroblast-derived matrix was largely composed of fibronectin. 
Fibroblasts were also found to be the main producers of collagen VII, a feat which could be enhanced by MMC in vitro. This Protocol could have implications in therapies pertaining to the skin disorder, recessive dystrophic epidermolysis bullosa, where there is defective collagen VII.
Finally, the application of MMC in a 3D model to generate organotypic skin co-cultures, provides a useful tool in the advancement of skin research. Since MMC allows for the generation of skin equivalents with complete stratification, a mature dermal-epidermal junction delivered in a condensed time frame, this would be largely beneficial in the modeling of skin diseases, the study of skin biology in vitro, as well as the production of skin grafts for burn and chronic wounds.

皮膚は、水の損失及び病原体侵入を防止することにより、保護バリアを形成します。これは、3つの主要コンポーネントで構成されています。間質豊富な真皮1、その上に層状表皮2,3,4、および5,6間における真皮表皮接合部。真皮は主にコラーゲンと弾性繊維で構成されており、まばら線維芽細胞7が入力されます。対照的に、細胞が豊富な表皮ケラチノサイトの複数の層で構成されています。最も内側の層のケラチノサイトは、増殖され、更新し、常に皮膚の最も外側の層に移動して受ける角質層で、その結果、それらの核と細胞質材料を失った末端分化するケラチノサイトを置き換える新たな基底細胞を提供します落屑。 真皮 - 表皮接合部、基底膜の特定のタイプは、tethe相互接続マトリックス分子からなる複合構造体であります真皮に表皮をRS。真皮のコラーゲンI繊維は、コラーゲンIV豊富な緻密層に固定されているコラーゲンVIIアンカリングフィブリルと交錯します。順番にアンカリングフィラメント（ラミニン5、コラーゲンXVIIおよびインテグリン）は、基底ケラチノサイトのヘミデスモゾームに緻密層を接続します。基底ケラチノサイト（基底層）が分化し、基底上層を形成するために階層化だけでなく、増殖して更新する能力を持っています。有棘層、顆粒層、および環境と肌の接触面を示し、最終的に角質層、。角質層の基底層からエンルート 、ケラチノサイトは、サイトケラチンの発現パターンを切り替えて、最終的にアポトーシスを受けるし、角質化で自分自身を包みます封筒、共有結合トランスグルタミナーゼ活性によって架橋されている特定のタンパク質の殻。 の複雑な構造を含む、in vitroで皮膚とその層の再作成<html>真皮 - 表皮接合部および真皮細胞外マトリックス、および角化プロセスをエミュレートするには、やりがいのある仕事であれば、興味をそそら科学者や生体工学を持っています。例えば、患者の生検から皮膚細胞の正常な抽出および患者由来の皮膚細胞8を用いて皮膚の器官培養物の生成皮膚組織工学における重要な進歩がありました。しかし、未解決の問題は、皮膚細胞自体による細胞外マトリックスタンパク質の悪い分泌に関係し、次善の皮膚モデルで得られたままです。さらに、現在のプロトコルを使用して3次元器官型皮膚共培養物を生成するのに必要な時間は、4〜8週間、潜在的に巨大分子crowdersの取り込みと短縮することができる時間枠の間で変化します。培養時間を減少させること、試薬コストを節約し、細胞老化の発生率を低減し、製品が臨床で使用されるべき患者の待ち時間を減少させます。 トン&quot;&gt;高分子は、（MMC）混雑は排除体積効果。これらは、標準的な水性の培養条件9-13の下で遅刻であるプロコラーゲンのタンパク質分解切断を含む酵素反応速度に影響を与える。MMCの下では、酵素反応を生成するために、培養培地に特定の高分子を導入することを含みます空い対照10と比較して、混雑した培養物中のIは分子コラーゲンの量の増加、プロコラーゲン切断の場合には、その結果、試薬14,15の量を増加させることなく早めている。コラーゲンとプロコラーゲンの変換は、コラーゲンの形成を可能にするようにアセンブリ、48時間MMCを用いて培養した線維芽細胞は、最大4週間11,16,17のために監視混雑していない線維芽細胞培養物と比較して有意に多くのコラーゲンIを得た。ECMの形成、安定化およびリモデリングに影響を与える酵素活性への影響のほか、MMCも直接コラーゲンを増強し、調節することが示されています繊維形成18,19。 我々はここでは特に、真皮線維芽細胞と表皮角化細胞における細胞外マトリックスの皮膚細胞による（ECM）の生産を増強する方法を提示します。また、我々は、単層培養でMMCで製造富化ECMは、脱細胞化し、純粋な細胞由来のマトリックス（CDM）として使用することができることを示しています。 私たちは、視覚化するために非従来型のアプローチを使用し、完全にMMCで培養皮膚細胞によって堆積ECMを感謝しています。干渉反射顕微鏡は、典型的には、細胞 - マトリックス相互作用または細胞 - ガラス接触点を研究するために使用されます。この技術は、ガラス表面上に堆積されたマトリックスの合計量を表示するために、我々のシステムで利用されました。干渉反射顕微鏡は、MMCの存在下および非存在下で、細胞外マトリックス組成物及びパターンの点でほとんどの情報量を得るために、蛍光免疫染色を用いてカップリングさせました。 OrganotypiC皮膚共培養は、三次元の文脈において、インビトロで皮膚をモデル化するための古典的な方法です。二次元の共培養は、重要な情報を提供することができるが、このデータを変換し、バック本質的に三次元構造であり、インビボ環境にそれを適用する場合、それは限られています。皮膚ケラチノサイトは、特に、偏光であり、恒常性と細胞接着のために必須である頂端および基底セグメントを含みます。また、このようなケラチン1、ケラチン10およびフィラグリンのように基底層以上のケラチノサイト、中の典型的な基底上のタンパク質の発現は、層化およびケラチノサイトの分化の過程で唯一の存在です。分化は、典型的な単層培養物中にほとんど存在するように、基底層直上タンパク質の発現は、通常、この培養系では達成されません。したがって、器官培養物は、培養培地中に浸漬開始、その後keratinocytを駆動するための気液界面に持ち上げられます電子分化。これは、層別マーカーの発現、さえ角化および表皮生理学の一般より良い反射が生じます。他のグループが以前に成功した器官皮膚共培養を生成しているが、機能的真皮 - 表皮接合ゾーンの確立が課題となっています。ここでは、凝縮された時間枠内で、これらの構築物の成熟度を損なうことなく、強化された基底膜と器官皮膚共培養を培養するための新しい方法を提示します。これは、in vitroでのモデリングのための皮膚模倣薬、皮膚生物学の研究およびスクリーニングアッセイの品揃えを提供するであろう。

<table border="0" cellpadding="0" cellspacing="0" width="1154">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ascorbic acid</td>
			<td style="width:181px;">Wako</td>
			<td style="width:175px;">013-12061</td>
			<td style="width:499px;">Cell culture media additive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture inserts&#160; (6-well format)</td>
			<td style="width:181px;">Greiner Bio-One</td>
			<td style="width:175px;">657610</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citrate solution</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">S2369</td>
			<td style="width:499px;">DakoCytomation Target Retrieval Solution Citrate, pH 6 (x10)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Collagen (rat tail)</td>
			<td style="width:181px;">Corning</td>
			<td style="width:175px;">354236</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">CnT-57&#160;</td>
			<td style="width:181px;">CELLnTEC</td>
			<td style="width:175px;">CnT-57</td>
			<td>Cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DAB Substrate + chromogen kit</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">K3468</td>
			<td>Histology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Deep-well plate (6-well format)</td>
			<td style="width:181px;">Corning</td>
			<td style="width:175px;">355467</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">ECL detection reagent</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">RPN2106</td>
			<td>Amersham ECL Western Blotting Detection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Electron microscope (TEM)</td>
			<td>&#160;JEOL&#160;</td>
			<td>&#160;JEM-1010 (100kV)</td>
			<td>Transmission electron microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fibroblast media (FM)</td>
			<td></td>
			<td></td>
			<td>High Glucose-DMEM + 10% Fetal Bovine Serum + 1% Penicillin Streptomycin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Ficoll PM70</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">17-0310-05</td>
			<td>Macromolecular crowder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Ficoll PM400</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">17-0300-05</td>
			<td>Macromolecular crowder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Keratinocyte serum-free media (KSFM)</td>
			<td style="width:181px;">Life Technologies</td>
			<td style="width:175px;">17005-042</td>
			<td>Cell culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Lysis buffer</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">89900</td>
			<td style="width:499px;">Lysis buffer for protein extraction. A protease inhibitor (Roche, #11836170001) was added to this lysis buffer.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;">LSM510</td>
			<td style="width:499px;">Red, green and blue channels to visualize AF594, AF488 and DAPI fluorescent immunostainings (40X mag).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mounting media (Hydromount)</td>
			<td style="width:181px;">National Diagnostics</td>
			<td style="width:175px;">HS-106</td>
			<td>Fluorescent staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mounting media (Cytoseal)</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">8310-16</td>
			<td>Histology (HRP)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">OCT compound (Tissue Tek)</td>
			<td style="width:181px;">Sakura</td>
			<td style="width:175px;">4583</td>
			<td>Embedding for cryotomy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-streptomycin antibiotics</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:175px;">A5955</td>
			<td>Cell culture media additive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Primary antibodies</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Collagen I antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:175px;">#ab6308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Collagen Type IV</td>
			<td style="width:181px;">Novocastra</td>
			<td style="width:175px;">#NCL-COLL-IV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-collagen VII</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>LH7.2 (in house)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Fibronectin antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:175px;">#ab2413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Reducing agent&#160;</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">NP0009</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sample buffer</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">NP0008</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Secondary antibodies (Immunostaining)</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:175px;">#D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 594 goat-anti-rabbit</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-11037&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 594 goat-anti-mouse</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">&#160;#A-11005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 488 chicken-anti-rabbit</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-21441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 488 goat-anti-mouse</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-11001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">Secondary antibodies (HRP)</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">Envision + System - HRP Labelled Polymer Anti-Rabbit and Anti-Mouse</td>
			<td>undiluted</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Sodium deoxycholate</td>
			<td style="width:181px;">Prodotti Chimicie Alimentari</td>
			<td style="width:175px;"></td>
			<td>Decellularization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Stratification media</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dulbecco&#39;s Modified Eagle&#39;s Medium&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ham&#8217;s F12&#160;&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">10% fetal bovine serum&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">100U/mL penicillin and 100U/mL streptomycin&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">0.4mg/mL hydrocortisone&#160;&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5mg/mL insulin</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">1.8&#183;10-4 M adenine&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5mg/mL transferrin&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">2&#183;10- 11 M triiodothyronine&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Trypsin</td>
			<td style="width:181px;">Biopolis Shared Facilities</td>
			<td style="width:175px;">0.125% Trypsin/Versene&#160;&#160; pH 7.0 + 0.3&#160;</td>
			<td>Used to trypsinize cells</td>
		</tr>
	</tbody>
</table>

improving 2d and 3d skin in vitro models using macromolecular crowding

コラーゲンの糖タンパク質ファミリーは、ヒトの体内での主な構造タンパク質を示しており、近代的な組織工学で使用される生体材料の主要コンポーネントです。それは、特に皮膚モデルにおいて、結合組織およびその後の組織の凝集の次善の形成をもたらす、悪名高い遅いような技術ボトルネックは、in vitroでのコラーゲンの沈着です。ここでは、コラーゲン沈着の劇的な向上をもたらす分子クラウディング（MMC）を生成するために、皮膚培養物に差次サイズスクロース共重合体を添加することを含む方法を記載します。特に、皮膚線維芽細胞は、対照と比較して、MMCの下のI / IV / VIIおよびフィブロネクチン、コラーゲンを大量に堆積しました。 プロトコルはまたimmunocyによって証明されるように培養表面上に保持された細胞外マトリックス（ECM）の著しい量を露光、混雑した細胞層を脱細胞化する方法を記載します組織化学。トータルマトリックス質量および分布パターンは、干渉反射顕微鏡を用いて調べました。興味深いことに、線維芽細胞、ケラチノサイトおよび共培養は、様々な組成や形態の細胞由来のマトリックス（CDM）を生産しました。 CDMは、より臨床的に関連するアプリケーションに向けて移動させる、コーティングまたは足場の現在の使用は、典型的には、異種動物源から、回避することができる二次電池の播種用「バイオ足場」として使用することができます。 また、このプロトコルは、特に、真皮 - 表皮接合部（DEJ）にECMの沈着を増強するのに十分であった3次元器官型皮膚共培養モデル、コラーゲンVII、主成分の浸漬段階中にMMCの適用を記載しますフィブリルを固定します。電子顕微鏡は、対照と比較して、MMCを使用して開発の文化にフィブリルを固定するの存在を確認しました。アンカーフィブリル従って、表皮と真皮をつなぐので、これは重要です予め形成された成熟DEJは、移植片の安定性と全体的な創傷治癒の点で皮膚移植片レシピエントの利益を得ることができる持ちます。また、培養時間は、コストを削減、MMCを使用する場合に、成熟した構築物を得るために、3週間、5週間から濃縮しました。

分子クラウディングは、特に、ECMの沈着を向上させることができた、線維芽細胞培養物対照と比較してより多くのコラーゲンI、IVおよびフィブロネクチン堆積（ 図1、細胞層を、 図1A、コラーゲンI、1B、コラーゲンIV、1C、フィブロネクチン）。脱細胞化の際には、線維芽細胞はケラチノサイト（ 図1、マトリックス）と比較して、コラーゲンI、IVおよびフィブロネクチンの主要な預金であったことが明らかでした。 図2においては、線維芽細胞ではなく、ケラチノサイトは、コラーゲンVIIの主な生産者であったことが観察されました。これは、in vitro（ベニーら 、2015） で正常に堆積したコラーゲンVIIの最初の報告です。 細胞由来のマトリックスは、特定の不格好なやつを使用して免疫蛍光法を介して特徴付けられましたIES。これは古典的なアプローチがあるが、その全体でECMを視覚化し、完全に高分子crowdersの効果を理解することが重要でした。干渉反射顕微鏡（IRM）を使用して、マトリックスの完全な範囲は、（ 図3）を捕捉しました。 IRMイメージで抗体染色のオーバーレイは、ECMの合計量との関係でそのECM成分の相対量を示しています。 3D のin vitro皮膚モデルでは、MMCは、成熟した器官、皮膚の共培養（ 図4）を得るために3週間に5 ​​週間の培養時間を凝縮しました。ヘマトキシリン​​およびエオシン染色は完全に分化した表皮を欠いていた混雑していない対照培養物と比較して3週間目に、高分子crowdersと文化は、多能層状表皮および間質豊富な真皮から成っていることを示しました。また、強​​烈な、連続コラーゲンVIIの免疫染色は、真皮で検出されました対照培養物に弱いとむらコラーゲンVIIの免疫染色と比較して、混雑した細胞培養物のリットル - 表皮接合部、。透過型電子顕微鏡法（ 図 5）の機能コラーゲンVIIを示す、器官培養において線維アンカーの存在を示しました。 <img alt="図1" src="/files/ftp_upload/53642/53642fig1.jpg" /> 図1：混合分子クラウディング（MMMC）は、in vitroでの真皮-表皮接合成分の付着を強化する （A）コラーゲンI蒸着のみのfi broblastsにMMMC（細胞層およびマトリックス）によって強化されます。共培養の混雑はほとんどコラーゲンIを生成し、ケラチノサイトがのfi broblastsによるコラーゲンIの産生を刺激したことを示しています。 （B）のfi broblastsによりIV型コラーゲンの沈着は混雑によって強化されています。これは、混雑した共培養物に一層明確に示されています。注目すべきは、keratinocyt主に細胞関連コラーゲンIV番組や細胞内のコラーゲンIVはなく、細胞周囲マトリックスのために染色エス。共培養では、両方の細胞型は、主にケラチノサイト島を温存のfi broblastsに関連付けられているコラーゲンIVで分離します。 （C）フィブロネクチン沈着のみのfi broblastsで見られ、そこに強く（細胞層およびマトリックス）を混雑によって増強されました。共培養では、のfiフィブロネクチンの網状のメッシュは、ケラチノサイトの島々を温存、のみのfi broblastsと関連していました。スケールバー=20μmです。この図は、ベニーらから変更されている。2015年は、 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53642/53642fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/53642/53642fig2.jpg" /> 図2：混合分子クラウディング（MMMCは）の堆積を促進しますfi BRIL建物コラーゲンVIIを固定する。コラーゲンVII沈着の（A）網状蒸着パターンのみMMMC下のfi broblastsと明らかです。共培養では、細胞外コラーゲンVIIを強くケラチノサイトの島の間でのfi broblastコロニーに関連付けられています。ケラチノサイトはより強くMMMCの存在下で発現細胞周囲および細胞内コラーゲンVIIを示しています。細胞溶解後、コラーゲンVIIの足跡はMMMC処理されたのfi broblast培養における微細粒状層に見られているが、識別可能なのfi brillar堆積は共培養から取得されます。溶解した細胞層の（B）イムノブロット分析は、混雑のfi broblastsとケラチノサイト培養の両方が混雑していない対照と比較して著しくより多くのコラーゲンVIIを含んでいることを示しています。検索されたコラーゲンVIIは、主に細胞周囲導出されます。 （C）Bのデンシトメトリー分析はMMMCが細胞会合の量を増加させることを示していますfi broblastsで8倍とケラチノサイトにおける2倍のDコラーゲンVII。スケールバー=20μmです。この図は、ベニーらから変更されている。2015年は、 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53642/53642fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/53642/53642fig3.jpg" /> 。図3：干渉再FL ection顕微鏡（IRM）は、個々 の ECM成分（コラーゲンIVとのfiフィブロネクチン）の分析では、MMMC下の文化が強化された細胞外沈着により可視化線維芽細胞の足跡は、より多くの合計ECMが含まれています。合計ECM堆積を視覚化するために、IRMは、抗体染色は限界があったように、すべてのECMを定量しました。 IRMは明らかに対照条件と比較してMMMC下の総マトリックス量やパターンを示しました。 =20μmのスケールバー。この図は、されていますベニーらから変更された。2015年には、 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53642/53642fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/53642/53642fig4.jpg" /> 図4：水没フェーズの間（MMMC）の混雑混合高分子は、皮膚同等物における真皮-表皮接合部（DEJ）の成熟を ​​増強する線維芽細胞含有コラーゲンゲルを上にケラチノサイトを播種し、1週間のために水没し続け、その後に持ち上げました。気液界面。古典的なプロトコルでは、コラーゲンVII（緑）は、培養3週間の合計後に不在だったが、5週間後に皮膚同等物に登場しました。これとは対照的に、MMMCの下で、コラーゲンVIIは3週間後にすでに強く明白であったことがさらに強く標準培養物と比較して5週間後に染色しました。ヘマトキシリン​​およびエオシン（20X倍率）染色CFiの上で、この急速なプロトコルで、皮膚同等物のstratusの複数形のfiカチオンと成熟度が維持され、加速されたことrmed。スケールバー=100μmです。この図は、ベニーらから変更されている。2015年は、 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53642/53642fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図5" src="/files/ftp_upload/53642/53642fig5.jpg" /> 図5：MMMCの下で生成された皮膚同等物でのfi brilsを固定するのデノボ形成の証拠器官の共培養の新生真皮-表皮接合部の微細構造の研究MMMC（A、B）で3週間培養プロトコルはアンカーに構造が似示唆した後、培養3週間後の非混雑皮膚同等物（C）には存在しないのfi brils（矢印）。この図は、FRが変更されましたオムベニーら 、2015年には、 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53642/53642fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding at JoVE.com

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

我々は、高分子crowders（MMC）を使用して、細胞外マトリックスタンパク質に富む細胞由来のマトリックスを得るためのプロトコルを提示します。また、我々は、コンストラクトの成熟度を維持しながら、培養時間を減少させる3D器官型皮膚共培養世代でMMCを組み込むプロトコルを提示します。

2Dと3Dの肌の改善<em&gt;インビトロ</em分子クラウディングの使用&gt;モデル

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

improving-2d-and-3d-skin-in-vitro-models-using-macromolecular-crowding

Nanyang Technological University

Research

JoVE Journal

Bioengineering

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

12.4K ビュー.  National University of Singapore. 我々は、高分子crowders（MMC）を使用して、細胞外マトリックスタンパク質に富む細胞由来のマトリックスを得るためのプロトコルを提示します。また、我々は、コンストラクトの成熟度を維持しながら、培養時間を減少させる3D器官型皮膚共培養世代でMMCを組み込むプロトコルを提示します。 

ビデオ: Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

ネガティブステインEMによるタンパク質と高分子複合体の可視化：グリッドの準備から画像取り込みに

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

生物工学

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2Dと3D環境での神経前駆細胞の電界制御監督移行

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

水性二相系を用いた細胞の共培養パターニング

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

粒子追跡マイクロレオロジーを用いて3次元腫瘍モデルにおける細胞外マトリックス剛性の縦測定

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

再構成ヒト角膜上皮様（RHCE）組織モデルを使用して眼刺激性化学物質の有害性の同定のための眼刺激性試験（EIT）

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

線状インベーゾームの形成および活性を研究するための2Dおよび3Dマトリックス

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

軟骨と皮膚組織類縁ミキシング ユニット Bioink Precellularization の小説のパッシブ バイオプリンティング

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

高分子群集を用いたヒト皮萎縮性瘢痕化のインビトロモデル

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

長期ナノ材料暴露後のインビトロ遺伝子毒性試験のための高度な3D肝モデル

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

ヒドロゲルベースの3D培養モデルを自動化および標準化された方法で製造するためのオープンソース技術プラットフォーム

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...