作者要感谢:弗兰克博士姜大卫林博士，伯纳德Yurke博士和乌代Chippada博士为他们开发的DNA凝胶技术的贡献;诺雷尔Hadzimichalis博士，斯密特·沙阿，金佰利彼得曼，罗伯特·阿特尔的意见和这份手稿的编辑;资金来源包括新泽西州委员会对脊髓损伤研究（批准＃07A-019-SCR1，NAL）和新泽西州神经科学研究所（MLP）;与组织工程的出版商，A部分为许可转载图2和图4和生物材料的许可转载如图3所示 。

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作者什么都没有透露。

DNA凝胶的能力来软化或硬化之前和之后细胞粘附使得他们研究的动态组织硬度在细胞功能中的作用的理想模型。所有三种设计中采用了机械和生物学研究。然而，所有三个设计具有各种交联百分数类似弹性，表明交联长度不影响DNA的凝胶弹性（ 表2）。与此相反，丙烯酰胺浓度影响的弹性。这些设计可以不同交联动力学自凝胶破裂的倾向，或凝胶的倾向，在媒体或者缓冲溶解，随增?...

静态和动态的基材是两类生物材料的开发，以研究组织的弹性或刚性对细胞功能的影响的。静态基板无法它们被制造之后和/或一旦细胞被镀改变其物理性质。聚丙烯酰胺（PA）的凝胶进行合成的力学生物学研究5,17的被所述第一二维的，静态的基板。 PA凝胶是易于制备，价廉，通用性，并且可以与宽范围的弹性模量来制造。虽然这些技术的优点使PA胶凝共同施加的基板，静基板并不表明细胞外基质（ECM）和周围的细胞环境在体内的动态特性。例如，细胞外基质经历刚度的改变如损伤，发展，或疾病的结果。因此，动态的衬底作为力学生物学研究仿组织基底模型青睐<sup&gt; 22,24,25。 许多合成的，天然的，二维的，三 ​​维的，静态的和动态的生物材料已经被开发，以模仿组织硬度1,3,6,16,23,26。一些动态基板需要加热，紫外线，电流，离子和pH值的变化，以改变其机械性能2,4,7,8,12,15,16，但这些刺激可以限制水凝胶的生物应用。 DNA的交联聚丙烯酰胺水凝胶（DNA凝胶）是动态的二维弹性基材。 DNA的交联允许进行DNA凝胶刚度通过加入单链DNA（ssDNA）的时间，空间，和可逆调制到媒体或缓冲9-11,13,14,18,21。不像其中施加于弹性调制刺激上述的动态凝胶，将DNA凝胶依赖于应用的ssDNA为弹性的改变的扩散。因此，上部凝胶表面，其中细胞生长，是调制的，因为速度ø第一区域˚F弹性调制取决于凝胶的厚度。 DNA凝胶相似，其PA凝胶同行，他们有一个聚丙烯酰胺的骨干，但是双丙烯酰胺交联被替换为交联的DNA（ 图1）组成。两个的ssDNAs（SA1和SA2）杂交与交联剂链（L2），以弥补凝胶的DNA交联。 SA1和SA2有两个包含Acrydite修饰的5&#39;末端进行有效的结合到PA的网络不同序列。用于制备凝胶，SA1和SA2的单独聚合成PA骨架，随后聚合的SA1和SA2一起混合。 L2，交联剂，将被添加到SA1和SA2混合物。在L2的碱基序列互补的两个SA1和SA2的序列和二级杂交与SA1加上SA2以形成DNA交联。最初，DNA琼脂糖凝胶的弹性是由两个二级浓度和交联（ 表1确定</强&gt;和2）。含有L2，SA1，SA2和相等化学计量的量的DNA的凝胶是硬的凝胶，因为SA1和SA2是100％的交联用L2（被指定为100％的凝胶）。对二级导致DNA交联的比例较低，因此较低的浓度，柔和的DNA凝胶。凝胶低至50％的交联的（指定为50％凝胶）已经构建9-11。 <img alt="图1" src="/files/ftp_upload/51323/51323fig1highres.jpg" /> 图1中的DNA凝胶的交联和uncrosslinking概略9-11,13,14,18,21步骤1:SA1（红色）和SA2（蓝色）分别聚合成聚丙烯酰胺主链（黑色）。聚合后，SA1和SA2的聚合溶液混合在一起。步骤2:L 2（绿色），并将与SA1加上SA2杂交以形成凝胶的交联。第3步:R2的杂交与T他立足点L2的。步骤4:R2的立足点杂交推动L2的从SA1和SA2的解压。 与PA的凝胶，DNA凝胶可以变硬和合成后软化。出于这个原因，生长在DNA凝胶的细胞可以进行动态刚度的改变。变硬细胞粘附凝胶，L2可以加入到低百分比的凝胶培养基中，以增加交联点的百分比。软化细胞粘附凝胶，L2可以除去以减少交联点10,13,21的百分比。 L2具有在3&#39;端附加的立足点序列以允许L2从SA1和SA2中（ 表1）uncrosslink。去除L2的被逆转链称为R2的杂交来实现的。 R2是互补的L2的全长，并与L2立足点第一杂交。立足点杂交推动L2的从SA1和SA2的解压，这消除了交联，降低了凝胶的硬度。 在这份报告中，并视频，一步接一步提供了用于加固和软化的DNA凝胶的制备中说明。而100％和80％的凝胶制剂进行了描述，此协议可定制，以创建其他的最初和最后的交联百分比的DNA的凝胶。在一般情况下，100％和80％凝胶剂的制备，固定于盖玻片上，官能化的，并且接种有细胞。 L2加至80％凝胶介质和R2加至100％的凝胶剂，电镀后48小时的媒体。加L2的媒体变硬80％凝胶，以100％的交联，而加入R2到媒体软化100％的凝胶，以80％的交联。硬化的凝胶被指定为80→100％的凝胶和软凝胶中指定的文本100→80％的凝胶。为控制或静态凝胶，单链DNA组成的Ts或由于被传递到另一组的100％和80％的凝胶。至少两天下列弹性调制后，细胞可以进行处理和分析。 <table border="0" cellpadding="0" cellspacing="0" fo:kEEP-together.within页="总是"&gt; <tbody align="center"><tr><td rowspan="2"></td><td rowspan="2"> DNA交联 </td><td rowspan="2"> ＃基地 </td><td> 序列（ 立足点 ） </td><td rowspan="2"> 修改 </td><td rowspan="2"> 熔化温度（Tm值，℃） </td><td rowspan="2"> 评论 </td></tr><tr><td> 5&#39;→3&#39; </td></tr><tr><td rowspan="4"> 设计1 </td><td> SA1 </td><td> 10 </td><td> GCA CCT TTGç </td><td> 5&#39;Acrydite </td><td> 34.9 </td><td></td></tr><tr><td> SA2 </td><td> 10 </td><td> GTC AGA ATGá </td><td> 5&#39;Acrydite </td><td> 23.6 </td><td></td></tr><tr><td> L2 </td><td> 30 </td><td> TCA TTC TGAç 气相色谱AAA级转肽酶的GC 摹CTA CAC得志 </td><td></td><td> 56 </td><td>10 bp的立足点序列包括在内。 </td></tr><tr><td> R2 </td><td> 30 </td><td> CAA GTG豪CGC ACC TTT GCG TCA GAA TGA </td><td></td><td></td><td> R2是互补的二级</td></tr><tr><td rowspan="4"> 设计2 </td><td> SA1 </td><td> 14 </td><td> CGT GGC ATA GGA CT </td><td> 5&#39;Acrydite </td><td> 46.​​9 </td><td></td></tr><tr><td> SA2 </td><td> 14 </td><td> GTT TCC民航局TCA GA </td><td> 5&#39;Acrydite </td><td> 40.2 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> TCT GAT TGG GAA交流 教学专业议会注册税务师TGC CAC摹 GT交铁通卡特彼勒C </td><td></td><td> 65.9 </td><td>一个12碱基序列的立足点是包括在内。 </td></tr><tr><td> R2 </td><td> 40 </td><td> GAT GAA GGT AAC CGT GGC ATA GGA CTG TTT CCC AAT CAGá </td><td></td><td> 65.9 </td><td> R2是互补的二级</td></tr><tr><td rowspan="3"> DESIGñ3 </td><td> SA1 </td><td> 20 </td><td> ACG GAG GTG达GCA ATG TC </td><td> 5&#39;Acrydite </td><td> 55 </td><td></td></tr><tr><td> SA2 </td><td> 20 </td><td> 猫GCT TAG GGA CGA CTG GA </td><td> 5&#39;Acrydite </td><td> 56.6 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> 台泥AGT CGT CCC认证TAA贲门癌的TG 摹ACA TTG猫ACA建CCG牛逼 </td><td></td><td> 68.8 </td><td>立足点不包括在内。 </td></tr><tr><td rowspan="2"> 控制 </td><td rowspan="2">控制</td><td rowspan="2"> 20-40 </td><td> AAA AAA（ 等 ），或</td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td></tr><tr><td> TTT TTT（ 等等 ） </td></tr></tbody></table> 对单链DNA 9-11,13,14,18,21。细胞和机械的研究中采用了多种ð 表1中相应序列ifferent交联的设计，产生了一系列的静态和动态力学性能的DNA凝胶。调制的交联点的设计参数是碱基序列和序列长度或交联的长度。粗体和斜体字体说明SA1和L2之间和SA2和L2，分别之间的碱基配对。 <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody align="center"><tr><td rowspan="2"></td><td colspan="8"> 设计 </td></tr><tr><td colspan="3"> 1 </td><td colspan="3"> 2 </td><td colspan="2"> 3 </td></tr><tr><td> 丙烯酰胺浓度（％） </td><td colspan="3"> 10 </td><td colspan="3"> 10 </td><td> 10 </td><td> 4 </td></tr><tr><td> SA1加上SA2杂交到二级（％交联） </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 100 </td><td> 100 </td></tr><tr><td> &lt;STRONG&gt;弹性（千帕，平均值±标准差） </td><td> 6.6±0.6 </td><td> 17.1±0.8 </td><td> 29.8±2.5 </td><td> 5.85±0.62 </td><td> 12.67±1.33 </td><td> 22.88±2.77 </td><td> 25.2±0.5 </td><td> 10.4±0.6 </td></tr></tbody></table> 表2 DNA的凝胶9-11,13,14,18,21。丙烯酰胺浓度，交联百分比，和交联长度的杨氏模量（E）的可调制中的DNA凝胶。设计1，2，和3具有20，28和40碱基的交联​​长度，分别为。 100％的凝胶对于所有的设计也有类似的弹性模量表示的交联长度，不影响凝胶的弹性。但是，不同的丙烯酰胺浓度改变DNA的凝胶弹性。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ssDNA</td>
			<td>Integrated DNA Technologies (Coralville, Iowa) idtdna.com</td>
			<td></td>
			<td>Do not vortex ssDNA. Gentle invert the vial and/or pipette solution to mix.</td>
		</tr>
		<tr>
			<td>PBS with calcium and magnesium</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>100X Tris-EDTA buffer (TE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO) sigmaldrich.com</td>
			<td>T9285</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Borate-EDTA buffer (TBE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>93290</td>
			<td>TBE is a reproductive toxin.</td>
		</tr>
		<tr>
			<td>40% Acrylamide solution</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>BP14021</td>
			<td>Acrylamide is a toxin.</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>A3678</td>
			<td>Prepare a 2% solution in TE buffer. APS is a toxin and irratant.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>T9281</td>
			<td>Prepare a 20% solution in TE buffer. TEMED is flammable, a corrosive, and a toxin.</td>
		</tr>
		<tr>
			<td rowspan="2">12-mm diameter round coverglass</td>
			<td rowspan="2">Fisher Scientific (Pittsburg, PA) fishersci.com</td>
			<td rowspan="2">12-545-82</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Norland optical adhesive 72</td>
			<td>Norland Products (Cranbury, NJ) norlandprod.com</td>
			<td>NOA72</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">24-well tissue culture plate</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td rowspan="2">Microcentrifuge tubes</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH</td>
			<td>ProteoChem or Thermo Fisher, (Rockland, IL) proteochem.com or thermofisher.com</td>
			<td>C111 or 22589</td>
			<td>Prepare a 0.315 mg/ml solution in water immediately before use. Dissolve at 37&#176;C and filter sterilze. It is normal to observe undisolved sulfo-SANPAH in the filter. Sulfo-SANPAH is light sensitive and, therefore, the solution should be protect from light until UV exposure.</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>P6407</td>
			<td>Prepare a 0.2 mg/ml solution in water and filter sterilize.</td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Affymetrix (Santa Clara, CA) affymetrix.com</td>
			<td>13813</td>
			<td>Prepare a 0.2 mg/ml solution in 0.2 N acetic acid. Solution needs to remain cold at all times to avoid polymerization. Acetic acid is a flammable, toxic, and corrosive.</td>
		</tr>
		<tr>
			<td>22 X 60 cover glass</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive-displacement pipette</td>
			<td>Gilson, Inc (Middletown, WI) gilson.com</td>
			<td>F148504</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>11-718</td>
			<td></td>
		</tr>
		<tr>
			<td>UV light source</td>
			<td></td>
			<td></td>
			<td>Place gels as close as possible to the UV light. UV light can cause skin or eye injury.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>GTS-Welco (Flemington, NJ) www.praxairmidatlantic.com/</td>
			<td>NI 5.0UH-R</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of dna-crosslinked polyacrylamide hydrogels

力学生物学是一个新兴科学领域，解决物理线索中指导细胞的形态和功能中的关键作用。例如，组织的弹性对细胞功能的影响是力学生物学研究的一个主要领域，因为组织硬度调制与疾病，发展和损伤。静态仿组织材料，或材料，可以不改变刚度一旦细胞接种，都主要是用于研究组织刚度对细胞功能的影响。虽然从静态研究收集到的信息是有价值的，这些研究并不表示体内细胞微环境的动态性质。为了更好地解决对细胞功能的动态劲度的影响，我们开发了一种DNA的交联聚丙烯酰胺水凝胶系统（DNA凝胶）。不像其他动态基底，DNA的凝胶具有降低或制造之后增加刚度而不刺激的能力。 DNA凝胶组成的DNA crossl的被聚合成聚丙烯酰胺骨干油墨。加入并经由输送的单链DNA的去除交联允许的时间，空间，和凝胶弹性的可逆控制。我们已经表明在以前的报告中说动态DNA琼脂糖凝胶弹性的影响调制成纤维细胞和神经细胞的行为。在这份报告和视频，我们提供的示意图描述的DNA制备凝胶的DNA琼脂糖凝胶的交联机理，并一步一步的指示。

在此之前我们的研究，细胞外基质的相互作用，观察静态兼容生物材料或不可逆的单向动态基板。这些衬底不准确地反映细胞微环境的动态性质。我们的工作提供了研究的细胞外基质相互作用的软化和硬化动态生物材料更生理模型转移现有的技术范式。在以往的研究中，我们通过研究各种细胞的形态和表型上这些凝胶上分析细胞活力衬底的效果。在一个例子中，我们通过评价成纤维细胞形态学上?...

Watch this Scientific Journal Video about Preparation of DNA-crosslinked Polyacrylamide Hydrogels at JoVE.com

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

我们的实验室已开发的DNA交联聚丙烯酰胺水凝胶，动态水凝胶体系，以更好地了解调节组织硬度对细胞功能的影响。这里，我们提供了示意图，说明和协议来制备这些凝胶。

DNA交联聚丙烯酰胺水凝胶的制备

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

preparation-of-dna-crosslinked-polyacrylamide-hydrogels

Research

JoVE Journal

Biology

14.5K 次观看.  JFK Medical Center. 我们的实验室已开发的DNA交联聚丙烯酰胺水凝胶，动态水凝胶体系，以更好地了解调节组织硬度对细胞功能的影响。这里，我们提供了示意图，说明和协议来制备这些凝胶。 

视频: Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

视网膜水平切片制备

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

科研

生物学

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

游离的小鼠皮层神经元文化的制备

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

快速，灵敏的胶体考马斯亮G - 250染色蛋白聚丙烯酰胺凝胶

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

变性尿素聚丙烯酰胺凝胶电泳（尿素页）

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

组织工程细胞在三维凝胶封装

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

蓝原生聚丙烯酰胺凝胶电泳（BN - PAGE）分析，从细胞裂解液多蛋白复合物

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

编制质量肌醇Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

机械性刺激软骨细胞琼脂糖凝胶

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...