1. 准备步骤
<ol><li>准备穆勒-欣顿汤（MHB），将25克MH汤加入1升蒸馏水中混合。高压灭菌在121°C为2.5小时。然后，在室温下或冰箱中存储自动切割的介质。</li>
	<li>使用四象限条纹法在阿加介质上对有关细菌（大肠杆菌 ESBL）进行亚培养，并在37°C处过夜孵化。<ol>
<li>使用无菌循环，采取一个殖民地，并通过做密切的平行条纹在麦康基阿加板的上半部分传播它。</li>
		<li>使用循环，通过从第一个象限中条纹来传播第二个象限中的细菌。</li>
		<li>从第二个象限，使用紧密的平行条纹延长第三个象限中的条纹。</li>
		<li>通过从第三象限中条纹细菌，在第四象限的中心传播细菌。</li>
	</ol>
</li>
</ol>2. 面板准备
<ol><li>将四个 96 井板放在一起，形成一个正方形。使用磁带，将底部粘合在一起。</li>
	<li>重复此步骤以获取四个面板，每个面板包含 4 个板，并命名为 A1、A2、A3 和 A4。</li>
	<li>在四个面板的 16 96 井板中，将 50μL 的 MH 汤添加到第 2 列和第 11 列之间的井中。</li>
	<li>在四个面板的 16 96 井板中，将 200 μL 的 MH 汤添加到井 H12 中，作为负控制井。</li>
	<li>在四个面板的 16 96 井板中，在井 A1 和 H1 中加入 150 μL 的 MH 汤作为正控制井。</li>
</ol>3. 药物 1， 塞福塔西姆， 串行稀释
<ol><li>在圆锥管中，加入 15 毫升无菌 dH2O。<ol>
<li>按照公式 C1V 1 = C 2 V2计算从药物库存溶液中去除的体积。因此，V1 = （C2 x 15 mL） / C1。 注意：在我们的情况下，Cefotaxime 库存解决方案为 10 5μg/mL，C2为 256 μg/mL;因此，V 1=38.4μL。</li>
	</ol>
</li>
	<li>从 15 毫升的无菌 dH2O 中取出计算的体积，然后添加药物。</li>
	<li>除 H12 外，将制备药物溶液的 Pipette 50 μL 放入第 11 列和第 12 列的每口井中。</li>
	<li>开始串行稀释，从列 11 中去除 50 μL，并将其放入第 10 列中的相应井中，然后从 10 到到达列 2，其中从列 2 中获取的 50 μL 将被丢弃。</li>
	<li>对于四个面板的所有 16 个 96 井板，重复步骤 3.4 和 3.5。</li>
</ol>4. 药物 2， 安卡辛， 串行稀释
<ol><li>在圆锥管中，加入 10 毫升无菌 dH2O。</li>
	<li>按照公式 C1V 1 = C 2 V2计算从药物库存溶液中去除的体积。因此，V1 = （C2 x 10 mL） / C1。 注：在我们的情况下，阿米卡辛股票解决方案是103微克/mL，C2是64微克/mL;因此，V 1=64μL。</li>
	<li>从无菌 dH2O 的 10 毫升中取出计算的体积，然后添加药物。</li>
	<li>取8个独立的96井板。</li>
	<li>在每个板中，在 G 行和 B 行之间的井中加入 100 μL 的 MHB。</li>
	<li>将先前准备的药物 2 溶液中的 100 μL 添加到 G 排的井中。</li>
	<li>从每口井中取出 100 μL，最后从 B 排的井中丢弃 100 μL，从而从 G 排到 B 排进行连续稀释。</li>
	<li>重复步骤 4.5、4.6 和 4.7 以准备 8 个板。</li>
</ol>5. 将药物2转移到四个面板
<ol><li>Pipette 50 μL 的药物 2 从 G 排和 B 行之间的井进入四个面板中每个板中的相应井中。一个准备好的96井板含有100μL的药物2，足以在一个面板中两个板。</li>
</ol>6. 药物 3， 利沃弗洛沙辛， 添加
<ol><li>在四个不同的圆锥管中，添加 14 毫升无菌 dH2O。</li>
	<li>按照公式 C1V 1 = C 2 V2计算从药物库存溶液中去除的体积。因此，V1 = （C2 x 14 mL） / C1。 注意：在我们的情况下，利沃弗洛沙辛是准备在四个不同的浓度从股票溶液5 x 10 3μg/mL。 C1 = 2 微克/升， V1 = 5.6 微升 C2 = 4 微克/升， V1 = 11.2 μL C3 = 8 微克/升， V1 = 22.4 μL C4 = 16 微克/升， V1 = 44.8 微升</li>
	<li>从每个管子的 14 毫升无菌 dH2O 中取出计算的体积，然后添加药物。</li>
	<li>在准备第三种药物所需的浓度后，取 50 μL 并将其添加到 B 行和 G 行之间的相应井中，并在每个面板中 C1 与四个面板中的四个 P1 板相对应的相应板中将第 2 和第 12 列添加到相应的井中，C2 与四个面板中的四个 P2 板相对应C3 与四个面板中的四个 P3 板相对应，C4 对应于四个面板中的四个 P4 板。</li>
</ol>7. 药物 4， 特里梅托普林 - 磺酰硫磺酰， 添加
<ol><li>在圆锥管中，加入 14 毫升无菌 dH2O。</li>
	<li>按照公式 C1V 1 = C 2 V2计算从药物库存溶液中去除的体积。因此，V1 = （C2 x 14 mL） / C1。 注意：在我们的情况下，三氯苯丙酮-磺酰二甲酰从48 x 10 3μg/mL的库存溶液中以四种不同的浓度制备。 C1 = 512 微克/升， V1 = 149.33 微升 C2 = 1024 微克/升， V1 = 298.66 μL C3 = 2048 微克/升， V1 = 597.33 微升 C4 = 4096 微克/升， V1 = 1 194.66 μL</li>
	<li>从每个管的 14 毫升无菌 dH2O 中取出计算的体积，然后添加药物。</li>
	<li>在准备第四种药物所需的浓度后，服用 50 μL 并将其添加到 B 行和 G 行之间的相应井中，并在相应面板中的四个板中将第 2 和第 12 列添加到相应的面板中，其中 C1 与面板 1 相对应，C2 对应于面板 2，C3 对应于面板 3，C4 对应于面板 4。</li>
</ol>8. 大 肠杆菌大肠杆菌 的制备和添加
<ol><li>使用无菌循环，将以前在盘子上培养的细菌分离 物大肠杆菌 ESBL的一个菌落转移到2mL的无菌MHB和涡流中。</li>
	<li>使用密度计检查浑浊度，它应该是0.5麦克法兰。</li>
	<li>在无菌尿杯中加入 80 mL 的无菌 MH 汤。</li>
	<li>在 C 1V 1= C 2 V2之后，将 0.5McFarland 接种的细菌接种加入尿杯中，其中 V1 = （106 x 80 mL） / 108 = 800 μL。</li>
	<li>除H12外，每口井的接种液50μL为106 CFU/mL，即无菌控制井。</li>
	<li>在 37 °C 的夜间孵化面板。</li>
	<li>孵化后，加入50微升的二硝酸二醇，以记录油井的生长。</li>
</ol>9. FIC 模板协议（补充文件）
<ol><li>在面板 A 中的黄色细胞中写上药物 1 （Cefotaxime） 的最高浓度。</li>
	<li>在面板 B 中的黄色细胞中写上药物 2 （阿米卡辛） 的最高浓度。</li>
	<li>在面板 C 中的黄色细胞中写上药物 3 （利沃弗洛沙辛） 的最高浓度。</li>
	<li>在面板 D 中写下药物 4 的最高浓度 （三氯苯甲酰磺酰二甲苯甲酰） 。</li>
	<li>通过突出显示以红色增长的油井来跟踪红线（增长/无增长界面）。</li>
	<li>将药物 1 的 MIC 写在药物 1 （ATB1） 的表中。</li>
	<li>将药物 2 的 MIC 写在药物 2 （ATB2） 的表中。</li>
	<li>将药物 3 的 MIC 写在药物 3 （ATB3） 的表中。</li>
	<li>将药物 4 的 MIC 写在药物 4 （ATB4） 的表中。</li>
	<li>计算ATB1的图片<ol>
<li>确定生长/无增长界面上的油井。</li>
		<li>在表 ATB1 中，双击 FIC1 旁边的单元格，并将面板 A 左侧的黄色单元格拖至第一个选定的井。</li>
		<li>重复步骤 9.10.2 对于每个选定的井， 其中井 1 对应于 FIC1， 井 2 对应于 FIC2， 等等。</li>
	</ol>
</li>
	<li>计算ATB2的传真<ol>
<li>在表 ATB2 中，双击 FIC1 旁边的单元格，并将面板 B 左侧的黄色单元格拖至第一个预选井。</li>
		<li>对于每个预先选定的井重复步骤 9.11.1。</li>
	</ol>
</li>
	<li>计算ATB3的传真<ol>
<li>在表 ATB3 中，双击 FIC1 旁边的单元格，并将面板 C 左侧的黄色单元格拖至第一个预选井。</li>
		<li>对于每个预先选定的井重复步骤 9.12.1。</li>
	</ol>
</li>
	<li>计算ATB4的传真<ol>
<li>在表 ATB4 中，双击 FIC1 旁边的单元格，并将面板 D 左侧的黄色单元格拖至第一个预选井。</li>
		<li>对于每个预先选定的井重复步骤 9.13.1。</li>
	</ol>
</li>
	<li>在标有 ATB1+2+3+4 的表中，自动将每个 ATB 的 FIC1 相和。其他 FIC（FIC2、FIC3 等）也会发生同样的情况。<ol>
<li>在包含 FIC 的单元格中，以黄色突出显示，双击它，然后从表 ATB1+2+3+4 中选择总结的ΣFICs。</li>
	</ol>
</li>
	<li>代表九个板的九张纸中的每一张重复这些步骤。 注：在表FIC中，表将显示最终总结的FIC与获得的价值的解释。</li>
</ol>10. 使用微分检测对这四种药物进行MIC测定
<ol><li>用每个测试药物的缩写标记四个不同的行。例如，Ctx 用于塞福塔西姆， AMK 用于阿米卡辛， LEVO 用于利沃弗洛沙辛， SXT 用于特里梅托普林 - 磺胺佐尔。</li>
	<li>派珀特 200 μL 的无菌穆勒欣顿汤成井 1 号和井号 12 在每个用过的行。12号井将作为负控制井。</li>
	<li>派珀特 100 μL 的无菌穆勒欣顿汤到井 2 到 11 在每个用过的行。</li>
	<li>使用公式 C1V 1 = C2V 2计算要添加的每种药物的体积。因此，V1 = （C2 x 4 x V2）/ C1。V2是井中的最终体积，为 200 μL，C1是库存溶液的浓度，C2是我们需要在第一口井中的初始浓度。 注：此处，我们使用以下内容： Cefotaxime： C1 = 10 5μg/mL，在那里我们稀释到 104 μg/mL，C 2 = 256μg/mL;因此，V1= 20.48μL 阿米卡辛： C1 = 10 3μg/mL， C2 = 64μg/mL;因此，V1= 51.2μL 利沃福沙辛： C1 = 5 x 10 3μg/mL， 在那里我们稀释到 5 x 102μg/mL， C2 = 16μg/mL;因此，V 1=25.6μL 三甲苯丙酮-硫磺酰硫化物：C1 = 48 x 10 3μg/mL，C2 = 4096 微克/升;因此，V1= 68.26μL</li>
	<li>从 200 μL 汤中取出相同体积后，在相应行的第一口井中，每种药物所需的体积在添加药物后获得总量为 200 μL。</li>
	<li>通过将 100 μL 从井 1 移入井 2 等，然后连续稀释，直到达到 10 井，从井 10 中去除的 100 μL 将被丢弃。请注意，井11作为积极的控制井。</li>
	<li>Pipette 100 μL 的 106 种预制细菌接种到每个使用行中的每口井中，但 12 号油井用作负控制。</li>
	<li>在37°C的夜间孵化。</li>
</ol>

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四重棋盘方法类似于棋盘和协议中的三维棋盘。但是，应考虑某些关键步骤，以避免在实验过程中出现错误。
在启动协议之前，请务必对每种药物的MIC进行针对测试分离物的测试，以了解使用药物 1 和药物 2 开始稀释所需的浓度，这些浓度需要在盘子中连续稀释。关于药物 3 和药物 4，还应知道 MIC 计算需要测试的浓度（1/8 MIC、1/4 MIC、1/2 MIC 和 MIC）。有很多方法可以确定MIC?...

由于过度使用和滥用抗生素1，2，耐药性增加，开发治疗细菌感染的新药和制剂的必要性变得至关重要。开发新药等新方法对于克服耐药性危机非常重要。然而，制药业对开发新的抗菌剂不感兴趣。此外，如果开发新药，细菌将继续进化和发展对这些新药的耐药性3，4。因此，耐药性问题将无法解决，因此需要另一种方法，必须考虑和研究，以克服细菌耐药性。
药物组合是治疗细菌感染的一个非常重要的概念，主要是那些由耐多药病原体5，6引起的。它减少了治疗过程，减少了给予的剂量：因此，降低给定药物的毒性，有助于降低耐药性发展速度，并在某种程度上使细菌对附带敏感性5、7、8、9概念中描述的给定药物敏感。
一种药物的耐药性发展需要单一的突变：然而，针对多种途径的药物组合的耐药性发展需要几个独立的突变，这些突变会因这种组合而减慢。使用联合疗法时抵抗力下降的一个例子是10号分枝杆菌对里法姆平的抵抗率下降。另一个例子是Gribble等人所做的一项研究，该研究表明，单独服用皮拉西林的患者中耐药菌株的出现率高于服用卡博西霉素和阿米诺糖苷10组合的患者。研究表明，在进化的细菌中对阿米糖苷的耐药性发展使这些菌株对各种其他药物敏感。β-乳酸类药物阿莫西林与乳糖酶抑制剂克拉武拉尼酸的结合表明，在治疗耐药细菌菌株8方面取得了成功。
减少治疗时间是药物组合产生的一个很好的优势。例如，一种将青霉素或西夫特里亚松与根塔米辛结合2周的疗法，在给予4周11时，将单独给予青霉素或西夫特里亚松相同的疗效。组合药物允许使用剂量较低的药物，这些药物在单独服用时（如子 MIC）无效。硫化物的例子可以给出，其中使用三磺酰平减小，在较低的剂量，产生的毒性是晶体形成或结晶时，使用不溶性磺酰平地全剂量12。
因此，减少剂量和治疗时间最终将减少药物对身体的毒性。开发评估联合药物相互作用的方法的想法非常重要。在一项研究中，结果表明，联合疗法对治疗抗药性抗性阿 西诺菌 和 P.阿鲁吉诺萨8种更为有效。
联合给药 研究药物组合的方法有多种，如棋盘法、消磨曲线法、电子测试法等。棋盘方法可以在一个实验中研究两种药物之间的所有可能组合。此外，它被开发为研究三种药物的组合14。现在，我们扩展这一点，以研究四种药物的组合，主要用于治疗耐多药病原体。
时间杀死曲线检测通常用于测试某种药物的杀菌效果。它也被用来测试药物组合的效果，其中几个药物在特定浓度下结合。该协议要求准备几个无菌管或杯子，在每杯中，我们添加汤，药物的组合，和所需的细菌菌株。经过几个时间点的光密度的孵化和记录，结果与用过的菌株的正常增长率进行比较，看增长率是增、减，还是不变。
电子测试方法通常用于测试最低抑制浓度 （MIC），其中含有有关药物梯度浓度的条状被放在接种板上。它也被用来测试两种药物之间的组合，其中两条条被添加到板垂直的方式相交在他们的MIC13。
根据文献，没有黄金标准来定义和研究协同作用：因此，很难评估哪种方法用于研究组合更好，哪种方法产生更好、更可靠的结果，主要是13。然而，时间杀人检测是劳动密集型的，耗时的，昂贵的15，16，而电子测试方法的开发，以研究两种药物之间的组合只。棋盘可以研究测试的两种药物之间的所有可能组合，这就是为什么这种技术已被选择开发。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1000 &#181;L tips</td>
			<td>Citotest</td>
			<td>4330000402</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L tips</td>
			<td>Citotest</td>
			<td>4330-0013-17</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>corning</td>
			<td>430828</td>
			<td>For drug 3 and 4 preparation</td>
		</tr>
		<tr>
			<td>5 mL polysterene round-bottom Tube</td>
			<td>Falcon</td>
			<td>352058</td>
			<td>For 0.5 MacFarland bacterial inoculum preparation</td>
		</tr>
		<tr>
			<td>90mm petri dishes</td>
			<td>JRZ Plastilab</td>
			<td></td>
			<td>As bed for the solutions to be added using the multichannel pipette</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>corning</td>
			<td>3596</td>
			<td>For serial diltuion and combining drugs</td>
		</tr>
		<tr>
			<td>Bactrim 200, 40 mg (Trimethoprim-sulamethoxazole</td>
			<td>By CRNEXI SAS Fontenay-sous-Bois, France</td>
			<td>10177403</td>
			<td>Drug 4</td>
		</tr>
		<tr>
			<td>Ceforane, 1 g (Cefotaxime)</td>
			<td>PHARCO Pharmaceuticals</td>
			<td>24750/2006</td>
			<td>Drug 1</td>
		</tr>
		<tr>
			<td>Densitometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. Coli ESBL strain</td>
			<td>Retreived as a medical strain from the Saint-George Hospital Lebanon</td>
			<td></td>
			<td>Bacterial strain</td>
		</tr>
		<tr>
			<td>Mac Conkey + crystal violet agar</td>
			<td>BIO-RAD</td>
			<td>64169508</td>
			<td>For making agar plates used for subculturing</td>
		</tr>
		<tr>
			<td>Miacin 500 mg/2 mL (Amikacin)</td>
			<td>HIKMA Pharmaceuticals</td>
			<td>2BXMIA56N-AEF</td>
			<td>Drug 2</td>
		</tr>
		<tr>
			<td>Muller-Hinton Broth</td>
			<td>BIO-RAD</td>
			<td>69444</td>
			<td>For making bacterial media</td>
		</tr>
		<tr>
			<td>Multichannel Pipette</td>
			<td>Thermo Scientific</td>
			<td>GJ54761</td>
			<td>For serial dilution and addition of media, bacteria and drugs</td>
		</tr>
		<tr>
			<td>Paper Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel pipettes</td>
			<td>Thermo Scientific</td>
			<td>OH19855 HH40868</td>
			<td>For the addition of media, bacteria and drugs</td>
		</tr>
		<tr>
			<td>Tavanic, 500 mg (Levofloxacin)</td>
			<td>sanofi aventis</td>
			<td>221937/2009</td>
			<td>Drug 3</td>
		</tr>
	</tbody>
</table>

quadruple-checkerboard: a modification of the three-dimensional checkerboard for studying drug combinations

药物组合疗法的概念正变得非常重要，主要是随着对药物的抵抗力的急剧增加。四重棋盘也称为 Q-棋盘，旨在最大限度地增加一个实验中四种药物之间可能获得的组合数量，以最大限度地减少与其他协议实现相同结果所需的时间和工作。此协议基于简单的微稀释技术，其中药物被稀释并组合在几个 96 井板中。
在第一组96井板，穆勒-欣顿汤被添加，其次是第一个所需的药物（如Cefotaxime这里）连续稀释它。第一步完成后，另一组96井板用于稀释第二种药物（例如阿米卡奇），通过去除特定量的药物2进行转移，并在第一组含有药物的96井板中放入相应的井中。第三步是将第三种药物（如利沃弗洛沙辛）的所需浓度添加到包含药物1和2组合的初始组的适当板中。第四步是在第一组中将第四种药物（如三聚丙酮-磺酰硫磺醇）的所需浓度添加到适当的盘子中。然后，将准备和添加 大肠杆菌 ESBL细菌。
此方法对于评估所有可能的组合非常重要，并且具有更广泛的可能性，可进一步进行 体内 测试。尽管是一项需要大量关注的累人技术，但结果非常出色，而且节省了时间，许多组合可以在一个实验中进行测试。

图2A 表示通过将塞福塔西姆和阿米卡辛与特定浓度的利沃福沙辛和三氯环己烷-磺胺佐尔结合而获得的结果。我们可以在图的左侧看到图右侧显示药物浓度的四个板。箭头表示增长/无增长界面上的井。彩色油井是包含生长的井。我们注意到，第四板不包含包含组合的象限中的生长。这是因为在这个板块中，我们有利沃弗洛沙辛的MIC，将抑制增长。在这个数字中，我们可以看?...

Watch this Scientific Journal Video about Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations at JoVE.com

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

该协议描述了如何研究在一个实验中四种药物之间可以获得的所有可能的组合。该方法基于标准的96井板微稀释检测和分数抑制浓度（FICs）的计算来评估结果。

四重检查板：研究药物组合的三维棋盘的修改

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The ...

quadruple-checkerboard-modification-three-dimensional-checkerboard

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Biology

Unit 1

Pharmacodynamics

SCIENTISTS IN ACTION

4.3K 次观看.  University of Balamand. 该协议描述了如何研究在一个实验中四种药物之间可以获得的所有可能的组合。该方法基于标准的96井板微稀释检测和分数抑制浓度（FICs）的计算来评估结果。

视频: Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

基于芯片的三维细胞培养，在灌注微生物反应器

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

科研

生物学

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

使用高解析度电脑断层可视化的三维结构和功能的植物血管

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

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 ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

无源清清除小鼠卵巢血管构筑的三维重建

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

使用CyanoGate模块化克隆工具包进行共生对蓝藻的遗传改造

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

单个细胞分辨率三维成像的有机体

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...