这项工作得到了浙江树人大学研究项目 （2023R053 和 2023KJ237） 的支持。

实验中使用的血液来自体重 20-25 g、大约 7 周龄的 SPF 级 BALB/c 雌性小鼠。浙江树仁学院动物实验伦理委员会批准了所有动物护理和实验程序。
1. 溶液制备
<ol><li>称取 ALG 并在 50 °C 的水浴中搅拌，溶于超纯水中，得到 2% ALG 溶液。</li>
	<li>在超纯水中分别制备含有 5%（或摩尔质量浓度为 0.3 M）的 CaCl2、FeCl3、ZnSO4 和 CuSO4 溶液的质量分数。将每种溶液分别倒入收集皿中。</li>
</ol>2. 微流体电喷雾装置
<ol><li>取玻璃毛细管，将其放在喷灯火焰上软化，然后将其拉伸成各种直径 （50 μm-500 μm）。用玻璃切割机切掉精细部分，然后用优质碳化硅砂纸轻轻抛光喷嘴端口。清洁后，将毛细管的粗端连接到长管上，将管子的另一端连接到分配针和 5 mL 注射器（补充图 1）。 注意：玻璃管切口的锋利边缘很难插入软管;因此，在火焰边缘处，中断应呈圆角。</li>
	<li>将注射器连接到微流体注射泵上，并将毛细管的一端连接到支架上。将高压电源的红色高压端夹连接到注射器的分配针头上，将银色夹子放入收集液中。这种配置创建了一个简单的微流体电喷雾装置（图 1A）。 注意：将毛细管垂直于工作台放置。</li>
</ol>3. ALG 微球制备
<ol><li>将含有不同金属离子的单独收集皿直接放在毛细管下方。打开微流体注射泵并选择 Fast Forward 以使用 ALG 灌注管，然后设置适当的流速 （2 mL/h）。</li>
	<li>打开高压电源开关并转动旋钮以设置合适的电压 （5-7 kV）。</li>
	<li>向每个 90 mm 培养皿中加入 20 mL 溶液，以收集 ALG 微球以形成与不同金属离子交联的 ALGMS（图 1B）。</li>
	<li>ALG 在电场和重力的作用下，在几秒钟内在不同的离子收集溶液中形成微球。用消毒的移液管吸出微球，并将其转移到单独的 1.5 mL 离心管中，以获得 Zn2+、Ca2+、Cu2+和 Fe3+ -ALG 水凝胶微球（图 1C）。标记 Zn-ALGMS、Ca-ALGMS、Cu-ALGMS 和 Fe-ALGMS 管。 注：通过调整电压、针尖直径、流速和溶液浓度等参数，可以获得不同直径的微球。</li>
	<li>取少量制备好的微球，在 PBS 中尽可能均匀地分散，并将它们置于显微镜下进行观察。</li>
	<li>在同一批次的微球中随机选取 10 个观察场，将需要测量的图像导入 ImageJ 软件，将图像转换为合适的灰度模式，并使用阈值分割技术准确定义目标颗粒的面积并启动分析颗粒的功能，以便 ImageJ 能够识别和测量目标颗粒。获取微球的数据，然后将数据导入 Origin 软件进行分析和粒度映射。</li>
</ol>4. 抗菌性能测试
<ol><li>使用液体 Luria-Bertani （LB） 培养基制备 5 mL 初始细菌浊度为 0.2 mcF 的 金黄色葡萄球 菌（金黄色葡萄球菌）和 大肠杆菌 （大肠杆菌）悬浮液。</li>
	<li>将 10 mL 每种细菌溶液添加到 4 个 15 mL 无菌离心管中。然后，向每个试管中加入 0.5 mL 的 Zn-ALGMS、Ca-ALGMS、Cu-ALGMS 和 Fe-ALGMS ALG，并将它们置于 37 °C 和 200 rpm 的恒温振荡器中 12 小时。向对照组中加入等量的盐水。</li>
	<li>用无菌水将每种细菌溶液稀释至 105 倍。使用无菌玻璃珠将 200 μL 细菌溶液铺布在 LB 固体培养基上。</li>
	<li>将溶液快速均匀地铺在包衣板上，避免在同一区域来回涂抹。然后，将板置于 37 °C 的培养箱中 12 小时。观察不同群体的菌落。 注意：所有无菌程序都应在酒精灯附近进行。对微生物培养容器、接种器具和培养基进行消毒。</li>
</ol>5. 药物释放测试
<ol><li>为了评估不同微球的药物释放能力，使用牛血清白蛋白 （BSA） 作为负载模型药物。将 500 μL 的每个微球（Ca-ALGMS、Cu-ALGMS、Zn-ALGMS 和 Fe-ALGMS）添加到 1 mg/mL BSA 悬浮液中 24 小时。</li>
	<li>对于药物释放测定，将每个饱和样品加入 5.0 M 磷酸盐缓冲盐水 （PBS;pH 7.4） 中，然后在 37 °C 下搅拌 15 分钟，并以 80 rpm 的速度连续振荡。</li>
	<li>使用 100 μL 溶液，并在 1、3、6、12、24、36 和 48 小时用新鲜的培养基更换培养基。</li>
	<li>按照制造商的说明，使用 BCA 试剂盒在特定时间定量上清液蛋白浓度。经酶标定量测定释放的药物，得到相应的吸光度，并按标准曲线将实测值转换为实际药物浓度。使用以下公式计算药物释放速率： 药物释放百分比 = （特定时间点释放的药物浓度 / 初始药物浓度） x 100%</li>
	<li>定期从每个孔中取出等体积的 PBS，并用等体积的新鲜培养基替换。</li>
</ol>6. 溶血试验
<ol><li>将制备好的 Ca-ALGMS、Cu-ALGMS、Zn-ALGMS 和 Fe-ALGMS 放入 PBS 中，并在 37 °C 下孵育 24 小时以配制孵育溶液。</li>
	<li>通过眼静脉切开术从健康的 BALB/c 小鼠获得全血，收集在含有柠檬酸钠的抗凝管中。涡旋并以 1509 x g 离心 15 分钟以获得红细胞。</li>
	<li>用 PBS 冲洗红细胞 2 次至 3 次，并用 PBS 制备 10% 红细胞溶液。</li>
	<li>各种 ALGMS 的制备：实验组每个微球 500 μL，阳性对照组 1 mL 超纯水 （ddH2O），阴性对照组 1 mL PBS 溶液，每个与 20 μL 血细胞溶液混合。</li>
	<li>将样品在 37 °C 下孵育 4 小时，以 1509 x g 离心，15 分钟。将上清液放在一边，并在溶血后拍摄每组中的红细胞。</li>
	<li>取阳性对照组溶血率为100%，阴性对照组为0%。测量每组中上清液的吸光度值，并使用以下公式计算溶血速率： 溶血 （%） =（每组上清液的吸光度值 - 单独蒸馏水的吸光度值）/（阳性对照组的吸光度值 - 单独蒸馏水的吸光度值）x 100</li>
</ol>7. 细胞生物相容性试验
<ol><li>用 PBS 洗涤不同的 ALGMS 2 次，放入 5 mL 培养皿中 15 分钟，弃去上清液。</li>
	<li>在含有 10% FBS 的完全培养基 （DMEM） 中分别加入 0.2 mL 的不同 ALGMS，在 37 °C 下孵育 24 小时，并过滤溶液以获得渗滤液。</li>
	<li>在 24 孔板中将 NIH3T3 细胞培养至约 70% 的细胞密度，用微球浸出溶液处理 NIH3T3 细胞，并继续用完全培养基再培养 24 小时。</li>
	<li>取出细胞培养基并用 PBS 洗涤细胞。</li>
	<li>使用钙黄绿素-AM/PI 测定法评估细胞活力。取 2.5 μL 钙黄绿素-AM 溶液 （4 mM） 和 12.5 μL PI 溶液 （2 mM），将它们加入 5 mL 的 1x PBS 中并充分混合，以获得含有 2 μM 钙黄绿素-AM 和 5 μM PI 的工作溶液。</li>
	<li>将工作溶液以每孔 500 μL 的浓度添加到先前接种的细胞培养板中，在 37 °C 下避光孵育 15 分钟，并在倒置荧光显微镜下观察和拍照。为每个组设置三个仿行。</li>
	<li>根据以下公式计算细胞活力： 细胞活力 （%） = 活细胞数 / （活细胞数 + 死细胞数）</li>
</ol>

开发用于生物医学的海藻酸钠水凝胶微球

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A., Shum, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+future+of+droplet+microfluidics.">Development and future of droplet microfluidics.</a> LOC. 24 (5), 1135-1153 (2024).</li></ol>

不得披露任何利益冲突。

在该协议中，我们提出了一种基于微流体电喷雾技术制备 ALGMS 的方法。该方法操作简单，可产生大量圆度均匀、直径可控的微球。这种方法为研究人员提供了便利，可以促进水凝胶微球的研究和应用。此外，通过与不同的金属离子交联，ALGMS 的稳定性和生物活性得到提高。在抗菌实验中，Cu-ALGMS 和 Zn-ALGMS 表现出显著的细菌抑制作用。
水凝胶微球是具有出色可塑性的柔性微结...

药物递送系统是生物组织工程领域的研究热点，旨在提高药物递送效率和疗效，减少不良反应和副作用1.在这些系统中，水凝胶微球具有良好的生物相容性、可调的机械性能和功能性可塑性，是最常用的药物加载和递送载体之一2。它们可用于药物的缓释和受控释放，为药物提供良好的保护作用，避免或尽量减少药物在其他组织中的非特异性作用，并将药物靶向递送到特定的组织结构3。因此，水凝胶微球已成为一种新的高效药物递送系统，该领域的研究逐渐显现4.
水凝胶微球通常由可生物降解的材料合成，包括多糖、蛋白质和天然聚合物5。其中，ALG 是从海洋褐藻中提取的生物相容性、可生物降解的多糖6。其分子链包含游离羟基和羧基，可与大多数二价或多价阳离子交联，形成具有三维网络的水不溶性水凝胶结构5。ALG 形成的水凝胶微球可以在中性和碱性溶液中转化为带负电荷的聚电解质。负电荷之间的这种排斥导致微球膨胀，从而释放封装的活性成分或药物。这些特性导致人们认为 ALG 微球是广泛用于载药和控释的有前途的药物载体7。
制备水凝胶微球的方法多种多样。传统的 ALGMS 制备方法通常包括 sol-gel 法或 emulsion-sol 法。这些方法涉及沉淀、共沉淀和凝胶化反应等步骤，以获得目标微球8。近年来，随着微流控技术的不断发展，微流控电喷雾法逐渐成为一种高效、精密的微球制备方法9.该方法利用微流体技术，通过超细喷嘴电喷涂聚合物溶液，在随后的固化或交联过程中形成微米级的液滴和微球10。与传统方法相比，微流体电喷雾通过调整溶液流速、电压和细喷嘴尺寸等参数来精确控制微球粒径和形态11。它还能够高速连续制备微球，提高制备效率并保持温和的反应条件。此外，ALGMS 可以制备成具有多种功能，例如控释药物和负载催化剂，使其易于在各个领域应用。
在这里，我们提出了一种使用微流体电喷雾法制备 ALG 微球的方案。该过程包括将 ALG 溶液通过微流体装置并对其进行电喷雾。将所得液滴收集在含有不同金属离子（Ca2+、Cu2+、Zn2+ 和 Fe3+）的溶液中，以引发交联反应。该反应提高了微球的稳定性和粘附性，并赋予它们不同的功能。这种方法易于执行，并且合成的微球在其形态上表现出良好的尺寸均匀性。此外，我们还研究了它们的抗菌特性、缓慢的药物释放能力和生物相容性。该协议将有助于进一步的药物开发和生产。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>120 mesh screen</td>
			<td>Solarbio,China</td>
			<td>YA0946</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol burner</td>
			<td>Solarbio,China</td>
			<td>YA2320</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c mice</td>
			<td>Wukong Biotechnology,China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bicinchoninic Acid Assay reagent</td>
			<td>Meilunbio,China</td>
			<td>MA0082</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Lablead,China</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#160; powder</td>
			<td>Aladdin,China</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein-AM/PI</td>
			<td>Biosharp,China</td>
			<td>BL130A</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Corning,America</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4&#160; powder</td>
			<td>Jnxinyuehuagong,China</td>
			<td>7758-99-8</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibicol,China</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>FeCl3&#160; powder</td>
			<td>Aladdin,China</td>
			<td>7705-08-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HAKATA,China</td>
			<td>HN-FBS</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tubes</td>
			<td>Sartorius,Germany</td>
			<td>CC0028</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscopy</td>
			<td>Evidentscientific,Japan</td>
			<td>BX53(LED)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic syringe pump</td>
			<td>Longerpump,England</td>
			<td>LSP01-3A</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH3T3</td>
			<td>HyGyte,China</td>
			<td>TCM-C752</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Thermofisher,America</td>
			<td>150464</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Thermofisher,America</td>
			<td>3002</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Thermofisher,America</td>
			<td>Axia ChemiSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium alginate powder&#160;</td>
			<td>Bjbalb,China</td>
			<td>Y13095</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4 powder</td>
			<td>Jnxinyuehuagong,China</td>
			<td>7733-02-0</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of cross-linked sodium alginate microspheres with different metal ions using the microfluidic electrospray technology

微球是微米大小的颗粒，可以通过物理封装或吸附将药物加载并逐渐释放到聚合物表面和内部。在生物医学领域，水凝胶微球作为药物载体的应用已被广泛研究，因为它们能够减少给药频率、最大限度地减少副作用并提高患者依从性。海藻酸钠 （ALG） 是一种天然存在的线性多糖，具有三个主链糖苷键。聚合物的每个部分都存在两个辅助羟基，它们具有醇羟基部分的特性。合成的 ALG 单元可以与金属离子发生化学交联反应，形成聚合物堆栈的交联网络结构，最终形成水凝胶。水凝胶微球可以使用涉及 ALG 的离子交联特性的简单工艺制备。在这项研究中，我们使用微流体电沉积策略制备了基于 ALG 的水凝胶微球 （ALGMS）。由于精确控制了微流体电喷雾流，制备的水凝胶微球尺寸均匀且分散良好。采用微流控和高电场相结合的微流控电喷雾技术制备与不同金属离子交联的 ALGMS，并研究了其抗菌性能、缓慢药物释放能力和生物相容性。该技术有望应用于高级药物开发和生产。

与不同金属离子交联的 ALGMS 的表征 Ca-ALGMS、Cu-ALGMS、Zn-ALGMS 和 Fe-ALGMS 的光学形貌如图 2 所示，表现出良好的球形度、光滑的表面、均匀的粒径分布（补充图 2）和优异的单分散性。我们进一步使用扫描电子显微镜 （SEM） 和能量色散光谱 （EDS） 分析进行了显微表征。如图 3 所示，微球通常是球形的，具有明确的圆度。Zn-ALGMS...

作者聚焦：使用生物相容性海藻酸钠水凝胶微球推进治疗

该方案描述了使用用于药物载体设计的微流体装置制备与不同金属离子交联的海藻酸钠微球。还研究了这些微球的抗菌特性和缓慢的药物释放。

利用微流控电喷雾技术制备具有不同金属离子的交联海藻酸钠微球

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within ...

preparation-cross-linked-sodium-alginate-microspheres-with-different

Research

JoVE Journal

Bioengineering

Preparation of Cross-Linked Sodium Alginate Microspheres with Different Metal Ions Using the Microfluidic Electrospray Technology

1.4K 次观看.  Zhejiang Shuren University. 氢微球的特点是相容性好、设计灵活性高、定制化程度高。它们在生物医学领域具有广泛的应用保护器，例如治疗药物递送和 TC 修复发现的构建。在这项研究中，我们使用简单的微流控装置制备了氢微球。最近，我们一直在尝试使用载有一些中医小分子的氢微球来促进所有愈合。在这项研究中，我们发现锌和铜离子交联海藻酸钠微球具有良好?...

视频: 作者聚焦：使用生物相容性海藻酸钠水凝胶微球推进治疗

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

科研

生物工程

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

解吸电喷雾电离质谱成像的生物组织

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

的系统分析<em&gt;体外</em&gt;细胞滚动使用多孔板微流控系统

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

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

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

人体软骨组织制作采用三维喷墨打印技术

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

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Reconfigurable Microfluidic Channel with Pin-discretized Sidewalls

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or reactions. A microfluidic channel that deforms even after fabrication while retaining the channel shape enables high spatiotemporal reconfigurability. This reconfigurability is required in such key microfluidic functions that are difficult to achieve in existing "reconfigurable" or "integrated" microfluidic systems. We describe a method for the fabrication of a microfluidic channel with a deformable sidewall consisting of a laterally aligned array of the ends of rectangular pins. Actuating the pins in their longitudinal directions changes the pins' end positions, and thus, the shape of discretized channel sidewalls.Pin gaps can cause unwanted leakage or adhesion to adjacent pins caused by meniscus forces. To close the pin gaps, we have introduced hydrocarbon-fluoropolymer suspension-based gap filler accompanied by an elastomeric barrier. This reconfigurable microfluidic device can generate strong temporal in-channel displacement flow, or can stop the flow in any region of the channel. This feature will facilitate, on demand, the handling of cells, viscous liquids, gas bubbles, and non-fluids, even if their existence or behavior is unknown at the time of fabrication.

A microfluidic channel with deformable sidewalls offers flow control, particle handling, channel dimension customization and other reconfigurations while in use. We describe a method for fabricating a microfluidic channel with sidewalls made of an array of pins that allows their shape to change.

基于 Pin 离散壁的可重构微流控通道

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

超薄聚二甲基硅氧烷微氟化片在保水作用下固定化活线虫个体

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...