1。含量的设置<ol><li>冰在4°C（约2小时），解冻的基底膜小等分（&lt;500μL）。 </li><li>准备凝胶配方（看到表中的例子卷以下）：10倍PBS（总体积的1倍），1N钠氢（相当于0.023补充胶原蛋白的量，或每的胶原蛋白制造商的建议，如适用），基底膜和胶原键入我来终浓度为1毫克/毫升和1.3毫克/毫升（可用于其他基质配方，根据细胞类型和实验）。 </li></ol> 例如凝胶配方 <table border="1"><tbody><tr><td> 组件 </td><td> 股权集中度 </td><td> 终浓度 </td><td> 添加量 </td></tr><tr><td> 10X PBS </td><td> 10X </td><td> 1X </td><td> 0.090毫升</td></tr><tr><td>无菌水</td><td></td><td></td><td> 0.346毫升</td></tr><tr><td> 1N氢氧化钠</td><td></td><td></td><td> 0.008毫升</td></tr><tr><td>基底膜</td><td> 9.90毫克/毫升</td><td> 1毫克/毫升</td><td> 0.101毫升</td></tr><tr><td>鼠尾I型胶原</td><td> 3.66毫克/毫升</td><td> 1.3毫克/毫升</td><td> 0.355毫升</td></tr></tbody></table><ol start="3"><li>上述孵育1小时的最终解决方案在同一顺序的混合冰凝胶的组成部分，在4°C根据我们的经验，1小时的孵化前更均匀的胶原凝胶细胞种植结果。确保在任何时候都保持冰基底膜和胶原工作，尽可能快，以防止凝胶。 </li><li>地方12毫米直径8微米孔细胞成12井板，用消毒镊子文化插入。 </li><li>在无血清培养基，在5×10 6细胞/ ml（凝胶溶液总体积应该是10％），计数细胞，重悬。 </li><li>加入100μL细胞苏spension凝胶溶液900μL（最终细胞浓度5×10 5细胞/ ml），调匀移液器轻轻上下。 </li><li>最后的混合物中加入150μL，每个插入和30分钟到37℃，5％ 二氧化碳培养箱，直到凝胶聚合。 </li><li>使用无血清培养基， </li></ol><ul><li>加入100μL插入静止状态下的凝胶和1200μL上。内插入和良好的外部流体的水平应该大致相等，从而导致整个凝胶，无间隙流的最小压差。 </li><li>插入和650μL上述凝胶的水流条件下，加入100μL。要小心，以避免任何气泡下方的插入，因为他们将阻止细胞通过膜在这些地方迁移。这些卷所产生的压力差约1.3厘米高2 O（或1毫米汞柱）。 </li></ul><ol start="9"><li>现浇板在37℃，5％CO2培养箱24小时。 </li></ol> 2。细胞染色和计数<ol><li>加入500μL的1X PBS，每到新的24孔板，这将被用来洗插入。 </li><li>取出介质留在的流动transwells的上部和确定音量。从最初的增加，650μL（这将用于流量计算，见下文）总量减去剩余量计算总的洗脱体积。用棉签从插入凝胶和擦拭膜的顶面，以消除非侵入细胞。 24 15秒以及含有1X PBS洗插入板放入插入。 </li><li>除去PBS和添加500μl4％多聚甲醛（PFA）的下孵育30分钟室温，修复transmigrated的细胞和每个插入的。 </li><li>删除的煤灰，用500μl1×PBS冲洗一次，以去除残留的固定液。 </li><li>加入500μLØf 0.5％的Triton X-100的解决方案下的插入和室温孵育10分钟通透细胞。 </li><li>切膜插入到2微克/毫升的DAPI 1X PBS溶液中加入100μl使用刀片和地方，小心地将膜朝下的底面（transmigrated细胞朝下）。 </li><li>保鲜膜板在铝箔上在室温为30分钟150转的振动筛的地方。 </li><li>在500μL1X PBS（重复3次，每次10分钟），以消除自由的DAPI对振动筛洗膜。 </li><li>广场膜transmigrated细胞朝上玻片上，加安装的解决方案和盖玻片。 </li></ol> 3。数据分析<ol><li>依靠5随机选择每个膜的位置（远离边缘和使用10X或20X物镜）DAPI染色细胞核。 </li><li>使用下列公式计算平均细胞计数，并取得百分之入侵： 入侵百分比= 100％×（平均细胞计数x膜面积）/（X号的显微图像细胞种子表面面积） ％的入侵可以归到一些控制条件（通常是静态的条件）允许独立的实验之间的比较。 </li><li>计算平均流速：孵化时间（如24小时），总分为洗脱体积流量条件。 </li><li>计算平均流速：凝胶/膜（在这种情况下，60毫米2）截面积除以平均流速。 </li></ol> 4。代表结果以质流量下测量肿瘤细胞的侵袭，我们执行我们的3-D流使用的MDA-MB-435S转移性黑色素瘤细胞侵袭实验。先前已被证明这些细胞侵入间质流体流动13-14。细胞包埋在1.3毫克/毫升大鼠大组成的矩阵升肌腱I型胶原和1毫克/毫升基底膜基底膜矩阵，最终细胞浓度在5×10 5细胞/毫升。两个不同的条件进行了比较：（1）= 0.1微米s-1和（2）静止状态=没有可测量的流量平均间隙流（ 图1）。 24小时后，用DAPI染色的膜，毛孔侵入细胞，通过促 ​​进细胞计数。 图2显示了一个入侵细胞的形象代表。根据明，只有膜孔是可见的。使用荧光，细胞核DAPI染色细胞计数和鬼笔环肽染色F-肌动蛋白的结构，用于可视化的细胞体（可选）。使用学生的t检验假设方差相等时，我们发现，间质性流量显着增加的MDA-MB-435S细胞浸润，由静态条件下的2.3倍（P = 0.003）（ 图3）。这corrobora工商业污水附加费类似的结果（但不同基质条件，因此流速），使用这种细胞线13-14。 <img alt="图1" src="/files/ftp_upload/4159/4159fig1.jpg" /> 图1。的3-D间质流体流动的入侵检测的原理图。首先准备凝胶溶液使用适当的浓度和体积。再加入细胞凝胶溶液，并转移到细胞培养插入。最后加入适当体积的媒体每个条件和孵化。间质流体流动是由流体压头。 <img alt="图2" src="/files/ftp_upload/4159/4159fig2.jpg" /> 图2。Transmigrated膜的MDA-MB-435S细胞。入侵细胞被固定后，间质流体流动的入侵检测和染色DAPI和的Alexa Fluor 488标记的鬼笔，以方便侵入细胞的计数;图片的亮场下膜），B）的DAPI STA独立非执行董事的细胞核（蓝色），C）的Alexa Fluor 488-鬼笔环肽染色的F-肌动蛋白（绿色）。比例尺代表50微米。 <img alt="图3" src="/files/ftp_upload/4159/4159fig3.jpg" /> 图3。增加流量下的MDA-MB-435S细胞的入侵。 24小时后的细胞入侵质流（P = 0.003）显着提高。结果正常化平均静态条件下的值代表平均值±SEM 6细胞培养插入。

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没有利益冲突的声明。

在这里，我们所描述的量化对肿瘤细胞浸润间流动的影响，使用一个3-D矩阵嵌入细胞内的细胞培养插入方法。这和其他类似的方法已被用来研究多种细胞类型，13日至15日，17间流动的影响。我们的做法，部分模仿使用类型的肿瘤基质微环境，我胶原和基底膜含有蛋白质发现在基底膜上皮组织和周围基质21-22。这个系统是相对容易的设置了，简单，更具成本比大多数微流体装置，用?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>胶原蛋白（鼠尾） </td><td>屋宇署</td><td> 354236 </td><td>保持无菌</td></tr><tr><td> Millicell小细胞培养插入</td><td> Millipore公司</td><td> PI8P01250 </td><td> 8微米孔径，聚碳酸酯膜</td></tr><tr><td>基底膜</td><td>屋宇署</td><td> 354234 </td><td>保持无菌</td></tr><tr><td> PBS </td><td> Sigma Aldrich公司</td><td> 100M-8202 </td><td> 10倍的准备凝胶溶液，1个洗涤步骤</td></tr><tr><td>氢氧化钠，1.0N解决方案</td><td> Sigma Aldrich公司</td><td> S2770 </td><td>保持无菌</td></tr><tr><td> DMEM培养液1X </td><td> CellGro </td><td> 10-013-CV </td><td>保持无菌</td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> 511150 </td><td>保持无菌</td></tr><tr><td>青霉素链霉素</td><td> CellGro </td><td> 30002CI </td><td>保持无菌</td></tr><tr><td>的Triton X-100 </td><td> Sigma Aldrich公司</td><td> X100-500毫升</td><td>在PBS的0.5％ </td></tr><tr><td>多聚甲醛</td><td> Fisher Scientific则</td><td> 04042-500 </td><td> 4％的PBS </td></tr><tr><td>去离子水</td><td></td><td></td><td>保持无菌</td></tr><tr><td> 4&#39;，,6-diaminido-2-苯基吲哚（DAPI） </td><td> MP的Biomedicals </td><td> 0215757401 </td><td> 1毫克/毫升原液</td></tr><tr><td>安装解决方案</td><td> Thermo Scientific的</td><td> TA-030调频</td><td></td></tr><tr><td>胰蛋白酶EDTA </td><td> CellGro </td><td> 25-052-CI </td><td>保持无菌</td></tr></tbody> &lt;/ TABLE&gt;

three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion

大多数实体肿瘤的生长和发展依赖于初始变换的癌细胞，他们的反应基质的相关信号在肿瘤微环境1。此前，在肿瘤微环境的研究主要集中在肿瘤基质的相互作用1-2。然而，肿瘤微环境，还包括各种生物物理力量，其影响仍然知之甚少。这些部队是肿瘤生长的生物力学的后果，导致基因表达的变化，细胞分裂，分化和入侵3。矩阵密度4，5-6，刚度和结构6-7，组织间液压力8，8间质流体流动都改变在癌症的发展。 在肿瘤间质流体流动特别是高于正常组织8-10。估计间质液FLOW速度进行了测量，发现是在0.1-3微米S -1的范围内，根据肿瘤大小和分化9，11。这是由于肿瘤诱导的血管生成和血管通透性增加12所造成的升高间质流体压力。间质流体流动已被证明能增加入侵的癌细胞13-14血管成纤维细胞和平滑肌细胞15。这次入侵可能是由于自体细胞趋化细胞周围建立3 - D 16的梯度或增加15基质金属蛋白酶（MMP）的表达，分泌趋化因子和细胞粘附分子表达17。然而，细胞感受到流体流动的机制，不能很好地理解。此外，改变肿瘤细胞的行为，间质流体流动的调节其他细胞在肿瘤微环境的活动。它与（一）驾驶的成纤维细胞分化成肿瘤促进myofibr州18，（B）抗原的搬运和其他可溶性因子，淋巴结肿大19，（三）调节淋巴管内皮细胞形态20。 这里介绍的技术对体外细胞间质流体流动和量化入侵（ 图1）的影响。这种方法已经公布在基质和癌细胞侵袭13日至15日，17多个研究测量流体流量的影响。通过改变基质组成，细胞类型和细胞浓度，此方法可以适用于其他疾病和生理系统的研究侵袭，分化，增殖，基因表达，如细胞过程间流动的影响。

Watch this Scientific Journal Video about Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion at JoVE.com

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

在实体肿瘤间质流体流动升高，可以调节肿瘤细胞的侵袭。在这里，我们描述了技术应用嵌入在一个矩阵的细胞间质流体流动，然后测量其细胞浸润的影响。这种技术可以很容易地适应学习其他系统。

三维细胞培养模式的测量肿瘤细胞浸润间质流体流动的影响

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

three-dimensional-cell-culture-model-for-measuring-effects

Research

JoVE Journal

Bioengineering

16.4K 次观看.  Drexel University. 在实体肿瘤间质流体流动升高，可以调节肿瘤细胞的侵袭。在这里，我们描述了技术应用嵌入在一个矩阵的细胞间质流体流动，然后测量其细胞浸润的影响。这种技术可以很容易地适应学习其他系统。

视频: Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

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

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

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

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

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

科研

生物工程

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

研究模型声乐折叠聚合物引起的三维流量分离

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

肿瘤微环境基质与应用癌细胞侵袭组织工程

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

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

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

在一个三维胶原蛋白矩阵分析细胞迁移

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

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

基因转染向着文化微模式球体板细胞基因修饰的细胞移植

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

倒胶体晶体聚（乙二醇）脚手架制作：肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

二维和三维 i 型胶原蛋白基质角质形成细胞增殖的评价

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

在三维生物打印中使用多层水凝胶生物油墨，实现均匀细胞分布

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

使用声学分离器对CHO收获的细胞培养液进行初步澄清

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...