1。从皮肤外植体的成纤维细胞培养的建立<ol><li> 4 mm打孔活检放置在从人力前臂获取的MEM培养基的100单位/ ml青霉素，100μg/ ml链霉素，0.25微克/毫升两性霉素B。 </li><li>修剪任何的皮下脂肪和切碎活检摇动一些24对陪替氏培养皿底部的手术刀刀片切成小块。 </li><li>将组织的淤浆中的25cm 2组织培养瓶中与初级成纤维细胞生长介质（MEM培养基与10％FCS，100单位/ ml青霉素，100μg/ ml链霉素，和25微克/毫升两性霉素B）补充，直到烧瓶中的表面覆盖，但液体的深度是不够的组织浮动。 </li><li>经过3天的培养，在37°C在5％CO 2培养箱中，加入3毫升的主要成纤维细胞生长介质。 </li><li>另外3天的潜伏期变化的新鲜的成纤维细胞生长介质的媒体，或者CELLS是近汇合，他们可能会被分割到新鲜的培养基1:4。 （两性霉素B可以省略从这个点开始）。 </li></ol> 2。第一阶段 - 从鼠尾I型胶原的制备注:协议约12-14青少年（新鲜或冷冻）大鼠的尾巴<ol><li>准备大鼠尾通过在70％乙醇洗涤，并去除肌腱如下: </li><li>删除从鼠尾皮肤切片，从顶部到底部，在中间的尾巴手术刀和敲打沿尾部的长度。 </li><li>分离从芯的近侧区的尾部肌腱。 </li><li>删除肌腱朝向远侧区的尾部避免鞘使用齿镊。 </li><li>通过在4℃下搅拌48小时提取肌腱的1克/ 250毫升的0.5M乙酸。 </li><li>提取物（7,500 xg离心）离心30分钟，弃沉淀。 </li><li>向上清液中加入等体积的10％（重量/体积）NaCl的，​​并搅拌30-60分钟。 </li><l&gt;离心（10,000 xg离心30分钟），弃去上清液，并重新溶解在0.25M乙酸中的沉淀物搅拌24小时，在4〜1:1的比例℃。 </li><li>透析的胶原蛋白溶液对6-8变化6L〜17.5 mM乙酸（1毫升冰乙酸的每公升的冷水，改变2 x每日）。 </li><li>离心透析的胶原蛋白以30,000 xg 1.5小时。 </li><li>去除上清液和在无菌烧瓶中的地方。 </li><li>调整的I型胶原〜2毫克/毫升的浓度在4℃下使用0.5 mM乙酸</li></ol> 3。第二阶段 - 建立3D矩阵与的嵌入式成纤维细​​胞（允许8天的合同）。 <ol><li>组装在一个预冷冻瓶使用冷的试剂在4℃下混合。保持Ⅰ型胶原冰。 </li><li> T75烧瓶融合的主要成纤维细胞（细胞数1×10 6）使用方法: <ul><li> 25毫升鼠尾胶原蛋白（约浓度2mg/ml） </li><li> 3毫升10倍MEM </li><li> 0.22 M氢氧化钠:第一，加入2ml，然后下降明智的同时搅拌，直到胶原变为橙色， </li><li>但不是粉红色（通常高达3毫升）。近似P H = 7.2。 </li><li>注:这是重要的，以确保该介质/凝胶保持中性或微酸性的成纤维细胞也就不会收缩如果暴露于碱性条件下即使是略微的凝胶。 </li></ul></li><li> Trypsinise成纤维细胞，旋转400×g离心5分钟，弃上清。 </li><li>在旋转5分钟以上:准备12×35毫米的塑料菜 - 通常达到12道菜，从一个烧瓶的成纤维细胞/胶原蛋白组合。 </li><li>重悬，立即加入3毫升FCS的胶原蛋白混合，搅拌成纤维细胞。 </li><li>板约2.5毫升/成纤维细胞的胶原蛋白每道菜尽快。尽量避免气泡和胶原蛋白的设置。 </li><li>让胶原蛋白设定在37℃下，在湿润的5％CO 2气氛下在空气中10分钟。 </li><li>加入1ml的成纤维细胞生长个介质（DMEM +10％FCS）。 </li><li>分离矩阵的菜用移液管从两侧的胶原蛋白/成纤维细胞。 </li><li>第二天:成纤维细胞生长培养基（DMEM +10％FCS）加入1ml。 </li><li>媒体每一天。允许胶原蛋白/成纤维细胞基质合同约8天，直到他们适合在24孔板（〜3.5厘米至1.5厘米，直径收缩，参见图1）。 </li></ol>注:胶原蛋白的浓度应进行调整，以满足不同应用。较稀的胶原溶液，将承包的成纤维细胞。收缩率的轻微差异，将经历不同批次的胶原蛋白在相同的浓度。同样，不同的纤维母细胞胶原凝胶收缩以不同的速率，而目前更多的成纤维细胞，更快的收缩率。胶原蛋白的浓度和成纤维细胞密度因此可以通过调整，修改的收缩率，的胶原蛋白凝胶的最终密度。 4。第二阶段 - 电镀利息细胞矩阵顶部注意:在使用前消毒用乙醇钳和设备。 <ol><li>使用钝镊子，轻轻将24孔板合同矩阵。确保不折。 </li><li>准备感兴趣的细胞的悬浮液中，在约4×10 4个 / ml和的板1毫升（后摆脱的胰蛋白酶的trypsinising，旋细胞）上的矩阵的顶部。实际所需的细胞数量会有所不同，这取决于所使用的细胞类型。细胞培养基中应该是所关注的细胞正常生长培养基。 </li><li>允许细胞生长至汇合矩阵顶部，约3-5天。 </li></ol> 5。第三阶段 - 转移矩阵的一个网格入侵（约0-21天） <ol><li>切割不锈钢网格，建立一个三脚架和高压灭菌器在使用之前（参见图2）。 </li><li>将无菌的网格中6 cm培养皿中，生长培养基添加到网格（约10.5毫升）以上的水平。放置在网格上的矩阵，并轻轻地吸出介质，使底部的矩阵是与媒体接触，但不被水淹没。这被称为作为气/液界面，它创建了一个梯度，促进入侵。更换中等每隔两天（参见图2）。 </li><li>活细胞，使用荧光细胞进行成像的上下文成像，在这个阶段或更早的版本。合同或纤维状的胶原蛋白，我可以成像，二次谐波产生（SHG，参见图3）。使用多光子激发与宽视场的检测相结合，我们发现，SHG可以被成像到矩阵的至少为100微米，而表达细胞质绿色荧光蛋白的细胞可以被成像至少200微米深。 </li></ol>注:对于量化的入侵，被放置在网格在这一天matricies定义0天。对电网产生位置的梯度，促进细胞培养基入侵诠释<html> Ø矩阵。样品可以进行成像，在未来的1 - 21天（或更长），以评估生物过程，如的侵入，增殖，存活和分化（见参考文献列表）。 6。第四阶段 - 枷<ol><li>加入5毫升的4％PFA中的Falcon管中。 </li><li>转移到平坦的表面上的矩阵。切矩阵与一个新鲜干净的手术刀（因为你将染色的横截面）的一半，解除矩阵，用解剖刀和转让4％PFA固定过夜。 </li><li>器官的矩阵现在已经准备好与抗体染色/染色的选择（参见图3）。 </li></ol> 7。代表性的成果: <img alt="图1" src="/files/ftp_upload/3089/3089fig1.jpg" /> 图1实施例的成纤维细胞承包胶原I.混合料的成纤维细胞与大鼠尾胶原脱离陪替氏培养皿，并允许收缩超过6-8天。 图2"SRC ="/ files/ftp_upload/3089/3089fig2.jpg"/&gt; 图2器官的试验进展。 A，器官和进展示意图，B，胶原/成纤维细胞顶部不锈钢，无菌的网格建立气/液界面上的矩阵的实施例。 <img alt="图3" src="/files/ftp_upload/3089/3089fig3.jpg" /> 图3的器官型分析中的应用。 A，B非侵入性和侵入性胰腺导管腺癌（PDAC）细胞侵入随着时间的推移与器官矩阵。C，生活呈现出分层表皮的皮肤替代物，胶原真皮成纤维细胞承包组件。D，C8161黑色素瘤细胞的成纤维细胞侵入签约胶原真皮相当于E，入侵PDAC细胞（绿色）与成纤维细胞（红色）的器官矩阵内的互动和入侵。F </stron克，可视化创PDAC细胞（绿色）与有辱人格的周围细胞外基质（紫色）使用多光子基于二次谐波的产生。

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我们什么都没有透露。

在这里，我们提出了一种方法，用于生产适合于研究入侵细胞迁移6-8的一个3 -维的矩阵。基质由第一型胶原蛋白纤维，收缩超过数天的期间，由初级人表皮成纤维细胞。胶原蛋白的制备通过酸提取，而不是酶消化，它保留在聚肽两端的反应性，并促进交联的胶原纤维成更大的聚集体中和时的缓冲液条件9。这将导致在重新构成的凝胶更密切模仿的胶原蛋白在体内的功能，并具有重要的后果，在凝胶中培养的细胞的浸润性能。不要紧，不论是新鲜或冷冻的起始材料的使用，但使用青春期的老鼠的尾巴是很重要的，因为在年轻的动物胶原蛋白的交联更不稳定。因此，Ⅰ型胶原，从年幼的动物是比较容易提取，并重新构成瓦特第i个更高的保真度。 的胶原基质可以是固定的，和与对任何侵入性肿瘤细胞或基质成纤维细胞2,10抗体染色，是非常有用的，用于测试的功效的药物，这可能抑制入侵5,6。 虽然此协议建议使用初级人皮肤成纤维细胞中，可行的是使用主要从其他类型的组织的成纤维细胞，根据接受调查的组​​织类型。另外，也可以建立使用永生化的成纤维细胞，包括已稳定转染的细胞与荧光的蛋白质结合物的文化。以这种方式，基质细胞和肿瘤细胞可以标记为可视化入侵11期间的细胞-细胞相互作用。

<table border="1"><tbody><tr><th>的试剂的名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> 10倍MEM </td><td> Gibco公司</td><td> 21430 </td><td> - </td></tr><tr><td>氢氧化钠</td><td>西格玛</td><td> 367176-500G </td><td>准备0.22 M的股票在水</td></tr><tr><td> FCS </td><td> PAA实验室</td><td> A15-101 </td><td> - </td></tr><tr><td> 35 mm培养皿</td><td>鹰</td><td> 353001 </td><td>对于步骤3.4 </td></tr><tr><td> 60 mm培养皿</td><td>鹰</td><td> 353004 </td><td>对于步骤5.2 </td></tr><tr><td>弹簧钳钝</td><td>萨姆肯</td><td> E003/02 </td><td>锯齿，不平滑</td></tr><tr><td> 16％的多聚甲醛</td><td>电子显微镜</td><td> 15710 </td><td>在PBS中稀释至4％，在使用前</td></tr><tr><td>屏幕为CD-1，大小为40目</td><td>西格玛</td><td> S07707-5EA </td><td>不锈钢网格，每包5 </td></tr><tr><td>透析管</td><td> Medicell国际</td><td> 7607 2295 </td><td> 12 - 14 kD的</td></tr><tr><td> PBS </td><td> Oxoid公司</td><td> BR0014G </td><td> - </td></tr><tr><td>醋酸</td><td>西格玛</td><td> 242853 </td><td> - </td></tr><tr><td> 24孔板</td><td>鹰</td><td> 353047 </td><td>对于步骤4.1 </td></tr><tr><td>两性霉素B </td><td>在vitrogen </td><td> 15290018 </td><td> - </td></tr></tbody></table>

organotypic collagen i assay: a malleable platform to assess cell behaviour in a 3-dimensional context

细胞迁移是生物学的许多方面，包括发育，伤口愈合，对免疫系统的细胞反应，肿瘤细胞转移的基础。迁移已研究盖玻片上，用光学显微镜，以使适合进行调查的细胞动力学。然而，现在已经很清楚，许多方面的细胞迁移取决于功能对本地环境，包括它的弹性，蛋白质的组合物，和孔径大小，没有忠实地表示的刚性的二维基板，如玻璃和塑料1。此外，互动与其他类型的细胞，包括间质成纤维细胞和免疫细胞3，已被证明发挥了关键作用，促进肿瘤细胞的侵袭。分子动力学，包括对药物治疗反应，相同的细胞时有显著差异相比，越来越显示出在分子水平上的调查<em在体外和在体内 4。 理想的情况下，这将是最好在其在生物体中的自然发生的上下文来研究细胞迁移，但是这并不总是可能的。中级组织培养系统，如细胞衍生的基质，基质胶，器官培养（这里描述的）组织外植体，类器官，以及异种移植物，因此，重要的实验中间体。这些系统近似的体内环境的某些方面，但更适合于实验操作，如使用稳定转染的细胞株，药物的治疗方案，长期和高分辨率成像。这种中间的系统是特别有用的试验场验证探头和建立所需的图像之前进行成像在体内 5细胞和荧光记者的动态响应的参数。因此，它们可以起到重要的作用，在减少实验活着需要克动物。

Watch this Scientific Journal Video about Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context at JoVE.com

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

描述的方法，用于制备一个3 - 维的矩阵组成的I型胶原蛋白和初级人成纤维细胞。器官凝胶作为一个有用的基体，以评估侵入细胞的迁移，因为它模仿组织基质的基本功能，是适合各种形式的显微镜。

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

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22.4K 次观看.  University of Glasgow. 描述的方法，用于制备一个3 - 维的矩阵组成的I型胶原蛋白和初级人成纤维细胞。器官凝胶作为一个有用的基体，以评估侵入细胞的迁移，因为它模仿组织基质的基本功能，是适合各种形式的显微镜。

视频: Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

在三维自组装多肽水凝胶的人类神经祖细胞的培养

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

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 ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

平面梯度扩散系统的调查趋化三维胶原基质

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

在压缩的胶原组织糜:含细胞Biotransplant单上演重建修复

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...