我们想感谢所有成员的香港实验室和邓焰博士为的显微镜核心，办公室协同科学，NYUMC他们的援助和技术支持。这项工作是支持的试点补助金从纽约大学艾滋病研究中心（CFAR，美国国立卫生研究院授予AI027742）。

1。质粒DNA纯化<ol><li>变换到DH5α 大肠杆菌感受态细胞的真核质粒DNA编码EGFP（PCX-EGFP）。空PCX载体也可以使用作为阴性对照。 </li><li>文化和收获DH5α 大肠杆菌细胞和纯化的质粒DNA根据到的Qiagen公司EndoFree的质粒纯化手册"。 </li><li>筛选pDNA的溶液通过0.22μm的PVDF无菌过滤器，并将其储存在-20℃直到使用。 </li></ol> 2。纹身系统准备<ol><li>手持式单元和控制踏板连接到电源单元，按照制造商的说明书。 </li><li>设置针的振荡频率约为100赫兹。我们选择了纹身的设备（隐形旋转纹身系统），表盘上的电源应设置为4伏。请参阅用户手册或其他纹身系统的技术支持。 </li><li>消毒针头AR使用光前。我们建议您使用一个针阵列的每个动物。用肥皂水清洗和高压灭菌后可重复使用的针。 </li><li>针阵列安装到手持设备和松散连接的塑料把手。 </li><li>针深度调整到一个适当的设置，通过向上或向下移动的塑料的抓地力。我们使用我们的小鼠实验深度大约为0.5毫米的针。 </li><li>正确设置时，进针深度，拧紧塑料把手和纹身系统是可以使用的。 </li></ol> 3。动物剃毛<ol><li>选择网站纹身的治疗。该网站应该是一个相对稳固，平坦的，和肉质部位的皮肤，例如，动物的后腿侧。 </li><li>与氯胺酮（90毫克/千克）和赛拉嗪（5毫克/公斤），根据动物的体重的混合物麻醉的BALB / c小鼠。对于其他动物，请参阅您的机构的麻醉指引。 </li><li>检查动物的反射，按捏它的脚，以确认动物是适当的麻醉。 </li><li>使用电剪修剪下来的头发，动物的后肢慢慢地，仔细地。从计划的纹身现场及周边地区的去除毛发。 </li><li>去除残留的短发的一次性使用的安全剃须刀。注意不要剪动物的皮肤。需要注意的是，可以使用脱毛膏脱毛作为一种替代方法;然而，我们宁愿剃须超过脱毛膏。 </li><li>删除杂散切下的毛发从皮肤如有必要，用压缩空气。 </li></ol> 4。质粒DNA的纹身交货<ol><li>如果先前已建立的免疫协议，剂量和要应用的DNA溶液的体积应据此计算。我们建议您使用尽可能小的体积为实际使纹身的过程中更易于管理。在这EGFP示范，我们使用7.5μlDNA溶液在CON对中的0.25毫克/毫升，施加到一个纹身面积约1厘米2。用于免疫的实验中，更高的质粒DNA浓度可能需要诱导强烈的免疫反应1,2。 </li><li>纹身的网站上移液直接应用DNA解决方案。可替换地，加载到塑料把手的DNA溶液通过移液，因此针和塑料把手的前端之间的液体悬浮。一个用于演示的视频。我们已观察到的两种方法之间没有明显的区别。然而，加载溶液倒入塑料把手可能是有利的垂直的皮肤表面上，例如。 </li><li>启动针振荡和放置针阵列与动物的皮肤纹身的站点上的压力的光。然后将振荡针轻轻地，慢慢地以线性方式提供质粒DNA在整个纹身区。维持一个90度角之间的针和皮肤上，以免皮肤割伤。 </li><li>观察皮肤。磨损和炎症是正常的。如果出血情况时，停止纹身的过程中，减少进针深度。 </li><li>继续针运动约1分钟，然后停止纹身的过程。 </li><li>用干净的棉签涂抹一层薄薄的局部止痛纹身的网站上。如磺胺嘧啶银乳膏（SSD霜）或抗生素软膏软膏外用止痛药可以帮助减轻疼痛或痛苦的动物，因此建议申请前的麻醉消退。第二天观察动物的疼痛或痛苦的步态变化，缺乏运动的迹象。如果仍然存在的迹象，重新应用止痛膏。 </li></ol> 5。确认抗原的表达<ol><li>纹身的治疗后48小时内（注意，不同的质粒DNA结构可能需要不同的表达时间），鼠标CO 2麻醉安乐死。对于其他动物安乐死的指引，你的机构。 </li><li>剖析在纹身的部位，并修复皮肤组织4％多聚甲醛4℃过夜。 </li><li>用70％乙醇冲洗组织，并将其转移到的1X PBS中，用于成像。 </li><li>检查整个组织使用激光共聚焦显微镜检查EGFP信号。另外，皮肤组织可以被嵌入在石蜡中，并在荧光显微镜下检查节。 </li></ol>

<ol>
	<li>Bins, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+potent+DNA+vaccination+strategy+defined+by+in+vivo+monitoring+of+antigen+expression.">A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression.</a> Nat Med. 11, 899-904 (2005).</li><li>Pokorna, D., Rubio, I., Muller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-vaccination+via+tattooing+induces+stronger+humoral+and+cellular+immune+responses+than+intramuscular+delivery+supported+by+molecular+adjuvants.">DNA-vaccination via tattooing induces stronger humoral and cellular immune responses than intramuscular delivery supported by molecular adjuvants.</a> Genet. Vaccines Ther. 6, 4 (2008).</li><li>van den Berg, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intradermal+vaccination+by+DNA+tattooing+in+human+skin.">Optimization of intradermal vaccination by DNA tattooing in human skin.</a> Hum. Gene Ther. 20, 181-189 (2009).</li><li>Liu, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccines:+a+review.">DNA vaccines: a review.</a> J. Intern. Med. 253, 402-410 (2003).</li><li>Koide, Y., Nagata, T., Yoshida, A., Uchijima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccines.">DNA vaccines.</a> Jpn. J. Pharmacol. 83, 167-174 (2000).</li><li>Peachman, K. K., Rao, M., Alving, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+DNA+through+the+skin.">Immunization with DNA through the skin.</a> Methods. 31, 232-242 (2003).</li><li>Verstrepen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+HIV-1+specific+T-cell+responses+by+short-interval+DNA+tattooing+as+compared+to+intramuscular+immunization+in+non-human+primates.">Improved HIV-1 specific T-cell responses by short-interval DNA tattooing as compared to intramuscular immunization in non-human primates.</a> Vaccine. 26, 3346-3351 (2008).</li><li>Potthoff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+and+efficacy+of+intradermal+tattoo+immunization+with+adenoviral+vector+vaccines.">Immunogenicity and efficacy of intradermal tattoo immunization with adenoviral vector vaccines.</a> Vaccine. 27, 2768-2774 (2009).</li><li>Quaak, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+tattoo+vaccination:+effect+on+plasmid+purity+and+transfection+efficiency+of+different+topoisoforms.">DNA tattoo vaccination: effect on plasmid purity and transfection efficiency of different topoisoforms.</a> J. Control Release. 139, 153-159 (2009).</li><li>Pokorna, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+with+human+papillomavirus+type+16-derived+peptides+using+a+tattoo+device.">Vaccination with human papillomavirus type 16-derived peptides using a tattoo device.</a> Vaccine. 27, 3519-3529 (2009).</li></ol>

没有利益冲突的声明。

DNA疫苗比传统的疫苗接种策略，被认为是更安全，因为它不要求操作，或暴露疫苗，活的或减毒的病原体。然而，DNA疫苗接种的结果，在很大程度上依赖于配送路线。皮肤是丰富的抗原呈递细胞，如朗格汉斯细胞和树突状细胞1，从而在一个理想的站点用于免疫的免疫原性和易于访问5,6。其结果是，皮内接种策略是用于DNA疫苗的最流行的选择之一。在这个视频中，DNA纹身是一个简?...

<table border="1"><tbody><tr><td>的试剂/材料名称</td><td>公司</td><td>目录编号</td><td>评论</td></tr><tr><td> PCX-EGFP质粒DNA </td><td> Clontech公司</td><td></td><td></td></tr><tr><td> STEALTH旋转纹身系统</td><td>全球纹身器材</td><td> STEALTH-L </td><td></td></tr><tr><td>纹身针</td><td>全球纹身器材</td><td> 1207RSB </td><td></td></tr><tr><td> EndoFree质粒试剂盒</td><td> Qiagen公司</td><td> 12362 </td><td></td></tr><tr><td> 0.22微米PVDF无菌过滤器</td><td>密理博</td><td> SLGV013SL </td><td></td></tr><tr><td>电发剪</td><td>市售</td><td></td><td></td></tr><tr><td>一次性使用的安全剃须刀</td><td>市售</td><td></td><td></td></tr><tr><td>银磺胺嘧啶霜</td><td>沃森</td><td> NDC 0591-0810-55 </td><td></td></tr><tr><td>氯胺酮HCL </td><td></td><td> NDC 0856-2012-01 </td><td></td></tr><tr><td> Zylazine无菌溶液</td><td></td><td> NADA 139-236 </td><td></td></tr></tbody></table>

skin tattooing as a novel approach for dna vaccine delivery

基于核酸的疫苗接种是一个主题的日益增长的兴趣，特别是质粒DNA（pDNA的）编码免疫学上重要的抗原。工程质粒DNA疫苗给药后，它被转录和翻译成免疫原的蛋白质，可以从免疫系统引起的反应。提供DNA疫苗的方法很多被调查，但每次送货路线都有自己的优点和缺陷。皮肤纹身是一种新的技术是安全的，符合成本效益，方便的。此外，穿刺针造成，也可以作为一种有效的佐剂。在这里，我们a）表现出增强型绿色荧光蛋白的质粒DNA编码的皮内交付（PCX-EGFP）在小鼠模​​型中，使用纹身的移动设备和b）在皮肤细胞中，使用共聚焦显微镜，确认有效的EGFP表达。

在小鼠皮肤细胞中可以观察到在488nm和发射峰在509 nm处的激发峰的EGFP的表达。从1.875μg剂量的DNA，含有约3×10 17个拷贝的质粒，我们通常观察到10-20的EGFP信号在1厘米2纹身面积。这种相对较低的转染细胞的数量是先前的研究3的结果相一致。 EGFP表达（ 图1）提供的证据表明，EGFP质粒被输送到动物的皮肤细胞，使用的DNA纹身技术。 <p class="jove_content" fo:keep-togethe...

Watch this Scientific Journal Video about Skin Tattooing As A Novel Approach For DNA Vaccine Delivery at JoVE.com

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

皮肤纹身是一个强大的和安全的方式来传递DNA疫苗皮内。在这里，一个DNA质粒EGFP交付纹身实验室小鼠的皮肤，和EGFP在皮肤细胞中的表达，然后通过激光共聚焦显微镜检查。

皮肤纹身作为一种新的方法DNA疫苗交货

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

skin-tattooing-as-a-novel-approach-for-dna-vaccine-delivery

Research

JoVE Journal

Bioengineering

15.8K 次观看.  New York University School of Medicine. 皮肤纹身是一个强大的和安全的方式来传递DNA疫苗皮内。在这里，一个DNA质粒EGFP交付纹身实验室小鼠的皮肤，和EGFP在皮肤细胞中的表达，然后通过激光共聚焦显微镜检查。

视频: Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

科研

生物工程

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. ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

烟草中瞬时表达的蛋白作为一个案例研究:复杂系统采用的实验方法设计的表征

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

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 ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.