我々は彼らの支援と技術的なサポートのための共同研究、NYUMCの顕微鏡コア、オフィスで香港ラボ博士とヤン鄧小のすべてのメンバーに感謝したいと思います。この作品は、エイズ研究のためニューヨーク大学センター（CFAR、NIHの助成金AI027742）からパイロット助成金によって支えられている。

1。プラスミドDNA精製<ol><li> 大腸菌 DH5αコンピテントセルに真核生物のプラスミドDNAエンコードEGFP（PCX-EGFP）を変換します。空のPCXベクトルもまたネガティブコントロールとして使用することもできる。 </li><li>文化や収穫DH5α 大腸菌細胞およびQiagen EndoFreeプラスミド精製ハンドブックによるとpDNAを清める。 </li><li>フィルタのpDNA0.22μmのPVDFの滅菌フィルターを通して溶液、使用するまで-20℃で保管してください。 </li></ol> 2。タトゥーシステム準備<ol><li>メーカーの指示に従って電源ユニットにハンドヘルドユニットとコントロール·ペダルを接続します。 </li><li>約100 Hzに針発振周波数を設定します。我々が選んだの入れ墨装置（ステルスロータリータトゥーシステム）については、電源装置上のダイヤルは4ボルトに設定する必要があります。ユーザー·マニュアルまたは他の刺青システムのテクニカルサポートを参照してください。 </li><li>針ARを殺菌使用前検査。私たちは、動物あたり1針アレイの使用をお勧めします。針は、石鹸水で洗浄し、オートクレーブ処理された後に再使用することができます。 </li><li>ハンドヘルドユニットに針アレイをインストールして、緩くプラスチックグリップを取り付けます。 </li><li>上下プラスチックグリップを移動することによって適切な設定に針の深さを調整します。我々は、マウスの実験では約0.5mmの針の深さを使用していました。 </li><li>針の深さが適切に設定されているプラ​​スチック製のグリップを締めて、刺青システムを使用する準備ができている。 </li></ol> 3。動物シェービング<ol><li>刺青治療のためのサイトを選択してください。このサイトは比較的、しっかり平らで、肉質の皮膚の領域、例えば、動物のhindlegの側でなければなりません。 </li><li>動物の体重によるとケタミン（90 mg / kg）およびキシラジン（5 mg / kg）の混合物を用いてBALB / cマウスを麻酔。他の動物の場合も、あなたの機関の麻酔のガイドラインを参照してください。 </li><li>チェック動物を確認するために、その足をつまんで動物の反​​射神経は正常に麻酔をかける。 </li><li>ゆっくりと慎重に、動物の後肢で髪をスリム化するために、電気トリマーを使用しています。計画的な刺青サイトおよびその周囲から毛を削除します。 </li><li>使い捨ての安全カミソリの残留短い髪を削除します。動物の皮膚を切らないように注意してください。脱毛クリームは、代替脱毛方法として使用されるかもしれないことに注意しますが、我々は、脱毛クリームの上にシェービング好む。 </li><li>必要に応じて圧縮空気で肌からの迷光カット毛を取り除きます。 </li></ol> 4。タトゥーによるプラスミドDNAの送達<ol><li>免疫化プロトコルが以前に確立されている場合は、適用されるべきDNA溶液の投与量とボリュームは、それに応じて計算されるべきである。私たちは、刺青のプロセスを管理しやすくするための実用的なような小さなボリュームとして使用することをお勧めします。このEGFPのデモでは、詐欺でDNA溶液7.5μlを使用約1cm 2の刺青面積上に塗布した後、0.25 mg / mlでのセンタリング。予防接種の実験のために、より高いpDNAの濃度が強い免疫応答1,2を誘導する必要がある場合があります。 </li><li>刺青のサイトに直接ピペッティングによりDNA溶液を適用します。あるいは、液体が針とプラスチックグリップの先端との間に中断されているので、ピペッティングによりプラスチックグリップにDNA溶液をロードします。デモ用のビデオを参照してください。私たちは2つの方法の間には顕著な差は認められなかった。しかし、プラスチックグリップに溶液をロードすると、たとえば、垂直皮膚表面上で有利であるかもしれません。 </li><li>針の振動を開始し、刺青のサイトで動物の皮膚に軽い圧力で針アレイを配置します。そして、全体の刺青領域上プラスミドDNAを提供するために直線的に優しく、ゆっくりと振動針を動かす。針とを避けるために皮膚との間に90度の角度を維持する皮膚に裂傷。 </li><li>肌を守ってください。摩耗や炎症は正常である。出血が発生した場合は、刺青のプロセスを停止し、針の深さを減少させる。 </li><li>約1分間、針の動きを続行し、入れ墨のプロセスを停止します。 </li><li>刺青のサイトに局所鎮痛剤の薄層を適用するためにきれいな綿棒を使用してください。スルファジアジン銀クリームSSD（クリーム）またはネオスポリン軟膏などの局所鎮痛薬は、動物の痛みや苦痛を軽減することができます、そして、したがって、それは麻酔が切れる前にそれらを適用することをお勧めします。痛みや苦痛の徴候（歩行の変化、非アクティブ）の翌日に動物を観察。徴候が持続する場合は、鎮痛クリームを再適用します。 </li></ol> 5。抗原発現の確認<ol><li>刺青治療（別のpDNAコンストラクトは異なる発現時間を必要とするかもしれないことに注意してください）48時間後に、マウスをCO 2麻酔により安楽死させています。他の用動物は、貴機関の安楽死のガイドラインを参照してください。 </li><li>刺青部位の皮膚を切開し、4℃で一晩、4％パラホルムアルデヒド中で組織を固定します。 </li><li> 70％エタノールを用いて組織をすすぎ、イメージングのための1×PBSにそれを転送します。 </li><li> EGFPの信号をチェックするために、共焦点顕微鏡を用いて組織全体を調べます。代わりに、皮膚組織をパラフィンワックスに埋め込まれており、蛍光顕微鏡下のセクションで調べることができます。 </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>

特別な利害関係は宣言されません。

それを操作する必要があり、または、ライブまたは弱毒病原体4にワクチンを公開していないとして、DNAワクチンは、従来のワクチン接種戦略よりも安全と考えられている。しかし、DNAワクチン接種の結果は送達経路に大きく依存します。皮膚は、ランゲルハンス細胞、樹状細胞1、従って、免疫原性およびアクセス5,6の容易さの点で予防接種のための理想的なサイト?...

<table border="1"><tbody><tr><td>試薬/材料の名称</td><td>会社</td><td>カタログ番号</td><td>注釈</td></tr><tr><td> PCX-EGFPプラスミドDNA </td><td>クロンテック</td><td></td><td></td></tr><tr><td> STEALTHロータリータトゥーシステム</td><td>ワールドワイドタトゥーサプライ</td><td> STEALTH-L </td><td></td></tr><tr><td>入れ墨の針</td><td>ワールドワイドタトゥーサプライ</td><td> 1207RSB </td><td></td></tr><tr><td> EndoFreeプラスミドマキシキット</td><td>キアゲン</td><td> 12362 </td><td></td></tr><tr><td> 0.22μmの滅菌フィルターPVDF </td><td>ミリポア</td><td> SLGV013SL </td><td></td></tr><tr><td>電気毛のトリマー</td><td>市販の</td><td></td><td></td></tr><tr><td>使い捨て安全カミソリ</td><td>市販の</td><td></td><td></td></tr><tr><td>シルバースルファジアジンクリーム</td><td>ワトソン</td><td> NDC 0591-0810-55 </td><td></td></tr><tr><td>ケタミンHCL </td><td></td><td> NDC 0856-2012-01 </td><td></td></tr><tr><td> Zylazine滅菌溶液</td><td></td><td> NADA 139から236 </td><td></td></tr></tbody></table>

skin tattooing as a novel approach for dna vaccine delivery

核酸ベースのワクチン接種は、成長、興味のあるトピック、免疫学的に重要な抗原をコードする、特にプラスミドDNA（pDNA比）です。人工のpDNAがワクチンに投与された後、それは転写され、免疫システムからの応答を誘発することができる免疫原性タンパク質に翻訳されています。 DNAワクチンを提供する多くの方法が研究されてきたが、各配信ルートは独自の利点と落とし穴があります。皮膚の入れ墨は、安全で費用対効果の高い、そして便利で斬新な手法です。また、針を招いパンクはまた強力なアジュバントとして役立つかもしれません。ここで、我々は）刺青デバイスを使用してマウスモデルにおけるプラスミドDNAのエンコーディング強化緑色蛍光タンパク質（PCX-EGFP）の皮内送達を実証し、b）の共焦点顕微鏡を用いた皮膚細胞におけるEGFPの効果的な発現を確認。

509 nmでの488nmおよび発光ピークの励起ピークとEGFPの発現は、マウスの皮膚細胞で観察することができます。 DNAの1.875μgの用量から、プラスミドの約3×10 17コピーを含む、我々は一般的に2エリアに入れ墨1センチメートルで10月20日EGFPの信号を観測しました。トランスフェクトされた細胞のこの比較的低い数は以前の研究3の結果と一致している。 EGFP発現（ 図1）?...

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

皮膚の入れ墨は、皮内送達DNAワクチンへの強力かつ安全な方法です。ここでは、DNAプラスミドエンコードEGFPは実験用マウスの皮膚に刺青で配信されており、皮膚細胞におけるEGFPの発現は、その後共焦点顕微鏡で検査されます。

DNAワクチンの送達のための新しいアプローチとしてタトゥー皮膚

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 ビュー.  New York University School of Medicine. 皮膚の入れ墨は、皮内送達DNAワクチンへの強力かつ安全な方法です。ここでは、DNAプラスミドエンコードEGFPは実験用マウスの皮膚に刺青で配信されており、皮膚細胞におけるEGFPの発現は、その後共焦点顕微鏡で検査されます。

ビデオ: 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.

堅牢、マイクロ流体細胞内送達プラットフォームとしての細胞絞る

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

生物工学

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.

皮膚からのI型コラーゲンの調製のための改良された方法

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.

ケーススタディとして、タバコにおける一過性タンパク質発現：実験アプローチのデザインを使用した複雑系のキャラクタリゼーション

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

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

生体材料の血管新生を評価するために、完全な皮膚欠損モデル<em&gt;インビボ</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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.

マイクロスケール渦アシストシーケンシャル分子送達用エレクトロポ

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

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.

微小重力下で増殖させたヒト腸粘膜の多細胞三次元器官型モデルの開発

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.

自家条件で間葉系幹細胞の培養および配信：整形外科アプリケーションのためのスマートなアプローチ

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.

セミフレキシブル高分子を研究する多目的ツールとして DNA ナノチューブ

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.

軟骨と皮膚組織類縁ミキシング ユニット Bioink Precellularization の小説のパッシブ バイオプリンティング

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.