نود أن نشكر جميع أعضاء مختبر هونغ يان والدكتور دنغ في مكتب المجهري، العلوم الأساسية التعاونية، NYUMC على مساعدتهم وتقديم الدعم التقني. وأيد هذا العمل من خلال منحة التجريبية من مركز جامعة نيويورك للبحوث الإيدز (CFAR، NIH منحة AI027742).

1. بلازميد الحمض النووي تنقية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحويل البلازميد DNA حقيقية النواة ترميز EGFP (PCX-EGFP) في الخلايا القولونية DH5α المختصة. ويمكن أيضا للناقلات الفارغة PCX استخدامها كعنصر تحكم سلبية. </li><li style=";text-align:right;direction:rtl"> الثقافة وحصاد الخلايا القولونية DH5α وتنقية PDNA وفقا لكتيب EndoFree QIAGEN تنقية البلازميد. </li><li style=";text-align:right;direction:rtl"> مرشح PDNA حل من خلال 0،22 ميكرون تصفية العقيمة PVDF، وتخزينها في -20 درجة مئوية حتى الاستخدام. </li></ol> 2. الوشم نظام إعداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> توصيل الوحدة المحمولة ودواسة التحكم إلى وحدة التزويد بالطاقة في إرشادات الشركة المصنعة. </li><li style=";text-align:right;direction:rtl"> تعيين تيرة التذبذب الإبرة إلى ما يقرب من 100 هرتز. للجهاز الوشم اخترنا (نظام الشبح الوشم الروتاري)، يجب تعيين الطلب على إمدادات الطاقة إلى 4 فولت. الرجوع إلى دليل المستخدم أو الدعم الفني لأنظمة الوشم الأخرى. </li><li style=";text-align:right;direction:rtl"> تعقيم الإبرة عراي قبل الاستخدام. نوصي باستخدام أحد مجموعة إبرة لكل حيوان. يمكن إعادة استخدامها الإبر بعد تطهيرها بالماء والصابون وتعقيمها. </li><li style=";text-align:right;direction:rtl"> تثبيت مجموعة إبرة في يده وحدة وإرفاق فضفاضة قبضة بلاستيكية. </li><li style=";text-align:right;direction:rtl"> ضبط عمق الإبرة إلى الإعداد المناسب عن طريق نقل قبضة بلاستيكية أعلى أو لأسفل. كنا على عمق الإبرة حوالي 0.5 مم لدينا تجربة الماوس. </li><li style=";text-align:right;direction:rtl"> تشديد قبضة بلاستيكية عندما يتم تعيين بشكل صحيح عمق الإبرة، ونظام الوشم هو جاهز للاستخدام. </li></ol> 3. حلاقة الحيوان <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اختيار موقع لعلاج الوشم. يجب أن يكون الموقع في منطقة شركة نسبيا، شقة، وحمي من الجلد، على سبيل المثال، إلى جانب hindleg الحيوان. </li><li style=";text-align:right;direction:rtl"> تخدير ماوس BALB / ج مع خليط من الكيتامين (90 ملغ / كلغ) وزيلازين (5 ملغ / كلغ) وفقا لوزن جسم الحيوان. لحيوانات أخرى، الرجوع إلى المبادئ التوجيهية التخدير من مؤسستكم. </li><li style=";text-align:right;direction:rtl"> تحقق منردود الفعل من قبل الحيوان معسر قدم لها للتأكد من الحيوان للتخدير بشكل صحيح. </li><li style=";text-align:right;direction:rtl"> استخدام الانتهازي الكهربائية لخفض الشعر في hindlegs الحيوان ببطء وحذر. إزالة الشعر من موقع المخطط والوشم والمنطقة المحيطة بها. </li><li style=";text-align:right;direction:rtl"> إزالة الشعر أقصر المتبقية مع الحلاقة سلامة التخلص منها. يجب الحرص على عدم قطع الجلد الحيوان. ملاحظة التي يمكن أن تستخدم كريم مزيل الشعر كطريقة بديلة إزالة الشعر، ولكن، ونحن نفضل كريم الحلاقة على مزيل الشعر. </li><li style=";text-align:right;direction:rtl"> إزالة الشعر طائشة قطع من الجلد مع الهواء المضغوط إذا لزم الأمر. </li></ol> 4. تسليم DNA البلازميد بواسطة الوشم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إذا كان قد تم إنشاء بروتوكول التحصين سابقا، ينبغي أن تحسب الجرعة وحجم حل DNA ليتم تطبيقها وفقا لذلك. نوصي باستخدام وحجم صغير ممكن عمليا لجعل عملية الوشم أكثر قابلية للإدارة. في هذه المظاهرة EGFP، استخدمنا 7،5 ميكرولتر من محلول الحمض النووي في يخدعوحدانية من 0.25 ملغ / مل، والتي تم تطبيقها على منطقة الوشم من حوالي 1 سم 2. التحصين للتجارب، قد يكون مطلوبا تركيزات أعلى PDNA للحث على الاستجابات المناعية قوية 1،2. </li><li style=";text-align:right;direction:rtl"> تطبيق حل DNA من قبل pipetting مباشرة على موقع الوشم. بدلا من ذلك، تحميل حل DNA في قبضة بلاستيكية بواسطة pipetting، لذلك تم تعليق السائل بين الإبرة وغيض من قبضة بلاستيكية. الرجوع إلى الفيديو لمظاهرة. وقد لاحظنا يوجد فرق ملحوظ بين الطريقتين. ومع ذلك، قد حل في تحميل قبضة بلاستيكية يكون من المفيد على السطوح العمودية الجلد، على سبيل المثال. </li><li style=";text-align:right;direction:rtl"> بدء التذبذب الإبرة ووضع مجموعة الإبرة مع الضغط الخفيف على جلد الحيوان في موقع الوشم. ثم نقل إبرة تتأرجح بلطف وببطء بطريقة خطية لإيصال الحمض النووي البلازميد فوق منطقة الوشم بالكامل. الحفاظ على زاوية 90 درجة بين الإبرة والجلد لتجنبجروح على الجلد. </li><li style=";text-align:right;direction:rtl"> مراقبة الجلد. الكشط والتهاب طبيعية. إذا كان النزيف يحدث، ووقف عملية الوشم وتقلل من عمق الإبرة. </li><li style=";text-align:right;direction:rtl"> تواصل حركة إبرة لحوالي 1 دقيقة، ثم إيقاف عملية الوشم. </li><li style=";text-align:right;direction:rtl"> استخدام مسحة قطنية نظيفة لتطبيق طبقة رقيقة من مسكن موضعي على موقع الوشم. يمكن المسكنات الموضعية مثل كريم سلفاديازين الفضة (SSD كريم) أو مرهم Neosporin تساعد على التخفيف من الألم أو الضيق من الحيوانات، وبالتالي ينصح تطبيقها قبل التخدير يلبس. مراقبة الحيوانات في اليوم التالي بحثا عن علامات على الألم أو الضيق (تغيير من الخمول والمشية). تطبيق كريم مسكن إذا علامات لا تزال قائمة. </li></ol> 5. تأكيد التعبير مستضد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 48 ساعة بعد العلاج الوشم (لاحظ أن يبني PDNA مختلفة قد تتطلب أوقات مختلفة التعبير)، والموت الرحيم الماوس من الخدر 2 CO. لأخرىالحيوانات، تشير إلى المبادئ التوجيهية القتل الرحيم من مؤسسة الخاصة بك. </li><li style=";text-align:right;direction:rtl"> تشريح الجلد في موقع الوشم، وإصلاح الأنسجة في بارافورمالدهيد 4٪ بين عشية وضحاها في 4 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> شطف الأنسجة مع الايثانول 70٪، وتحويلها إلى PBS 1X للتصوير. </li><li style=";text-align:right;direction:rtl"> دراسة الأنسجة كله باستخدام مجهر مبائر للتحقق من الإشارات EGFP. بدلا من ذلك، تكون جزءا لا يتجزأ من نسيج الجلد في شمع البارافين ودرست في أقسام تحت المجهر الفلورسنت. </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>

الإعلان عن أي تضارب في المصالح.

ويعتبر التطعيم DNA أكثر أمانا من استراتيجيات التطعيم التقليدية لأنها لا تتطلب التلاعب، أو فضح لقاحات ومسببات الأمراض الموهنة الحية أو 4. ومع ذلك، ونتيجة للتطعيم DNA يعتمد اعتمادا كبيرا على طريق التسليم. الجلد هو وفرة في الخلايا مستضد تقديم مثل خلايا لانغرهانس وال...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف / المواد </td><td> شركة </td><td> كتالوج رقم </td><td> تعليقات </td></tr><tr><td> PCX-DNA البلازميد EGFP </td><td> Clontech </td><td></td><td></td></tr><tr><td> نظام STEALTH الوشم الروتاري </td><td> في جميع أنحاء العالم الوشم التموين </td><td> STEALTH-L </td><td></td></tr><tr><td> الوشم والإبر </td><td> في جميع أنحاء العالم الوشم التموين </td><td> 1207RSB </td><td></td></tr><tr><td> EndoFree كيت ماكسي البلازميد </td><td> QIAGEN </td><td> 12362 </td><td></td></tr><tr><td> 0،22 ميكرون تصفية العقيمة PVDF </td><td> ميليبور </td><td> SLGV013SL </td><td></td></tr><tr><td> الشعر الانتهازي الكهربائية </td><td> المتاحة تجاريا </td><td></td><td></td></tr><tr><td> يمكن التخلص منها سلامة شفرات الحلاقة </td><td> المتاحة تجاريا </td><td></td><td></td></tr><tr><td> فضةسلفاديازين كريم </td><td> واتسون </td><td> NDC 0591-0810-55 </td><td></td></tr><tr><td> الكيتامين HCL </td><td></td><td> NDC 0856-2012-01 </td><td></td></tr><tr><td> محلول معقم Zylazine </td><td></td><td> NADA 139-236 </td><td></td></tr></tbody></table>

skin tattooing as a novel approach for dna vaccine delivery

الحمض النووي القائم على التطعيم هو موضوع اهتمام متزايد، وخاصة DNA البلازميد (PDNA) ترميز المستضدات المناعية الهامة. بعد إدارة PDNA هندسيا للقاحات، هو كتب وترجم إلى البروتينات التي يمكن أن مستمنع الحصول على ردود من الجهاز المناعي. وقد تم التحقيق في العديد من الطرق لإيصال اللقاحات DNA، ولكن كل طريق تسليم مزاياه الخاصة والمزالق. الوشم الجلد هو أسلوب الرواية التي هي آمنة، فعالة من حيث التكلفة، ومريحة. وبالإضافة إلى ذلك، يمكن للثقوب من جراء إبرة بمثابة مساعد قوية. هنا، ونحن أ) إثبات الدفع من داخل الأدمة ترميز الحمض النووي البلازميد تعزيز الفلورسنت الأخضر البروتين (PCX-EGFP) في نموذج الفأر باستخدام جهاز الوشم وب) تأكيد التعبير الفعال للEGFP في خلايا الجلد باستخدام المجهر متحد البؤر.

ويمكن ملاحظة التعبير عن EGFP مع ذروة الإثارة في 488 نانومتر والتي تبلغ 509 ذروة الانبعاثات نانومتر في خلايا الجلد الماوس. من جرعة ميكروغرام من الحمض النووي 1،875، تحتوي على ما يقرب من 3 × 10 17 نسخ من البلازميد، لاحظنا عادة إشارات EGFP 10-20 سم في 1 2 وشم المنطقة. هذا العد?...

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

الوشم الجلد هو وسيلة فعالة وآمنة لقاح الحمض النووي تسليم intradermally. هنا، يتم تسليم EGFP DNA البلازميد الترميز عن طريق الوشم على الجلد على فأرة الحاسوب المختبر، ويتم تفتيش ثم التعبير عن EGFP في خلايا الجلد عن طريق الفحص المجهري متحد البؤر.

الجلد الوشم ونهجا جديدا للتسليم اللقاحات DNA

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 Views.  New York University School of Medicine. الوشم الجلد هو وسيلة فعالة وآمنة لقاح الحمض النووي تسليم intradermally. هنا، يتم تسليم EGFP DNA البلازميد الترميز عن طريق الوشم على الجلد على فأرة الحاسوب المختبر، ويتم تفتيش ثم التعبير عن EGFP في خلايا الجلد عن طريق الفحص المجهري متحد البؤر.

Video: 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.

الضغط خلية باعتبارها قوية، ميكروفلويديك بين الخلايا توصيل منصة

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

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.

طريقة محسنة لإعداد النوع الأول الكولاجين من الجلد

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.

توصيف النظم المعقدة باستخدام تصميم التجارب النهج: عابر التعبير البروتين في التبغ كدراسة حالة

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.

جلد كامل عيب نموذج في تقييم الأوعية الدموية من الحيوية<em&gt; في فيفو</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.

الميكروسكيل Electroporator لتوصيل متسلسل الجزيئية بمساعدة دوامة

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.

تطوير عضوي نموذج متعددة الخلايا ثلاثي الأبعاد من الأمعاء الغشاء المخاطي الإنسان نمت في ظل الجاذبية

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.

الوسيطة اللحمية خلية الثقافة و التوصيل في حالة ذاتي: مقاربة ذكية لتطبيقات العظام

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.

النانومترية الحمض النووي كأداة مرنة لدراسة البوليمرات سيميفليكسيبلي

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.

بيوبرينتينج الغضاريف والجلد الأنسجة النظير استخدام سلبية رواية خلط وحدة تقنية بريسيلولاريزيشن بيوينك

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.