وأيد هذا العمل من قبل جبهة الخلاص الوطني منح RUI MCB-0950372 و 1105310 DMR-بعنوان &quot;التوصيف الجزيئي من الحرائر عنكبوت الأرملة السوداء، والسلوك الميكانيكي للالحرائر الغراء العنكبوت&quot;، على التوالي.

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الإعلان عن أي تضارب في المصالح.

ألياف تركيبية من نسج هذه المنهجية هي ميكانيكيا على نفس الترتيب من حيث الحجم مقارنة مع الألياف الطبيعية. من خلال خفض كمية من خطأ بشري من قبل ميكنة التخزين المؤقت وعمليات السحب آخر زيادة ونقصان، والاختلاف بين العينات التجريبية هي أكثر رقابة وتقلص إلى حد كبير. <p class="jov...

حرير العنكبوت لديه الخصائص الميكانيكية غير العادية التي يقوم بها مواد من صنع الإنسان عدة، بما فيها عالية الشد الصلب، كيفلر والنايلون. 1 العناكب تدور على الأقل 6-7 أنواع الألياف المختلفة التي تعرض الخواص الميكانيكية المختلفة، تم تصميم كل منها مع كميات متفاوتة من الشد والتمدد لتنفيذ مهام محددة البيولوجية. العلماء البحث 2 يسعون بسرعة استخدام الحرير العنكبوت كما الحيوية الجيل القادم بسبب خصائصها الميكانيكية المعلقة، توافق مع الحياة الخاصة بهم، وطبيعتها غير السامة والمواد الخضراء. 3،4 بسبب أكل لحوم البشر و طبيعة السامة من العناكب، حصاد الحرير العنكبوت من خلال الزراعة ليست استراتيجية عملية لتلبية المطالب اللازمة للتصنيع على نطاق صناعي. لذلك، تحولت العلماء الى انتاج بروتينات الحرير المؤتلف في الكائنات المعدلة وراثيا في المختبر إلى جانب الغزل من ألياف تركيبية منحد ذاتها تنقية البروتينات. وكان 5-8 التعبير عن كامل مدة المؤتلف بروتينات حرير العنكبوت من الصعب من الناحية الفنية نظرا للخصائص الذاتية للتسلسل الجيني، والتي تشمل طبيعتها المتكررة جدا وأطوال المادية (&gt; 15 كيلوبايت)، GC-غنية المحتوى ومنحاز ألانين وجليكاين كودون الاستخدام. 9-11 وحتى الآن، ركزت معظم المختبرات في التعبير عن أشكال اقتطاعها من البروتينات أمبولي الشكل الحرير الرئيسية MaSp1 أو MaSp2 باستخدام تسلسل [كدنا] جزئي أو الجينات الاصطناعية. 12-15 غزل الحرير الاصطناعي العنكبوت هي عملية صعبة تتطلب ولم يتم التمكن والمعرفة في عدة تخصصات علمية، وتعقيدات عملية الغزل وكشف تماما لعامة الناس عن طريق التمثيل الفيديو. في الواقع، سوى عدد قليل من المختبرات في جميع أنحاء العالم لديهم الخبرة للتعبير عن cDNAs حرير العنكبوت، تنقية بروتينات الحرير، وتدور الألياف الاصطناعية، وتأدية ما بعد تدور التعادل، ثم أخيرا اختبار خواصها مادة بيولوجية. 8،16،17 مناهج مختلفة للألياف تركيبية الغزل وشملت الغزل الرطب والجاف وكذلك أساليب electrospinning 16،18،19 جميع الإجراءات لديها هدف واحد مشترك - وضع بروتوكول الاصطناعية التي تنتج حرير العنكبوت مع الخواص الميكانيكية التي الطبيعية المواضيع منافس على نطاق واسع عمليات التصنيع التجاري. نحن هنا وصف الإجراء لتوليد الحرير العنكبوت الاصطناعي على نطاق المختبر باستخدام منهجية الرطب الغزل. بالنسبة لأساليب الغزل وغيرها، وقد أنتجت الغزل الرطب أكثر النتائج متسقة لتحليل الألياف. نحن الخطوط العريضة بداية هذا الإجراء مع التعبير عن بروتينات الحرير المؤتلف في البكتيريا، تليها تنقية بهم، ومن ثم وصف الخطوات إعداد بروتين للغزل، بما في ذلك منهجية التعادل بعد تدور تطبق على الألياف &quot;، كما نسج&quot; يمكن أن ينتج عن المواضيع مع خصائص المواد التي تقترب من جودة الحرير العنكبوت الطبيعية. لدينا منهجيهتم تصميم Y لمحاكاة عن كثب عملية طبيعية غزل من ألياف الحرير، وأنه يعتمد بشدة على خبرتنا في الهندسة المعمارية وظيفة من وظائف الغدد المنتجة للحرير من محجر العين، وقطعة خبز نسيج العنكبوت. 20-22 وعلاوة على ذلك، فإننا نستنتج مع ما يلزم من خطوات لتحديد خصائص المواد من الألياف الاصطناعية باستخدام مقياس التوتر لرسم منحنيات الإجهاد والانفعال، والتي تسمح للمحققين لحساب قوة في نهاية المطاف، السلالة في نهاية المطاف، وصلابة من الألياف. وأخيرا، ولكن ذات قيمة كبيرة، لا يمكن للأجهزة الغزل، التخزين المؤقت، ورسم يكون في المنزل التي تم إنشاؤها باستخدام أجزاء المتاحة تجاريا، بدلا من شراء معدات مخصصة متقنة ومكلفة.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> كاشف / معدات </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> pBAD / TOPO كيت ThioFusion التعبير </td><td> إينفيتروجن </td><td> K370-01 </td><td></td></tr><tr><td> الكاشف FastBreak تحلل الخلية، 10X </td><td> Promega </td><td> V857C </td><td></td></tr><tr><td> NI-NTA الاغاروز </td><td> QIAGEN </td><td> 30210 </td><td> وتشمل تعليمات للمخازن </td></tr><tr><td> ProteoSilver الفضة عدة اللطخة </td><td> سيغما الدريخ </td><td> PROTSIL1-1KT </td><td></td></tr><tr><td> المناطق الحرة Lyophilizer </td><td> Labconco </td><td> 7960041 </td><td> المناطق الحرة 12Plus </td></tr><tr><td> Hexafluoroisopropanol (HFIP) </td><td> سيغما الدريخ </td><td> 52512 </td><td></td></tr><tr><td> محقنة </TD&gt; <td> هاميلتون </td><td> 7657-01 </td><td> 250 ميكرولتر </td></tr><tr><td> إبرة </td><td> هاميلتون </td><td> 7780-01 </td><td> 26S المقياس، بلانت إبرة قابل للنقل نهاية </td></tr><tr><td> المحاقن مضخة </td><td> هارفارد جهاز </td><td> 702208 </td><td> 11Plus </td></tr><tr><td> الفرجار الرقمية </td><td> كاريرا </td><td> CP5906 </td><td> 0-150 ملم نطاق </td></tr><tr><td> الفولاذ المقاوم للصدأ ملقط </td><td> الآلات الدقيقة العالم </td><td> 501764 </td><td> مصغرة دومون # M5S </td></tr><tr><td> محرك </td><td> طبيعة طاحونة </td><td> 7090529 </td><td> 12VDC، وسرعة دورة في الدقيقة 2 </td></tr><tr><td> خطي المحرك </td><td> نذير الكهربائية </td><td> 01-D024-0050-A06-LP-IP65 </td><td> 24VDC، مجموعة 6 بوصة </td></tr><tr><td> تشريح المجهر </td><td> لايكا مايكروسيستمز </td><td> لايكا MZ16 </td><td></td></tr><tr><td> الرقمية microscمكتب مستشار رئيس الوزراء كاميرا </td><td> لايكا مايكروسيستمز </td><td> DFC320 </td><td> البرنامج: لايكا تطبيق جناح v2.8.1 </td></tr><tr><td> Vannas مقص </td><td> الآلات الدقيقة العالم </td><td> 500260 </td><td></td></tr><tr><td> Microtensometer </td><td> أورورا العلمية </td><td> 310C </td><td> 5N وضع مزدوج نظام </td></tr></tbody></table>

synthetic spider silk production on a laboratory scale

كما تقدم المجتمع، وأصبحت أكثر ندرة الموارد، يصبح من المهم على نحو متزايد لزراعة التكنولوجيات الجديدة التي الحيوية مهندس الجيل القادم مع خصائص عالية الأداء. يجب تطوير مواد هذه الهيكلية الجديدة ستكون سريعة وفعالة من حيث التكلفة وتنطوي على منهجيات المعالجة والمنتجات التي هي صديقة للبيئة والتنمية المستدامة. العناكب تدور العديد من أنواع مختلفة من الألياف مع الخواص الميكانيكية المختلفة، وتقدم مصدرا غنيا للجيل المقبل المواد الهندسية لتقليد الطبيعة أن المنافس أفضل المواد الاصطناعية والطبيعية. منذ جمع كميات كبيرة من حرير العنكبوت الطبيعية أمر غير عملي، الاصطناعية إنتاج الحرير لديه القدرة على توفير العلماء من أجل الحصول على امدادات غير محدودة من المواضيع. ولذلك، إذا كانت عملية الغزل ويمكن أن تكون مبسطة والكمال، والألياف الاصطناعية العنكبوت لديهم إمكانية استخدام لمجموعة واسعة من التطبيقات التي تتراوح بين الدروع الواقية للبدن، خياطة جراحيةق والحبال والكابلات، والإطارات، وسلاسل للآلات الموسيقية، والمواد المركبة للطيران وتكنولوجيا الطيران. وضعنا من أجل المضي قدما في إنتاج حرير صناعي وعملية لانتاج الألياف التي تعرض التباين المنخفض في خصائص المواد الخاصة بهم من زيادة ونقصان لزيادة ونقصان، وبروتوكول الرطب الغزل يدمج التعبير عن بروتينات حرير العنكبوت المؤتلف في تنقية والبكتيريا وتركيز البروتينات ، تليها قذف الألياف ومعالجة ميكانيكية في مرحلة ما بعد زيادة ونقصان. وهذا هو أول تمثيل المرئية التي تكشف عن عملية خطوة بخطوة لزيادة ونقصان وتحليل ألياف الحرير الصناعي على نطاق المختبر. كما يوفر تفاصيل لتقليل اعتماد التباين بين ألياف نسج من مخدر الغزل نفسه. بشكل جماعي، وهذه الأساليب دفع عملية إنتاج الحرير الصناعي، مما أدى إلى ألياف عالية الجودة التي تفوق العنكبوت الحرير الطبيعي.

Watch this Scientific Journal Video about Synthetic Spider Silk Production on a Laboratory Scale at JoVE.com

Synthetic Spider Silk Production on a Laboratory Scale

على الرغم من الخصائص المتميزة الميكانيكية والكيميائية الحيوية للعنكبوت الحرير، لا يمكن أن تحصد هذه المواد بكميات كبيرة بالوسائل التقليدية. نحن هنا وصف استراتيجية فعالة لزيادة ونقصان ألياف اصطناعية حرير العنكبوت، والتي هي عملية هامة بالنسبة للمحققين الذين يدرسون العنكبوت إنتاج الحرير واستخدامها على النحو الجيل المقبل من المواد الحيوية.

الاصطناعية إنتاج حرير العنكبوت على نطاق المختبر

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

synthetic-spider-silk-production-on-a-laboratory-scale

Research

JoVE Journal

Bioengineering

26.6K Views.  University of the Pacific. على الرغم من الخصائص المتميزة الميكانيكية والكيميائية الحيوية للعنكبوت الحرير، لا يمكن أن تحصد هذه المواد بكميات كبيرة بالوسائل التقليدية. نحن هنا وصف استراتيجية فعالة لزيادة ونقصان ألياف اصطناعية حرير العنكبوت، والتي هي عملية هامة بالنسبة للمحققين الذين يد?...

Video: Synthetic Spider Silk Production on a Laboratory Scale

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly &alpha;-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable &beta;-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.

Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.

تصفية الهواء بما فى ذلك الأجهزة تنسجم محبوكة من Electrospun المؤتلف بروتينات حرير العنكبوت

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

تصميم بروتين الحرير الحرير المواد سبيكة للتطبيقات الطبية الحيوية

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

الإنتاج واستهداف الأحادي التكافؤ الكم النقاط

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

الاستنساخ واسع النطاق إنتاج ذات قدرة عالية ناقلات الفيروسة الغدانية استنادا إلى نوع اتش 5 الإنسان

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

استنادا الحرير الكولاجين المهندسة 3D نموذج الاستقطاب العصبية الأنسجة

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

قياسات الطاقة الإدخال في المفاعلات الحيوية المقلبة في المختبرات

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

تشكيل المواد من الحرير العنكبوت المؤتلف من خلال الإفراز المائي باستخدام الحرارة والضغط

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

الإنتاج الآلي على نطاق واسع من الخلايا الجذعية المشتقة من الدهون كروية للطباعة الحيوية 3D

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

إنتاج نظام مغلق للخلايا الملتصقة على أساس ناقلات دقيقة قابلة للذوبان

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To address the primary shortcomings of the two-dimensional (2D) planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on a GMP-grade, dissolvable, and porous microcarrier for the 3D culture of adherent cells, including human mesenchymal stem/stromal cells (hMSCs), HEK293T cells, and Vero cells. To achieve large-scale expansion, a two-stage expansion was conducted with 5 L and 15 L stirred-tank bioreactors to yield 1.1 x 1010 hMSCs with an overall 128-fold expansion within 9 days. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous-flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Compared with 2D planar culture, there are no significant differences in the quality of hMSCs harvested from 3D culture. We have also applied these dissolvable porous microcarriers to other popular cell types in the CGT sector; specifically, HEK293T cells and Vero cells have been cultivated to peak cell densities of 1.68 x 107 cells/mL and 1.08 x 107 cells/mL, respectively. This study provides a protocol for using a bioprocess engineering platform harnessing the characteristics of GMP-grade dissolvable microcarriers and advanced closed equipment to achieve the industrial-scale manufacturing of adherent cells.

تسليط الضوء على المؤلف: تطوير تصنيع العلاج بالخلايا باستخدام ناقلات دقيقة قابلة للذوبان 

Here, we present a protocol to achieve the large-scale manufacturing of adherent cells through a fully closed system based on GMP-grade dissolvable microcarriers. The cultivation of human mesenchymal stem cells, HEK293T cells, and Vero cells was validated and met both quantity demands and quality criteria for the cell and gene therapy industry.

إنتاج الخلايا على نطاق واسع على أساس الناقلات الدقيقة المسامية القابلة للذوبان من الدرجة GMP

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To ...