This work was funded by a New Faculty Award II from the California Institute of Regenerative Medicine (CIRM; RN2-00921-1), NIH-funded National Research Award (F32-HL104924), and CIRM Training Grant (TG21163). Materials were provided by: Glycosan Biosystems Inc / BioTime and Dr. Stephen Badylak (University of Pittsburgh)

1. إنشاء لوحات pNIPAAM المغلفة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حل 2.6 غرام من pNIPAAM في 2 مل من / 40٪ محلول الهكسان 60٪ التولوين. </li><li style=";text-align:right;direction:rtl"> حرارة الخليط إلى 60 درجة مئوية لمدة 10 دقيقة أثار، حتى يتم حل pNIPAAM. </li><li style=";text-align:right;direction:rtl"> قطع ورق الترشيح في دائرة قطرها 60 ملم ورقة مكان في قمع بوخنر. </li><li style=";text-align:right;direction:rtl"> تصفية حل من خلال بوخنر القمع في وزنه قبل دورق زجاجي (لا تستخدم البلاستيك، والهكسان سوف تذوب البلاستيك). </li><li style=";text-align:right;direction:rtl"> ضع الكأس ومحتوياتها في فراغ جرس (24 رطل) O / N (16 ساعة). ملاحظة: حتى يتفاعل مع بقايا الآيزوبروبيل سيكون أكسدة لذلك تأكد من أنها لا تأتي في اتصال مع الأوكسجين. </li><li style=";text-align:right;direction:rtl"> تزن الكأس لتحديد وزن pNIPAAM. </li><li style=";text-align:right;direction:rtl"> إضافة ايزوبروبيل إلى pNIPAAM، وخلق 50/50 ث / ث الحل. </li><li style=";text-align:right;direction:rtl"> ضع 2 مل من محلول على سطح لوحة زراعة الأنسجة، ومعطف لمدة 5 دقائق تحت ضوء الأشعة فوق البنفسجية. </li><li style=";text-align:right;direction:rtl"> غسل لوحة مع 2 مل من برنامج تلفزيوني الدافئ مرتينقبل استخدام لثقافة الخلية. </li></ol> 2. إنشاء خلية صفائح ملاحظة: يمكن إنشاء أوراق خلية من الخلايا الأولية للجهاز الهدف باستخدام عدد من الأساليب المختلفة، أو طلاء الأسطح زراعة الأنسجة مع الحرارية استجابة البوليمر كما هو موضح هنا. وتقدم لوحات الحرارية الحساسة قبل المغلفة أيضا عدد من البائعين. ملاحظة: هذا البروتوكول هو لثقافة استخدام طبق 35 ملم. باختصار، يتم تحضين الخلايا أولا عند 37 درجة مئوية لمدة لا تقل عن 24 ساعة عند التقاء لتأسيس اتصالات جانبية بين الخلايا المجاورة. للافراج عن صحائف خلية، يتعرض لوحات لدرجات حرارة أقل من 32 درجة مئوية. ثم يتم نقل ورقة الخلية إلى قاعدة مصفوفة ليفية قوية تحتوي على هيدروجيل متساهل التوعية المستحدثة مع الخلايا البطانية في الأوعية الدموية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عزل السكان الخلية. ملاحظة: هذه الطريقة تعتمد على إجراءات اشتقاق الفردية ونوع من الخلايا. الفئران مو سلس الأبهروتستخدم خلايا scle (RASMC) في هذا المثال. هذه هي خلايا العضلات الملساء معزولة الأولية من الشريان الأورطي البطني من الفئران. </li><li style=";text-align:right;direction:rtl"> غسل الخلايا مع 2 مل من PBS الدافئ. </li><li style=";text-align:right;direction:rtl"> إضافة 3 مل من التربسين (أو الشق الآخر / حل يفصل) إلى الخلايا لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> تمنع التربسين بإضافة 3 مل من وسائل الإعلام والثقافة، أو حل العازلة الفوسفات (PBS) التي تحتوي على 10٪ مصل بقري جنيني (FBS). </li><li style=";text-align:right;direction:rtl"> جمع الخلايا في أنبوب مخروطي الشكل والاعتماد قسامة. </li><li style=";text-align:right;direction:rtl"> تدور الخلايا في 1،000 دورة في الدقيقة (228 x ج) لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> نضح وطاف resuspend الخلايا في وسائل الإعلام نموها (يتم استخدام SmGM2 بالإضافة إلى عدة رصاصة مستنبت للRASMC). </li><li style=";text-align:right;direction:rtl"> وضع وسائل الإعلام التي تحتوي على الخلايا على 35 ملم الحرارية الحساسة وحة - pNIPAAM لوحة المغلفة بتركيز التي من شأنها تحقيق 100٪ التقاء. ملاحظة: للحصول RASMCs تم تحديد هذا العدد ليكون 100،000 خلية / سم 2. ولكن نظرا لفقدان الخلايا أثناء وفاة، و 120٪ من ذلك فا<html> يستخدم LUE. </li><li style=";text-align:right;direction:rtl"> وضع في حاضنة عند 37 درجة CO / N. ملاحظة: من المهم للحفاظ على الخلايا عند 37 درجة مئوية للحفاظ على التصاق الخلية إلى لوحة. </li></ol> 3. إعداد مصفوفة التأسيسية ملاحظة: مختلف المصفوفات ليفية 3D يمكن استخدامها لطبقة مصفوفة ليفية قوية بين الأوراق الخلايا الحساسة. وتشمل بعض الأمثلة: جلفوم، bioglass والمواد الطبيعية acellularized 26 أو nanospun المواد 27،28 وقدمت المثانة البولية الخنزير مصفوفة (UBM) المستخدمة في هذه الدراسات بسخاء من وجهة نظرنا متعاون، الدكتور Badylak 29. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل استخدامها، وتحديد خصائص المصفوفة بما في ذلك عدم وجود محتوى الخلوي إذا تم استخدام مصفوفة decellularized، 27،28 بقاء الخلية المحددة، والفضاء باطل 22 </li><li style=";text-align:right;direction:rtl"> قطع مصفوفة تعقيمها قبل إلى الحجم المطلوب والشكل. ملاحظة: وهنا، يستخدم حفرة لكمة لقطع دائرة قطرها 4 مم. </li></ol>ve_title &quot;&gt; 4. الخلايا البطانية لحصد في حديث التوعي متساهل هيدروجيل ملاحظة: يمكن الحصول على الخلايا البطانية من مجموعة متنوعة من المصادر، بما في ذلك التمييز من الخلايا الجذعية أو السلف. هنا، وتستخدم HuVECs. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام أي هيدروجيل متساهل (الليفين، والمواد الهلامية الكولاجين) لفترة طويلة كما المرة عبر ربط قصيرة بما يكفي للسماح للخلايا تبقى قابلة للحياة. ملاحظة: هنا، هلام لHyaluronan (HA) على ربط عبر مع يستخدم جسر ثاني كبريتيد. </li><li style=";text-align:right;direction:rtl"> إعداد هيدروجيل HA وفقا لبروتوكول الشركة. </li><li style=";text-align:right;direction:rtl"> جمع الخلايا البطانية وتفريق في حل وحيد الخلية باستخدام 1X التربسين. ملاحظة: يمكن أيضا Accutase أو خلية التفكك العازلة استخدامها لتشتت خلية واحدة. </li><li style=";text-align:right;direction:rtl"> تنشيط انزيم التربسين باستخدام مبلغ مساو من مثبط التربسين فول الصويا (إذا كان من المهم أن الخلايا لا تتلامس مع المصل) أو 10٪ FBS في برنامج تلفزيوني، وجمع الحل / الخلايا في أنبوب مخروطي 15 مل. </li><li style=";text-align:right;direction:rtl"> Count الخلايا، وحساب حجم الحاجة للأبعاد التصحيح (كميا سابقا). ملاحظة: للحصول على 4 ملم التصحيح، وتستخدم هنا من 2 مليون الخلايا البطانية. </li><li style=";text-align:right;direction:rtl"> استخراج خلايا من 2 مليون، والمكان الى 15 مل أنبوب مخروطي الشكل الجديد. </li><li style=";text-align:right;direction:rtl"> تدور في (228 x ج) لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> نضح طاف، وترك الخلايا كما بيليه في أنبوب مخروطي. </li><li style=";text-align:right;direction:rtl"> مزيج من المواد السائلة HA والجيلاتين في 1: الحصة 1. ثم إضافة 80٪ من حجم التداول الكلي في أنبوب مخروطي يحتوي على بيليه. </li><li style=";text-align:right;direction:rtl"> resuspend الخلايا البطانية في 1: 1 HA / خليط الجيلاتين </li><li style=";text-align:right;direction:rtl"> وضع خلايا معلقة في خليط HA / الجيلاتين في قاعدة مصفوفة ليفية من الخطوة 2. </li><li style=";text-align:right;direction:rtl"> إضافة 1/5 (20٪) من حجم التداول الكلي المطلوب للرابط عبر </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 1 ساعة على 37 درجة مئوية. </li></ol> 5. عزل صفائح خلية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة لوحات 35 ملم pNIPAAM المعاملة التي تحتوي على خلايا من الحاضنة ومكان في غطاء ثقافة الخلية فيRT. </li><li style=";text-align:right;direction:rtl"> نضح بسرعة وسائل الإعلام من الخلايا، وإضافة 2 مل من 6٪ الجيلاتين العادية الذي تم تسخينه إلى 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> في حين أن الجيلاتين لا تزال دافئة، ضع شعرية معدنية في الجيلاتين، غمر تحت سطح الجيلاتين العادية (فيلم 1). </li><li style=";text-align:right;direction:rtl"> وضع لوحة كاملة على الجليد لمدة 5 إلى 7 دقائق، والسماح للالجيلاتين لتتصلب. </li><li style=";text-align:right;direction:rtl"> بعد 7 دقائق، واستخدام ملعقة لفصل بعناية حواف الجيلاتين من الجانب من لوحة، ومن ثم استخدام ملقط لرفع شعرية معدنية من لوحة ملاحظة: الجيلاتين 6٪، ورقة خلية يجب أن ترفع مع شعرية. </li><li style=";text-align:right;direction:rtl"> نقل ورقة الخلية إلى الطبق ووضع على رأس قاعدة ليفية مصفوفة هيدروجيل الجمع، ووضع بعناية شعرية على أعلى بناء. ملاحظة: الجانب القمي من ورقة خلية ستبقى في هذا المنصب. </li><li style=";text-align:right;direction:rtl"> إضافة 2 مل من وسائل الإعلام دافئ (37 درجة مئوية). </li><li style=";text-align:right;direction:rtl"> احتضان O / N السماح للخلايا ورقة التمسك السطح هيدروجيل. </li><li style=";text-align:right;direction:rtl"> إزالة رانه شعرية المعادن بعد حل ارتفاع درجة حرارة (حوالي 1 ساعة)، أو في اليوم التالي. </li></ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+extracellular+matrix+as+an+inductive+scaffold+for+the+partial+replacement+of+functional+myocardium.">The use of extracellular matrix as an inductive scaffold for the partial replacement of functional myocardium.</a> Cell Transplant. 15, S29-S40 (2006).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restriction+of+human+hepatic+stem+cells+to+mature+fates+is+made+efficient+by+tissue-specific+biomatrix+scaffolds.">Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.</a> Hepatology. 53, 293-305 (2011).</li><li>Gilbert, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fiber+alignment+and+biaxial+mechanical+behavior+of+porcine+urinary+bladder+derived+extracellular+matrix.">Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix.</a> Biomaterials. 29, 4775-4782 (2008).</li><li>Luna, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+biomimetic+topography+for+the+alignment+of+neonatal+and+embryonic+stem+cell-derived+heart+cells.">Multiscale biomimetic topography for the alignment of neonatal and embryonic stem cell-derived heart cells.</a> Tissue Eng Part C Methods. 17, 579-588 (2011).</li></ol>

We have nothing to disclose.

الخطوات الحاسمة في البروتوكول ما يلي: طلاء أسطح لوحة مع البوليمر thermoresponsive والتلاعب في أوراق الخلية بعد التبريد لوحات. لأن خلايا مختلفة تظهر الخصائص الفيزيائية المختلفة، مثل adhesivity، يجب أن يكون الأمثل الوقت لرفع كل نوع من الخلايا المختلفة. العنصر الثاني، والأكثر تحدي...

Injection of cells and/or single materials alone has shown variable success in other organ systems and limited success in cardiac regeneration.5,7-12 Currently, stem cell-derived cells are delivered to damaged tissue using a variety of delivery methods including: direct cell injection into tissue and perfusion into the blood supply.13-17 Others have implanted cells alone, materials alone and/or in combination with material carriers to help regenerate damaged organs.18-21 This design combines multiple strategies that provide material strength, patterning in multiple materials and multiple cell types. 
Specifically, the base acellularized fibrous matrix provides the foundational physical strength to the construct, making it suitable for suturing in into the patient, if necessary. The void spaces in the base matrix are filled with endothelial cells in a neovascular permissive hydrogel22 for rapidly establishing vascularization of the implanted construct. This composite is then integrated with pre-patterned cell sheets that allow enhanced cell-to-cell communication, more closely mimic the native tissue.1,23-25 The overall production process for the layered cellular patch is outlined by the flowchart in Figure 1.

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tissue engineering: construction of a multicellular 3d scaffold for the delivery of layered cell sheets

العديد من الأنسجة، مثل قلوب البشر البالغين، غير قادرين على تجديد كاف بعد الضرر. 2،3 استراتيجيات في هندسة الأنسجة تقترح الابتكارات لمساعدة الجسم على الشفاء والإصلاح. على سبيل المثال، قد يكون النهج TE قادرة على تخفيف إعادة القلب بعد احتشاء عضلة القلب (MI)، وربما زيادة إجمالي وظائف القلب إلى القريب العادي قبل MI مستوى 4 كما هو الحال مع أي نوع من الأنسجة وظيفية، وتجديد أنسجة القلب الناجح ينطوي على تسليم السليم لل أنواع الخلايا متعددة مع العظة البيئية لصالح التكامل وبقاء الكسب غير المشروع زرع خلية / الأنسجة. يجب أن تعالج الأنسجة المهندسة معلمات متعددة بما في ذلك: الإشارات القابلة للذوبان، خلية الى خلية التفاعلات، والمواد مصفوفة تقييم وسائل إيصالها، آثارها على بقاء الخلية، والقوة المادية، وتسهيل وتنظيم الخلايا إلى الأنسجة. الدراسات باستخدام الحقن المباشر للخلايا الكسب غير المشروع فقط تجاهل هذه العناصر الأساسية. 2،5،6لم يتم بعد إعداد تصميم النسيج الجمع بين هذه المكونات. هنا، نقدم مثالا لتصاميم متكاملة باستخدام طبقات من ألواح الخلايا منقوشة مع نوعين متميزين من المواد البيولوجية المشتقة من نوع من الخلايا التي تحتوي على الجهاز المستهدف والخلايا البطانية لتعزيز تكوين الأوعية جديد في &quot;نسيج&quot;. على الرغم من أن هذه الدراسات تركز على توليد أنسجة تشبه القلب، وهذا التصميم الأنسجة يمكن تطبيقها على العديد من الأجهزة الأخرى من القلب مع تصميم المواد والحد الأدنى من التغييرات، ومن المفترض أن يكون المنتج قبالة الجاهزة للاستخدام للعلاجات التجدد. بروتوكول يتضمن خمس خطوات مفصلة. ويستخدم البولي درجة الحرارة حساسة (N -isopropylacrylamide) (pNIPAAM) لأطباق زراعة الأنسجة معطف. ثم، وخلايا أنسجة محددة يتم تربيتها على سطح لوحات مغلفة / أسطح micropattern لتشكيل خلية ورقة مع التصاقات جانبية قوية. ثالثا، يتم إنشاء مصفوفة قاعدة للأنسجة عن طريق الجمع بين مسامية مصفوفة مع permissi التوعية المستحدثةلقد الهلاميات المائية والخلايا البطانية. وأخيرا، يتم رفع الأوراق خلية من الأطباق pNIPAAM المغلفة ونقل إلى قاعدة عنصر، مما يجعل بناء كاملة.

مخطط التدفق (الشكل 1) يبين طريقة الكلي لجعل التصحيح متعدد الطبقات. يتم فصل الخلايا من ورقة لوحة تعامل pNIPAAM بإسقاط درجة حرارة أقل من 32 درجة مئوية. ثم يتم وضع ورقة خلية على رأس هيدروجيل عبر ربط تحتوي على الخلايا البطانية المصنف في مصفوفة ليفية الأساسية (الشك?...

Watch this Scientific Journal Video about Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets at JoVE.com

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

هندسة الأنسجة: بناء ومتعددة الخلايا 3D سقالة لتسليم الطبقات صفائح خلية

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

tissue-engineering-construction-multicellular-3d-scaffold-for

Research

JoVE Journal

Bioengineering

14.4K Views.  University of California, Merced. For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Video: Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

تصميم مفاعل حيوي ذو محورين الميكانيكية جار للهندسة الأنسجة

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

هندسة الأنسجة ل3D الإنسان<em&gt; في المختبر</em&gt; نظام ورم اختبار

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

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

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

سقالات الإبلاغ الذاتي للخلية ثقافة 3 الأبعاد

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

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

الهندسة 3D Cellularized الهلام الكولاجين لتجديد الأنسجة الوعائية

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

تكييف عملية Electrospinning لتوفير ثلاث بيئات فريدة من نوعها لثلاثي الطبقات<em&gt; في المختبر</em&gt; نموذج جدار مجرى الهواء

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

بناء وحدات صفائح هيدروجيل لتحجيم الكلي Micropatterned 3D الخلوية العمارة

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

إعداد 3D الهلام الكولاجين وMicrochannels لدراسة التفاعلات 3D<em&gt; في فيفو</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

تصنيع مقلوب الغروية كريستال بولي (جلايكول الإثيلين) سقالة: وثلاثي الأبعاد منصة الثقافة خلية للهندسة الأنسجة الكبد

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

تصنيع كسوات وناقل دقيق مستمد مصفوفة خارج الخلية حيث الثقافة خلية الأنسجة محددة ومنصات التوصيل

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

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 ...

هندسة الأنسجة: بناء ومتعددة الخلايا 3D سقالة لتسليم الطبقات صفائح خلية

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