وأيد هذا العمل من خلال منحة بحثية من المعاهد الوطنية للصحة (HL068915 إلى DBC)، وجائزة الباحث جديد من صندوق البحوث الدراسه (لCAP)، منحة في والمساعدات من جمعية القلب الأمريكية (12GRNT11910008 إلى DBC) ، منحة بحثية من مؤسسة القلب للأطفال (لDBC)، والتبرعات لمستشفى صندوق الأطفال في بوسطن القلب التوصيل، والوقف ريان الأسرة، وديفيد بولمان.

هنا سوف علينا أن نظهر عزلة COL1 من الجلد الضأن. كما هو مكتوب، وهذا البروتوكول يمكن أن تستخدم أيضا لعزل بنجاح COL1 من الأرانب وجلد الإنسان. 1. إعداد الجلدية عينة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تتوازن جميع الكواشف إلى 4 درجة مئوية قبل استخدامها. </li><li style=";text-align:right;direction:rtl"> شطف العينة الجلدي (25-50 غ) في DH الجليد الباردة 2 O وإزالة أي الصوف والفراء، أو الشعر مع كريم مزيل الشعر. </li><li style=";text-align:right;direction:rtl"> استخدام شفرة حلاقة واحدة الحافة لكشط نظيفة عينة من النسيج الضام والدهون. </li><li style=";text-align:right;direction:rtl"> شطف العينة في الجليد الباردة درهم 2 O. </li><li style=";text-align:right;direction:rtl"> شريحة عينة الجلد إلى 1 سم × 1 سم قطع مع شفرة حلاقة واحدة الحافة. </li></ol> 2. إزالة المواد غير كولاجيني Solubilized <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تزن من 5 غرام من العينة في أنبوب 50 مل المخروطية وإضافة 30 مل من الجليد الباردة M 0.5 خلات الصوديوم. </li><li style=";text-align:right;direction:rtl"> خلط الأنابيب لمدة 1 دقيقة في 6 إعداد م / ثانية باستخدام محول أنبوب 50 مل في الخالط مقعد بين كبار. </li><li style=";text-align:right;direction:rtl"> تجاهل سوبernatant وتكرار لما مجموعه 7 الصوديوم خلات دورات الغسيل. </li><li style=";text-align:right;direction:rtl"> شطف العينة في DH الجليد الباردة 2 O وتخلط مرة واحدة لإزالة خلات الصوديوم المتبقية. </li><li style=";text-align:right;direction:rtl"> استخدام ملعقة لضغط العينة ضد الجانب من الأنبوب لإزالة السائل الزائد، ثم نقل إلى أنبوب 50 مل المخروطية الطازجة. </li></ol> 3. النوع الأول الكولاجين استخراج <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> غسل العينة مرتين في 2 مل / ز 0.075 M الصوديوم العازلة سيترات لمدة 1 دقيقة في 6 متر / ثانية في الخالط مقعد بين كبار ضغط العينة والتخلص من طاف بعد كل غسل. </li><li style=";text-align:right;direction:rtl"> إضافة جديدة 2 مل / ز قسامة من 0.075 M الصوديوم العازلة سترات للعينة. </li><li style=";text-align:right;direction:rtl"> تنفيذ 6 متتابعة 1 دقيقة، 6 م / ثانية دورات مقعد بين كبار مزيج من الانفعالات الخالط دون إزالة المنطقة العازلة بين كل دورة. </li><li style=";text-align:right;direction:rtl"> نقل، طاف واضحة سميكة إلى أنبوب جمع. </li><li style=";text-align:right;direction:rtl"> إضافة مبلغ إضافي 1 مل / ز 0.075 M الصوديوم العازلة سيترات إلى عينة واحدة وأداء النهائي مقعد بين كبار الخالط agitaدورة نشوئها. </li><li style=";text-align:right;direction:rtl"> إضافة هذا طاف النهائي إلى أنبوب جمع. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في أنبوب جمع 3،200 x ج لمدة 10 دقيقة في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> نقل طاف إلى المقصورة العلوي من 100،000 الوزن الجزيئي قطع جهاز تصفية الطرد المركزي. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في 3،200 x ج لمدة 30 دقيقة في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> نقل COL1 تنقيته من المقصورة العلوية لأنبوب مخروطي نظيفة وتخزينها في 4 درجات مئوية. </li></ol>

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والكتاب ليس لديهم مصالح مالية تتنافس في الكشف عنها.

وثمة عنصر حاسم لهذا الأسلوب هو ضغط كفاءة المتكررة من العينة عن طريق الجلد لإزالة السائل الزائد. فمن الأهمية بمكان لإزالة أكبر قدر ممكن خلات الصوديوم في الخطوة 2.5 عن طريق ضغط العينة. نستخدم ملعقة ضغط الجلد ضد الجانب من الأنبوب، ولكن يمكن أن تستخدم أيضا القماش القطني، ع...

على مدى عقود، وقد عزل الباحثون والباعة التجارية solubilized COL1 من مجموعة متنوعة من المصادر بما في ذلك الجلد والأنسجة وتر استخدام بعض الاختلاف من بروتوكول استخلاص الحمض بسيطة تليها تحييد، مما يؤدي الى إعادة تعليق من مصفوفة من الألياف COL1 المنظمة التي يمكن استخدامها لعدد وافر من التطبيقات الطبية الحيوية 1-4. في حين أن هناك العديد من الأمثلة على التطبيقات السريرية لCOL1، عدد قليل من هذه توظف COL1 المستمدة autologously لأن إعداد هذا البروتين يتطلب قلع مطولة مع الأخذ أيام أو أسابيع لأداء 5-7. ونتيجة لذلك، والمحققين البحوث والأطباء عموما استخدام، المستحضرات التجارية باهظة الثمن من COL1 التي يتم إعدادها باستخدام الأنسجة غير البشرية مثل الفئران والأبقار، أو أدمة الخنازير أو باستخدام إزالة الجثث من الجلد أو بعد ختان الذكور. للتطبيقات التجدد، وكثير من هذه المنتجات المعرض التباين فيما يتعلق قدرتها على تشكيل الصلبةمصفوفات أو يبني الأنسجة قادرة على دعم مرفق الخلية والنمو. وبالتالي، هناك حاجة واضحة لطريقة موحدة جديدة مصممة لاستخراج بسرعة COL1 من مصادر الأنسجة ذاتي الوصول إليها وفيرة. أن مثل هذا الأسلوب توفير الوقت والمال في إعداد البحوث التي يمكن استخراجها COL1 من نموذج حيواني من الفائدة وتستخدم لاختبار قبل السريرية الأنسجة المهندسة وتجميعها على السقالات القائمة على الكولاجين. في الوقت نفسه، فإن وجود خيار استخدام معزولة بسرعة، COL1 المستمدة autologously في العيادة زيادة السلامة العامة للمرضى من شأنها أن تلقي خلاف ذلك خيفي أو مستحضرات أعضاء غير بشرية. للمرضى الذين يتلقون استعدادا ذاتي المنشأ، فإن هذا الأسلوب مبسطة يقلل كثيرا من الفاصل الزمني بين جمع خزعة وتطبيق الكولاجين. لهذه الأسباب، سعينا لتحسين بناء على COL1 العزلة وتنقية الأسلوب الراسخة باستخدام عالية السرعة AGitation والحجم الاستبعاد الطرد المركزي. هذه طريقة بسيطة لأداء باستخدام مخازن القياسية والمعدات الموجودة في العديد من المختبرات. من خلال توظيف عالية السرعة والإثارة، لدينا بروتوكول يقلل من الوقت اللازم لعزل القابلة للذوبان، الجلد COL1 من ما يقرب من 10 يوما إلى أقل من 3 ساعة 8. الأهم من ذلك، هذا الأسلوب لا يمكن أن يؤديها بسهولة في الإعداد السريرية من أجل إعداد COL1 المستمدة autologously للاستخدام في المرضى خلال مجموعة واحدة من الإجراءات.

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			<td height="43" style="height:43px;width:123px;">FastPrep-24 System</td>
			<td style="width:136px;">MP Biomedicals</td>
			<td style="width:147px;">116004500</td>
			<td style="width:139px;">-</td>
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			<td height="190" style="height:190px;width:123px;">Centrifuge</td>
			<td style="width:136px;">Beckman</td>
			<td style="width:147px;">J6-MI</td>
			<td style="width:139px;">Swing bucket rotor to accommodate 50 mL conical tubes at 3200 g.</td>
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an improved method for the preparation of type i collagen from skin

ويستخدم نوع قابل للذوبان 1 الكولاجين (COL1) على نطاق واسع باعتبارها الركيزة لاصقة لمزارع الخلايا وكما سقالة الخلوية للتطبيقات التجدد. سريريا، ويستخدم على نطاق واسع هذا البروتين للجراحة التجميلية، والحقن الجلدية، تطعيم العظام، والجراحة الترميمية. مصادر COL1 لهذه الإجراءات هي غير البشرية عادة، مما يزيد من احتمال التهاب والرفض وكذلك انتقال المرض أجنبي بيولوجيا. في ضوء ذلك، فإن الأسلوب بكفاءة وبسرعة تنقية COL1 من كميات محدودة من أنسجة مستمدة autologously-تحايل العديد من هذه القضايا، ولكن العزلة البروتوكولات القياسية هي طويلة وتتطلب كميات كبيرة من الأنسجة كولاجيني كثير من الأحيان. هنا، علينا أن نظهر طريقة استخراج COL1 فعالة أن يقلل من الوقت اللازم لعزل وتنقية هذا البروتين من حوالي 10 أيام إلى أقل من 3 في الساعة. اخترنا الأدمة كمصدر الأنسجة لدينا بسبب توافره خلال العديد من الإجراءات الجراحية. Tيستخدم طريقته مخازن استخراج التقليدية جنبا إلى جنب مع الانفعالات قوية والترشيح الطرد المركزي للحصول على درجة عالية من محض، قابل للذوبان COL1 من كميات صغيرة من أدمة. لفترة وجيزة، يتم غسلها الخزعات الجلدية تماما في DH الجليد الباردة 2 O بعد إزالة الدهون، والنسيج الضام، والشعر. يتم تجريد عينات الجلد من البروتينات والسكريات غير كولاجيني باستخدام 0.5 M خلات الصوديوم وسرعة عالية مقعد بين كبار الخالط. يتم استخراج الكولاجين من المواد الصلبة المتبقية في وقت لاحق مع 0.075 M الصوديوم العازلة سيترات باستخدام الخالط. يتم تنقية هذه المقتطفات باستخدام 100،000 ميغاواط وقف انتاج المواد الانشطارية مرشحات الطرد المركزي التي تحقق الاستعدادات COL1 من نوعية مماثلة أو متفوقة على المنتجات التجارية أو تلك التي تم الحصول عليها باستخدام إجراءات التقليدية. نتوقع أن هذه الطريقة تسهل استخدام المستمدة autologously-COL1 للعديد من البحوث والتطبيقات السريرية.

هذا البروتوكول العزلة COL1 يتطلب حوالي 2 ساعة و 15 دقيقة لإكمال. ويبين الشكل 1 رسما تخطيطيا يحدد الخطوات الرئيسية في هذا الإجراء. إعداد العينة وتنفيذ 7 دورات من الثورات عالية السرعة ويشطف مع خلات الصوديوم يستغرق حوالي 35 دقيقة (أرقام 1A-C). أداء التحريض واحدة وشطف دو...

Watch this Scientific Journal Video about An Improved Method for the Preparation of Type I Collagen From Skin at JoVE.com

An Improved Method for the Preparation of Type I Collagen From Skin

تتطلب الإجراءات التقليدية لعزل نوع قابل للذوبان 1 الكولاجين (COL1) حوالي 10 يوما من البداية الى النهاية بسبب حضانات عازلة طويلة وشاقة resuspensions من الألياف. هنا، نحن تصف وسيلة لتنقية COL1 الخزعات الجلدية الصغيرة في أقل من 3 دقائق.

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. ...

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Bioengineering

22.7K Views.  Boston Children's Hospital.  تتطلب الإجراءات التقليدية لعزل نوع قابل للذوبان 1 الكولاجين (COL1) حوالي 10 يوما من البداية الى النهاية بسبب حضانات عازلة طويلة وشاقة resuspensions من الألياف. هنا، نحن تصف وسيلة لتنقية COL1 الخزعات الجلدية الصغيرة في أقل من 3 دقائق. 

Video: An Improved Method for the Preparation of Type I Collagen From Skin

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

عضوي النمط I الكولاجين الفحص: A منهاج طيع لتقييم سلوك الخلية في سياق الأبعاد 3-

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

تحسين طريقة لإعداد القائم على الخليوي، الاتصال نموذج الإنسان من حاجز الدم في الدماغ

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

لتحسين طرق الاختبارات الميكانيكية لتقييم العظم زرع مرسى

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

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. ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

الأنسجة المفروم في المضغوط الكولاجين: وBiotransplant التي تحتوي على خلية لإصلاح الترميمية نظموا وحيد

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

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 ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

تقييم انتشار Keratinocyte على عمليات ثنائية وثلاثية الأبعاد في النوع الأول من ركائز الكولاجين

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

القياس الكمي المتزامن لـ Kynurenines المختارة التي تم تحليلها بواسطة الكروماتوغرافيا السائلة- القياس الطيفي الكتلي في المتوسط الذي تم جمعه من ثقافات الخلايا السرطانية

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

طريقة حقن بطيئة طفيفة التوغل لتقليل إصابة الأنسجة

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

تسليط الضوء على المؤلف: طريقة حقن خلية جديدة مع الحد الأدنى من الغزو  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

طريقة جديدة لحقن الخلايا مع الحد الأدنى من الغزو

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...