This project was funded by the National Science Foundation ECCS grant 10-02165, and the Interdisciplinary Innovation Initiative Program, University of Illinois grant 12035. M.Y. A. was funded at UIUC from NIH National Cancer Institute Alliance for Nanotechnology in Cancer &#8216;Midwest Cancer Nanotechnology Training Center&#8217; Grant R25 CA154015A. Immnunostaining and confocal microscopy imaging were carried out at the Institute for Genomic Biology (IGB), UIUC. M.Y.A. acknowledges the discussions with B. J. Williams of UIUC regarding the isolation experiments. M.Y.A. acknowledges C. Nemeh and Abdul Bhuiya of UIUC for assistance in schematic and materials list preparation.

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The authors have nothing to disclose.

وقد ظهرت الإجهاد الجر الخلوية مؤخرا كمؤشر الفيزيائية الحيوية المحتمل للدولة النقيلي 13. ومع ذلك، لا توجد بيانات الجر تجريبية مع خلايا الورم الرئيسي موجود في الأدب حتى الآن. أيضا، لا يتم الإبلاغ عن زراعة خلايا القولون مباشرة الأساسي معزولة على مختلف المواد الهل?...

في السنوات الأخيرة أصبح من الواضح بشكل متزايد أن الميكانيكية البيئة الصغيرة، بالإضافة إلى العوامل البيولوجية والكيميائية، ويلعب دورا هاما في تنظيم وظائف الخلية. الخلايا يمكن الشعور والاستجابة للصلابة الركيزة التي تقوم على الالتزام لهم (كما هو الحال في الثقافة 2D) أو تحيط بها (كما في الثقافة 3D) 3-7. بذلك، يمكن خلايا تعدل التمايز بها 3، 4 التشكل، والهجرة / حركية 5، البيولوجية الخصائص الفيزيائية 6، 7 النمو، وغيرها من العمليات. تستجيب الخلايا السرطانية أيضا إلى 2D و 3D مصفوفة الصلابة باستخدام منسقة الهرمي الجمع والميكانيكية والكيميائية للمستقبلات الالتصاق والبروتينات المرتبطة الغشاء نقل الإشارة، والهندسة المعمارية هيكل الخلية، والمحركات الجزيئية 1، 2. على سبيل المثال، الخلايا الظهارية الثديية (MECs) النموذج العادي حمة عنيبية عندما مثقف على 150 ركائز با مشابه لصلابةنسيج الثدي صحية. ومن المثير للاهتمام، ويحمل بصمات ورم النامية، على حد سواء الهيكلية والنسخي، عندما مثقف على ركائز أكثر صرامة (&gt; 5000 باسكال) التي تحاكي صلابة من سدى الورم 8. بالإضافة إلى ذلك، تظهر تجربة أخرى أن تكون الأورام الثدي يترافق يشابك الكولاجين وECM تشنج 9. وتبين التجارب الحديثة أن سرطان القولون البشري (HCT-8) خلايا عرض ورم خبيث مثل النمط الظاهري (MLP) عندما تكون مثقف على ركائز 2D وجود تصلب ذات الصلة من الناحية الفسيولوجية (20-47 كيلو باسكال)، ولكن ليس على قاسية جدا (3.6 جيد جدا) ركائز 10- 12. هذه الخلايا شكل أول مجموعات الخلايا تشبه الورم ومن ثم تنأى عن بعضها البعض، بدءا من المحيط. لأن هذا الظهارية لتقريب الصرفي (E إلى R الانتقالية) يحدث تغير، فإنها تتكاثر، والحد من خلية خلية وخلية ECM التصاق، وتصبح المهاجرة. HCT-8 الخلايا المستزرعة على ركائز البوليسترين صعبة للغاية لا تظهر هذهالصفات الخبيثة. وبالتالي فقد تم الافتراض أن HCT-8 تصبح الخلايا المتنقل وذلك بسبب تعرضها لمناسبة البيئة الصغيرة. ومن الجدير بالذكر أن هذه التجارب التي أجريت مع خطوط الخلايا السرطانية مخلد أو الفئران المستمدة الخلايا الأولية، وليس مع خلايا سرطانية بشرية الأولية. تقترح دراسة حديثة أن المعزز الإجهاد الجر الخلوية يمكن أن تستخدم كتوقيع الفيزيائية الحيوية المحتملة للخلايا النقيلي 13. ودراسة ينطوي على قياس قوة الجر لمختلف خطوط الخلايا السرطانية البشرية على المواد الهلامية بولي أكريلاميد. تبين أن الخلايا السرطانية المنتشر يمكن أن تمارس أعلى بكثير الإجهاد الجر مقارنة مع الخلايا غير المنتشر في جميع الحالات (13). ومع ذلك، فإن هذه النتائج تتناقض بشكل مباشر على نتائج الدراسة التي نشرت في وقت سابق على الفئران المستمدة خطوط خلايا سرطان الثدي 14. أيضا، تبرز الدراسة الأخيرة وجود فروق ملحوظة بين الخلايا البشرية مخلد والابتدائية في هيكل الخلية على إعادة المواليةالتنميط البروتين وبقاء الخلية البروتين التعبير (15). وبالتالي، فإنه من المهم إعادة النظر في العديد من فحوصات الفيزيائية الحيوية بما في ذلك الجر للخلايا السرطانية البشرية الأولية. وسوف يعالج مسألة ما إذا كانت الخلايا الأولية ألخص خلد خطوط الخلايا السرطانية الجر الاتجاه. تم تحسين البروتوكول الموصوفة هنا لعزل الخلايا الأولية البشرية القولون (على حد سواء صحية والسرطانية)، وللزراعة منها على ركائز الناعمة (الهلاميات المائية بولي أكريلاميد)، وكذلك في أطباق بتري. ويستند هذا البروتوكول على الهضم ويترتب على ذلك تفارق الأنزيمية من عينة الأنسجة جراحية في تعليق وحيد الخلية 16. على حد علمنا، وهذا هو أول مظاهرة من زراعة معزولة ورم القولون الابتدائي والخلايا الطبيعية مباشرة على ركائز هيدروجيل لينة مع بلي الفلورسنت المدمجة للالجر الخلوي. ركائز هلام شفاف يسمح أيضا المناعية. وكشف هذا الاختبار الاختلافات في المؤسسة F-أكتين والالتصاقات البؤرية في خلايا القولون البشري الأولية باعتبارها الركيزة التغييرات صلابة. هذه المنصة زراعة الخلايا يفتح إمكانية استكشاف مختلف الخصائص الفيزيائية الحيوية من خلايا الإنسان الأولية مثل تصلب الخلايا والجر كمعلمات لprognostics السرطان.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td ></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr >
			<td >HBSS</td>
			<td>Life technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin</td>
			<td>Worthington</td>
			<td>LS003736</td>
			<td></td>
		</tr>
		<tr >
			<td >Collagenese</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr >
			<td >3- Aminopropyltrymethoxysilane (ATS)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td >281778</td>
			<td></td>
		</tr>
		<tr >
			<td >Glutaraldehyde</td>
			<td>Polysciences, Inc.</td>
			<td>01201-5</td>
			<td></td>
		</tr>
		<tr >
			<td >Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A4058</td>
			<td></td>
		</tr>
		<tr >
			<td >N- methylenebisacrylamide (bis)</td>
			<td>Sigma-Aldrich</td>
			<td>M1533</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES buffer solution</td>
			<td>Sigma-Aldrich</td>
			<td >83264</td>
			<td></td>
		</tr>
		<tr >
			<td >Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr >
			<td >N&#714;-tetramethylethylenediamine (TEMED)</td>
			<td>Bio-Rad</td>
			<td>161-0801</td>
			<td></td>
		</tr>
		<tr >
			<td >Human fibronectin</td>
			<td>BD biosciences</td>
			<td >354008</td>
			<td></td>
		</tr>
		<tr >
			<td >Hydrazine hydrate</td>
			<td>Sigma-Aldrich</td>
			<td >18412</td>
			<td>Hazardous</td>
		</tr>
		<tr >
			<td >Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT15710</td>
			<td></td>
		</tr>
		<tr >
			<td >Signal enhancer</td>
			<td>Life technologies</td>
			<td>I36933</td>
			<td></td>
		</tr>
		<tr >
			<td >Monoclonal anti vinculin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>V9131</td>
			<td></td>
		</tr>
		<tr >
			<td >Alexa fluor 488 goat anti-mouse IgG</td>
			<td>Life technologies</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr >
			<td >TRITC phalloidin conjugates&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr >
			<td >0.1 &#181;m fluroscent beads</td>
			<td>Life technologies</td>
			<td>F8801</td>
			<td></td>
		</tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Life technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	
		<tr >
			<td ></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr >
			<td >12 mm2 glass cover slips</td>
			<td>Corning</td>
			<td>2865-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of primary human colon tumor cells from surgical tissues and culturing them directly on soft elastic substrates for traction cytometry

الخلايا السرطانية تستجيب إلى matrix صلابة ميكانيكية بطريقة معقدة باستخدام، نظام هرمي للتنسيق والميكانيكية والكيميائية تتألف من مستقبلات الالتصاق والبروتينات المرتبطة الغشاء نقل الإشارة، والهندسة المعمارية هيكل الخلية، والمحركات الجزيئية 1 و 2. Mechanosensitivity الخلايا السرطانية المختلفة في المختبر ل التحقيق في المقام الأول مع خطوط الخلايا خلد أو الفئران المستمدة الخلايا الأولية، وليس مع خلايا سرطانية بشرية الأولية. وبالتالي، لا يعرف إلا القليل عن mechanosensitivity من خلايا سرطان القولون الإنسان الأساسية في المختبر. هنا، تم تطوير بروتوكول الأمثل الذي يصف عزل الخلايا القولون الإنسان الأساسية من العمليات الجراحية عينات الأنسجة البشرية السليمة والسرطانية. خلايا القولون معزولة ثم يتم تربيتها بنجاح في الناعمة (2 كيلو باسكال صلابة) وقاسية (10 كيلو باسكال صلابة) الهلاميات المائية بولي أكريلاميد والبوليسترين جامدة (~ 3.6 جيغا صلابة) ركائز functionalized من قبل المصفوفة خارج الخلية (فبرونيكتينفي هذه الحالة). هي جزء لا يتجزأ بلي الفلورسنت في المواد الهلامية اللينة قرب السطح ثقافة الخلية، ويتم تنفيذ الجر فحص لتقييم الضغوط مقلص الخلوية باستخدام البرمجيات الحرة ومفتوحة. بالإضافة إلى ذلك، المناعي المجهري على ركائز صلابة مختلفة ويوفر معلومات مفيدة عن التشكل الأساسي الخلية، وتنظيم الهيكل الخلوي وvinculin تحتوي على الالتصاقات البؤرية بوصفها وظيفة من الركيزة صلابة.

ويعمل البروتوكول المذكورة أعلاه بنجاح للحصول على عينات الأنسجة المتعددة (ن = 12) من أربعة مرضى مختلفة في إطار المبادئ التوجيهية للمجلس المراجعة المؤسسية. ويوضح الشكل 1A ورم القولون والمستقيم ممثل الحق بعد الجراحة التي يتم الحصول على أقسام الأنسجة لمزارع الخل?...

Watch this Scientific Journal Video about Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry at JoVE.com

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

عزل خلايا ورم القولون الإنسان الأساسية من الأنسجة الجراحية وزراعة بشكل مباشر على لينة مرنة ركائز للالجر الخلوي

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

isolation-primary-human-colon-tumor-cells-from-surgical-tissues

Research

JoVE Journal

Bioengineering

15.4K Views.  University of Illinois at Urbana-Champaign. A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses. 

Video: Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

سريع عزل الخلايا السرطانية قابلة للحياة اعارة من عينات الدم من المريض

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

مناديل الهندسة العضلات الهيكلية من الفئران الخلايا الاصلية Myoblast وتطبيق تحفيز الكهربائية

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

الخلوي الجماعي: بروتوكول لضبط اليومية وعينات الخلايا تشغيل عداد الكريات الشامل على CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

الزخرفة الخلية على Photolithographically معرفة Parylene-C: شافي<sub&gt; 2</sub&gt; ركائز

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

استرجاع الجراحية، عزل و<em&gt; في المختبر</em&gt; توسيع الأمامي الإنسان الصليبي الخلايا المشتقة من الرباط لتطبيقات هندسة الأنسجة

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

توصيف النشاط الحيوي للأنسجة لينة الإنسان عن طريق المسافة البادئة واختبار الشد

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

عزل الخلايا العقدة الشبكية الفئران الأولية (رغس) عن طريق التدفق الخلوي

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

عزل الخلايا الجذعية الوسيطة من السمحاق السنخية البشرية وآثار فيتامين د على نشاط الخلايا المستمدة من السمحاق أوستيوجينيك

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

قياس صلابة من ركائز السيليكون لينة للدراسات ميتشانوبيولوجي باستخدام مجهر الأسفار ويديفيلد

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.