Name: a 3d system for culturing human articular chondrocytes in synovial fluid
Uploaded: 2012-01-31T13:00:00
Description: Watch this Scientific Journal Video about A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid at JoVE.com

Мы хотели бы поблагодарить Робина Най (Тафтс Медицинский центр), Tomoya Uchimura и Дана Кэрнс (Университет Тафта) за оказание помощи в синовиальной жидкости хранения и центрифугирования. Эта работа финансировалась NIH (1R01AR059106-01A1) для LZ

3D системы для культивирования человеческих хондроцитов суставного в синовиальной жидкости В этой работе мы инкапсулированные человеческих хондроцитов суставного в альгинат бисера с использованием модифицированного производства, предложил протокол инкапсуляции (Lonz...

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H., Chen, S. L., Bao, C. D., Du, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degeneration+of+normal+articular+cartilage+induced+by+late+phase+osteoarthritic+synovial+fluid+in+beagle+dogs.">Degeneration of normal articular cartilage induced by late phase osteoarthritic synovial fluid in beagle dogs.</a> Tissue Cell. 41, 13-22 (2009).</li><li>Kruger, J. P., Endres, M., Neumann, K., Haupl, T., Erggelet, C., Kaps, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+differentiation+of+human+subchondral+progenitor+cells+is+impaired+by+rheumatoid+arthritis+synovial+fluid.">Chondrogenic differentiation of human subchondral progenitor cells is impaired by rheumatoid arthritis synovial fluid.</a> J. Orthop. Res. 28, 819-827 (2010).</li><li>Steinhagen, J., Bruns, J., Niggemeyer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+culture+system:+Synovial+fibroblasts+modulate+articular+chondrocyte+matrix+synthesis+in+vitro.">Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro.</a> Tissue Cell. 42, 151-157 (2010).</li><li>Yang, K. G., Saris, D. B., Verbout, A. J., Creemers, L. B., Dhert, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+synovial+fluid+from+injured+knee+joints+on+in+vitro+chondrogenesis.">The effect of synovial fluid from injured knee joints on in vitro chondrogenesis.</a> Tissue Eng. 12, 2957-2964 (2006).</li><li>Skoog, V., Widenfalk, B., Ohlsen, L., Wasteson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+growth+factors+and+synovial+fluid+on+chondrogenesis+in+perichondrium.">The effect of growth factors and synovial fluid on chondrogenesis in perichondrium.</a> Scand. J. Plast. Reconstr. Surg. Hand. Surg.. 24, 89-95 (1990).</li><li>Nuver-Zwart, I., Schalkwijk, J., Joosten, L. A., van den Berg, W. B., van de Putte, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synovial+fluid+and+synovial+fluid+cells+on+chondrocyte+metabolism+in+short+term+tissue+culture.">Effects of synovial fluid and synovial fluid cells on chondrocyte metabolism in short term tissue culture.</a> J. Rheumatol. 15, 210-216 (1988).</li><li>Beekhuizen, M., Bastiaansen-Jenniskens, Y. M., Koevoet, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+tissue+inhibits+proteoglycan+production+in+human+osteoarthritic+cartilage;+Establishment+and+characterisation+of+a+long-term+coculture.">Osteoarthritic synovial tissue inhibits proteoglycan production in human osteoarthritic cartilage; Establishment and characterisation of a long-term coculture.</a> Arthritis Rheum. , (2011).</li><li>Rodrigo, J. J., Steadman, J. R., Syftestad, G., Benton, H., Silliman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+human+knee+synovial+fluid+on+chondrogenesis+in+vitro.">Effects of human knee synovial fluid on chondrogenesis in vitro.</a> Am. J. Knee. Surg. 8, 124-129 (1995).</li><li>van den Hoogen, B. M., van de Lest, C. H., van Weeren, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loading-induced+changes+in+synovial+fluid+affect+cartilage+metabolism.">Loading-induced changes in synovial fluid affect cartilage metabolism.</a> Br. J. Rheumatol. 37, 671-676 (1998).</li><li>Webb, G. R., Westacott, C. I., Elson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+fluid+and+synovium+supernatants+up-regulate+tumor+necrosis+factor+receptors+on+human+articular+chondrocytes.">Osteoarthritic synovial fluid and synovium supernatants up-regulate tumor necrosis factor receptors on human articular chondrocytes.</a> Osteoarthritis Cartilage. 6, 167-176 (1998).</li><li>Kook, S. H., Son, Y. O., Lee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+affects+positively+the+proliferation+of+bovine+satellite+cells+and+their+myogenic+differentiation+through+up-regulation+of+MyoD.">Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD.</a> Cell. Biol. Int. 32, 871-878 (2008).</li><li>Knobloch, T. J., Madhavan, S., Nam, J., Agarwal, S., Agarwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chondrocytic+gene+expression+by+biomechanical+signals.">Regulation of chondrocytic gene expression by biomechanical signals.</a> Crit. Rev. Eukaryot. Gene. Expr. 18, 139-150 (2008).</li><li>Guo, J. F., Jourdian, G. W., MacCallum, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+growth+characteristics+of+chondrocytes+encapsulated+in+alginate+beads.">Culture and growth characteristics of chondrocytes encapsulated in alginate beads.</a> Connect. Tissue. Res. 19, 277-297 (1989).</li><li>Toegel, S., Huang, W., Piana, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+reliable+reference+genes+for+qPCR+studies+on+chondroprotective+action.">Selection of reliable reference genes for qPCR studies on chondroprotective action.</a> BMC. Mol. Biol. 8, 13-13 (2007).</li><li>Lin, Z., Fitzgerald, J. B., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+human+chondrocytes+during+passaged+monolayer+cultivation.">Gene expression profiles of human chondrocytes during passaged monolayer cultivation.</a> J. Orthop. Res. 26, 1230-1237 (2008).</li><li>Goessler, U. R., Bieback, K., Bugert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chondrocytes+differentially+express+matrix+modulators+during+in+vitro+expansion+for+tissue+engineering.">Human chondrocytes differentially express matrix modulators during in vitro expansion for tissue engineering.</a> Int. J. Mol. Med. 16, 509-515 (2005).</li><li>Anat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 121, 107-118 (1976).</li></ol>

В этом докладе, мы разработали метод, который позволяет культуре человеческих хондроцитов суставного в 3D среде в среде, содержащей высокие концентрации человеческого синовиальной жидкости. Синовиальная жидкость является одним из основных компонентов, составляющих природной среды в ...

Таблица специфических реагентов и оборудования: <table border="1"><tr><th> Название реагента </th><th> Компания </th><th> Номер по каталогу </th><th> Комментарии </th></tr><tr><td> Альгинат (альгиновая кислота натриевая соль) </td><td> Сигма </td><td> A2158-250G </td><td> 2,4% раствор хранится при температуре 40 ° C </td></tr><tr><td> Дигидрат хлорида кальция, гранулированный </td><td> JT Baker </td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" > Хондрогенного СМИ Роста </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3156 (база СМИ) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4409 (дополнение) </td></tr><tr><td width="239" rowspan="2" > Хондрогенного СМИ Дифференциация </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3226 (база СМИ) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4408 (дополнение) </td></tr><tr><td> Человек суставные хондроциты </td><td> Lonza </td><td > CC-2550 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-Diamidino-2-фенилиндола дигидрохлорид) </td><td> Sigma-Aldrich </td><td > D9542 </td><td></td></tr><tr><td> RNeasy мини-комплект (для экстракции РНК) </td><td> Qiagen </td><td > 74104 </td><td></td></tr><tr><td> ПЦР реагентов: SYBR-зеленый </td><td> Quanta </td><td > 95053-500 </td><td></td></tr><tr><td> 12 мл шприц </td><td> Tyco-Kendall-Monoject </td><td > 512852 </td><td></td></tr><tr><td> 22-Gague шприц </td><td> Tyco-Кендалл-Monoject </td><td > 8881 </td><td></td></tr><tr><td> Микроскоп </td><td> Олимп </td><td > IX71 </td><td></td></tr><tr><td> Платформа рокер </td><td> Thermoscientific thermolyne </td><td > Vari-микс </td><td></td></tr><tr><td></td><td></td><td ></td><td></td></tr><tr><td> Грунтовки последовательности </td><td></td><td ></td><td></td></tr><tr><td> Коллаген IIa вперед </td><td> 5&#39;-TTC CCC ATC ОСО TCT CAC АГТ-3 &#39; </td><td ></td><td></td></tr><tr><td> Коллаген IIa-реверс </td><td> 5&#39;-CCTCTGCCTTGACCCGAA-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13 вперед </td><td> 5&#39;-TGT GCC СТТ СТТ CAC ACA GAC ACT-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13-реверс </td><td> 5&#39;-GAG AGC AGA СТТ TGA GTC ATT GCC-3 &#39; </td><td ></td><td></td></tr><tr><td> Каспазы 3-вперед </td><td> 5&#39;-TCA TTA TTC AGG CCT GCC GTG GTA-3 &#39; </td><td ></td><td></td></tr><tr><td> Каспазы 3-обратный </td><td> 5&#39;-TGG ATG AAC CAG GAG ОСО TCC ТТТ -3 &#39; </td><td ></td><td></td></tr></table>

a 3d system for culturing human articular chondrocytes in synovial fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

Watch this Scientific Journal Video about A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid at JoVE.com

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

3D системы культивирования человеческих хондроцитов суставного к высоким уровням синовиальной жидкости описывается. Синовиальная жидкость отражает наиболее естественный микросреду для суставного хряща, и может быть легко получена и сохранена. Эта система при этом может быть использована для изучения регенерации хряща и для скрининга терапии для лечения артрита.

3D системы для культивирования правам суставные хондроциты в синовиальной жидкости

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

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A System for ex vivo Culturing of Embryonic Pancreas

A System for ex vivo Culturing of Embryonic Pancreas

Система<em&gt; Бывших естественных условиях</em&gt; Культивирования эмбриональных поджелудочной железы

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Анализ функций генов и визуализации реснички, созданного потока жидкости в Vesicle Купфера

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Культивирование носа человека эпителиальных клеток в Военно-воздушной жидкость

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Method for Culturing Embryonic C. elegans Cells

A Method for Culturing Embryonic C. elegans Cells

Метод для культивирования эмбриональных<em&gt; С. Элеганс</em&gt; Клетки

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

Gap Junctional межклеточной коммуникации: Функциональная биомаркеров для оценки неблагоприятного воздействия токсикантов и токсинами, и Польза для здоровья натуральных продуктов

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Ассамблея и применение &quot;Shear колец&quot;: Роман эндотелиальной Модель для Orbital, однонаправленные и периодического потока жидкости и напряжения сдвига

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Магнитные активированный ячейку Сортировка стратегии изоляции и очищают синовиальной жидкости производные мезенхимальных стволовых клеток из кролика модели

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...