وأيد هذا العمل من قبل المعاهد الوطنية الأميركية للصحة منح HD051998 # المعاهد الوطنية للصحة / معاهد الصحة القومية (لCAM).

1. الكولاجين الخرزة التحضير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل EVTs التحميل لمدة 3 - D خلية ثقافة ، ويحتاج المرء لتحضير حبات microcarrier Cytodex - 3 : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تزن خارج القدر المناسب من Cytodex - 3 حبات المطلوبة لهذه التجربة. ويتم تكييف هذا البروتوكول للسفينة 10ML المراكز المناخية الإقليمية ، والتي هي بحاجة 0.05g من الخرز. لسفينة 50ml المراكز المناخية الإقليمية ، وفقا لمقياس. في أنبوب 50mL autoclavable المخروطية ، مزيج من 250 ملغ Cytodex - 3 حبات مع الفوسفات Dulbecco 12mL مخزنة الحل (DPBS). وهذا المبلغ غير كاف بالنسبة للسفن المراكز المناخية الإقليمية 5. </li></ol></li><li style=";text-align:right;direction:rtl"> ضمان وجود كمية كافية في أنبوب مخروطي الشكل ، وهذه العملية ستؤدي في الأوتوكلاف التبخر. قبعة فضفاضة الأنبوب والأوتوكلاف لمدة 10 دقيقة في 110 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> إزالة أنبوب مخروطي الشكل عند الانتهاء من دورة الأوتوكلاف والسماح للحل Cytodex - 3 حبة لتبرد. </li><li style=";text-align:right;direction:rtl"> بعد أنبوب مخروطي الشكل ويتم تبريده الى درجة حرارة الغرفة ، واستخدام تقنية معقمة لتحقيق الحجم الكلي ل12.5mL 1X به موانئ دبيBS. </li><li style=";text-align:right;direction:rtl"> قبعة وتخزين أعد Cytodex - 3 حبات في درجة حرارة الغرفة. السماح للالخرز على استعداد لتنتفخ وتبرد لدرجة حرارة الغرفة قبل استخدامها. سيتم إعداد أعلاه Cytodex - 3 حبات توفير خمس سفن 10ML المراكز المناخية الإقليمية. وقد لاحظنا أن التخزين الموسعة Cytodex - 3 حبات في درجة حرارة الغرفة سوف تؤدي إلى تراجع الأداء ، كما سيؤدي إلى زعزعة الاستقرار الكولاجين بعد شهر واحد تقريبا. Cytodex - 3 حبات يتراوح حجمها 133-215 ميكرون. </li></ol> 2. إعداد وسائل الاعلام <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحضير 1L من المراكز المناخية الإقليمية الأمثل GTSF - 2 وسائل الاعلام (مقتبس من 22) ، الذي يتكون من 40 ٪ MEM ألفا ، بالإضافة إلى ملاحق و 60 ٪ L - 15 ليبوفيتز وسائل الاعلام (مقتبس من 22). أولا ، إعداد 400mL في إجمالي حجم ألفا MEM تستكمل مع : 21.2mM بيكربونات الصوديوم 0.06 ٪ ببتون 0.7mM الفركتوز 1.4mM الجالاكتوز 5.6mM الجلوكوز 1 ٪ HEPES 1 ٪ L - الجلوتامين 0.5 ٪ ITS 10 ٪ FBS </li><li style=";text-align:right;direction:rtl"> لإعداد محلول المخزون من ITS ، تذوب في 5mL العقيمة O 2 H يحمض أعدها إضافة حمض الخليك الجليدي (حوالي 0.05mL). دوامة حل ، اتبع مع الماء المعقم 45mL. </li><li style=";text-align:right;direction:rtl"> تزن ما يكفي من L - 15 ليبوفيتش لمسحوق 600ml المتوسط ​​عن طريق إذابة الأنسجة في الثقافة O. الصف 2 H </li><li style=";text-align:right;direction:rtl"> جلب المتوسطة الحجم الكلي للثقافة الخلية ل1L مع المتوسط ​​L - 15 ليبوفيتز. فلتر تعقيم وتخزين عند 4 في الظلام درجة مئوية. إضافة 1 ٪ البنسلين ، الستربتوميسين فردي لكل قسامة من الوسيلة المستخدمة. </li><li style=";text-align:right;direction:rtl"> وقد تم العديد من وسائل الاعلام الصيغ الأخرى من وسائل الاعلام - 2 GTSF اختبرت بنجاح في المراكز المناخية الإقليمية. يجب المختبرات الفردية التي تقرر لنفسها المتوسط ​​هو الأمثل ليتم تنفيذ التجربة. </li></ol> 3. الخلايا والحضانة الخرزة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نشر EVTs لconfluency 80 ٪ ~ ، ويعرض للتريبسين العد باستخدام الممارسات المقبولة ثقافة الخلية. </li><li style=";text-align:right;direction:rtl"> تعليق 1x10 6 EVTs في 4mL الدافئةاد وسائل الإعلام. </li><li style=";text-align:right;direction:rtl"> المزيج بلطف على استعداد Cytodex - 3 حبات. باستخدام تقنيات معقمة ، إزالة 2.5mL من الخرز إعدادها باستخدام تلميح واسعة ، 10ML ماصة المصلية ونقل إلى أنبوب مخروطي 15mL غير المستخدمة. علما أنه قد تكون هناك خسارة صغيرة من الخرز والتي يعلقونها على ماصة. </li><li style=";text-align:right;direction:rtl"> السماح لل- 3 حبات Cytodex لتسوية في الجزء السفلي من الأنبوب. بعد الترسيب ، واستخدام pipettor لإزالة الطبقة العليا من دون إزعاج DPBS حبات Cytodex - 3. </li><li style=";text-align:right;direction:rtl"> مزيج 1x10 6 EVTs أعدت في وسائل الإعلام مع حبات Cytodex - 3 المعدة. </li><li style=";text-align:right;direction:rtl"> احتضان الخليط خلية حبة في درجة حرارة الغرفة لمدة 30 دقيقة. المزيج بلطف دوريا. احتضان لمزيد من 30 دقيقة عند 37 درجة مئوية و 5 ٪ CO 2. المزيج بلطف دوريا. </li></ol> 4. تحميل RWV <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في مجلس الوزراء تدفق الصفحي ، إزالة المراكز المناخية الإقليمية 10ML من التعبئة والتغليف ووضعه في العقيمة ، لوحة الثقافة 6 - جيدا لتحقيق الاستقرار. الرجوع إلى الشكل (1) رسم تخطيطي لتسمية المنسق المقيمCS. </li><li style=";text-align:right;direction:rtl"> إزالة سدادة كبيرة من الميناء. </li><li style=";text-align:right;direction:rtl"> جعل إجمالي حجم الخليط خلية حبة لاحتضان 10ML تحسنت مع وسائل الاعلام. </li><li style=";text-align:right;direction:rtl"> تحميل خلية / حبة الخليط في المراكز المناخية الإقليمية ، من خلال ميناء كبير. وينبغي إمالة المراكز المناخية الإقليمية في زاوية 45 درجة (كبير يصل الميناء) عند تحميل للمساعدة في إزالة فقاعات الهواء المحتملة. منع تراكم الضغط الايجابي عن طريق ملء السفن ببطء ولكن بثبات. </li><li style=";text-align:right;direction:rtl"> استبدال سدادة في ميناء كبير. </li><li style=";text-align:right;direction:rtl"> إزالة بيستونز من 3 مل الحقن والمحاقن مكان فارغ على المنافذ الصغيرة. إضافة 1 - 3mL من وسائل الإعلام إلى كل حقنة. فتح ببطء الصمامات اثنين. استبدال المحاقن بيستونز على المحاقن بلطف. إضافة وسائط من الحقنة حتى تتم إزالة كافة الفقاعات من داخل الغرفة. </li><li style=";text-align:right;direction:rtl"> التقط المراكز المناخية الإقليمية والاستفادة من الجانب بلطف حين تناوب في أمامك للتحقق من الفقاعات. إذا كان هناك أي فقاعات ، ويجب اخراجهم من الغرفة ، وفقاعات الهواء سوف تتداخل مع تشكيل سالمجاميع و الخلايا وإدخال قوى القص. إزالة الفقاعات من خلال تناوب المراكز المناخية الإقليمية حتى فقاعات تحت ميناء صغير. ثم ادفع برفق لأسفل على حقنة في الجانب المقابل لإجبار فقاعات في الميناء والخروج من القاعة. </li><li style=";text-align:right;direction:rtl"> إغلاق ميناء واحد من الجانبين. ادفع برفق لأسفل على مكبس الحقنة أخرى لإدخال كمية صغيرة من الضغط الإيجابي في السفينة ، والذي يمنع من تشكيل فقاعات. إغلاق صمام الثانية. </li><li style=";text-align:right;direction:rtl"> تحميل المراكز المناخية الإقليمية على الدوار. بدء الدوران في 19rpm في 37 درجة مئوية حاضنة 2 CO. </li></ol> 5. تغيير وسائل الإعلام <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تغيير وسائل الاعلام كل يوم على مدى الأيام الثلاثة الأولى ، ثم بعد ذلك كل يوم. </li><li style=";text-align:right;direction:rtl"> إيقاف الدوار وإزالة المراكز المناخية الإقليمية. سحب المكابس لخلق بعض الشفط ، إزالة المحاقن من كل ميناء صغير ووضع المراكز المناخية الإقليمية على زاوية بحيث الخرز تسوية المقابل من ميناء كبير. </li><li style=";text-align:right;direction:rtl"> بعد الخرز وقد استقر كل شيء ، فتح أحد الصمامات الصغيرة والسماح لوسائل الإعلام في التدفق من المراكز المناخية الإقليمية وداخل حاوية النفايات. إزالة 2 / 3 من وسائل الإعلام بهذه الطريقة. يجب التأكد من عدم إزعاج الخرز وعدم تجاهل أي المجاميع. </li><li style=";text-align:right;direction:rtl"> إغلاق صمام الصغيرة وفتح ميناء كبير. إضافة وسائل الإعلام مرة أخرى إلى المراكز المناخية الإقليمية من خلال ميناء كبير. استبدال سدادة في ميناء كبير. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 4،6-4،9. </li><li style=";text-align:right;direction:rtl"> كما المجاميع الزيادة في الحجم ، وتحتاج إلى زيادة سرعة دوران للاحتفاظ بها في تعليق العمل في جميع الأوقات ، من الناحية المثالية في نمط الدورة الدموية الصغيرة في السرعة المثلى. عموما ، وزيادة السرعة بين 0،3-0،7 بعد كل دورة في الدقيقة التغذية مرة واحدة في المجاميع تبدأ في النمو بشكل ملحوظ. </li></ol> 6. نشر جمع خلايا <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة المراكز المناخية الإقليمية من الدوار. إزالة المحاقن من كل ميناء صغير ووضع المراكز المناخية الإقليمية على زاوية بحيث يمكن تسوية الخرز العكس من ميناء كبير. </li><li style=";text-align:right;direction:rtl"> بعد الخرز وقد استقر كل شيء ، فتح أحد الصمامات الصغيرة والسماح لوسائل الإعلام لتتدفقمن المراكز المناخية الإقليمية وداخل حاوية النفايات. إزالة 1 / 3 من وسائل الإعلام بهذه الطريقة. </li><li style=";text-align:right;direction:rtl"> إغلاق صمام الصغيرة وفتح ميناء كبير. بلطف دوامة السفينة لتفريق التجمعات مرة أخرى إلى الحل. الخليط السائل في أنبوب فارغ 50ml العقيمة المخروطية. السماح للمجاميع التي جمعت لتستقر في أنبوب مخروطي طاف به قبل أن يغسل جيدا وعاء الثقافة لتحقيق أقصى قدر من الانتعاش من الركام. ويمكن استخدام المجاميع عن المقايسات المصب على الفور ، مثل التدفق الخلوي ، المقايسات الغزو المناعي وغيرها. </li></ol> 7. ممثل النتائج ويظهر مثال EVT تشبه الخلايا (خلية SGHPL - 4 الأرومة الغاذية خط) التي تزرع في المراكز المناخية الإقليمية على Cytodex - 3 حبات في الشكل 2. خط خلية EVT مثل يعرض توقعات بتمديد بعيدا عن الكتلة الرئيسية ، وربط على كتل المجاورة. مغطاة بالكامل العديد من الخرز مع خلايا التكاثر. إزالة مرة واحدة من المراكز المناخية الإقليمية وجيش التحرير الشعبى الصينىتيد على المصفوفة خارج الخلية ، وEVT مثل 3 - D غزو الخلايا المزروعة بقوة و / أو ترحيل (الشكل 3). RT - PCR البيانات تؤكد على التعبير زيادة MMPs ينظر في المجاميع 3 - D بدلا من الثقافة التقليدية monolayers الخلية (الشكل 4). ومن المثير للاهتمام ، هي أيضا upregulated الجينات لا ترتبط مع الغزو في المراكز المناخية الإقليمية (الجدول 1) و قد تساعد في تحديد مجالات مثل تفاعلات المناعية مع trophoblasts غزو الخلايا. <img alt="الشكل 1" src="/files/ftp_upload/3367/3367fig1.jpg" /> الشكل 1. تصوير أفلام كارتون لنظام خلية الثقافة الدوارة (المراكز المناخية الإقليمية). <img alt="الشكل 2" src="/files/ftp_upload/3367/3367fig2.jpg" /> الشكل 2. المرحلة مجهرية على النقيض من إجمالي النمو ممثل بعد 5 أيام في المراكز المناخية الإقليمية. تشير الأسهم إلى غزو الخلايا و* يدل على حبات microcarrier Cytodex - 3. <imgALT = &quot;شكل 3&quot; SRC = &quot;/ files/ftp_upload/3367/3367fig3.jpg&quot; /&gt; كانت جزءا لا يتجزأ من الرقم 3. المجاميع المراكز المناخية الإقليمية يزرع في المواد الهلامية الليفين. ولوحظ من خلال غزو الجل الليفين في أقرب وقت (أ) تضمين بعد الليفين 24 ساعة و (ب) واصلت خلال 48 ساعة. تشير الأسهم الخلايا الغازية ويدل على Cytodex * 3 حبات microcarrier (بتصرف ، بإذن من المرجع 7). <img alt="الشكل 4" src="/files/ftp_upload/3367/3367fig4.jpg" /> نشر الرقم 4. zymogram الجيلاتين المقارن المونولاير والمراكز المناخية الإقليمية SGHPL - 4 - EVT مثل الخلايا. ويفرز أشكالا المؤيدة ونشطة من MMP - 2 و MMP - 9 من الأرومة الغاذية المراكز المناخية الإقليمية الخلايا المزروعة (بتصرف ، بإذن من المرجع 7). <img alt="الجدول 1" src="/files/ftp_upload/3367/3367table1.jpg" /> الجدول 1. ملخص النتائج ميكروأري من خلايا - 4 SGHPL نمت في المراكز المناخية الإقليمية (بتصرف ، بإذن من المرجع 7). <ol class="lalpha" style=";text-align:right;direction:rtl"><liوقد حسبت&gt; الحث طية في المجاميع 3 - D على النحو التالي : تطبيع 3 - D الجينات القيمة / الجين monlayer تطبيع القيمة. </li><li style=";text-align:right;direction:rtl"> يشير الجينات التي كانت أقل من الحد من الكشف في monolayers. </li></ol>

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Cell. Sci. 116, 2377-2377 (2003).</li><li>Bentrup, H. ?. ?. n. e. r. z. u., K, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+organotypic+models+of+human+colonic+epithelium+to+study+the+early+stages+of+enteric+salmonellosis.">Three-dimensional organotypic models of human colonic epithelium to study the early stages of enteric salmonellosis.</a> Microbes. Infect. 8, 1813-1813 (2006).</li><li>Carterson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A549+lung+epithelial+cells+grown+as+three-dimensional+aggregates:+alternative+tissue+culture+model+for+Pseudomonas+aeruginosa+pathogenesis.">A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis.</a> Infect. Immun. 73, 1129-1129 (2005).</li><li>Myers, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closing+the+phenotypic+gap+between+transformed+neuronal+cell+lines+in+culture+and+untransformed+neurons.">Closing the phenotypic gap between transformed neuronal cell lines in culture and untransformed neurons.</a> J. Neurosci. Methods. 174, 31-31 (2008).</li><li>Hjelm, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+three-dimensional+organotypic+human+vaginal+epithelial+cell+model.">Development and characterization of a three-dimensional organotypic human vaginal epithelial cell model.</a> Biol. Reprod. 82, 617-617 (2010).</li><li>Straub, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+culture+infectivity+assay+for+human+noroviruses.">In vitro cell culture infectivity assay for human noroviruses.</a> Emerg. Infect. Dis. 13, 396-396 (2007).</li><li>Nickerson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tissue+assemblies:+novel+models+for+the+study+of+Salmonella+enterica+serovar+Typhimurium+pathogenesis.">Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis.</a> Infect. Immun. 69, 7106-7106 (2001).</li><li>Carvalho, H. M., Teel, L. D., Goping, G., O'Brien, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+tissue+culture+model+for+the+study+of+attach+and+efface+lesion+formation+by+enteropathogenic+and+enterohaemorrhagic+Escherichia+coli.">A three-dimensional tissue culture model for the study of attach and efface lesion formation by enteropathogenic and enterohaemorrhagic Escherichia coli.</a> Cell. Microbiol. 7, 1771-1771 (2005).</li><li>Sainz, B., TenCate, V., Uprichard, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+Huh7+cell+culture+system+for+the+study+of+Hepatitis+C+virus+infection.">Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection.</a> Virol. J. 6, 103-103 (2009).</li><li>Lelkes, P. I., Ramos, E., Nikolaychik, V. V., Wankowski, D. M., Unsworth, B. R., Goodwin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTSF-2:+a+new,+versatile+cell+culture+medium+for+diverse+normal+and+transformed+mammalian+cells.">GTSF-2: a new, versatile cell culture medium for diverse normal and transformed mammalian cells.</a> In Vitro Cell. Dev. Biol. Anim. 33, 344-344 (1997).</li><li>Lelkes, P. I., Ramos, E., Nikolaychik, V. V., Wankowski, D. M., Unsworth, B. R., Goodwin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTSF-2:+a+new,+versatile+cell+culture+medium+for+diverse+normal+and+transformed+mammalian+cells.">GTSF-2: a new, versatile cell culture medium for diverse normal and transformed mammalian cells.</a> In Vitro Cell. Dev. Biol. Anim. 33, 344-344 (1997).</li><li>GE Healthcare. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcarrier+Cell+Culture+-+Principles+and+Methods.">Microcarrier Cell Culture - Principles and Methods.</a> Handbooks. , (2005).</li></ol>

ليس لدينا شيء في الكشف عنها.

ثقافة تقنية المعروضة هنا يقدم المحققون مع الخلايا EVT مثل الغازية للغاية. وقد تم الآن من المسلم به أن فقدان التمايز يحدث في monolayers بسبب تثبيط الاستجابات الخلوية لمنبهات الكيميائية والجزيئية في ثلاثة أبعاد (سطوح الخلايا القمي ، القاعدية ، والجانبية) 10،13. هذا الأسل...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> Cytodex الخرز microcarrier </td><td> سيغما الدريخ </td><td> C3275 </td><td></td></tr><tr><td> نظام التناوب الثقافة الخلية (المراكز المناخية الإقليمية) </td><td> Synthecon </td><td> المراكز المناخية الإقليمية - D </td><td> ويشمل العرض الدوار قاعدة السلطة ، و 4 وحدات للتصرف المراكز المناخية الإقليمية </td></tr><tr><td> وحدات المراكز المناخية الإقليمية يعاد </td><td> Synthecon </td><td> الاتصال Synthecon </td><td></td></tr><tr><td> 3ml حقنة غيض Luer لوك </td><td> دينار بحريني </td><td> 309585 </td><td></td></tr><tr><td> 10ML اسعة طرف المصلية ماصة </td><td> دينار بحريني </td><td> 357504 </td><td></td></tr><tr><td> MEM ألفا </td><td> Invitrogen </td><td> 12561-072 </td><td></td></tr><tr><td> L - 15 ليبوفيتش المتوسطة ، ومسحوق </td><td> Invitrogen </td><td> 41300-039 </td><td></td></tr><tr><td> H 2 O ، الذيفان الداخلي مجانا </td><td> الصياد </td><td> MT - 25 - 055 - CM </td><td></td></tr><tr><td> بيكربونات الصوديوم </td><td> سيغما الدريخ </td><td> S - 7795 </td><td></td></tr><tr><td> الببتون مادة تنشأ عن البروتينات </td><td> فيشر العلمية </td><td> BP1420 - 100 </td><td></td></tr><tr><td> سكر الفاكهة </td><td> سيغما الدريخ </td><td> F3510 - 100 </td><td></td></tr><tr><td> الجالاكتوز </td><td> سيغما الدريخ </td><td> G5388 - 100 </td><td></td></tr><tr><td> جلوكوز </td><td> سيغما الدريخ </td><td> G7528 - 250 </td><td></td></tr><tr><td> HEPES </td><td> Invitrogen </td><td> 15630-080 </td><td></td></tr><tr><td> L - الجلوتامين </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> الانسولين ترانسفيرين - زيلونيت الصوديوم (ITS) </td><td> سيغما الدريخ </td><td> I1884 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10437 </td><td></td></tr><tr><td> البنسلين الستربتوميسين </td><td> Invitrogen </td><td> 15140 </td><td></td></tr></tbody></table>

rotating cell culture systems for human cell culture: human trophoblast cells as a model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

التقليدية ، واثنين من خلية ثقافة تقنيات الأبعاد غالبا ما تؤدي إلى تغيير الخصائص فيما يتعلق علامات التمايز ، السيتوكينات وعوامل النمو. ثلاثي الأبعاد ثقافة خلية في نظام خلية ثقافة الدورية (المراكز المناخية الإقليمية) استعادت التعبير عن كثير من هذه العوامل كما هو موضح هنا مع خط الأرومة الغاذية extravillous الخلية.

نظم الدورية الثقافة خلية خلية للثقافة الإنسان : خلايا الأرومة الغاذية الإنسان نموذجا

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

rotating-cell-culture-systems-for-human-cell-culture-human

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Bioengineering

16.4K Views.  Tulane University Medical School. التقليدية ، واثنين من خلية ثقافة تقنيات الأبعاد غالبا ما تؤدي إلى تغيير الخصائص فيما يتعلق علامات التمايز ، السيتوكينات وعوامل النمو. ثلاثي الأبعاد ثقافة خلية في نظام خلية ثقافة الدورية (المراكز المناخية الإقليمية) استعادت التعبير عن كثير من هذه العوامل كما هو ...

Video: Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Microfabricated المنصات الحيوي للثقافة الخلية ميكانيكيا

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

البوليمرات الذاكرة شكل خلية للثقافة أحدث

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Microcapsule الجينات كمنصة 3D لإكثار وتمايز الخلايا الجذعية الجنينية البشرية (hESC) إلى أنساب مختلفة

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

نموذج ثلاثي الأبعاد للخلية ثقافة قياس آثار تدفق السائل الخلالي على غزو الخلايا السرطانية

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

خلية المشارك الثقافة الزخرفة باستخدام مائي مرحلتين أنظمة

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

في المختبر خلية الثقافة نموذج لاستنشاق الفحوصات الكيميائية السامة

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

سامسونج بوراتيد مطاطا الهلاميات المائية كغشاء الطابق السفلي المحاكاة البيولوجية لثنائي خلية ثقافة

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

ثقافة الخلايا الجذعية Pluripotent البشرية على حمض البولي فينيل الكحول-Co-إيتاكونيك الهلاميات المائية مع صلابة متفاوتة تحت ظروف خالية من كرة

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

A الإنسان 3D مصفوفة-الخلايا الشحمية نموذج ثقافة لدراسة مصفوفة خليه الأيض الأيضي

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

قياس إمكانات العمل البصري أحادي الخلية في خلايا القلب المشتقة من الخلايا الجذعية المتعددة القدرات

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...