نشكر الجهود التعاونية الدكتور ثاو دانغ وأعضاء مختبر لها لاستخدام معدات التصوير والمجهر بهم. كما نشكر Biorepository والأنسجة مرفق البحوث (BTRF) الأساسية، جيف هاربر، والمقيمين في جامعة فيرجينيا لتزويدنا أنسجة الرحم. نشكر كارول Szlachta للمساعدة في تخطيطي لمحة عامة.

<ol>
	<li>Heller, D., Heller, 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> in The endometrium: a clinicopathologic approach. , 56-75 (1994).</li><li>Emera, D., Romero, R., Wagner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+menstruation:+a+new+model+for+genetic+assimilation:+explaining+molecular+origins+of+maternal+responses+to+fetal+invasiveness.">The evolution of menstruation: a new model for genetic assimilation: explaining molecular origins of maternal responses to fetal invasiveness.</a> Bioessays. 34, 26-35 (2011).</li><li>Gudjonsson, T., Villadsen, R., Ronnov-Jessen, L., Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+protocols+used+in+cell+culture+models+of+human+breast+morphogenesis.">Immortalization protocols used in cell culture models of human breast morphogenesis.</a> Cell Mol Life Sci. 61, 2523-2534 (2004).</li><li>Brosens, J. J., Hayashi, N., White, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+receptor+regulates+decidual+prolactin+expression+in+differentiating+human+endometrial+stromal+cells.">Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells.</a> Endocrinology. 140, 4809-4820 (1999).</li><li>Jones, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+SUMO+pathway+sensitizes+differentiating+human+endometrial+stromal+cells+to+progesterone.">Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone.</a> Proc Natl Acad Sci U S A. 103, 16272-16277 (2006).</li><li>Salamonsen, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+the+human+endometrium+and+uterine+fluid:+a+pathway+to+biomarker+discovery.">Proteomics of the human endometrium and uterine fluid: a pathway to biomarker discovery.</a> Fertil Steril. 99, 1086-1092 (2013).</li><li>Haouzi, D., Dechaud, H., Assou, S., De Vos, J., Hamamah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+human+endometrial+receptivity+from+transcriptomic+and+proteomic+data.">Insights into human endometrial receptivity from transcriptomic and proteomic data.</a> Reprod Biomed Online. 24, 23-34 (2012).</li><li>Chen, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+characterization+of+midproliferative+and+midsecretory+human+endometrium.">Proteomic characterization of midproliferative and midsecretory human endometrium.</a> J Proteome Res. 8, 2032-2044 (2009).</li><li>Talbi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+phenotyping+of+human+endometrium+distinguishes+menstrual+cycle+phases+and+underlying+biological+processes+in+normo-ovulatory+women.">Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.</a> Endocrinology. 147, 1097-1121 (2006).</li><li>Punyadeera, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oestrogen-modulated+gene+expression+in+the+human+endometrium.">Oestrogen-modulated gene expression in the human endometrium.</a> Cell Mol Life Sci. 62, 239-250 (2005).</li><li>Ponnampalam, A. P., Weston, G. C., Susil, B., Rogers, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+human+endometrium+during+the+menstrual+cycle.">Molecular profiling of human endometrium during the menstrual cycle.</a> Aust N Z J Obstet Gynaecol. 46, 154-158 (2006).</li><li>Zhang, L., Rees, M. C., Bicknell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+long-term+culture+of+normal+human+endometrial+epithelium+and+stroma.+Expression+of+mRNAs+for+angiogenic+polypeptides+basally+and+on+oestrogen+and+progesterone+challenges.">The isolation and long-term culture of normal human endometrial epithelium and stroma. Expression of mRNAs for angiogenic polypeptides basally and on oestrogen and progesterone challenges.</a> J Cell Sci. 108 (1), 323-331 (1995).</li><li>Richards, R. G., Brar, A. K., Frank, G. R., Hartman, S. M., Jikihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+cells+from+term+human+decidua+closely+resemble+endometrial+stromal+cells:+induction+of+prolactin+and+insulin-like+growth+factor+binding+protein-1+expression).">Fibroblast cells from term human decidua closely resemble endometrial stromal cells: induction of prolactin and insulin-like growth factor binding protein-1 expression).</a> Biol Reprod. 52, 609-615 (1995).</li><li>Gellersen, B., Brosens, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+and+progesterone+receptor+cross-talk+in+human+endometrium:+a+decidualizing+affair.">Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair.</a> J Endocrinol. 178, 357-372 (2003).</li><li>Marsh, M. M., Hampton, A. L., Riley, S. C., Findlay, J. K., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+endothelin+released+by+human+endometrial+epithelial+cells+in+culture.">Production and characterization of endothelin released by human endometrial epithelial cells in culture.</a> J Clin Endocrinol Metab. 79, 1625-1631 (1994).</li><li>Siegfried, J. M., Nelson, K. G., Martin, J. L., Kaufman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+identification+of+cultured+cells+from+human+endometrium.">Histochemical identification of cultured cells from human endometrium.</a> In Vitro. 20, 25-32 (1984).</li><li>Rawdanowicz, T. J., Hampton, A. L., Nagase, H., Woolley, D. E., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+production+by+cultured+human+endometrial+stromal+cells:+identification+of+interstitial+collagenase,+gelatinase-A,+gelatinase-B,+and+stromelysin-1+and+their+differential+regulation+by+interleukin-1+alpha+and+tumor+necrosis+factor-alpha.">Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B, and stromelysin-1 and their differential regulation by interleukin-1 alpha and tumor necrosis factor-alpha.</a> J Clin Endocrinol Metab. 79, 530-536 (1994).</li><li>Dimitriadis, E., Robb, L., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+11+advances+progesterone-induced+decidualization+of+human+endometrial+stromal+cells.">Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells.</a> Mol Hum Reprod. 8, 636-643 (2002).</li><li>Sakai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+histone+acetylation+in+ovarian+steroid-induced+decidualization+of+human+endometrial+stromal+cells.">Involvement of histone acetylation in ovarian steroid-induced decidualization of human endometrial stromal cells.</a> J Biol Chem. 278, 16675-16682 (2003).</li><li>Logan, P. C., Ponnampalam, A. P., Steiner, M., Mitchell, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cyclic+AMP+and+estrogen/progesterone+on+the+transcription+of+DNA+methyltransferases+during+the+decidualization+of+human+endometrial+stromal+cells.">Effect of cyclic AMP and estrogen/progesterone on the transcription of DNA methyltransferases during the decidualization of human endometrial stromal cells.</a> Mol Hum Reprod. 19, 302-312 (2013).</li><li>Tamura, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+IGFBP-1+expression+by+cAMP+is+associated+with+histone+acetylation+status+of+the+promoter+region+in+human+endometrial+stromal+cells.">Induction of IGFBP-1 expression by cAMP is associated with histone acetylation status of the promoter region in human endometrial stromal cells.</a> Endocrinology. 153, 5612-5621 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American Society for Reproductive Medicine: Quick facts about infertility. , (2013).</li><li>Salker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+selection+of+human+embryos:+impaired+decidualization+of+endometrium+disables+embryo-maternal+interactions+and+causes+recurrent+pregnancy+loss.">Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss.</a> PLoS One. 5, (2010).</li></ol>

والكتاب ليس لديهم ما الإفصاح.

وقد وصفت جماعات أخرى وتكييفها منهجية لإعداد الثقافات انسجة بطانة الرحم، وهي في معظمها تستخدم كولاجيناز 4،12،13،15-18. في هذا المخطوط، قدمنا ​​المنهجية والدليل على طريقتين مبسطة الأولية بطانة الرحم الثقافة اللحمية، وكلاهما تستخدم من قبل مختبرنا لأسباب اقتصادية ?...

ويتكون جسم الإنسان الرحم من ثلاث طبقات، وperimetrium (أو المصلية)، وعضل الرحم، وبطانة الرحم. تميز كل من هذه الطبقات هي خطوة مهمة لإنشاء خطوط الخلايا بطانة الرحم. وperimetrium هو الأكثر الخارجي طبقة من الرحم ويتكون من خلايا المصلية رقيقة. عضل الرحم هو، طبقة وسطى سميكة من الرحم وتتألف من خلايا العضلات الملساء. يتم التعرف على بطانة الرحم والطبقة الداخلية للرحم، ويشمل الظهارية وخلايا انسجة السكان. تنقسم بطانة الرحم الى مزيد من الطبقة القاعدية التي هو الافتراض السكان الخلية لإعادة ملء طبقة طبقة وظيفية كل ما يقرب من 28 أيام 1 الجذعية. طبقة من بطانة الرحم وظيفي الإنسان يخضع لتغيرات كيميائية حيوية والصرفية كبير في استجابة لتعميم الهرمونات. وتستمد هذه الهرمونات من الغدة النخامية والمبايض. الإنتاج منسقة والإفراج عن نتائج الهرمونات في دورة الإنجابية. تم تصميم دورة التكاثر لتحضير بطانة الرحم لغرس الجنين الأحداث المحتملة. في البشر، ومن المعروف أن دورة التكاثر باسم &quot;الدورة الشهرية&quot; وينقسم إلى ثلاث مراحل - التكاثري، إفرازية، والحيض. المرحلة التكاثري ينطوي على انتشار وظيفي طبقة بطانة الرحم في حين وضعت المرحلة إفرازية بواسطة ظيفي النضج. على وجه التحديد، والتعديلات خارج الخلية، إفرازات، وتمايز الخلايا يشير إلى غرس المحتملة. إذا لم تحدث زرع قبل نهاية المرحلة إفرازية، وتسليط طبقة بطانة الرحم وظيفي أثناء مرحلة الحيض. لا تزال قيد المناقشة على أهمية الحيض والأحداث التي تؤدي إلى سفك طبقة طبقة وظيفية. في البشر، وقد طرحت أن الحيض هو نتيجة لحدث معين منتصف المرحلة إفرازية التمايز المعروفكما &quot;decidualization عفوية&quot; 2. في هذا المخطوط، ونحن نقدم منهجية مفصلة لكلتا الطريقتين العزلة خلية انسجة بطانة الرحم، واستخدام مزيج من المناعي والصور الرقمية لإثبات فعالية هذه النهج. بالإضافة إلى ذلك، نحن نطبق التي يشيع استخدامها في نموذج المختبر من decidualization عفوية لتأكيد بطانة الرحم العزلة الخلايا اللحمية.

<table><tbody><tr><td>0.25 Trypsin or 0.05% Trypsin&amp;nbsp;</td><td>Hyclone</td><td>SH3023602 or SH30004202</td><td></td></tr><tr><td>1.7 micro Centrifuge Tube&amp;nbsp;&amp;nbsp;</td><td>Genesee Scientific</td><td>22-272A</td><td></td></tr><tr><td>1 &amp;micro;l, 20 &amp;micro;l, 200 ml and 1,000 &amp;micro;l Pipette&amp;nbsp;&amp;nbsp;</td><td>Genesee Scientific</td><td>24-401,24-402, 24-412, 24-430</td><td></td></tr><tr><td>15 ml Conical Tube&amp;nbsp;</td><td>Hyclone</td><td>339650</td><td></td></tr><tr><td>50 ml Conical Tube&amp;nbsp;</td><td>Hyclone</td><td>339652</td><td></td></tr><tr><td>6 cm Cell Culture Dish&amp;nbsp;</td><td>Thermo scientific</td><td>12-556-002</td><td></td></tr><tr><td>8 well Chambers&amp;nbsp;</td><td>Thermo Scientific</td><td>AB-4162</td><td></td></tr><tr><td>Acetate&amp;nbsp;</td><td>Fisher scientific</td><td>C4-100</td><td></td></tr><tr><td>AMV RT Enzyme/Buffer&amp;nbsp;</td><td>Bio Labs</td><td>M077L</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)&amp;nbsp;</td><td>Fisher Scientific</td><td>BP-1605-100</td><td></td></tr><tr><td>Buffered Zinc Formalin&amp;nbsp;</td><td>Thermo</td><td>59201ZF</td><td></td></tr><tr><td>Charcoal strip FBS&amp;nbsp;</td><td>Fisher</td><td>NC9019735</td><td></td></tr><tr><td>Chloroform&amp;nbsp;</td><td>Fisher Scientific</td><td>BP1145-1</td><td></td></tr><tr><td>Cover slip&amp;nbsp;</td><td>Fisher Brand</td><td>12-544D</td><td></td></tr><tr><td>Cyclic AMP (cAMP)&amp;nbsp;</td><td>Sigma</td><td>B7880</td><td></td></tr><tr><td>DMEM/High Glucose&amp;nbsp;</td><td>Hyclone</td><td>SH30243FS</td><td></td></tr><tr><td>dNTP&amp;nbsp;</td><td>Bioline</td><td>BIO-39025</td><td></td></tr><tr><td>Donkey Anti Goat -TRITC&amp;nbsp;</td><td>Santa Cruz</td><td>SC-3855</td><td></td></tr><tr><td>Donkey Serum&amp;nbsp;</td><td>Jackson&amp;rsquo;s lab</td><td>017-000-002</td><td></td></tr><tr><td>E Cadherin Antibody&amp;nbsp;&amp;nbsp;</td><td>Epitomics</td><td>1702-1</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Fisher Scientific</td><td>BP2818-1</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)&amp;nbsp;</td><td>Fisher Scientific</td><td>03-600-511</td><td></td></tr><tr><td>Fungizone Amphotericin B&amp;nbsp;</td><td>Gibco</td><td>15290-018</td><td></td></tr><tr><td>GAPDH Probe&amp;nbsp;</td><td>Life Technologies</td><td>HS99999905</td><td></td></tr><tr><td>Glycogen&amp;nbsp;</td><td>5Prime</td><td>2301440</td><td></td></tr><tr><td>Goat Anti Mouse - FITC&amp;nbsp;</td><td>Jackson&amp;rsquo;s Lab</td><td>115-096-003</td><td></td></tr><tr><td>Isopropanol&amp;nbsp;</td><td>Fisher Scientific</td><td>BP2618-1</td><td></td></tr><tr><td>Kanamycin&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific</td><td>BP906-5</td><td></td></tr><tr><td>Medroxyprogesterone acetate (MPA)&amp;nbsp;</td><td>Sigma</td><td>M1629</td><td></td></tr><tr><td>MeOH (Methanol)&amp;nbsp;</td><td>Fisher Scientific</td><td>A4-08-1</td><td></td></tr><tr><td>Mounting Media (w/DAPI)&amp;nbsp;</td><td>Vector Labratories</td><td>H-1500</td><td></td></tr><tr><td>N6 DNA Oligos&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Number 15 Scraper&amp;nbsp;&amp;nbsp;</td><td>BD</td><td>371615</td><td></td></tr><tr><td>Pan Cytokeratin&amp;nbsp; Mouse mAB&amp;nbsp;</td><td>Cell Signaling</td><td>4545</td><td></td></tr><tr><td>PBS (phosphate buffered saline)&amp;nbsp;</td><td>Fisher Scientific</td><td>BP-399-4</td><td></td></tr><tr><td>Penicillin-Streptomycin Glutamine Solution 100X&amp;nbsp;&amp;nbsp;</td><td>Hyclone</td><td>SV30082.01</td><td></td></tr><tr><td>PML Anti Goat Anti body&amp;nbsp;</td><td>Santa Cruz</td><td>SC-9862</td><td></td></tr><tr><td>Primer(s)&amp;nbsp;</td><td>Eurofins</td><td></td><td></td></tr><tr><td>RPMI&amp;nbsp;</td><td>Hyclone</td><td>SH30027FS</td><td></td></tr><tr><td>RPMI (Phenol free)&amp;nbsp;</td><td>Gibco</td><td>11835</td><td></td></tr><tr><td>Sybr Green&amp;nbsp;&amp;nbsp;</td><td>Thermo Scientific</td><td>AB-4162</td><td></td></tr><tr><td>Taqman&amp;nbsp;</td><td>Thermo</td><td>AB-4138</td><td></td></tr><tr><td>Trizol&amp;nbsp;</td><td>Life Technologies</td><td>15596018</td><td></td></tr><tr><td>Vimentin Antibody&amp;nbsp;</td><td>Epitomics</td><td>4211-1</td><td></td></tr></tbody></table>

two methods for establishing primary human endometrial stromal cells from hysterectomy specimens

وقد كرس الكثير من الجهود لإنشاء نظم في خلية ثقافة المختبر. تم تصميم هذه النظم في تصميم نموذج لعدد كبير من العمليات في الجسم الحي. نظم زراعة الخلايا الناشئة من عينات بطانة الرحم الإنسان ليست استثناء. وتتراوح التطبيقات من العمليات الفسيولوجية دوري طبيعية لأمراض الرحم مثل سرطانات أمراض النساء، والأمراض المعدية، وأوجه القصور الإنجابية. هنا، ونحن نقدم طريقتين لإنشاء خلايا انسجة بطانة الرحم الأساسي من مقطوعة جراحيا العينات استئصال الرحم بطانة الرحم. يشار الأسلوب الأول باسم &quot;أسلوب تجريف&quot;، ويتضمن تجريف الميكانيكية باستخدام شفرات الحلاقة أو الجراحية في حين يسمى الطريقة الثانية &quot;طريقة التربسين.&quot; يستخدم هذا الأسلوب الأخير في النشاط الأنزيمي من التربسين لتعزيز الفصل بين الخلايا الأولية و ثمرة الخلية. نحن لتوضيح خطوة بخطوة منهجية من خلال الصور الرقمية والمجهري. ونحن كما قدمأمثلة الإلكترونية للتحقق من صحة بطانة الرحم خطوط الخلايا اللحمية في الوقت الحقيقي عبر تفاعلات البلمرة الكمية (QPCR) والمناعي (IF).

كما أكد في قسم البروتوكول، يجب التأكد من إجراء جميع الأساليب في ظل حكومة والمؤسسية، والمبادئ التوجيهية الأخلاقية عند التعامل مع وإعداد الأنسجة البشرية. المدرجة في هذا المخطوط هو التوضيح من سير العمل العام &quot;أسلوب تجر?...

Watch this Scientific Journal Video about Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens at JoVE.com

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

إنشاء نظم الثقافة الابتدائية بطانة الرحم انسجة الخلايا من عينات استئصال الرحم هي تقنية البيولوجية قيمة وخطوة حاسمة قبل متابعة مجموعة واسعة من ويهدف البحث. هنا، نحن تصف طريقتين تستخدم لإنشاء الثقافات اللحمية من أنسجة بطانة الرحم جراحيا مقطوعة من المرضى من البشر.

طريقتين لإنشاء أولي خلايا وانسجة بطانة الرحم الإنسان من استئصال الرحم العينات

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

two-methods-for-establishing-primary-human-endometrial-stromal-cells

Research

JoVE Journal

Medicine

22.6K Views.  University of Virginia.  إنشاء نظم الثقافة الابتدائية بطانة الرحم انسجة الخلايا من عينات استئصال الرحم هي تقنية البيولوجية قيمة وخطوة حاسمة قبل متابعة مجموعة واسعة من ويهدف البحث. هنا، نحن تصف طريقتين تستخدم لإنشاء الثقافات اللحمية من أنسجة بطانة الرحم جراحيا مق...

Video: Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

عزل ماوس بطانة الرحم طلائي واللحمية خلايا ل<em&gt; في المختبر</em&gt; Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Developmental Biology

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

الحصول على خلايا السرطان الجذعية من أورام أمراض النساء وسرطان الثدي

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Cancer Research

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

عزل الخلايا اللحمية الرحم البشرية في المختبر ديسيدواليزيشن

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

طريقة الحصول على خلايا سرطان المبيض الابتدائية من العينات الصلبة

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

توليد العضوية متعددة الخلايا الإنسان الاساسيه بطانة الرحم

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

نمذجة خطوات المبكر عن سرطان المبيض نشر في الثقافة عضوي النمط من البريتوني تجويف الإنسان

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Establishing 3D Endometrial Organoids from the Mouse Uterus

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of luminal and glandular epithelium, a stromal compartment, a vascular network, and a complex immune cell population. Mouse models have been a powerful tool to study the endometrium, revealing critical mechanisms that control implantation, placentation, and cancer. The recent development of 3D endometrial organoid cultures presents a state-of-the-art model to dissect the signaling pathways that underlie endometrial biology. Establishing endometrial organoids from genetically engineered mouse models, analyzing their transcriptomes, and visualizing their morphology at a single-cell resolution are crucial tools for the study of endometrial diseases. This paper outlines methods to establish 3D cultures of endometrial epithelium from mice and describes techniques to quantify gene expression and analyze the histology of the organoids. The goal is to provide a resource that can be used to establish, culture, and study the gene expression and morphological characteristics of endometrial epithelial organoids.

This protocol describes methodologies to establish mouse endometrial epithelial organoids for gene expression and histological analyses.

إنشاء عضويات بطانة الرحم 3D من رحم الفأر

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of ...

Biology

Generation and Characterization of Rat Uterus Organoids from Rat Endometrial Epithelial Stem Cells

Endometrial organoids offer valuable insights into the development and pathophysiology of endometrial diseases and serve as platforms for drug testing. While human and mouse endometrial organoids have been developed, research on rat endometrial organoids remains limited. Given that rats can better simulate certain endometrial pathologies, such as intrauterine adhesions, this study aimed to establish rat endometrial organoids. We present a detailed protocol for the isolation and culture of rat endometrial epithelial stem cells (reESCs) and the generation of rat endometrial organoids. Using a refined reESCs expansion medium, we successfully isolated and stably expanded reESCs, demonstrating their long-term culture potential. The reESC-generated organoids exhibited typical structural and functional characteristics of the endometrium, including hormone responsiveness. Our results showed that rat endometrial organoids could be cultured over a long term with stable proliferation, maintaining the glandular structure, cell polarity, and functional characteristics of the endometrial epithelium. This novel rat-derived endometrial organoid model provides a valuable platform for studying endometrial diseases and testing therapeutic interventions, with potential applications across various mammalian species.

Here, we present a protocol to isolate and culture rat endometrial epithelial stem cells (reESCs), generating rat endometrial organoids. This method facilitates in vitro studies of endometrial diseases, enabling gene editing and other cellular manipulations.

Endometrial organoids offer valuable insights into the development and pathophysiology of endometrial diseases and serve as platforms for drug testing. ...

<meta charset="utf8"></head><body>نموذج سقالة مطبوع ثلاثي الأبعاد للتحقيق في آليات زرع الأجنة

Co-culture Model Using Two Types of Adherent Cell Lines

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

<meta charset="utf8"></head><body>أضواء الكاتب: نماذج بطانة الرحم في المختبر لدراسة تفاعلات الظهارة الظهارية والجنين

The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

نموذج الزراعة المشتركة باستخدام نوعين من خطوط الخلايا الملتصقة

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications ...

طريقتين لإنشاء أولي خلايا وانسجة بطانة الرحم الإنسان من استئصال الرحم العينات

Summary

Explore More Videos

Chapters in this video

عزل ماوس بطانة الرحم طلائي واللحمية خلايا ل

الحصول على خلايا السرطان الجذعية من أورام أمراض النساء وسرطان الثدي

عزل الخلايا اللحمية الرحم البشرية في المختبر ديسيدواليزيشن

طريقة الحصول على خلايا سرطان المبيض الابتدائية من العينات الصلبة

توليد العضوية متعددة الخلايا الإنسان الاساسيه بطانة الرحم

نمذجة خطوات المبكر عن سرطان المبيض نشر في الثقافة عضوي النمط من البريتوني تجويف الإنسان

إنشاء عضويات بطانة الرحم 3D من رحم الفأر

Generation and Characterization of Rat Uterus Organoids from Rat Endometrial Epithelial Stem Cells

نموذج الزراعة المشتركة باستخدام نوعين من خطوط الخلايا الملتصقة

نموذج الزراعة المشتركة باستخدام نوعين من خطوط الخلايا الملتصقة