ونحن ممتنون للسيد روجر أكرويد، السيد أندرو وايمان والسيد كريس ستودارد، مستشار الجراحون في شيفيلد المستشفيات التعليمية NHS مؤسسة ترست، لمساعدتهم في الحصول على عينات الأنسجة المريء ودعمهم لعملنا. نشكر أشرف انعام الحق لمساعدته دمج خطوط الخلايا السرطانية في النموذج. نحن نعترف بامتنان الدعم المالي لهذه الدراسة من المنح المقدمة من باردهان البحوث والتعليم الثقة (BRET) ويوركشاير أبحاث السرطان (YCR).

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Cancer Res. 1 (10), 729-738 (2003).</li><li>Bhargava, S., Chapple, C. R., Bullock, A. J., Layton, C., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+buccal+mucosa+for+substitution+urethroplasty.">Tissue-engineered buccal mucosa for substitution urethroplasty.</a> BJU. Int. 93 (6), 807-811 (2004).</li><li>Ralston, D. R., 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=Keratinocytes+contract+human+dermal+extracellular+matrix+and+reduce+soluble+fibronectin+production+by+fibroblasts+in+a+skin+composite+model.">Keratinocytes contract human dermal extracellular matrix and reduce soluble fibronectin production by fibroblasts in a skin composite model.</a> Br. J. Plast. Surg. 50 (6), 408-415 (1997).</li><li>Green, N. H., 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=Pulsatile+exposure+to+simulated+reflux+leads+to+changes+in+gene+expression+in+a+3D+model+of+oesophageal+mucosa.">Pulsatile exposure to simulated reflux leads to changes in gene expression in a 3D model of oesophageal mucosa.</a> Int. J. Exp. Pathol. 95 (3), 216-228 (2014).</li><li>Wang, C. 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=Selective+culture+of+epithelial+cells+from+primary+breast+carcinomas+using+irradiated+3T3+cells+as+feeder+layer.">Selective culture of epithelial cells from primary breast carcinomas using irradiated 3T3 cells as feeder layer.</a> Pathol. Res. Pract. 197 (3), 175-181 (2001).</li><li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsinizing+and+subculturing+mammalian+cells.">Trypsinizing and subculturing mammalian cells.</a> J. Vis. Exp. (16), e755 (2008).</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=Using+a+Hemacytometer+to+Count+Cells.">Using a Hemacytometer to Count Cells.</a> Basic Methods in Cellular and Molecular Biology. , (2014).</li><li>Mahmood, T., Yang, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blot:+technique,+theory,+and+trouble+shooting.">Western blot: technique, theory, and trouble shooting.</a> N. Am. J. Med. Sci. 4 (9), 429-434 (2012).</li><li>Streit, S., Michalski, C. W., Erkan, M., Kleeff, J., Friess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Northern+blot+analysis+for+detection+and+quantification+of+RNA+in+pancreatic+cancer+cells+and+tissues.">Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues.</a> Nat. Protoc. 4 (1), 37-43 (2009).</li><li>Morgan, E., 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=Cytometric+bead+array:+a+multiplexed+assay+platform+with+applications+in+various+areas+of+biology.">Cytometric bead array: a multiplexed assay platform with applications in various areas of biology.</a> Clin. Immunol. 110 (3), 252-266 (2004).</li><li>Xu, 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=The+abnormal+expression+of+retinoic+acid+receptor-beta,+p+53+and+Ki67+protein+in+normal,+premalignant+and+malignant+esophageal+tissues.">The abnormal expression of retinoic acid receptor-beta, p 53 and Ki67 protein in normal, premalignant and malignant esophageal tissues.</a> World J. Gastroenterol. 8 (2), 200-202 (2002).</li><li>Moll, R., Divo, M., Langbein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+keratins:+biology+and+pathology.">The human keratins: biology and pathology.</a> Histochem. Cell Biol. 129 (6), 705-733 (2008).</li><li>Rice, R. H., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+in+human+epidermal+cells+of+a+soluble+protein+precursor+of+the+cross-linked+envelope:+activation+of+the+cross-linking+by+calcium+ions.">Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.</a> Cell. 18 (3), 681-694 (1979).</li><li>Eves, P., 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=Melanoma+invasion+in+reconstructed+human+skin+is+influenced+by+skin+cells+-+investigation+of+the+role+of+proteolytic+enzymes.">Melanoma invasion in reconstructed human skin is influenced by skin cells - investigation of the role of proteolytic enzymes.</a> Clin. Exp. Metastasis. 20 (8), 685-700 (2003).</li></ol>

الكتاب ليس لديهم ما يكشف.

تصف هذه المخطوطة إنتاج وتوصيف المريء البشري نموذج الغشاء المخاطي بيولوجيا مناسبة لاستخدامها بوصفها منصة تجريبية لدراسة تأثير التعرض للالضغوطات البيئية على ظهارة المريء. الخطوات الأكثر أهمية لإنتاج الناجح لنموذج الغشاء المخاط...

ويتكون الغشاء المخاطي المريء والطبقية، ظهارة الحرشفية فوق طبقة من النسيج الضام، الصفيحة المخصوصة، ويعد واحدا من المواقع الأولى لمواجهة الضغوطات البيئية بلعها. هو متورط التعرض للسموم الغذائية في تطور سرطان المريء الحرشفية، بينما-duodenogastro المريء الجزر هو العامل الحاسم في التسبب في حؤول باريت، الذي يرتبط مع زيادة خطر تطور الى غدية المريء. سرطان المريء هي الورم الخبيث الأكثر شيوعا ال 8 في الذكور UK وغدية المريء يتزايد بسرعة في العالم الغربي 1. وعلاوة على ذلك، كان هناك تحسن طفيف في تشخيص المرض، مع معدل البقاء على قيد الحياة لمدة 5 سنوات الإجمالي حوالي 15٪. ونتيجة لذلك هناك حاجة لمنصات التجريبية للتحقيق في تأثير التعرض للضغوطات البيئية على هذا ظهارة المريء ومشاركتها المحتملة في development من حؤول أو الأورام. على الرغم من خطوط الخلايا خلد أو ورم تسمح للباحثين لدراسة استجابة الخلايا الظهارية لهذه الضغوطات في المختبر، إلا أنها تظل التكاثري وتفشل في التمايز إلى خلايا الظهارية ناضجة وجدت على الطبقات العليا من الغشاء المخاطي المريء. وعلاوة على ذلك، وخطوط الخلايا التي خضعت بالفعل تكون الأورام قد توفر معلومات محدودة فقط بشأن الاستجابات الأولية من الخلايا الطبيعية داخل الظهارة إلى العوامل البيئية؛ وهذه هي المرحلة عندما إمكانية التدخل العلاجي قد تكون أعلى. أخيرا، تفشل نظم زراعة الخلايا التقليدية للقبض على التفاعلات المهمة المحتملة بين الخلايا الظهارية والوسيطة وبين هذه الخلايا والمصفوفة المحيطة التي تحدث داخل أنسجة في الجسم الحي. النماذج الحيوانية توفر المكروية أكثر واقعية لدراسة ردود epitheli المريءأم ويمكن أن تتضمن تحريض الاصطناعي لل-مرض الجزر المعدي المريئي (2). لكن يمكن أن يكون أكثر تحديا لمعالجة الضغوطات البيئية في هذه النماذج وأنها قد لا تمثل تماما على رد في غضون المريء البشري. وقد وضعت نماذج تجريبية المريء الإنسان الأخرى التي تستخدم الخلايا الأولية، وخلايا خلدت أو خطوط الخلايا السرطانية على الكولاجين، أو الجمع بين الكولاجين / Matrigel، سقالة التي تحتوي على الخلايا الليفية 3،4. أنها أقل كثافة عمالية لتوليد هذه الاطر من سقالة المريء ديكي وصفها في هذه المخطوطة، وهذه النماذج عضوي النمط توفر أداة مفيدة، ولا سيما في دراسة غزو الورم 5،6، حيث تسلل الخلايا السرطانية في هلام الكولاجين يمكن أن يكون بسهولة لوحظ. ولكن هذه المواد الهلامية الكولاجين لها خصائص ميكانيكية غير الأصلية وتفتقر إلى ميزات معينة من الأنسجة الأصلية، بما في ذلك الغشاء القاعدي محددة وappropriatالبريد تضاريس السطح. هذا يمكن أن تؤثر على سلوك الخلايا الناتجة، على سبيل المثال، التصاق فقرا بين الظهارة وسقالة عند استخدام الكولاجين هلام سقالة 7. ونتيجة لذلك تم تطوير سقالة المريء الخنازير ديكي، مع ميزة كونها سقالة أكثر واقعية من الناحية البيولوجية، وبالتالي أكثر ملاءمة للاستخدام كمنصة التجريبية. وقد تبين أيضا أنه من الأفضل أن دمج الخلايا الأولية في بنيات المريء من خطوط الخلايا الظهارية خلد المريء، مثل هيت-1A، لأن هذه الخلايا تشكل ظهارة متعددة الطبقات لكنه يفشل في تطبق أو تمييز 4،7،8 . ونتيجة لذلك، تم تكييف هذا البروتوكول من طريقة مستخدمة بالفعل في المختبر ماكنيل لصنع الأنسجة المهندسة الجلد والغشاء المخاطي للفم 9،10 ويشتمل على الخنازير سقالة دي cellularized المريء جنبا إلى جنب مع خلايا المريء الإنسان الأساسية الظهارية والخلايا الليفية. ثيالصورة بروتوكول ينتج ناضجة، ظهارة طبقية، مماثلة لتلك التي من المريء الإنسان العادي كما يتبين من CK4، CK14، Ki67 وinvolucrin تلطيخ. يوفر النموذج الناتج منصة تجريبية لدراسة الردود على الضغوطات البيئية، واستخدمت على نحو فعال لتحقيق تغييرات في التعبير الجيني في الظهارة المريء ردا على refluxate مكونات 11.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Trypsin</td>
			<td>BD Biosciences</td>
			<td align="right">215240</td>
			<td>Prepare 0.1% w/v solution in PBS and filter sterilise. Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Labtech</td>
			<td>LM-D1112</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Labtech</td>
			<td>LM-H1236</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Foetal Calf Serum</td>
			<td>Labtech</td>
			<td>FB-1090</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>R+D Systems</td>
			<td>236-EG-200</td>
			<td>Prepare 200 &#181;g/ml stock solution in 10 mM acetic acid, 1% FCS</td>
		</tr>
		<tr>
			<td>Hydorcortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0396</td>
			<td>Prepare stock solution in PBS and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma-Aldrich</td>
			<td>A2786</td>
			<td>Prepare stock solution in PBS and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I2767</td>
			<td>Prepare 10 mg/ml solution in 0.01M HCl, dilute 1:10 in distilled water and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T2036</td>
			<td>Prepare stock solution in distilled water and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Triiodothyronine</td>
			<td>Sigma-Aldrich</td>
			<td>T2752</td>
			<td>Prepare stock solution in distilled water and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td>Prepare stock solution in water</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15290-026</td>
			<td>Brand name Fungizone</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Oxoid</td>
			<td>BR0014</td>
			<td>Dissolve 1 tablet in 100 ml water and autoclave to sterilise</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td align="right">10103578001</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine solution</td>
			<td>Ecolab</td>
			<td>10830E</td>
			<td>Brand name Videne</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td align="right">433209</td>
			<td>Prepare 1M solution and autoclave to sterilise before use (121 &#730;C for 15 min)</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G2025</td>
			<td>Autoclave to sterilise before use (121 &#730;C for 15 min)</td>
		</tr>
		<tr>
			<td>Chelex 100</td>
			<td>Sigma-Aldrich</td>
			<td>C7901</td>
			<td></td>
		</tr>
		<tr>
			<td>Newborn calf serum</td>
			<td>Gibco</td>
			<td align="right">26010074</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma-Aldrich</td>
			<td>P8783</td>
			<td>Prepare stock solution in DMEM and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>E9508</td>
			<td>Prepare stock solution in DMEM and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>Prepare stock solution in DMEM and filter sterilise before use use</td>
		</tr>
		<tr>
			<td>O-phosphorylethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>P0503</td>
			<td>Prepare stock solution in DMEM and filter sterilise before use</td>
		</tr>
		<tr>
			<td>ITS (insulin, transferrin, selenium)</td>
			<td>Lonza</td>
			<td>17-838Z</td>
			<td>Used for composite media preparation</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T3924</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>EDTA 0.02% solution</td>
			<td>Sigma-Aldrich</td>
			<td>E8008</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>T75 culture flask</td>
			<td>VWR</td>
			<td>734-2313</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>Fisher</td>
			<td align="right">11819650</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml universal tube</td>
			<td>SLS</td>
			<td>SLS7504</td>
			<td></td>
		</tr>
		<tr>
			<td>180 ml pot</td>
			<td>VWR</td>
			<td>216-2603</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>SLS</td>
			<td align="right">150350</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>VWR</td>
			<td>734-2323</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel rings</td>
			<td></td>
			<td></td>
			<td>Manufactured in house - medical grade stainless steel, internal diameter 10 mm, external diameter 20 mm</td>
		</tr>
		<tr>
			<td>steel mesh grids</td>
			<td></td>
			<td></td>
			<td>Manufactured in house - sheets have 0.3 cm diameter holes, bent to produce grid 2cm (w) x2 cm (d) x 0.5 cm (h)</td>
		</tr>
		<tr>
			<td>ki67</td>
			<td>Novocastra</td>
			<td>KI67-MM1-L-CE</td>
			<td>Clone MM1 Use at 1:100</td>
		</tr>
		<tr>
			<td>CK4</td>
			<td>Abcam</td>
			<td>ab9004</td>
			<td>Clone 6B10 Use at 1:200</td>
		</tr>
		<tr>
			<td>CK14</td>
			<td>Novocastra</td>
			<td>LL002-L-CE</td>
			<td>Clone LL002 Use at 1:200</td>
		</tr>
		<tr>
			<td>Involucrin</td>
			<td>Novocastra</td>
			<td>INV</td>
			<td>Clone&#160; SY5 Use at 1:100</td>
		</tr>
		<tr>
			<td>OE21</td>
			<td>Sigma-Aldrich</td>
			<td align="right">96062201</td>
			<td></td>
		</tr>
		<tr>
			<td>OE33</td>
			<td>Sigma-Aldrich</td>
			<td align="right">96070808</td>
			<td></td>
		</tr>
		<tr>
			<td>Het-1A</td>
			<td>ATCC-LGC</td>
			<td>CRL-2692</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse 3T3 fibroblasts</td>
			<td>ATCC-LGC</td>
			<td>CRL-1658</td>
			<td>previously growth arrested by irradiation (60 Gy)</td>
		</tr>
	</tbody>
</table>

production, characterization and potential uses of a 3d tissue-engineered human esophageal mucosal model

وقوع كل من غدية المريء والسلائف، حؤول باريت، ترتفع بسرعة في العالم الغربي. وعلاوة على ذلك غدية المريء عموما لديها سوء التشخيص، مع تحسن طفيف في معدلات البقاء على قيد الحياة في السنوات الأخيرة. هذه هي ظروف صعبة للدراسة، وكان هناك نقص في منصات التجريبية المناسبة لتحقيق اضطرابات في الغشاء المخاطي المريء. وقد تم تطوير نموذج الغشاء المخاطي المريء البشري في المختبر ماكنيل الذي، على عكس الأنظمة خلية ثقافة 2D التقليدية، يلخص خلية خلية وخلية مصفوفة التفاعلات الموجودة في الجسم الحي وينتج ناضجة، ظهارة طبقية مماثلة لتلك التي للإنسان العادي المريء. لفترة وجيزة، ونموذج يستخدم خلايا غير طبيعية تحولت الخلايا الليفية المريء الإنسان الأساسية والظهارية نمت داخل ديكي سقالة المريء المشتقة من الخنازير. توصيف المناعى لهذا النموذجبواسطة CK4، CK14، Ki67 وinvolucrin تلطيخ يوضح خلاصة المناسب للأنسجة المخاطية المريء الإنسان العادي. يوفر هذا النموذج نموذج قوي، بيولوجيا التجريبية من الغشاء المخاطي المريء البشري. يمكن بسهولة أن يتم التلاعب بها لتحقيق عدد من الأسئلة البحثية بما في ذلك فعالية كلاء الدوائية وتأثير التعرض للعوامل البيئية مثل الكحول والسموم، وارتفاع في درجة الحرارة أو مكونات refluxate المعدي المريئي. يسهل هذا النموذج أيضا فترات ممتدة الثقافة لا يمكن تحقيقها مع خلية ثقافة 2D التقليدية، مما يتيح، في جملة أمور، ودراسة تأثير التعرض المتكرر للظهارة ناضجة لوكيل لمصلحة لمدة تصل إلى 20 يوما. وعلاوة على ذلك، ومجموعة متنوعة من خطوط الخلايا، مثل تلك المستمدة من أورام المريء أو حؤول باريت، يمكن إدراجها في النموذج للتحقيق في عمليات مثل غزو الورم وresponsivene المخدراتSS في بيئة أكثر ملاءمة من الناحية البيولوجية.

تصف هذه المخطوطة العملية المطلوبة، كما هو موضح في الشكل التخطيطي في الشكل 1، إلى ثقافة 3D نماذج من ظهارة المريء الإنسان بنجاح. لتأكيد مدى ملاءمة هذا النموذج بوصفه اتخذت لالنسيجية منصة تجريبية ودراسات مقارنة المناعى أنسجة مثقف مع العادي البشري مخاطية المريء ?...

Watch this Scientific Journal Video about Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model at JoVE.com

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

تصف هذه المخطوطة إنتاج وتوصيف والاستخدامات المحتملة لالأنسجة المهندسة 3D بناء المريء المعدة من المريء الطبيعية الأولية الخلايا الليفية والخلايا البشرية الظهارية الحرشفية المصنفة ضمن سقالة دي cellularized الخنازير. أظهرت النتائج وتشكيل ظهارة طبقية ناضجة مماثلة إلى المريء الإنسان العادي.

إنتاج وتوصيف والمحتملة استخدامات الأنسجة المهندسة-3D الإنسان المريء المخاطي نموذج

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

production-characterization-potential-uses-3d-tissue-engineered-human

Research

JoVE Journal

Bioengineering

10.4K Views.  University of Sheffield.  تصف هذه المخطوطة إنتاج وتوصيف والاستخدامات المحتملة لالأنسجة المهندسة 3D بناء المريء المعدة من المريء الطبيعية الأولية الخلايا الليفية والخلايا البشرية الظهارية الحرشفية المصنفة ضمن سقالة دي cellularized الخنازير. أظهرت النتائج وتشكيل ظهارة ?...

Video: Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

بروتوكول لتقييم هيدرودينامي النسبية للثلاثي النشرة بوليمر صمامات

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

جيل ومسمار سفن الهندسة الأنسجة في نموذج الفأر

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

نموذج الثقافة 3D-الهرمون تستجيب للالغدة الثديية الظهارة الإنسان

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

والأنسجة المشتركة 3D هندسيا<em&gt; في المختبر</em&gt; /<em&gt; في السيليكو</em&gt; الرئة نموذج الورم للتنبؤ فعالية الدواء في خلفيات الطفرات محددة

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

تطوير عضوي نموذج متعددة الخلايا ثلاثي الأبعاد من الأمعاء الغشاء المخاطي الإنسان نمت في ظل الجاذبية

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

ثلاثي الأبعاد الأنسجة المهندسة الانحياز شبكات Astrocyte الخص الآليات الإنمائية وتيسير التجدد العصبي

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

تقنية خياطة تينورهافي جديدة مع الأنسجة المهندسة الكولاجين الكسب غير المشروع لإصلاح عيوب وتر كبير

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

إنتاج بروتين الايلاستين مثل الهلاميات المائية للتغليف وإيمونوستينينج من الخلايا في 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

كشف الذيفان في تركيبات النانو باستخدام فحوصات ليستي (ال) أموبوسيتي ليمولوس

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

بذر وغرس الاختلاس الرغامى السكروز هندسة الأنسجة في طراز الماوس

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...