وأيد هذا العمل من قبل الدعم الحكومي بموجب منحة رقم EPS-0903795 تمنحها المؤسسة الوطنية للعلوم، NIH NIDCR R01-DE019355 (MJY PI)، ومنح 8P20 GM103444 (YM PI).

<ol>
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The authors have nothing to disclose.

التركيز الرئيسي لهندسة الأنسجة هو سد الفجوة بين نقص الأعضاء واحتياجات زرع من خلال تطوير بدائل بيولوجية قادرة على استعادة والحفاظ على، أو تحسين functio أنسجة الأم. وقد أدى هذا إلى تلفيق المباشر السقالات مع مجمع، هندسة الخارجية الصحيحة تشريحيا، ومراقبة دقيقة على مدى geometr...

يستخدم هندسة الأنسجة مبادئ علم الأحياء والهندسة في تطوير بدائل وظيفية للحفاظ على، استعادة أو تعزيز النسيج الأم و. القدرة على توليد بنيات المحاكاة البيولوجية ثلاثية الأبعاد عند الطلب من شأنه أن ييسر التقدم العلمي والتكنولوجي في هندسة الأنسجة وكذلك في أجهزة الاستشعار المعتمدة على الخلايا المخدرات / فحص السمية، والأنسجة أو الورم النماذج، وغيرها. المنظمة ثلاثية الأبعاد للبنيات الأنسجة المهندسة هو عنصر أساسي من أسلوب تلفيق لأنه يجب أن تحاكي بشكل وثيق التفاعل المنظم للغاية من الخلايا والمصفوفة خارج الخلية في الأنسجة الأصلية. السقالات ثلاثية الأبعاد القابلة للتحلل والتي تشكل شكل من العوامل الحاسمة في توليد بنيات الأنسجة الجديدة لأن الخلايا تهاجر لتشكيل طبقة ثنائية الأبعاد من الخلايا، ولكنها تفتقر إلى القدرة على النمو في فضل ثلاثي الأبعاد. تخدم سقالة كأساس مؤقت للخليةالمرفقات والانتشار، لذلك يجب أن تبنى من مواد ذات المسامية السيطرة عليها والتحلل البيولوجي، والنزاهه الميكانيكية الكافية. المواد سقالة لا ينبغي أن يكون السامة للخلايا أو إنشاء استجابة سلبية من المضيف. تم الهلاميات المائية التي يشيع استخدامها في تقنيات هندسة الأنسجة، ونظرا لhydrophilicity، وتسمح الهلاميات المائية الصرف السائل والغاز في جميع أنحاء متطوره. من خلال الجمع بين الهلاميات المائية المختلفة، خصائص هيدروجيل تصنيعه هي للتعديل لتلبية متطلبات تطبيق متميز. نهج هندسة الأنسجة التقليدية ينطوي على إنشاء السقالات الأضاحي التي يسهل اختراقها ديكي التي المصنف مع الخلايا آخر fabricatio. وقد استخدمت العديد من التقنيات، مثل الألياف الرابطة، صب المذيبات، وتذوب الصب، ولكن ثبت أن تكون ناجحة الحد الأدنى للتطبيقات هندسة الأنسجة. طرق الألياف الرابطة تسمح الألياف إلى الانحياز في أشكال محددة، ولكنها ليست سوى قادرة على المواليةducing سقالة رقيقة جدا. طرق الصب المذيبات أنتجت بنيات المسامية العالية، ولكن كان الغشاء أكبر تنتج سوى 3 ملم thic. لذلك، وخلق بنيات ثلاثية الأبعاد من غير الممكن استخدام هذه التقنيات. أثبتت تقنيات صب تذوب الناجحة في إنتاج السقالات ثلاثية الأبعاد، ولكنها تتطلب درجات حرارة عالية أن المواد البيولوجية لا يمكن إدراجها خلال بروسس الإنتاج. المصنفة السقالات بعد تلفيق-محدودة في قدرتها على تلبية متطلبات هندسة الأنسجة لإنتاج السقالات ثلاثية الأبعاد مع المجهرية ومحددة مسبقا أو السيطرة عليها. آخر قضية رئيسية مع التقنيات سقالة البذر الصلبة هي نقص الأوعية الدموية والفقراء الميكانيكية. ومنذ ذلك الحين تم تمديد Bioprinting إلى ثلاثة أبعاد من خلال استخدام غير سامة، قابلة للتحلل، والمواد الهلامية الحرارية عكسها للتغلب على عيوب التقليدية. وهناك عدد قليل من صلب حر تلفيق رechniques التي يجري استخدامها حاليا هي بمساعدة الليزر bioprinting والنافثة للحبر الطباعة. تقنيات bioprinting بمساعدة الليزر تستخدم مصدر نابض الليزر، لوحة الهدف، وركيزة المتلقي لتوليد ثلاثي الأبعاد. ومع ذلك، هذه التقنية محدودة بسبب الإنتاجية، وانخفاض بقاء الخلية، ويمكن أن تنتج فقط ترتيبات محدودة من الهياكل ملفقة فقط لأن prepolymers photocrosslinkable يمكن استخدامها لتشكيل هيدروجيل crosslinked. وقد وضعت الطباعة النافثة للحبر كمنهجية عدم الاتصال أن يستنسخ بيانات الصور الرقمية على الركيزة عن طريق إيداع الحبر بيكو لتر. ومع ذلك، الطباعة النافثة للحبر لا تنتج عالية الدقة بناء، يبني تجربة السريع تمسخ البروتين، والعديد من الخلايا هي lysed خلال الترسيب. حاليا، وقد تم تطوير طرق التصنيع المضافة bioprinting جديدة. في هذه الأنظمة يتم دمج الخلايا والبروتينات وعوامل النمو، والهلاميات المائية الجزيئية الحيوية عادة في مصفوفة الأمأمية أثناء عملية التصنيع وأودعت في نفس الوقت باستخدام المحركات الكمبيوتر التي تسيطر عليها لتوليد خلايا محملة يبني على أساس سقالة ثلاثية الأبعاد التي تحاكي عن كثب المعمارية المصغرة من الأم. الهلاميات المائية خلايا محملة تشكل bioink، التي يمكن أن تكون غير متجانسة، تتكون من أنواع خلايا متعددة، أو متجانسة. المضافات نظم التصنيع إيداع bioink قطرة تلو قطرة أو طبقة تلو طبقة عن طريق المحاقن ونصائح على مرحلة الكمبيوتر التي تسيطر عليها قادرة على التحرك في الاتجاهات X، Y، Z و. من خلال برامج الكمبيوتر، والهندسة المعمارية السقالات المطبوعة يمكن التلاعب بها بسهولة اعتمادا على متطلبات التطبيق. وخلافا للتقنيات التقليدية، ويمكن دمج التكنولوجيات الطبية ثلاثية الأبعاد (التصوير بالرنين المغناطيسي، والتصوير المقطعي الكمبيوتر) في التصاميم، وتوليد بناء المريض محددة. هذه الأساليب تسمح أيضا إمكانية إنتاج بدائل أوعية دموية لأن يتم إنتاج بنيات مع ارتفاع لكثافة الخلية OCAL، مما يسمح التفاعلات خلية خلية وتحسين احتمالات ما بعد الزرع بقاؤه. الطابعة بالميتو هي ثلاثية الأبعاد نظام تعدد موزع خصيصا بنيت يستخدم أساليب التصنيع الروبوتية قابلة للبرمجة لتوليد ثلاثية الأبعاد بنيات الأنسجة غير المتجانسة (الشكل 1). لأنها تتيح استخدام عدد وافر من المواد في مجموعات فريدة من نوعها لإنتاج هياكل غير المتجانسة. التهيئة للbioprinter هي واحدة من أهم الخطوات في bioprinting لأنه يسمح لك لتعيين مجموعة متنوعة من المعلمات لتحسين القابلية للبنيات bioprinted. تضم bioprinter عملية من النوع المتقطع مع تسلسل بدء التشغيل، التشغيل والاغلاق التي تسيطر عليها وحدة تحكم منطق برمجة (PLC)، التي تعمل على المستخدم من خلال لوحة تحكم تعمل باللمس التفاعلية (الشكل 1، A). لمنع التلوث الحيويالمواد منطقية ومرفق طيه وbioprinter في بولي إيجابيا لضغوط (ميتاكريليت الميثيل) (PMMA) غرفة مع الجسيمات arrestance عالية الكفاءة (HEPA) -filtered نظام دوران الهواء (الشكل 1، B، C). داخل الطابعة يمكن تعقيمها باستخدام مصادر الأشعة فوق البنفسجية المدمج في (الشكل 1، D). المكون الرئيسي للbioprinter هو الروبوت تحديد المواقع للبرمجة بشكل كامل يمكن أن تضع بتكاثر طرف موزع مع دقة 10 ميكرومتر (الشكل 1، E). هناك ثلاثة موزعات، والتي هي قادرة على أن تودع أحجام صغيرة مثل 230 NL باستخدام الدوارة (الشكل 1، F). هم برمجة بشكل مستقل باستخدام أجهزة كمبيوتر منفصلة التي تحكم المعلمات الطباعة لكل موزع (الشكل 1، G). دوار المسمار الاستغناء يستخدم دوران المسمار بالمحركات لنقل bioink أسفل حقنة ويخرجون من طرف الحقنة. هي التي شنت هذه موزعات على لpneumaticalأداة عش تسيطر لاي (الشكل 2A، B)، والسماح للروبوت للتبديل موزع شنت على الذراع الروبوتية Z المحور تحت السيطرة المبرمجة (الشكل 1، H). يتلقى روبوت XYZ تعليمات الطباعة من جهاز كمبيوتر يقوم بتشغيل برامج التصميم (الشكل 1، I). يحتوي كل برنامج مواقع صرفها، وإجراءات المعايرة، والبروتوكولات المتغيرة موزع. تصميم بنيات ولدت تتكون أساسا من XYZ ينسق حيث كل موزع ستودع المواد. تضم bioprinter اثنين من أجهزة الاستشعار البصرية ضوء (الشكل 2C) التي تحدد إحداثيات XYZ من نهاية حقنة طرف. هذه المجسات ترسل تنسيق المعلومات لالروبوت، والذي يستخدم هذه لحساب مواقف طرفي موزع طرف. هناك ليزر النزوح إضافية (الشكل 2D) التي تتوقع 633 نانومتر الصمام الثنائي الأحمر شعاع ليزر من حجم البقعة 30 × 100 ميكرومتر لقياس المسافة مع ACCURAقبرصي من 0.1 ميكرومتر. عندما تركز بشدة شعاع الروبوت يحدد المسافة Z من سطح الطباعة. هذا القياس، وقياس البصرية وأجهزة الاستشعار ضوء في نهاية طرف في Z، يسمح حساب Z دقة الإحداثيات المستخدمة لوضع طرف موزع فيما يتعلق سطح الطباعة. نصائح موزع تتحرك أفقيا وعموديا من خلال X-محور الموجهة ضوء استشعار بصري للعثور على مراكز Y و Z، وأفقيا من خلال جهاز استشعار العمودي للعثور على مركز للمحور X. تم تعيين سطح الطباعة باستخدام صيغة لمنبسط في الفضاء XYZ: الفأس + من قبل + تشيكوسلوفاكيا = د لتحديد مكان السطح هو نسبي لموقف نهاية الاستغناء طرف. المرحلة الطابعة (الشكل 1، J) يحمل طبق بتري عينة تصل إلى 80 مم في القطر، ويستخدم حمام مائي إعادة تدوير للحفاظ على درجة الحرارة المحددة (الشكل 1، K). يمكن ضبط درجة الحرارة المرحلة ضمن مجموعة من -20 و لا تزال مستقرة في الداخل. هناك كاميرا USB محمولةعلى الروبوت Z-الذراع لتقديم وجهة نظر تضخيم من طرف الاستغناء أثناء عملية الطباعة (الشكل 1، L). هناك كاميرا ثانية شنت نحو الجزء العلوي من الداخل غرفة التي توفر رؤية متكاملة للbioprinter أثناء عملية الطباعة (الشكل 1، L). وهناك برامج التصميم الرسم بمساعدة الكمبيوتر يحدد نمط ترسب ويسمح للمستخدم لتوليد قطرات متباعدة تدريجيا والهياكل المعقدة (الشكل 3). مسارات ثلاثي الأبعاد يمكن تلوينها يدويا في تصميم البرمجيات طابعة متوافقة أو استيرادها من برنامج منفصل التصميم بمساعدة الكمبيوتر الرسم (الشكل 4، الجدول 1). يسمح البرنامج للطابعة متوافقة الاختلافات المعلمات الطباعة مثل طريقة الترسيب (ترسب قطرة واحدة أو ترسب مسار مستمر)، والهندسة ثلاثية الأبعاد من المسارات، ومعدل الترسيب، والمسافة بين نهاية حقنة طرف وSUBSTيتم رفع معدل سطح الطباعة، وكمية من الوقت لإيداع انخفاضا الفردي، وارتفاع وسرعة الحقنة بين ترسب قطرات. يحتوي كل برنامج XYZ مواقع صرفها، والروتين طرف المعايرة، والبروتوكولات المتغيرة موزع لتوفير بيئة معقمة، دون تدخل المشغل، أثناء الطباعة. وحدة تحكم منطق برمجة (PLC) للروبوت يتلقى تعليمات من جهاز كمبيوتر يستخدم في تصميم البرمجيات ويتحكم في توقيت الأحداث من وحدات تحكم خارجية (على سبيل المثال، موزعات). للقيام بذلك، يستخدم PLC آلية تنفيذ الحلقات للسيطرة على موزعات ، جهاز تحديد المواقع الروبوتية، والعوامل البيئية. ثلاثي الأبعاد الكتابة المباشر bioprinting الاستفادة من دوار المسمار، نظام صرف السائل يسمح للعملية إيداع الخلايا لتكون أكثر كفاءة ودقة، وأسهل من الطرق السابقة. وتظهر هذه الدراسة bioprinter عرف بني قادر على توليد مبنيات هيدروجيل ليرة لبنانية محملة بقاء الخلية عالية.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Positioning Robot (JR2000 XYZ)</td>
			<td>Janome&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispensers: SDAV Linear Drive SmartDispensers</td>
			<td>Fishman Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Light Sensors:&#160;</td>
			<td>Keyensce</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Displacement Laser: OD Mini</td>
			<td>SICK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recirculating Water Bath: Polystat</td>
			<td>Cole-Parmer</td>
			<td>EW-12122-02</td>
			<td></td>
		</tr>
		<tr>
			<td>USB Cameras: Dino-Lite Premier 5MP</td>
			<td>AnMo Electrionics/YSC Technologies</td>
			<td>AD7013MT</td>
			<td></td>
		</tr>
		<tr>
			<td>Printer-Compatible Computer Design Software: JR-C Points</td>
			<td>Janome</td>
			<td></td>
			<td>Comes with purchase of Janome Robot</td>
		</tr>
		<tr>
			<td>Computer-Aided Design Drawing Software: Visual PathBuilder</td>
			<td>RatioServ</td>
			<td></td>
			<td>Can be downloaded at: www.ratioserv.com/index.php/downloads</td>
		</tr>
		<tr>
			<td>Printer 3 cc Syringes:&#160;</td>
			<td>Fishman Corporation</td>
			<td>122051</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Dispenser Tips</td>
			<td>Fishman Corporation</td>
			<td>Z520122&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>9000-70-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium Dioxide</td>
			<td>Sigma-Aldrich</td>
			<td>13462-67-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Protanal LF 20/40 Alginate (Sodium Alginate)</td>
			<td>FMC BioPolymer</td>
			<td>9005-38-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td>Mallinckrodt Baker, Inc</td>
			<td>9300-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Periodate</td>
			<td>Sigma-Aldrich</td>
			<td>7790-28-5</td>
			<td></td>
		</tr>
		<tr>
			<td>hADSC</td>
			<td>Lonza</td>
			<td>PT-5006</td>
			<td>Store in vials in liquid nitrogen until use.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Gibco Life Technologies</td>
			<td>11965-092</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Lonza</td>
			<td>CC-5012</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>Gibco Life Technologies</td>
			<td>C3100MP</td>
			<td>Store in the dark at -80&#176;C until use.</td>
		</tr>
		<tr>
			<td>Live/Dead Mammalian Viability Assay Kit</td>
			<td>Invitrogen Life Technologies</td>
			<td>L-3224</td>
			<td>Store in the dark at -80&#176;C until use.</td>
		</tr>
		<tr>
			<td>MES Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Hydroxysuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ethyl-(dimethylaminopropyl) carbodiimide (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>E1769&#160;</td>
			<td>10 G</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline, +Calcium, +Magnesium</td>
			<td>Life Technologies</td>
			<td>14040133</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline, -Calcium, -Magnesium</td>
			<td>Life Technologies</td>
			<td>14190144</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>RGD Peptides</td>
			<td>International Peptides</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 Phalloidin Stain</td>
			<td>Invitrogen Life Technologies</td>
			<td>A22283</td>
			<td>Store at -20&#176;C until use</td>
		</tr>
		<tr>
			<td>(4&#8217;, 6-Diamidino-2-Phenylindole, Dihydrochloride) (DAPI) Stain</td>
			<td>Life Technologies</td>
			<td>R37606</td>
			<td>Store at -20&#176;C until use</td>
		</tr>
	</tbody>
</table>

viability of bioprinted cellular constructs using a three dispenser cartesian printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

وتبين النتائج أن bioprinter قادر على إيداع الهلاميات المائية خلية لادن في المواقع الثلاثة الأبعاد محددة بدقة وباستمرار باستخدام البرمجيات بمساعدة الكمبيوتر. هذه البرمجيات تحدد وضع كل قطرة ومراقبة العديد من المعلمات للاستغناء عن (الشكل 3،4). والتكرار من bioprinter أن ...

Watch this Scientific Journal Video about Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer at JoVE.com

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

جدوى Bioprinted الخلوية التركيبات باستخدام طابعة ثلاثة الديكارتية موزع

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

viability-bioprinted-cellular-constructs-using-three-dispenser

Research

JoVE Journal

Bioengineering

9.9K Views.  Medical University of South Carolina. A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Video: Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

الجلد ثلاثي الأبعاد إعادة بناء الإنسان النموذجي : أداة لدراسة بشرة العادية والتقدم الميلانوما

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

خلق عابر المسامات غشاء الخلية باستخدام طابعة نافثة للحبر القياسية

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

في فيفو التقييم الكمي للبنية عضلة القلب، وظيفة، الإرواء والجدوى عن طريق احتساب الصغرى القلب المقطعي

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting Cellularized التركيبات باستخدام الأنسجة محددة هيدروجيل Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

الطبقات الجيني التركيبات: منصة لالمشارك ثقافة بالسكان خلية غير المتجانسة

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

Imaging Cell Viability on Non-transparent Scaffolds &amp;#8212; Using the Example of a Novel Knitted Titanium Implant

Here we present a fluorophore based imaging technique to detect cell viability on a non-transparent titanium scaffold as well as to detect glimpses of the scaffold impurities. This protocol troubleshoots the drawback of imaging cell-cell or cell-metal interactions on non-transparent scaffolds.

قابلية التصوير خلية على السقالات غير شفافة - استخدام مثال على رواية محبوك التيتانيوم زراعة الاسنان

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

علاج الرباط ينشأ مع مصل مكيفة مكيفة: نموذج هندسة الأنسجة الانتقالية

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

تصنيع صفيف متعدد المكروية الخلوية اصطناعية

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

كالوريسبيروميتري: اتباع نهج موسع، قوية للتحقيق في استقلاب الطاقة الخلوية

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

تصميم طابعة بيوينك بيوينك مفتوحة المصدر ومنخفضة التكلفة وطابعة البثق الغذائي

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...