The authors acknowledge funding from the National Institutes of Health R21CA182333.

<ol>
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الكتاب ليس لديهم ما يكشف.

الأجسام الشبه الكروية تقديم نموذج واقعي لفهم أفضل لعلم وظائف الأعضاء الورم ونجاعة الأدوية وتوفير أداة مفيدة لمكافحة السرطان اكتشاف الأدوية. ان مثل هذه التطبيقات تستفيد كثيرا من التقنيات البسيطة جيل كروي والصيانة التي تتطلب سوى معدات المختبرات القياسية، والتعامل م...

توفر المقايسات خلية القاعدة أداة هامة لتطوير واكتشاف عقاقير جديدة مضادة للسرطان. 1،2 تاريخيا، وقد استخدمت الثقافات أحادي الطبقة من الخلايا السرطانية للتحقيق كفاءة مركبات مرشح ضد أنواع معينة من الخلايا السرطانية. سهولة الصيانة الثقافات أحادي الطبقة في لوحات ثقافة القياسية، والتوافق لوحات القياسية مع الأدوات الروبوتية التجارية لإضافة الكواشف، ومع معدات الفحص لتحليل المصب من الاستجابات الخلوية إلى المركبات الكيميائية هي الفوائد الرئيسية التي تجعل الثقافات 2D أداة جذابة لاختبار المخدرات 3. لسوء الحظ، غالبا ما تفشل المقايسات خلية أحادي الطبقة التنبؤ فعالية من المركبات في الجسم الحي، مما يجعل تطوير العقاقير واكتشاف عملية مكلفة للغاية. وعلى الرغم من 4،5 استثمارات كبيرة وجهد من قبل شركات الأدوية والوحدات الأكاديمية، فقط ~ 1٪ المضادة للسرطان تمت الموافقة على المخدرات في التجارب السريريةمن قبل FDA على مدى العقدين الماضيين. 6 التفاوت بين الثقافات 2D و 3D البيئة المعقدة من الخلايا السرطانية في الجسم الحي هو أحد أوجه القصور الرئيسية لنظم الاستزراع أحادي الطبقة. 7 لذلك، وفحص المركبات مرشح ضد الخلايا السرطانية في الإعداد الذي أكثر شبها البيئة الورم 3D قد تعجل تطوير عقاقير العلاج الكيميائي الرواية. 8 الأجسام الشبه الكروية الخلايا السرطانية تقديم نموذج ورم 3D ذات الصلة في المختبر. 9،10 الأجسام الشبه الكروية هي مجموعات المدمجة التي تشكل من خلال التجمع العفوي أو المتعمد من الخلايا السرطانية على الأسطح غير ملتصقة أو في تعليق باستخدام تقنيات مثل قارورة الدوار، تراكب السائل، والصغرى microfabricated . كذلك المصفوفات، على microfluidics، وقطرات شنقا 11-16 الأجسام الشبه الكروية تحاكي الملامح الرئيسية الأورام الصلبة بما في ذلك الهندسة والنقل محدود من الأكسجين والمغذيات، والمركبات المخدرات في المنطقة الوسطى. وبالتالي، فإنها تتجدد بشكل وثيق respon المخدراتحد ذاته من الأورام الصلبة مقارنة الثقافات أحادي الطبقة. 17-19 وعلى الرغم من هذا الاستحقاق ملحوظة، لا تستخدم الأجسام الشبه الكروية بشكل روتيني لفحص المركبات الكيميائية ضد الخلايا السرطانية. صعوبة إنتاج الأجسام الشبه الكروية موحدة الحجم في إعداد إنتاجية عالية القياسية التي تتوافق مع الروبوتات المتاحة تجاريا وأدوات الفحص / التصوير تعرقل إدماج ثقافة كروي في خط أنابيب تطوير الأدوية. على الرغم من أن المواد المخصصة لوحات وأصبحت في الآونة الأخيرة المتاحة تجاريا لتلبية هذه الحاجة، اعتبارات التكلفة ردع استخدامها على نطاق واسع. اثنين من التقنيات الرئيسية لديها القدرة على إنتاج الأجسام الشبه الكروية متناسقة الحجم في إنتاجية عالية استخدام منصة قطرة شنقا جديدة وmicrofabricated الآبار الصغيرة. 13،16،20 ومع ذلك، كلا النهجين تتطلب لوحات والأجهزة التي هي مكلفة لصنع وغير مريح للمستخدمين النهائيين الخاصة في مراكز البحوث الأساسية والصناعات الدوائية حيث EF معظم كبارمصنوعة الحصون لاكتشاف عقاقير جديدة مضادة للسرطان. وعلى الرغم من بعض التحسن في استقرار قطرات تحتوي على خلية مع تصميم حديث من شنقا لوحات قطرة، لا تزال تستخدم فقط كل حفرة الآخر من لوحة خلال ثقافة لتجنب انتشار / دمج قطرات. 16 هذا يقلل بشكل ملحوظ الإنتاجية التجريبية. إدمان المخدرات والتجديد صعبة مع دليل أو pipetting لالروبوتية وتحتاج إلى أن يتم تحويلها إلى لوحة القياسية لتحليل الكيمياء الحيوية الكروية لهذا التكوين لوحة غير متوافق بسهولة مع معدات الفحص التقليدية مثل لوحة القراء. ملفقة 21 مايكرو الآبار باستخدام الطباعة الحجرية الناعمة أيضا سماح حجم تسيطر إنتاج كروي. 13،20 ومع ذلك، عدم توافق هذه المنصة مع الأدوات القياسية pipetting ليمنع علاج من الأجسام الشبه الكروية الفردية مع مختلف المركبات الدوائية / تركيزات، وفضح كل الأجسام الشبه الكروية إلى حالة معاملة واحدة. وهكذا، وهذه الطريقة ليست مناسبة لارتفاعفحص مجمع الخرج الذي يتطلب الاختبار في وقت واحد من المركبات متعددة / التركيزات. للتغلب على هذه العقبات، وقد تم تطوير تقنية جديدة لإنتاج إنتاجية عالية من الحجم باستمرار الكروية الخلايا السرطانية في لوحات 96-جيدا القياسية. 22،23 ويستند هذا النهج على نظام مائي على مرحلتين البوليمر (ATPS) مع البولي ايثيلين جلايكول ( وقد تم مؤخرا استخدمت PEG) وديكستران (DEX)، والبوليمرات التي تشكل المرحلة 24 ATPSs في مجموعة متنوعة من التطبيقات البيولوجية خلية جديدة لتمكين micropatterning الخلية والمترجمة تسليم الكواشف البيولوجية للخلايا في الوسط المائي للغاية. 25-32 تشكيل ل كروي، يتم خلط الخلايا السرطانية مع المرحلة DEX المائية وانخفاض-ميكروليتر الفرعية لتعليق الناتجة من pipetted إلى تحتوي أيضا على الغمر المائي حل المرحلة PEG. يبقى قطرة إمتزاج من مرحلة الغمر والخلايا حدود لتسهيل تشكيل كروي. عفريتortantly، مرحلة الغمر المائي للغاية توفر المواد المغذية للخلايا كروي ويقلل من مشكلة معروفة من التبخر وسائل الإعلام المشتركة لبعض المقايسات الأخرى التي تسبب التغيرات في الأسمولية وسائل الإعلام وتقلبات تركيزات المخدرات. هذه التقنية تتيح إنتاج كروي والعلاج من تعاطي المخدرات فقط باستخدام الكواشف المتاحة تجاريا وأدوات pipetting لفي لوحات 96-جيدا القياسية. الأهم من ذلك، يتم إجراء تحليل الاستجابات الخلوية من الأجسام الشبه الكروية في نفس لوحة باستخدام فحوصات البيوكيميائية القياسية والقراء لوحة. سهولة العمل مع ATPS والقدرة على التكيف للنهج لمعالجة السائل الروبوتية يجعل الجيل إنتاجية عالية من كلا أحادية الثقافة وشارك في الثقافة الكروية تقنية المختبرات مباشرة. وهذا النهج الجديد سيكون خطوة كبيرة إلى الأمام نحو تكامل الكروية الخلايا السرطانية في عمليات التنمية المخدرات والاكتشاف مع تحسين الإنتاجية الاختبار وفعالية التكاليف (زيادة أعداد المركبات وredu اختباراستهلاك دائرة الهندسة المدنية كاشف) والكفاءة (خفض اليدين في الوقت المحدد). يوصف بروتوكول مفصل لإنتاج الروبوتية من الأجسام الشبه الكروية الخلايا السرطانية في لوحات 96-جيدا باستخدام نهج ATPS أدناه. وبالإضافة إلى ذلك، يتم عرض تفاصيل العلاج من تعاطي المخدرات من الناتج الكروية وتحليل المصب من الاستجابات الخلوية باستخدام مقايسة البيوكيميائية التجاري.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagents and Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol, Mw: 35,000</td>
			<td>Sigma-Aldrich</td>
			<td>94646</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Mw: 500,000</td>
			<td>Pharmacosmos</td>
			<td>5510 0500 9007</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12306C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Clacein AM</td>
			<td>Life Technologies</td>
			<td>C3100MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Life Technologies</td>
			<td>33342</td>
			<td></td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Spectrum Chemicals</td>
			<td>15663-27-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PrestoBlue</td>
			<td>Life Technologies</td>
			<td>A-13261</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-108</td>
			<td>Sigma Aldrich</td>
			<td>542342</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Tips (10 &#181;l)</td>
			<td>Fluotics</td>
			<td>C-P10V11.ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Tips (70 &#181;l)</td>
			<td>Fluotics</td>
			<td>C-P70V11.ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-bottom 96-well plates</td>
			<td>Corning</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Handler</td>
			<td>Agilent Technologies</td>
			<td>SRT Bravo</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>Biotek Instruments</td>
			<td>Synergy H1M</td>
			<td></td>
		</tr>
	</tbody>
</table>

robotic production of cancer cell spheroids with an aqueous two-phase system for drug testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

ويرد محطة العمل من معالج السائل الروبوتية في الشكل 1. وقال رئيس pipetting لوصفت جميع المحطات المستخدمة في الطباعة الروبوتية من الأجسام الشبه الكروية في القسم 4.6. تظهر الصورة استخدام اثنين من محطات مختلفة لمربعات طرف (مجموعة واحدة من النصائح لخلط والمجموعة الثاني...

Watch this Scientific Journal Video about Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing at JoVE.com

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

الإنتاج الآلي للسرطان خلية الأجسام الشبه الكروية مع نظام مائي مرحلتين لاختبار المخدرات

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

robotic-production-cancer-cell-spheroids-with-an-aqueous-two-phase

Research

JoVE Journal

Bioengineering

8.9K Views.  The University of Akron. A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Video: Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cell Co-culture Patterning Using Aqueous Two-phase Systems

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

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

خلية المشارك الثقافة الزخرفة باستخدام مائي مرحلتين أنظمة

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

Robotic Mirror Therapy System for Functional Recovery of Hemiplegic Arms

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is conducted by eliciting an illusion through use of a mirror as if the hemiplegic arm is moving in real-time while moving the healthy arm. It can facilitate brain neuroplasticity through activation of the sensorimotor cortex. However, conventional mirror therapy has a critical limitation in that the hemiplegic arm is not actually moving. Thus, we developed a real-time 2-axis mirror robot system as a simple add-on module for conventional mirror therapy using a closed feedback mechanism, which enables real-time movement of the hemiplegic arm. We used 3 Attitude and Heading Reference System sensors, 2 brushless DC motors for elbow and wrist joints, and exoskeletal frames. In a feasibility study on 6 healthy subjects, robotic mirror therapy was safe and feasible. We further selected tasks useful for activities of daily living training through feedback from rehabilitation doctors. A chronic stroke patient showed improvement in the Fugl-Meyer assessment scale and elbow flexor spasticity after a 2-week application of the mirror robot system. Robotic mirror therapy may enhance proprioceptive input to the sensory cortex, which is considered to be important in neuroplasticity and functional recovery of hemiplegic arms. The mirror robot system presented herein can be easily developed and utilized effectively to advance occupational therapy.

We developed a real-time mirror robot system for functional recovery of hemiplegic arms using automatic control technology, conducted a clinical study on healthy subjects, and determined tasks through feedback from rehabilitation doctors. This simple mirror robot can be applied effectively to occupational therapy in stroke patients with a hemiplegic arm.

نظام العلاج الروبوتية مرآة للانتعاش وظيفية للأسلحة مفلوج

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

تلفيق والتحقق من صحة نظام الجهاز على رقاقة مع أقطاب متكاملة لقياس المقاومة الكهربائية ترانسيندوثيليال مباشرة

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

قمع النسخ الجيني قبل إعادة توجيه الأجهزة الخلوية مع المعدلات جينية الكيميائية

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

نظام ميكروبيوريكتور لإنتاج IgG1 النموذجي في الخلايا شو الآلي استخدام الفائق

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

تنقية وتحليل الأجسام المضادة أحادية النسيلة من خلايا المبيض الهامستر الصينية باستخدام نظام مفاعل ميكروبيوم الآلي

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

التوضيح الأولي لسائل زراعة الخلايا المقطوعة CHO باستخدام فاصل صوتي

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

الإنتاج الآلي على نطاق واسع من الخلايا الجذعية المشتقة من الدهون كروية للطباعة الحيوية 3D

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

جيل قابل للتطوير من كرويات الخلايا الموحدة باستخدام جهاز تجميع صوتي 3D

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

تسليط الضوء على المؤلف: تطوير طريقة تجميع صوتية خالية من السقالات لثقافة كروية خلية 3D عالية الجودة 

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

جهاز تجميع صوتي ثلاثي الأبعاد للتصنيع الشامل للكرويات الخلوية

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...