فإن الكتاب يود أن يقر الدكتور توماس بولاند لفكرة استخدام طابعات منضدية HP للطباعة الخلية. تمويل هذا المشروع من جبهة الخلاص الوطني RII-EPS 0903795 والمعاهد الوطنية للصحة K25 HL0922280.

<ol>
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ليس لدينا ما يكشف.

عملية لتحويل سطح المكتب القياسية النافثة للحبر الطابعة لbioprinting ليست صعبة للغاية. الخطوة الأكثر تحديا هو تحديد كيفية تجاوز آلية تغذية الورق، والتي تعتمد على نوع وطراز الطابعة المستخدمة. ومع ذلك، وهذا هو بسيط نسبيا عند استشعار تغذية الورقة الميكانيكية كما هو موضح هنا. لنماذج مع أجهزة الاستشعار البصرية تغذية، قد تحتاج إلى غيرها من التقنيات التي ستستخدم لخداع الطابعة إلى التفكير في أنه يستخدم ورقة، على سبيل المثال، يمكن للمرء أن تشغيل قطعة صغيرة من الورق من خلال الطابعة في الوقت الذي يطبع على الشريحة المجهر. تجاوز آلية تغذية الورق من المرجح أن تكون الخطوة الأكثر صعوبة في تطبيق هذه الإجراءات على النماذج طابعة مختلفة. عند بناء مرحلة لعقد coverslips للطباعة، فمن المهم لضمان التوافق السليم وارتفاع. وينبغي في مرحلة تسمح ساترة لتوضع في وسط منطقة الطباعة. وبالإضافة إلى ذلك، ينبغي له ررالآس وساترة في 1 ارتفاع كاف للسماح لإزالة خرطوشة الطباعة لتمرير أكثر من الشريحة دون تعطيل ذلك. فإن ارتفاع الدقيق للمرحلة تعتمد على طراز الطابعة. للتأكد من أن لا تحصل خلايا المطبوعة جرفت، وضعت الشرائح في حاضنة مباشرة بعد الطباعة لمدة 30 دقيقة تقريبا للسماح لمرفق الخلية قبل أضيف إضافية وسائل الاعلام في نمو الخلايا. لأنه تم طبع الخلايا في التوصل إلى حل يتضمن كميات كبيرة من الخلايا في برنامج تلفزيوني لم تجف، وظلت قادرة على البقاء. تم تصوير الخلايا باستخدام المجهر مضان لتصور توزيع مونومرات G-الأكتين المفتاحية داخل الخلية (أرقام 1، 5-7). عند الطباعة مع الخلايا الليفية 3T3، ويبين الشكل 5 نتيجة ممثل، مع الأكتين يسبب الكثير من الخلايا ليتألق، ولكن تعرض خطوط سطوع المتزايدة. إدماج الموسومة fluorescently G-الأكتين في الهيكل الخلوي هومفيدة لدراسة ديناميات هيكل الخلية وميكانيكا الخلوية. 12-15 وجود قيود من هذا الأسلوب هو أنه لا ينطبق إلا لجزيئات البروتينات، والتي لديها ما يقرب من أقطار أصغر من 10 نانومتر. لضمان والواقع أن الخلايا التي يتم معالجتها بواسطة الطابعة تحويلها، وأضيف دابي (1:5000 في PBS) إلى bioink في مكان من نصف حجم برنامج تلفزيوني محض. ودابي فلوري وصمة عار بربط أجزاء من الأحماض الأمينية الغنية من الحمض النووي الموجود في نواة. ولذلك دابي وصمة مفيدة في نوى التصوير fluorescently من الخلايا المطبوعة. ويبين الشكل 6 صورة ممثل خلية عرض على حد سواء اليكسا فلور 488 (الأكتين) ودابي (نواة) مضان مع صورة تراكب على حد سواء. الشكل 7 يوضح أيضا كيف والخلايا يتألق مرة واحدة تم فيها دمج مونومرات G-الأكتين في حين غير البيولوجية المادية التي أودعت في العينة لن يتألق بسبب absenc(ه) من G-الأكتين مونومرات. أحد الاعتبارات المهمة لbioprinting هو الوسط المائي تستخدم لbioink. وحيث تبين ان وسائل الاعلام باستخدام معيار نمو الخلايا مع المصل أسفرت عن نتائج غير متسقة. وهذا هو الأرجح بسبب انسداد فتحات الطباعة الرأس بواسطة بروتينات مصل الدم. زيادة استخدام برنامج تلفزيوني اتساق أنماط المطبوعة وأودع عدد من الخلايا. عيب واحد لاستخدام برنامج تلفزيوني أنه لا ينبغي أن تترك الخلايا في التعليق لفترات طويلة من الزمن. ومع ذلك، كانت الخلايا الليفية في هذه الأمثلة قادرة على تحمل الظروف bioink لمدة ساعة على الأقل مع عدم وجود تغيير في بقاء الخلية. وهذا يتفق مع النتائج قبل عدة، والتي تفيد بأن ثبت الخلايا طباعتها من خلال آليات نفث الحبر الحرارية لديها معدلات عالية الجدوى. 2-8 ويمكن استخدام هذا إعداد الطابعة تعديل لتطبيقات أخرى من الطباعة الخلية. البروتينات مصفوفة 16-22، مثل الكولاجين أو fibronectin، يمكن طباعتها بسهولة على ركائز مع هذه التقنية، التي يمكن أن تكون مفيدة لتنميط الخلية. لنوع المثال الكولاجين، وأنا المطبوعة في أنماط الخط سيؤدي إلى ركائز الكولاجين الانحياز التي يمكن استخدامها لمدة 17. في الدراسات المختبرية ثقافة خلية بالإضافة إلى مصفوفة من البروتينات، والجزيئات الأخرى، بما فيها عوامل النمو، ويمكن ترجمة بشكل صحيح على ركائز للدراسات الخلية والعلاجية التطبيقات المحتملة. 18 وجود قيود الرئيسية للتصميم هو موضح في الخطوات المذكورة أعلاه هو أن هذه الطابعة غير قادر على الطباعة في أكثر من بعد واحد. وهذا يحد من إمكانية للاستخدام في التطبيقات مثل نمط الطباعة سقالة. للسماح للطباعة 3D، والمتخصصة في مرحلة تحتاج لاستخدامه. المرحلة يحتاج الى تعديلات ارتفاع تدريجي للطبقة تلو طبقة ترسب bioink. وقد أظهرت Bioprinting وعد كطريقة فعالة وفعالة من حيث التكلفة لهندسة الأنسجة، الجينات ترنسفكأيشن، micropatterning وميكروأري تلفيق 1-12، 16-22 والتطبيقات المستقبلية لهذا النوع من جهاز عديدة، بما في ذلك:. خلق microenvironments الخلوية تسيطر عليها ومنقوشة، دمج الجزيئات في السيتوبلازم الخلية، وترسب الخلايا على السقالات والهياكل التي لا تحدث بصورة طبيعية أو كفاءة.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> HP DESKJET 500 </td><td> هيوليت باكارد </td><td> C2106A </td><td> توقف من الشركة المصنعة. اشترى التاجر مجدد ديسمبر. </td></tr><tr><td> إتش بي 26 خرطوشة الحبر الأسود </td><td> هيوليت باكارد </td><td> 51626A </td><td></td></tr><tr><td> الأكتين من عضلة أرنب، اليكسا فلور 488 المتقارن، 200 ميكروغرام </td><td> إينفيتروجن </td><td> A12373 </td><td></td></tr><tr><td> التخزين المؤقت فوسفات المالحة (PBS) </td><td> النائب Biomedicals </td><td> ICN1860454 </td><td></td></tr><tr><td> Dulbeccos معدلة النسور متوسطة (DMEM) </td><td> الحرارية العلمية </td><td> SH3002201 </td><td></td></tr><tr><td> الأبقار مصل الجنين </td><td&gt; سيغما الدريخ </td><td> F4135 </td><td></td></tr><tr><td> الأمفوتريسين B </td><td> سيغما الدريخ </td><td> A2942 </td><td> تستخدم عند 0.5٪ في أوساط استزراع الخلايا </td></tr><tr><td> البنسلين الستربتوميسين </td><td> سيغما الدريخ </td><td> P4333 </td><td> تستخدم عند 0.5٪ في أوساط استزراع الخلايا </td></tr><tr><td> اتجاه ومقعد تدفق النظيفة </td><td> Envirco </td><td> VLF 797 </td><td> اختياري السكن لحفظ طابعة العقيم </td></tr></tbody></table>

creating transient cell membrane pores using a standard inkjet printer

Bioprinting لديها مجموعة واسعة من التطبيقات وأهميتها، بما في ذلك هندسة الأنسجة، ومباشرة تطبيق علاجات الخلية، وmicrofabrication جهاز الاستشعار البيولوجي. 1-10 في الآونة الأخيرة، كما تم الحرارية الطباعة النافثة للحبر تستخدم لترنسفكأيشن الجينات. 8،9 والطباعة النافثة للحبر الحرارية عملية تبين لل تعطيل مؤقتا في أغشية الخلايا دون التأثير على بقاء الخلية. ويمكن استخدام مسام عابرة في غشاء لتقديم الجزيئات، والتي من شأنها أن يكون الأمر خلاف ذلك كبيرة جدا لتمر عبر الغشاء، في سيتوبلازم الخلية. 8،9،11 التطبيق يتم شرحها هنا هو استخدام الطباعة النافثة للحبر الحرارية لدمج مونومرات G-الأكتين fluorescently المسمى في الخلايا. وميزة استخدام الحرارية نافثة للحبر الطباعة لحقن الجزيئات في الخلايا هو أن التقنية هي حميدة نسبيا للخلايا. وقد تبين 8، بقاء الخلية 12 بعد الطباعة لتكون مماثلة لجيش التحرير الشعبى الصينى الخلية القياسيةطرق تينغ 1،8. وبالإضافة إلى ذلك، يمكن الطباعة النافثة للحبر معالجة الآلاف من الخلايا في غضون دقائق، وهو أسرع بكثير من microinjection دليل. وقد ثبت أن المسام التي أنشأتها الطباعة ليغلق في غضون ساعتين تقريبا. ومع ذلك، هناك حد لحجم المسام خلق (~ 10 نانومتر) مع هذا الأسلوب والطباعة، الأمر الذي يحد من تقنية لحقن الخلايا مع البروتينات الصغيرة و / أو جزيئات. 8،9،11 تم تعديل معيار HP DESKJET 500 الطابعة للسماح للطباعة الخلية. 3، 5، 8 غطاء الطابعة تم إزالة وآلية تغذية الورق وتجاوز استخدام رافعة ميكانيكية. تم إنشاء المرحلة للسماح لوضع الشرائح المجهر وcoverslips مباشرة تحت رأس الطباعة. فتحت خراطيش الحبر، وتمت إزالة الحبر وتم تنظيفها قبل استخدامها مع الخلايا. تم إنشاء نمط الطباعة باستخدام معيار برامج الرسم، والتي تسيطر وقتها على الطابعة من خلال أمر الطباعة بسيط. 3T3 الليفيوقد نمت الانفجارات إلى نقطة التقاء، trypsinized، ومعلق ثم في الفوسفات مخزنة المالحة مع ذوبان مونومرات G-الأكتين fluorescently المسمى. وكان pipetted تعليق خلية في خرطوشة الحبر وطبعت خطوط من الخلايا على زلات غطاء زجاجي المجهر. تم تصوير الخلايا الحية باستخدام المجهر مضان وعثر الأكتين في جميع أنحاء السيتوبلازم. إدماج الأكتين فلوري في الخلية يسمح للتصوير من لوقت قصير ديناميات هيكل الخلية وغير مفيدة لطائفة واسعة من التطبيقات. 13-15

Watch this Scientific Journal Video about Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer at JoVE.com

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

وصفا للأساليب المستخدمة لتحويل منضدية HP 500 الطابعة إلى bioprinter. الطابعة غير قادرة على معالجة الخلايا الحية، والذي يسبب مسام عابرة في غشاء. ويمكن استخدام هذه المسام لدمج الجزيئات الصغيرة، بما في ذلك فلوري G-الأكتين، إلى خلايا المطبوعة.

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 ...

creating-transient-cell-membrane-pores-using-a-standard-inkjet-printer

Research

JoVE Journal

Bioengineering

38.8K Views.  Clemson University. وصفا للأساليب المستخدمة لتحويل منضدية HP 500 الطابعة إلى bioprinter. الطابعة غير قادرة على معالجة الخلايا الحية، والذي يسبب مسام عابرة في غشاء. ويمكن استخدام هذه المسام لدمج الجزيئات الصغيرة، بما في ذلك فلوري G-الأكتين، إلى خلايا المطبوعة.

Video: Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

مخصص الصينية النامية الهامستر بروتين الخلية المبيض في استضافة فحوصات باستخدام التكنولوجيا الصوتية Microparticle الغشائية

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

زيادة غلال [كدنا] من خلية واحدة كميات مرنا في مختبر قياسي ردود الفعل الناسخ العكسي باستخدام Microstreaming الصوتية

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

إنشاء ثنائي الأبعاد ركائز منقوشة على الحبس البروتين والخلايا

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

الهندسة البكتيريا تمسكا من إنشاء واحدة الاصطناعية Curli OPERON

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

إنشاء تدرجات لاصق وقابل للذوبان للهجرة الخليوي مع التصوير مضان المجهري

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

المجهر الضوئي الكمية: قياس الخلوية البيوفيزيائية الميزات مع مجهر بصري قياسي

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

توصيف النظم المعقدة باستخدام تصميم التجارب النهج: عابر التعبير البروتين في التبغ كدراسة حالة

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

الغضروف الإنسان الأنسجة التصنيع باستخدام ثلاثي الأبعاد تكنولوجيا الطباعة النافثة للحبر

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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.

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 ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.