This work was supported by the Walther Cancer Foundation under the Advancing Basic Cancer Research program to the Harper Cancer Research Institute of the University of Notre Dame (DARW) and the 2011 Starter Grant from the Society for Analytical Chemists of Pittsburgh (ABH). Thanks also to the staff of the Mass Spectrometry and Proteomics Facility for useful discussions.

<ol>
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This article is part of a special issue on Multimodal Pre-Clinical Imaging, sponsored by Bruker Biospin. The MALDI-TOF system used to obtain the representative results is a Bruker AutoFlex III.

باستخدام الأساليب المذكورة أعلاه، قد تكون نمت مزارع الخلايا 3D وتحليلها مع MALDI-MSI. وكثير من خطوط الخلايا خلد تشكيل هياكل 3D عندما مثقف بشكل مناسب. وينبغي اتخاذ 9،10 العناية لزراعة الخلايا التي لم يتم مرت مرات كثيرة جدا، وهذا هو، لديهم أرقام مرور منخفضة. بشكل عام، والخلايا مع أرقام مرور عشر وأقل هي الأمثل لزراعة مزارع الخلايا 3D. نموا في مزارع الخلايا 3D في دعم الاغاروز التقليدي يوفر وسيلة فعالة من حيث التكلفة لمجموعات بحثية مهتمة في زراعة الخلايا 3D لمحاولة هذا النظام. هذه الطريقة تستخدم بعض المكونات لم تكن بالفعل في معظم المختبرات زراعة الخلايا الثديية. ويجب إيلاء اهتمام دقيق لإعداد لوحات الاغاروز للنمو بحيث مزارع الخلايا 3D تتفق في الحجم والشكل. وتشمل بعض الجوانب التي تتطلب عناية فائقة التحقق من أن عدم وجود فقاعات الهواء هي شرك إلى الخليط وأن يتم تشكيل الغضروف المفصلي السليم في الجزء السفلي من كل ثالذراع. إذا لم تشكل الغضروف المفصلي بشكل صحيح، فإن الخلايا قد تنمو في أشكال غير كروي أو في كتل صغيرة. أيضا، يجب الحرص على عدم وضع PBS في الجيلاتين مع الأجسام الشبه الكروية. إذا حدث هذا، إزالة برنامج تلفزيوني من الجزء العلوي من الجيلاتين باستخدام ماصة 200 ميكرولتر. إذا كانت التكلفة ليست عاملا، منخفضة للغاية لوحات مستديرة أسفل ملزمة هي تجاريا المتاحة وتقدم تبسيط هذه الخطوات. في حين أساليب تضمين الأنسجة الأخرى يمكن أيضا أن تكون مكلفة وغير الشامل الطيف متوافق، وقد تبين الجيلاتين التضمين على حد سواء غير مكلفة وتنوعا. وقد تم تحسين استراتيجيات إعداد المبينة أعلاه لمزارع الخلايا 3D للحصول على أعلى جودة البيانات. في حين فقد تبين الجيلاتين التضمين لتكون متوافقة مع تحليل الطيف الكتلي، وهذه العملية تضيق المصفوفات المتاحة بسبب شارك في التبلور. مرة واحدة المجمدة، ومزارع الخلايا 3D مستقرة لعدة أشهر طالما لم يتم إذابة تجميد هم. الجيلاتين تصبح معتمة عندما المجمدة، والخلية 3Dالثقافات هي من لون مشابه، لذلك فإنه من المستحسن قبل التجميد لتوثيق 3D ثقافة الخلية التنسيب للمساعدة في تشريح. عند إعداد الشرائح، مصفوفة يمكن تطبيقها مع عدة طرق مختلفة، ولكن تجدر الإشارة إلى أن بعض مصفوفات تم العثور على المشاركة في crystalize مع الجيلاتين، مثل حمض الفيرليك، مما يجعل عينة غير صالحة للاستعمال. بلورات المصفوفة أصغر تنتج أكثر اتساقا أطياف الكتلة. في حين handspotting هو أبسط طريقة لتطبيق مصفوفة، يمكن للحجم المذيب أكبر يؤدي إلى أحجام أكبر وضوح الشمس والهجرة تحليلها كبيرة. في مطياف الكتلة، والتقدم في التقنيات MALDI-MSI قد قلل من الخبرات اللازمة لتحلل البداية. الحصول على البيانات وتحليل واضح ومباشر على معظم أنظمة، مما يسمح حتى المبتدئ الحصول على معلومات ذات جودة عالية من الشرائح ITO المغلفة. الاختيار الدقيق للالمعلمات الصك، الأمثل على المجاورة غير صورة 3D يستحق خلية الثقافيهوفاق، أمر بالغ الأهمية لحصول على بيانات عالية الجودة. على سبيل المثال، وزيادة قوة الليزر قد تزيد من كثافة الذروة ولكن تقليل قوة الليزر قد يحسن القرار، وإعطاء الأشكال الذروة أكثر توازنا. مزارع الخلايا 3D ينبغي أن تستخدم بسرعة تشريح لضمان أفضل جودة العينة بعد. ويمكن اعتبار تدهور إشارة البروتين في أقل من أسبوع دون التخزين السليم (dessicator ل/ -80 ° C الفريزر)، وخاصة في المنطقة الجماهيرية العالية (فوق 20 كيلو دالتون). بسبب الطلب المتزايد، تم وضع العديد من البرامج المختلفة والتصور أحرار للجمهور البحوث. وتثبيت معظم الطيف كتلة التصوير البرامج اللازمة لتصور الجماهير واحدة مع صيغ الملفات الخاصة المستخدمة في كل وثيقة. ويمكن استخدام هذه البرامج للحصول على صور واحدة كتلة وتصدير البيانات. ومعظم البرنامج أيضا يسمح للتراكب من اثنين أو أكثر من جماهير الفائدة. بالإضافة إلى ذلك، الكثير من المشاهدين مختلفة للبيانات MSI لتحميل خالية، مثل MSiReader لد BioMap. 32،33 البيانات مطياف الكتلة التي تم الحصول عليها كما يمكن معالجتها في مرحلة ما بعد الاستحواذ بكل سهولة، وبذلك المزيد من المعلومات المفيدة الى الواجهة. من الأدوية إلى الدهون للبروتينات، ونظام المذكورة أعلاه يسمح لاقتناء السريع للبيانات لتقييم توزيع جزيئات من الفائدة عبر هيكل زراعة الخلايا 3D. يمكن أن تؤخذ أقسام التسلسلية لصورة هيكل 3D بأكمله من خلال بناء من كل صورة 2D من شريحة من ثقافة الخلية 3D. سوف التقنيات والملاحظات في البروتوكول أن تكون ذات فائدة للباحثين الذين يرغبون في بدء زراعة الخلايا ثلاثية الأبعاد كجسر بين الثقافة أحادي الطبقة والنماذج الحيوانية. مرة واحدة يتقن هذه التقنيات يمكن تطبيقها على أنواع متعددة من الثقافات 3D الخلية والأنسجة، والمستنبتات الخلوية، مما يتيح للباحثين صورة أيهما العينات التي تختارها.

Mass spectrometry imaging (MSI) is a powerful technique to examine the distribution of molecular species across samples of interest. Researchers have imaged biological samples from whole animals down to single cells with this technique.1&#8211;4 Using MSI hundreds of analytes may be imaged at a single time, rather than single-substrate techniques requiring the development of additional fluorophores for traditional confocal microscopy, or staining for histology.5&#8211;7 Additionally, MSI can be used in either a targeted or a discovery-based fashion, making it an attractive approach to look at the overlap between the distribution of chemical species. Presented within are methods for the growth, preparation, and matrix assisted laser desorption ionization/desorption (MALDI) imaging of small samples (~1 mm in diameter) of three-dimensional multicellular cell cultures.8&#8211;11
3D cell cultures are of particular interest to the biological community as an intermediary between traditional monolayer culture and in vivo tumors. As the name implies, 3D cell cultures are heterogeneous cellular aggregates that contain many of the chemical gradients in the microenvironment observed in small avascular tumor nodules, making them a more realistic model system to the native tumor environment than monolayer culture.10 Many human cell lines can be grown as 3D cultures, including cell lines derived from colon, breast, ovary, prostate, kidney and lung tissues.10 There are inherent challenges in imaging three dimensional cell cultures as they are smaller than many samples commonly used for mass spectrometry imaging and therefore require careful sample preparation.7 However, the advantages of these tractable model systems include lower cost and faster growth and analysis time compared to animal models. As a result, studies involving three dimensional cell cultures can be higher throughput than traditional animal studies.12&#8211;14
All of these advantages have resulted in increasing popularity for three dimensional cell cultures in biological research; there is a corresponding need to develop new analytical strategies to explore both the composition and spatial distribution of molecules in these systems.12,15&#8211;21 The goal of this manuscript is to describe the adaptation of mass spectrometry imaging methods and sample preparation to three dimensional cell cultures.

<table border="0" cellpadding="0" cellspacing="0" width="600">
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			<td height="21" style="height:21px;width:204px;">Name of Material/Equipment</td>
			<td style="width:103px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:175px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCT116 Cell line</td>
			<td>ATCC</td>
			<td>ATCC CCL-247</td>
			<td>Potential hazard, handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">McCoy&#39;s 5A media</td>
			<td>Gibco</td>
			<td>16600-108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine serum</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA solution</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin, unflavored</td>
			<td>Knox</td>
			<td>--</td>
			<td>Available at most grocery stores in single-packets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ITO-coated glass slides</td>
			<td>Delta Technologies, Limited</td>
			<td>CG-90IN-S115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-Cyano-4-hydroxycinnamic acid (CHCA)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C8982</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sinapic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>85429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.3mm Gravity Feed Dual-Action Airbrush Paint Spray Gun Kit Set</td>
			<td>RoyalMax Airbrush</td>
			<td>--</td>
			<td>Many options available on sites such as Amazon.com</td>
		</tr>
	</tbody>
</table>

sample preparation strategies for mass spectrometry imaging of 3d cell culture models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

<html> MSI التصوير لديه القدرة على كشف الكثير من التوزيع الجزيئي مختلفة في مزارع الخلايا 3D. باستخدام طريقة المبينة أعلاه، يمكن تتبع الأنواع من الجزيئات الصغيرة (الشكل 1) للبروتينات كبيرة (الشكل 2) عبر الثقافة. وتشير هذه النتائج التصور من أيون واحد مع أداة مجانية يجوز تحليل MSiReader 32 نتائج للأيونات واحدة من الفائدة عبر زراعة الخلايا 3D مع برنامج التصور سواء في المنطقة ذات الاهتمام أو المنطقة الممسوحة ضوئيا بأكملها. بالإضافة إلى ذلك فإن معظم البرامج تصور تلقائيا أيونات فوق عتبة معينة تحدد من قبل المستخدم، والتي قد تساعد جهود الاكتشاف. مرة واحدة ويتم الحصول على خرائط ايون واحدة، ويشمل معظم برامج القدرة على تراكب الصور لعرض colocalization من الأيونات. إما في النهج المستندة إلى اكتشاف أو بطريقة المستهدفة، والتصوير قياس الطيف الكتلي هو أداة قوية لتحليل الثقافات خلية 3D. في صفحة = &quot;دائما&quot;&gt; <img alt="الشكل (1)" src="/files/ftp_upload/52313/52313fig1highres.jpg" /> الشكل 1. ممثل النتائج من 3D نمو ثقافة الخلية وباجتزاء. اليسار) صورة ضوئية من القولون والمستقيم HCT-116 3D بنية الثقافة خلية سليمة كما تراه في يوم 14 قبل الحصاد. اليمين) صورة ضوئية من شريحة الاستوائية تقريبا من القولون والمستقيم 3D ثقافة الخلية ذوبان الجليد التي شنت على شريحة ITO المغلفة للتصوير. <a href="https://www.jove.com/files/ftp_upload/52313/52313fig1large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> <img alt="الشكل 2" src="/files/ftp_upload/52313/52313fig2highres.jpg" /> الشكل 2. ممثل النتائج من جزيء صغير MALDI-MSI من قسم الاستوائي ثقافة خلية 3D. الأعلى) متوسط ​​أطياف الشامل يتحقق مع α-CHCA في نطاق انخفاض كتلة (جزيء صغير). القاع) الصور التمثيلية للكتلة واحدة: 327.8 م / ض محلية في المقام الأول إلى المناطق الداخلية من زراعة الخلايا 3D و429.7 م / ض محلية في المقام الأول إلى حافة ثقافة الخلية 3D. خط متقطع يدل على حافة التقريبية للكروي. <a href="https://www.jove.com/files/ftp_upload/52313/52313fig2large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> <img alt="الشكل (3)" src="/files/ftp_upload/52313/52313fig3highres.jpg" /> الشكل 3. ممثل النتائج من التصوير البروتين MALDI-MSI من قسم الاستوائي ثقافة خلية 3D. الأعلى) متوسط ​​أطياف الشامل يتحقق مع حمض sinapic في أعلى (المدى البروتين) كتلة. أسفل) صور الممثل من كتلة واحدة: 10871 م / ض محلية في المقام الأول إلى حافة كروي، و12184 م / ض محلية أكثر نحو المناطق الداخلية من كروي. خط متقطع يدل على حافة التقريبية للكروي. <a href="https://www.jove.com/files/ftp_upload/52313/52313fig3large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a>

Watch this Scientific Journal Video about Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models at JoVE.com

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

خطوط الخلايا السرطانية خلد يمكن زراعتها، لأن الحضارات خلية 3D، نموذجا قيما لالأبحاث البيولوجية. يصف هذا البروتوكول التصوير قياس الطيف الكتلي من مزارع الخلايا 3D، بما في ذلك تحسينات في منصة إعداد العينات. والهدف من هذا البروتوكول هو لإرشاد المستخدمين لإعداد مزارع الخلايا 3D لتحليل التصوير قياس الطيف الكتلي.

عينة استراتيجيات التحضير للتصوير الطيف الكتلي لل3D الثقافة خلية نماذج

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

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JoVE Journal

Bioengineering

18.0K Views.  University of Notre Dame.  خطوط الخلايا السرطانية خلد يمكن زراعتها، لأن الحضارات خلية 3D، نموذجا قيما لالأبحاث البيولوجية. يصف هذا البروتوكول التصوير قياس الطيف الكتلي من مزارع الخلايا 3D، بما في ذلك تحسينات في منصة إعداد العينات. والهدف من هذا البروتوكول هو لإرشاد ا...

Video: Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

إسقاط خلية بسيطة الشنق بروتوكول الثقافة للجيل 3D الأجسام الشبه الكروية

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

إعداد الفايبرين 3D السقالات لتطبيقات الجذعية زراعة الخلايا

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

التصوير من الأنسجة البيولوجية بواسطة الامتزاز Electrospray التأين الطيف الكتلي

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

انتقائية القضاء خلية من الثقافة 3D المختلطة باستخدام تقنية الأشعة تحت الحمراء بالقرب من Photoimmunotherapy

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

كامل الجسم الطيف الكتلي التصوير من قبل بمساعدة مصفوفة الأشعة تحت الحمراء ليزر الامتزاز Electrospray التأين (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

إعداد 3D الهلام الكولاجين وMicrochannels لدراسة التفاعلات 3D<em&gt; في فيفو</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

الانضباطي الهلاميات المائية من الرئوي خارج الخلية مصفوفة للثقافة خلية 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

القياس الكمي المتزامن لـ Kynurenines المختارة التي تم تحليلها بواسطة الكروماتوغرافيا السائلة- القياس الطيفي الكتلي في المتوسط الذي تم جمعه من ثقافات الخلايا السرطانية

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Establishing the 3D Stromal-Endothelial Cell Co-Culture

To begin, take the pellet of the GFP-HUVECs grown to a confluence level of 80 to 100%and resuspend the cells in an appropriate volume of basal medium to achieve a concentration of one times 10 to the seventh cells per milliliter of stock solution. Prepare 50-milliliter conical centrifuge tubes containing the required volume of basal medium supplemented with the respective growth factors. Add the GFP-HUVECs from the stock solution at a dilution of one to 66.67 to achieve a concentration of 1.5 times 10 to the fifth cells per microliter.Then aspirate the medium from the plate containing the stromal monoculture and add 200 microliters per well of the prepared GFP-HUVECs suspension. Incubate at 37 degrees Celsius in 5%carbon dioxide in a humidified atmosphere, changing the medium every three to four days. Monitor the development of the culture by bright-field and fluorescence microscopy using a five times objective.Differences between the cultures could be observed from the bright-field and fluorescence images showing only the GFP-HUVECs. The cultures appeared less developed without any growth factor, However, faster development was observed in the presence of FGF-2. In contrast, the bright-field images showed the densest cultures in the presence of BMP-2.However, vascular like networks formed in both growth factor-containing conditions and the most extensive and interconnected networks were formed with FGF-2.

إنشاء ثقافة مشتركة للخلايا اللحمية البطانية 3D

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 ...