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.

שימוש בשיטות שתוארו לעיל, תרביות תאי 3D עשויות להיות מבוגרות וניתחו עם MALDI-MSI. שורות תאים הנציחו רבים יהוו מבני 3D כאשר בתרבית כראוי. יש להקפיד 9,10 לטפח תאים שלא עברו יותר מדי פעמים, כלומר, יש לי מספרי מעבר נמוכים. באופן כללי, תאים עם מספרי חלוף עשר ותחתונים הם אופטימליים לגידול תרביות תאי 3D. גידול תרביות תאי 3D בתמיכת agarose מסורתית מספק דרך חסכונית לקבוצות מחקר המתעניייינות בתרבית תאי 3D לנסות מערכת זו. בשיטה זו משתמשת כמה רכיבים לא כבר ברוב מעבדות תרבית תאי יונקים. תשומת לב קפדנית צריכה להיות משולם להכנת צלחות agarose לצמיחה, כך שתרביות תאי 3D עולות בקנה אחד בגודל ובצורה; כמה היבטים שדורשים תשומת לב קפדנית כוללים בדיקה שאין בועות אוויר וממולכד לתערובת וכי המניסקוס נכון נוצר בתחתית כל well. אם המניסקוס לא יתבצע כראוי, התאים עשויים לגדול בצורות שאינן אליפטית או בגושים קטנים. כמו כן, יש לקחת לא למקום PBS לתוך הג&#39;לטין עם spheroids טיפול. אם זה יקרה, להסיר את PBS מהחלק העליון של ג&#39;לטין בעזרת פיפטה 200 μl. אם העלות היא לא גורם, צלחות תחתונה נמוכות במיוחד מחייבות עגולות הם זמינות מסחריות ומציעות פישוט הפעולות הבאות. בעוד שיטות הטבעת רקמות אחרות יכולות להיות גם ספקטרומטריית יקרה ולא המונית תואמות, ג&#39;לטין-הטבעה הוכח להיות גם זול ורבים-תכליתי. אסטרטגיות הכנה שתוארו לעיל כבר מותאמות לתרביות תאי 3D להשיג נתונים באיכות הגבוהות ביותר. בעוד ג&#39;לטין-הטבעה הוכח להיות תואם לניתוח ספקטרומטריה, תהליך זה אמנם מצמצם מטריצות הזמינות בשל שיתוף התגבשות. ברגע קפוא, תרביות תאי 3D יציבות במשך חודשים, כל עוד הם לא בהקפאה מופשרות. ג&#39;לטין הופך אטום כאשר קפוא, ותא 3Dתרבויות הן בצבע דומה, ולכן רצוי לפני ההקפאה לתעד את מיקום תרבית תאי 3D כדי לסייע בחיתוך. בעת הכנת שקופיות, מטריצה ​​יכולה להיות מיושמת במספר שיטות שונות, אך יש לציין כי חלק מהמטריצות נמצאו לשתף להתגבש עם ג&#39;לטין, כגון חומצת ferulic, עיבוד המדגם לא שמיש. גבישי מטריצה ​​קטנים יותר לייצר ספקטרום מסה עקבי יותר. בעוד handspotting היא השיטה הפשוטה של ​​יישום מטריצה, נפח הממס הגדול יותר יכול לגרום לגדלי גביש גדולים יותר והגירה אנליטי משמעותית. בספקטרומטר המסה, התקדמות בטכנולוגיות MALDI-MSI צמצם את המומחיות הנדרשת לתחילת ניתוח. רכישת נתונים וניתוח הוא פשוט ברוב המערכות, המאפשרות אפילו טירון כדי להשיג מידע באיכות גבוהה משקופיות מצופות איטו. בחירה זהירה של הפרמטרים המכשיר, מותאמת בcultur תא הסמוך שאינה תמונה 3D הראויes, הוא קריטי לקבלת נתונים באיכות גבוהה. לדוגמא, הגדלת כוח הלייזר עלולה להגביר עוצמת שיא אבל ירידת כוח הלייזר עשויה לשפר את הרזולוציה ולתת צורות שיא סימטריות יותר. יש להשתמש בתרביות תאי 3D במהירות לאחר חיתוך על מנת להבטיח איכות מדגם אופטימלית. השפלה של אות חלבון ניתן לראות בפחות משבוע ללא אחסון נאות (dessicator / -80 ° C במקפיא), בעיקר באזור המסה הגבוה (מעל 20 KDA). בשל ביקוש ההולך וגדל, תוכנת הדמיה תוכניות רבות ושונות פותחו וחופשיים לציבור המחקר. רוב ספקטרומטרים מסת ההדמיה התקינו את התוכנה דרושה כדי לחזות המונים יחידים עם הפורמטים קנייניים בשימוש בכל מכשיר. תוכנות אלו עשויות לשמש כדי להשיג תמונות חדה-מסה ולייצא את הנתונים. רוב התוכנות תאפשר גם את השכבה של שניים או יותר המונים של עניין. בנוסף, צופים רבים ושונים לנתונים MSI הם להורדה בחינם, כגון MSiReaderד BioMap. 32,33 נתונים ספקטרומטר המסה שהושגו עשוי גם להיות מעובד לאחר רכישה בקלות, הבאת מידע שימושי יותר לחזית. מתרופות לשומנים לחלבונים, המערכת שתוארה לעיל מאפשרת רכישה מהירה של נתונים על מנת להעריך את ההפצה של מולקולות של עניין על פני מבנה תרבית תאי 3D. ניתן לקחת קטעי סדרתי לתמונת מבנה 3D כולו על ידי בנייה מכל תמונת 2D של פרוסת תרבית תאי 3D. הטכניקות והערות בפרוטוקול יהיו שימוש לחוקרים המעוניינים להתחיל תרבית תאים תלת ממדי כגשר בין תרבות monolayer ומודלים של בעלי חיים. ברגע שמשתלט עליו, יכולות להיות מיושמות בטכניקות אלה לסוגים שונים של תרבויות 3D תא, רקמות, ותרביות תאים, המאפשרות לחוקרים תמונה לפי דוגמאות שהם בוחרים.

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.

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			<td height="21" style="height:21px;width:204px;">Name of Material/Equipment</td>
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			<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>
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			<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>
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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> יש הדמיה MSI הפוטנציאל לחשוף הפצה מולקולרית רבות ושונה בתרביות תאי 3D. תוך שימוש בשיטה שתוארה לעיל, מינים ממולקולות קטנות (איור 1) לחלבונים גדולים (איור 2) עשויים להיות במעקב על פני התרבות. תוצאות אלו מראות הדמיה של יון יחיד עם כלי חינם MSiReader. 32 תוצאות עשויות להיות מנותחות ליונים בודדים של עניין על פני תרבית תאי 3D עם תוכנת הדמיה או באזור של עניין או האזור שנסרק כולו. בנוסף רוב התוכנות באופן אוטומטי לדמיין יונים מעל לסף מסוים שנקבע על ידי המשתמש, אשר עשוי לסייע במאמצי גילוי. ברגע שמפות יון בודדים מתקבלות, רוב התוכנות כוללות יכולת כיסוי התמונות לצפייה בcolocalization של יונים. כך או בגישות גילוי מבוסס או באופן ממוקד, ההדמיה ספקטרומטריית מסה היא כלי רב עוצמה כדי לנתח תרביות תאי 3D. בתוך-page = &quot;תמיד&quot;&gt; <img alt="איור 1" src="/files/ftp_upload/52313/52313fig1highres.jpg" /> איור 1. תוצאות נציג של צמיחת תרבית תאי 3D וחתך. תמונה אופטית של מבנה מעי גס שלם HCT-116 3D תרבית תאים כפי שנצפו ביום 14 לפני קציר שמאל). מימין) תמונה אופטית של הפרוסה כ משווני של הפשרה תרבית תאי 3D מעי גס רכובה על גבי שקופית מצופה איטו להדמיה. <a href="https://www.jove.com/files/ftp_upload/52313/52313fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 2" src="/files/ftp_upload/52313/52313fig2highres.jpg" /> איור 2. תוצאות נציג של מולקולה הקטנה MALDI-MSI של סעיף קו המשווה של תרבית תאי 3D. ספקטרה ההמונית הממוצעת שהושגה עם α-CHCA בטווח מסה נמוכה (מולקולה קטנה) למעלה). ) נציג תמונות תחתיתמסה אחת: 327.8 מ &#39;/ z מקומי בעיקר הפנים של תרבית תאי 3D ו429.7 מ&#39; / z מקומי בעיקר לקצה של תרבות תא 3D. קו מקווקו מציין את הקצה המשוער של אליפטית. <a href="https://www.jove.com/files/ftp_upload/52313/52313fig2large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 3" src="/files/ftp_upload/52313/52313fig3highres.jpg" /> איור 3. נציגי תוצאות של הדמיה חלבון על ידי MALDI-MSI של סעיף קו המשווה של תרבית תאי 3D. למעלה) ספקטרה ההמונית הממוצעת שהושגה עם חומצת sinapic בטווח מסת חלבון) (גבוה יותר. תחתון) נציג תמונות של מסה אחת: 10871 מ &#39;/ z מקומי בעיקר לקצה אליפטית, ו12,184 מ&#39; / z מקומי יותר לכיוון הפנים של אליפטית. קו מקווקו מציין את הקצה המשוער של אליפטית. <a href="https://www.jove.com/files/ftp_upload/52313/52313fig3large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</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

ניתן לגדל שורות תאי סרטן הונצחו כתרביות תאי 3D, מודל רב-ערך למחקר ביולוגי. פרוטוקול זה מתאר הדמיה ספקטרומטר מסה של תרביות תאי 3D, כולל שיפורים בפלטפורמת הכנת מדגם. המטרה של פרוטוקול זה היא להנחות את המשתמשים כדי להכין תרביות תאי 3D לניתוח הדמיה ספקטרומטר מסה.

לדוגמא הכנת אסטרטגיות להדמית ספקטרומטריית המסה של מודלי תרבית תאי 3D

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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Research

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Bioengineering

18.0K Views.  University of Notre Dame.  ניתן לגדל שורות תאי סרטן הונצחו כתרביות תאי 3D, מודל רב-ערך למחקר ביולוגי. פרוטוקול זה מתאר הדמיה ספקטרומטר מסה של תרביות תאי 3D, כולל שיפורים בפלטפורמת הכנת מדגם. המטרה של פרוטוקול זה היא להנחות את המשתמשים כדי להכין תרביות תאי 3D לניתוח הדמיה...

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.

תא פשוט תלוי תרבות Drop פרוטוקול הדור של Spheroids 3D

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.

הכנת 3D הפיברין פיגומים יישומים סלולריים גזע תרבות

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.

הדמיה של רקמות ביולוגיות על ידי Desorption electrospray יינון המוני ספקטרומטריית

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).

ביעור Cell סלקטיבי מן תרבות 3D מעורבת באמצעות טכניקה האדומה קרוב 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.

הדמיה של כל הגוף ספקטרומטריית מסה על ידי אינפרא אדום מטריקס בסיוע לייזר Desorption electrospray יינון (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.

הכנת ג&#39;לי 3D קולגן microchannels לחקר אינטראקציות 3D<em&gt; In vivo</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.

הידרוג מתכוונן מ מטריקס ריאתי עבור תרבית תאים 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.

כימות בו זמני של קינורנינים נבחרים שנותחו על ידי ספקטרומטריית כרומטוגרפיה-מסה נוזלית במדיום שנאסף מתרביות תאים סרטניים

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.

הקמת תרבית תאי סטרומה-אנדותל תלת-ממדית

ייצור מדרגי של כדורי תאים אחידים באמצעות התקן הרכבה אקוסטית תלת מימדית

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.

מחבר זרקור: פיתוח שיטת הרכבה אקוסטית ללא פיגומים לתרבית תאי תלת מימד באיכות גבוהה 

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.

התקן הרכבה אקוסטית תלת מימדי לייצור המוני של ספרואידים סלולריים

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