פרויקט זה מומן על ידי NIH (R21EB013717) וIUPUI OVCR (RSFG). המחבר מודה גב &#39;האן שי לקבלת הסיוע הטכני שלה.

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הפרוטוקול המתואר מציג פרטים על אנקפסולציה הקלה של תאים בידרוג thiol-ene נוצר על ידי photopolymerization שלב צמיחה. בעוד שיחס של 1:1 של stoichiometric norbornene לקבוצות פונקציונליות thiol היה בשימוש בפרוטוקול זה, היחס יכול להיות מותאם בהתאם לניסויים. בנוסף לניסוח נכון, חשוב לשמור על אחידות בפתרו?...

הידרוג הם פולימרי crosslinked הידרופילי עם פוטנציאל יוצא דופן כחומרי פיגומים לרקמות תיקון והתחדשות. 1-3 תכולת המים הגבוהה של הידרוג מאפשרת דיפוזיה קלה של חמצן וחומרים מזינים חילופים ומוצרים מטבוליות סלולריים, שכולם חיוניים לשמירה על כדאיויות תא. בנוסף, הידרוג הם נישא מצוין לשחרור מבוקר ומשלוח תא בשל tunability הגבוה שלהם. 2 הידרוג סינטתית כגון אלה שהוכנו מפולי (אתילן גליקול) (PEG) משמשים יותר ויותר ביישומי הנדסת רקמות, בעיקר בשל cytocompatibility, רקמות כמו אלסטיות, וtunability גבוה בחומר תכונות פיסיקליות ומכאניות. 4-6 למרות פלטפורמת הידרוג נפוץ, מחקרים הראו כי PEG diacrylate (PEGDA) הידרוג נוצרה על ידי photopolymerizations שרשרת צמיחה יש נטייה לפגוע בתאים במארז duricrosslinking ng הרשת ובאתרו אנקפסולציה תא. 7 ניזק לתאים יוחס בעיקר למינים רדיקליים שנוצרו על ידי מולקולות photoinitiator, אשר מתפשטות דרך הקבוצות ויניל על PEGDA לשרשרות פולימר לתוך Crosslink הידרוג. למרבה הצער, מינים רדיקליים אלה גם לגרום ללחצים וניזקים סלולריים בעת אנקפסולציה תאים, במיוחד לתאים קיצוניים רגישים כגון לבלב β-תאים. 8-10 על מנת לקבל את גודל רשת גבוהה יותר עבור דיפוזיה טובה יותר והישרדות תא, משקולות מולקולריות גבוהות יותר PEGDA משמש לעתים קרובות לאנקפסולציה תא. זה, לעומת זאת, פוגע בקינטיקה פולימריזציה וגורם למאפייני biophysical ג&#39;ל תת אופטימליים. 7,11,12 בנוסף לחסרונות שהוזכרו לעיל, זה מאוד קשה להתאושש ממבני תאי הידרוג PEGDA בשל ההטרוגניות ושאינן מתכלה, אופי רשתות crosslinked. בעוד פפטידים פרוטאז רגישים יכולים להיות משולביםלשדרת PEG macromer כדי להבהיר את הידרוג PEGDA אחרת אינרטיים הרגישה למחשוף האנזימטית, הנטייה לעתים קרובות משתמשת בריאגנטים יקרים ורשתות כתוצאה עדיין מכילות רמה גבוהה של הטרוגניות בשל האופי של פילמור בשרשרת צמיחה. 13-15 לאחרונה, הידרוג PEG-פפטיד שנוצרה באמצעות צעד צמיחת photopolymerization thiol-ene הוכחה להציג תכונות מועדפות לאנקפסולציה תא מעל הידרוג נוצרה על ידי photopolymerization שרשרת צמיחה. 7 קינטיקה gelation המעולה של thiol-ene הידרוג מיוחסת ל&#39;קליק &quot;טבעה של תגובה בין פונקציות Ene thiol ו. בהשוואה לפילמור בשרשרת צמיחה של PEGDA, תגובת thiol-ene פחות חמצן שתוצאתו שיעור gelation מהר יותר. עכבות 16,17 הידרוג Thiol-ene גם יעילות גבוהה יותר ופילמור מאפייני biophysical ג&#39;ל טובים יותר בהשוואה לשרשרת צמיחת PEGDA הידרוג, 7 , 18 &lt;/ Sup&gt; שתוצאתו ניזק לתאים הנגרם על ידי זנים מוגבלים קיצוניים במהלך photopolymerization. בעבר, הידרוג thiol-ENE הוקמה על ידי 4-זרוע PEG-norbornene (PEG4NB) macromer וcrosslinkers bis-ציסטאין המכיל פפטיד, כגון פרוטאז רגישים פפטידים כבר נוצל לאנקפסולציה תא. Tunability גבוה 7,18 של רשתות הידרוג PEG מציע microenvironment גמיש והשליטה 3D לחקירת הישרדות תא ופעילות, תוך השימוש ברצף פפטיד פרוטאז הרגיש מספק דרך קלה להתאוששות של מבני תא שנוצרו באופן טבעי בתוך הידרוג. בפרוטוקול זה אנו מנצלים צעד צמיחת photopolymerized thiol-ene הידרוג המפוברקת באמצעות 4-זרוע PEG-norbornene (PEG4NB) וcrosslinker פפטיד chymotrypsin רגיש (CGGY ↓ C) לאנקפסולציה של MIN6 תאי β. פרוטוקול זה מרחיב באופן שיטתי ללימוד טכניקות הישרדות, השגשוג ואליפטית היווצרות MIN6β-תאים בידרוג thiol-מז. בנוסף, אנו מספקים שיטה להתאוששות אליפטית β-תאים ואפיון ביולוגי של שספרואידים התאוששו.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> PEG 4-זרוע (20kDa) </td><td> Jenkem טכנולוגית ארה&quot;ב </td><td> 4ARM-PEG-20K </td><td></td></tr><tr><td> חומצות אמינו-Fmoc </td><td> Anaspec </td><td></td><td></td></tr><tr><td> ערכת כדאיויות תא חי / מלח </td><td> Invitrogen </td><td> L3224 </td><td> כולל Calcein AM וethidium homodimer-1 </td></tr><tr><td> מגיב AlamarBlue </td><td> עבד Serotec </td><td> BUF012 </td><td></td></tr><tr><td> מגיב CellTiter Glo </td><td> Promega </td><td> G7570 </td><td></td></tr><tr><td> DPBS </td><td> Lonza </td><td> 17-512F </td><td> ללא Ca 2 וMg 2 </td></tr><tr><td> HBSS </td><td> Lonza </td><td> 10547F </td><td> ללאCa 2 וMg 2 </td></tr><tr><td> גלוקוז DMEM הגבוה </td><td> Hyclone </td><td> SH30243.01 </td><td></td></tr><tr><td> FBS </td><td> Gibco </td><td> 16000-044 </td><td></td></tr><tr><td> אנטיביוטיקת Antimycotic </td><td> Invitrogen </td><td> 15240-062 </td><td></td></tr><tr><td> β-Mercaptoethanol </td><td> סיגמה אולדריץ </td><td> M7522-100 מ&quot;ל </td><td></td></tr><tr><td> טריפסין-EDTA </td><td> Invitrogen </td><td> 15400-054 </td><td></td></tr><tr><td> טריפסין ללא α-chymotrypsin </td><td> ורטינגטון קורפ יוכימית </td><td> LS001432 </td><td></td></tr><tr><td> עכבר Inusin ELISA ערכה </td><td> Mercodia </td><td> 10-1247-01 </td><td></td></tr><tr><td> מזרק 1 מ&quot;ל פנוי </td><td> BD Biosciences </td><td></td><td></td></tr></tbody></table>

generation and recovery of &beta;-cell spheroids from step-growth peg-peptide hydrogels

הידרוג הם פולימרי crosslinked הידרופילי המספקים microenvironment תלת ממדים עם רקמות כמו גמישות וחדירות גבוהות לתאים או רקמות טיפוליות רלוונטיים culturing. הידרוג הוכן מפולי (אתילן גליקול) (PEG) נגזרים משמשים יותר ויותר למגוון רחב של יישומי הנדסת רקמות, בחלקו בשל המאפיינים מתכוננים וcytocompatible. בפרוטוקול זה, אנו מנוצלים thiol-ene photopolymerizations שלב צמיחה לפברק הידרוג PEG-פפטיד לencapsulating לבלב MIN6 תאי B. הג&#39;לים נוצרו על ידי 4-זרוע PEG-norbornene macromer (PEG4NB) וcrosslinker פפטיד chymotrypsin רגיש (CGGYC). הטבע הידרופילי ולא עכירות של PEG מציע microenvironment cytocompatible להישרדות תא והתפשטותו ב3D, תוך השימוש ברצף פפטיד chymotrypsin הרגיש (C GGY ↓ C, חץ מציין אתר מחשוף אנזים, בעוד ציסטה מסוףשאריות eine נוספו לcrosslinking thiol-ene) מאפשר התאוששות מהירה של מבנים בתוך תא יוצרים הידרוג&#39;ל. הפרוטוקול הבא מפרט טכניקות ל: (1) Encapsulation של MIN6 β-תאים בthiol-ene הידרוג; (2) מבחני כדאיות סלולרית איכותיים וכמותיים לקביעת הישרדות תא ושגשוג; (3) שחזור של שספרואידים סלולריים באמצעות ג&#39;ל chymotrypsin בתיווך שחיקה; ו (4) ניתוח מבני ותפקודי של הספרואידים התאוששו.

<html> איורים 1-4 נציג תוצאות להראות לאנקפסולציה, הישרדות, התפשטות, היווצרות אליפטית, והתאוששות אליפטית בידרוג thiol-מז. איור 1 מציג את סכמטי התגובה (1) שלב צמיחת photopolymerization thiol-ene באמצעות PEG4NB וCGGYC, ו( 2) שחיקת chymotrypsin תיווך ג&#39;ל העוקב מנגנון שחיקת פני שטח. א?...

Watch this Scientific Journal Video about Generation and Recovery of Î²-cell Spheroids From Step-growth PEG-peptide Hydrogels at JoVE.com

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

הפרוטוקול הבא מספק טכניקות לencapsulating לבלב β-תאים בידרוג PEG-פפטיד שלב צמיחה שהוקם על ידי thiol-ene תגובות תמונה בלחיצה. פלטפורמת חומר זה לא רק מציעה microenvironment cytocompatible אנקפסולציה תא, אלא גם מאפשרת התאוששות מהירה למשתמש שליטה מלאה של מבני תא נוצרה בתוך הידרוג.

דור ושחזור של spheroids β-תא מהידרוג שלב צמיחת PEG-פפטיד

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

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Bioengineering

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

14.1K Views.  Indiana University - Purdue University at Indianapolis. הפרוטוקול הבא מספק טכניקות לencapsulating לבלב β-תאים בידרוג PEG-פפטיד שלב צמיחה שהוקם על ידי thiol-ene תגובות תמונה בלחיצה. פלטפורמת חומר זה לא רק מציעה microenvironment cytocompatible אנקפסולציה תא, אלא גם מאפשרת התאוששות מהירה למשתמש שליטה מלאה של מבני תא נוצרה בתוך הידרוג.

Video: Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

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 ...

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 ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

הייצור של חלבון כמו אלסטין Hydrogels כימוס, Immunostaining של תאים ב- 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

זן מבוקר של הידרוג'ל תלת מימדי תחת הדמיה מיקרוסקופית חיה

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

מיקרופריקציה ביולוגית בסיוע מיקרוסקופיית חמצן במחיר סביר של ספרואידים רב-תאיים

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

פיצול הידרוג'לים בתפזורת ועיבוד להידרוג'לים גרעיניים ליישומים ביו-רפואיים

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

ספרואידים של כבד אנושי מדם היקפי למחקרי מחלות כבד

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

ייצור, סינון בתפוקה גבוהה וביו-בנקאות של ספרואידים לבביים פלוריפוטנטיים שמקורם בתאי גזע המושרים על ידי בני אדם

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

מחקר השוואתי של טכניקות דה-צלולריזציה עבור הידרוג'לים ECM של ריאות בקר

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypic<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: black; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&#160;alternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

מחבר זרקור: אופטימיזציה של דה-צלולריזציה של ריאות בקר עבור הידרוג'לים אורגנוטיפיים

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

פיתוח ואפיון הידרוג'לים מטריקס חוץ-תאיים ריאתיים דה-צלולריים

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...