Der Autor dankt Herrn Dr. Dongxuan Jia für die technische Unterstützung danken. Einige der Bilder erscheinen in diesem Artikel wurden in Zusammenarbeit mit Dr. Malcolm S. Steinberg, Department of Molecular Biology, Universität Princeton. Der Autor möchte auch das Department of Defense Prostate Cancer Research Program (Zuschüsse PC-030482 und PC-991552) und dem NCI / NIH (Grant R01CA118755) für ihre großzügige Unterstützung danken.

1. Erstellung eines Single Cell Suspension <ol><li> Adhärenten Zellkulturen zu 90% Konfluenz sollten angebaut werden sollten, worauf Monoschichten zweimal mit PBS gespült werden. Nach dem Ablassen gut, fügen Sie 2 ml (für 100 mm-Platten) von 0,05% Trypsin-1 mM EDTA, und bei 37 ° C, bis die Zellen zu lösen. Stoppen Trypsinierung durch Zugabe von 2 ml Vollmedium und sanft mit einem 5 ml-Pipette auf die Mischung verreiben, bis die Zellen in Suspension. Transfer-Zellen auf eine 15 ml konischen Rohr. </li><li> Add 40 ul einer 10 mg / ml DNAse Lager und Inkubation für 5 Minuten bei RT. Vortex kurz und Zentrifuge bei 200 xg für 5 Minuten. </li><li> Überstand verwerfen und Pellet waschen mit 1 ml komplettem Zellkulturmedium. Wiederholen Sie dann die Zellen in 2 ml komplette Zellkulturmedium. </li><li> Zählen Sie die Zellen mit einem Hämacytometer oder automatisierte Zellzahl und passen Konzentration auf 2,5 x 10 6 Zellen / ml. Für diese Demonstration eine BioRad TC10 automatisierte Zelle Zähler verwendet wurde. </li></ol> 2. Die Bildung von hängenden Tropfen. <ol><li> Entfernen Sie den Deckel von einem 60 mm Gewebe-Kulturschale und Platz 5 ml PBS in den Boden der Schale. Dies wird als Hydratation Kammer handeln. </li><li> Drehen Sie die Deckel und die Verwendung eines 20 ul Pipette zu 10 ul Tropfen auf der Unterseite des Deckels zu hinterlegen. Stellen Sie sicher, dass die Tropfen ausreichend voneinander entfernt sind so platziert, wie nicht zu berühren. Es ist möglich, mindestens 20 Tropfen pro Teller platzieren. </li><li> Drehen Sie die Deckel wieder auf das PBS-gefüllten unteren Kammer und bei 37 ° C / 5% CO 2 / 95% Luftfeuchtigkeit, überwachen die Tropfen täglich und inkubieren, bis entweder Zellschichten oder Aggregate gebildet haben. Ein Stereo-Mikroskop kann verwendet werden, um Aggregatbildung zu beurteilen. </li><li> Sobald Blätter zu bilden, sie können, um mit rundem Boden Glas Schüttler übertragen werden Kolben mit 3 ml Vollmedium und inkubiert in einem Schüttel-Wasserbad bei 37 ° C und 5% CO 2 bis Sphäroide zu bilden. </li></ol> 3. Hinweise <ol><li> Je nach Größe der Zellen kann Konzentration müssen nach oben oder unten angepasst werden. </li><li> Die Zellen können mit Membran interkalierende Fluoreszenzfarbstoffe vor dem Aufhängen Tropfenbildung gefärbt werden. </li><li> Zwei verschiedene Zelltypen können unterschiedlich gefärbt sein und in einer 1:1 (oder andere)-Verhältnis vor der Bildung der hängende Tropfen gemischt. Diese können Krebs und Stromazellen, embryonalen Geweben oder Zellen gentechnisch verändert, um spezifische Kleber Signaturen haben. </li><li> Die Zellen können aus Gewebekulturschalen abgenommen werden mit 0,05% Trypsin / 2 mM Kalzium Cadherin-Funktion zu bewahren. </li><li> Je nach Experiment können Blattgrößen entweder gemessen werden oder Aggregate für die mechanische Prüfung oder für biochemische oder molekulare Analyse verwendet werden. </li></ol> 4. Repräsentative Ergebnisse: Blatt-oder Sphäroid Bildung tritt in der Regel innerhalb von 24 Stunden, kann aber länger dauern, je nach Zelltyp. Abbildung 1 zeigt Bilder nach 18 Stunden Inkubation von unbehandelten Prostatakarzinom MAT-LyLu (MLL)-Zellen in hängenden Tropfen Kultur (Abbildung 1A) oder Zellen mit 25 &amp;mgr; MEK-Inhibitor PD98059 behandelt eingefangen. Beachten Sie, dass Meki Behandlung führt zu einer deutlichen Verdichtung der Zellschicht. Verdichtung kann durch die Messung der Größe 10-20 (oder mehr) hängende Tropfen und Vergleich der durchschnittlichen Größe zwischen den Behandlungsgruppen quantifiziert werden. Wir analysieren in der Regel Bilder mit ImageJ Software Schwellwerte jedes Bild dann Konvertieren von Bildern in Binary-Modus, worauf wir Partikelanalyse den gesamten Bildbereich in Pixel offenbart hat. Dies kann dann leicht zu Quadrat-Mikrometer umgewandelt werden. Abbildung 2 stellt einen Größenvergleich für unbehandelte und Meki behandelten MLL-Zellen. Wie bereits erwähnt, Meki Behandlung deutlich reduziert Bogenformat als durch den Student-t-Test (P &lt;0,0001). Für diesen Vergleich wurden 10 Aggregate für jeden der unbehandelten und behandelten Zellen gemessen. Abbildung 3 stellt ein Küken embryonalen Leber Sphäroid nach 24 Stunden in hängenden Tropfen Kultur und 2 Tage in einem Schüttelbad gebildet. Die hängenden Tropfen Assay kann auch geändert werden, um mehr als einen Zelltyp zu offenbaren, die räumliche Positionierung verabschiedet wurden. Zum Beispiel kann chick embryonalen Leberzellen isoliert, gefärbt mit einem fluoreszierenden Membran-interkalierende Farbstoff und mit chick embryonalen Herzzellen, die mit verschiedenen fluoreszierenden Marker wurden gefärbt. 4A zeigt, dass anstatt verbleibenden vermischt, neigen Leber-und Herzzellen zu &quot;sort-out&quot; von einem anderen und nehmen eine Kugel-in-a-Kugel-Konfiguration in die Herzzellen sind umhüllt von Leberzellen. Diese Konfiguration kann durch Unterschiede in der interzellulären Adhäsion zwischen Leber und Herz-Zellen erklärt werden. Dieses Konzept ist in der Differential Adhesion Hypothese, die verwendet wurde, um Zellen zu erklären Sortierung Verhalten zwischen embryonalen Keimblatt Gewebe für Amphibien-3 und Knochenfische 4 Embryonen, für die Inselzellen der Bauchspeicheldrüse Organisation 5 und für Zellsortierung zwischen zwei Zellpopulationen identisch i kodifiziertn jeder Hinsicht anders als für die Expression des gleichen Typs von Cadherin (6 und Abbildung 4B). Dieses Ergebnis ist besonders wichtig für den Nachweis, wie Engineering einen einfachen Unterschied in der Haftung zwischen Zellpopulationen können bei der Erzeugung von Organstruktur führen, zum Beispiel, ein Inselzellen der Bauchspeicheldrüse 5. <img src="/files/ftp_upload/2720/2720fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Images von Zellen in hängenden Tropfen Kultur für 18 Stunden entweder in Abwesenheit (A) oder Anwesenheit inkubiert (B) von 25 pm der MEK-Inhibitor PD98059. Die Bilder wurden mit einer digitalen Nikon Eclipse TE 300 Epifluoreszenzmikroskop und Photometrics CoolSnap ES Digitalkamera. <img src="/files/ftp_upload/2720/2720fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Quantifizierung von Meki-induzierte Verdichtung Ratte Prostatakrebs MLL-Zellen. Hanging drop Kulturen von unbehandelten und behandelten Meki MLL-Zellen wurden für 18 Stunden inkubiert, worauf Aggregatgröße wurde bestimmt, wie oben beschrieben. Die statistische Analyse der aggregierten Größe war durch den Student-t-Test. Der Stern steht für einen statistisch signifikanten Unterschied in Körnung (P &lt;0,0001) zwischen unbehandelten und Meki-behandelten Zellen und regt an, Meki-Behandlung führt zu einem deutlich kleineren und kompakteren Aggregaten. <img src="/files/ftp_upload/2720/2720fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Ein Sphäroid durch Inkubation chick embryonalen Leberzellen in hängenden Tropfen Kultur für 18 Stunden dann in einen Inkubator / Schüttelbad für 48 Stunden gebildet. Dieses Bild wurde mit einer digitalen Nikon SMZ800 Stereo-Mikroskop und ein Sony Schwarz CCD-Kamera. <img src="/files/ftp_upload/2720/2720fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Hanging drop Kultur zeigt Aussortieren Verhalten zwischen verschiedenen Zelltypen. In Panel A, wurden chick embryonalen Leberzellen mit PKH-2 Membran interkalierende Farbstoff angefärbt und gemischt zu gleichen Teilen mit PKH-26 gefärbten Küken embryonalen Herzzellen. In Panel B wurden zwei N-Cadherin-transfizierten L-Zell-Klone (LN4 und LN2a), zum Ausdruck N-cad an ihrer Oberfläche im Verhältnis 2,4:1, auch mit dem fluoreszierenden Membran interkalierende Farbstoffe PKH-2 und PKH-26 gefärbten , (Sigma-Aldrich), zu gleichen Teilen gemischt und kultiviert wie hängende Tropfen 7. Nach 18 Stunden in Kultur wurden Zellschichten zu Schüttelkolben überführt und für weitere 48 Stunden. Aggregate wurden in 2% Paraformaldehyd in festen Phosphat-gepufferter Kochsalzlösung und betrachtete mit einem BioRad MRC600 Scanning-Laser-konfokalen Mikroskop-System an einem Nikon Optiphot-2 Mikroskop. Optische Schnitte von beiden grünen und roten Kanälen wurden gesammelt und ein Silicon Graphix purpurnen VGX-Workstation mit Vital Images ausgestattet Voxel anzeigen Ultra-Imaging-Software wurde benutzt, um falsche Farbe auf beiden Kanälen zuordnen und die Bilder verschmelzen und enthüllt die anatomischen Konfigurationen erzeugt. (A) Konfokale optische Schnitt durch ein Aggregat demonstriert Umhüllung der Herzzellen (hier in einer Pseudo-Farbe gelb) von Leberzellen in Falschfarben blau (ab 8). (B) Konfokale optische Schnitt durch ein Aggregat demonstriert Umhüllung der hohen N-Cadherin-exprimierenden Zellen (grün) von jenen Ausdruck niedrigeren N-Cadherin (rot) (ab 6).

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Studien haben gezeigt, dass die Kultivierung von Zellen in einem dreidimensionalen Kontext (3D) unterschiedliche zelluläre Morphologie und Signalisierung, wenn eine starre zweidimensionale (2D) Kultur System 9 verglichen produziert. Zum Beispiel zeigen Fibroblasten besiedelten Kollagen-Gelen, die Fibroblasten-Morphologie in 3D ganz verschieden von dem in 2D 10,11 beobachtet wird. Ebenso können 3D-Kultur induzieren Gewebe-spezifische Differenzierung von Brustepithelzellen. 3D-Kultur-Systeme wurden...

<ul><li> automatische Zellzahl (BioRad TC10) </li><li> Schüttelwasserbad mit CO 2 Schrägdeckel Modell 3540 (Lab-line) </li></ul>

a simple hanging drop cell culture protocol for generation of 3d spheroids

Studium der Zell-Zell-Kohäsion und Zell-Substrat Adhäsion haben in der Vergangenheit auf Monolayerkulturen Anhänger auf starren Substraten durchgeführt. Zellen innerhalb eines Gewebes, jedoch sind in der Regel innerhalb eines dicht gepackten Gewebemasse in die Zellen zu etablieren intimen Verbindungen mit vielen nahen Nachbarn und mit Komponenten der extrazellulären Matrix umgeben. Dementsprechend sind die chemischen Milieu und körperlichen Kräfte durch die Zellen in einer 3D-Gewebe erfahrenen grundlegend anders als die von den Zellen in Monolayer-Kultur gezüchtet erlebt. Dies hat sich gezeigt, dass deutlich Auswirkungen zelluläre Morphologie und Signalisierung. Es wurden verschiedene Methoden entwickelt, um 3D-Zellkulturen, einschließlich Verkapselung von Zellen in Kollagen-Gelen 1 oder in Biomaterial Gerüste 2 zu erzeugen. Solche Methoden, die brauchbar sind, nicht rekapitulieren die intime direkte Zell-Zell-Adhäsion Architektur in gesundem Gewebe. Vielmehr sie enger ungefähre Kultur Systemen, in denen einzelne Zellen sind locker innerhalb einer 3D-Geflecht von ECM-Produkte verteilt. Hier beschreiben wir eine einfache Methode, bei der Zellen in hängenden Tropfen Kultur gelegt und bebrütet unter physiologischen Bedingungen, bis sie echte 3D Sphäroide in die Zellen stehen in direktem Kontakt miteinander und mit Komponenten der extrazellulären Matrix zu bilden. Das Verfahren erfordert keine spezielle Ausrüstung und kann an Zusatz von biologischen in sehr geringen Mengen, die von Interesse bei der Aufklärung Auswirkungen auf Zell-Zell-oder Zell-ECM-Interaktion kann erweitert werden. Das Verfahren kann auch zur Co-Kultur von zwei (oder mehr) eingesetzt werden unterschiedliche Zellpopulationen, um so die Rolle der Zell-Zell-oder Zell-ECM-Interaktionen in der Angabe räumlichen Beziehungen zwischen den Zellen aufzuklären. Zell-Zell-Kohäsion und Zell-ECM Haftung sind die Eckpfeiler des Studiums der embryonalen Entwicklung, Tumor-Stroma-Zell-Interaktion im malignen Invasion, Wundheilung, und für Anwendungen auf Tissue Engineering. Diese einfache Methode wird ein Mittel zur Erzeugung von Tissue-like zelluläre Aggregate für die Messung der biomechanischen Eigenschaften oder für die molekulare und biochemische Analyse in einem physiologisch relevanten Modell.

Watch this Scientific Journal Video about A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids at JoVE.com

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

Wir beschreiben eine einfache, schnelle Methode zur Erzeugung von 3D-Gewebe-like Sphäroide und ihre mögliche Anwendung auf Unterschiede in der Zell-Zell-Interaktionen zu quantifizieren.

A Simple hängenden Tropfen Cell Culture Protokoll zur Generierung von 3D-Sphäroide

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

a-simple-hanging-drop-cell-culture-protocol-for-generation-3d

Research

JoVE Journal

Bioengineering

56.6K Aufrufe.  UMDNJ-Robert Wood Johnson Medical School. Wir beschreiben eine einfache, schnelle Methode zur Erzeugung von 3D-Gewebe-like Sphäroide und ihre mögliche Anwendung auf Unterschiede in der Zell-Zell-Interaktionen zu quantifizieren.

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

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.

Erstellung von 3D-Fibrin-Gerüste für Stem Cell Culture Anwendungen

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 ...

Forschung

Biotechnik

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

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

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

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Erzeugung und Verwertung von β-Zellen Spheroids Von Step-Wachstum PEG-Peptid-Hydrogele

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

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D-Hydrogel Scaffolds für Articular Chondrozyten-Kultur und Knorpel-Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

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.

Tunable Hydrogele aus Pulmonary Extrazellulärmatrix für 3D-Zellkultur

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

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Ein Protokoll für Echtzeit-3D Einzelkorn-Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Bau eines vielschichtigen mesenchymale Stammzellen-Blatt mit einem 3D dynamische Kultursystem

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Ein menschliches 3D extrazelluläres Matrix-Adipozyten-Kulturmodell zum Studium der Matrix-Zell Metabolischen Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

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

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

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

Großflächige, automatisierte Produktion von aus Fett gewonnenen Stammzellsphäroiden für den 3D-Bioprinting

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

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

Erzeugung von Multicue-Zellmikroumgebungen durch UV-Photostrukturierung von dreidimensionalen Zellkultursubstraten

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

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.

Generierung, Hochdurchsatz-Screening und Biobanking von human-induzierten pluripotenten Stammzell-abgeleiteten Herzsphäroiden

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