L&#39;autore desidera ringraziare il Dott. Dongxuan Jia per assistenza tecnica. Alcune delle immagini che appaiono in questo articolo sono stati, in collaborazione con il Dr. Malcolm S. Steinberg, Dipartimento di Biologia Molecolare, Università di Princeton. L&#39;autore desidera anche ringraziare il Dipartimento della Difesa Programma di Ricerca sul Cancro alla prostata (borse di PC e PC-030482-991552) e il NCI / NIH (borsa R01CA118755) per il loro generoso sostegno.

1. Preparazione di una sospensione singola cella <ol><li> Colture di cellule aderenti dovrebbe essere cresciuta al 90% di confluenza, monostrati dopo di che devono essere risciacquati due volte con PBS. Dopo aver svuotato bene, aggiungere 2 ml (per 100 piastre mm) del 0,05% tripsina-1 mM EDTA, e incubare a 37 ° C fino a staccare le cellule. Fermare tripsinizzazione aggiungendo 2 ml di terreno completo e delicatamente utilizzare un pipetta da 5 ml per triturare il composto fino a quando le cellule sono in sospensione. Trasferire le cellule in una provetta da 15 ml. </li><li> Aggiungere 40 ml di un 10 mg / ml di DNAsi fotografici e incubare per 5 minuti a temperatura ambiente. Vortex brevemente e centrifugare a 200 xg per 5 minuti. </li><li> Gettare il surnatante e lavare pellet con 1 media tessuto completo cultura ml. Ripeto, poi risospendere le cellule in 2 ml di terreno completo coltura dei tessuti. </li><li> Contare le cellule utilizzando un emocitometro o contatore di cellule automatizzati e regolare la concentrazione di 2,5 x 10 6 cellule / ml. Per questa dimostrazione uno TC10 contatore di cellule BioRad automatizzato è stato usato. </li></ol> 2. Formazione di gocce. <ol><li> Togliere il coperchio da un piatto tessuto 60 millimetri cultura e luogo 5 ml di PBS sul fondo del piatto. Questo fungerà da camera di idratazione. </li><li> Invertire il coperchio e utilizzare un pipettatore 20 microlitri di depositare 10 gocce microlitri sul fondo del coperchio. Assicurarsi che gocce sono posti sufficientemente distanziati in modo da non toccare. E &#39;possibile mettere almeno 20 gocce al piatto. </li><li> Invertire il coperchio sulla PBS camera piena basso e incubare a 37 ° C / 5% di CO 2 / 95% di umidità, monitorare le gocce giornaliere e incubare fino a quando i fogli di cellule o aggregati si sono formati. Un microscopio stereo può essere utilizzato per valutare la formazione di aggregati. </li><li> Una volta forma fogli, essi possono essere trasferiti a fondo rotondo in vetro shaker palloni contenenti 3 ml di terreno completo e incubate in un bagno d&#39;acqua agitazione a 37 ° C e 5% di CO 2 fino a formare sferoidi. </li></ol> 3. Note <ol><li> A seconda delle dimensioni delle cellule, la concentrazione potrebbe essere necessario essere regolata verso l&#39;alto o verso il basso. </li><li> Cellule possono essere colorate con tinture fluorescenti membrana intercalando prima formazione delle gocce sospese. </li><li> Due diversi tipi di cellule possono essere differentemente colorate e mescolato in una (o altro) rapporto di 1:1 prima formazione di gocce. Questi possono essere cellule tumorali e stromali, tessuti embrionali, o cellule geneticamente modificate per avere firme adesivo specifico. </li><li> Le celle possono essere staccato dai piatti coltura di tessuti con tripsina 0,05% / 2 di calcio mM di preservare la funzione caderina. </li><li> A seconda della sperimentazione, le dimensioni del foglio può essere misurato, o aggregati può essere utilizzato per il test meccanici o per analisi biochimiche o molecolari. </li></ol> 4. Rappresentante dei risultati: Foglio o la formazione di sferoide si verifica in genere entro 24 ore, ma può richiedere più tempo a seconda del tipo di cellula. La figura 1 rappresenta le immagini catturate dopo 18 ore di incubazione del tumore della prostata non trattati MAT-LyLu (MLL), le cellule in coltura appeso calo (Figura 1A) o cellule trattate con 25 micron MEK inibitori PD98059. Si noti che il trattamento con Meki in una marcata compattazione dello strato di cellule. La compattazione può essere quantificata misurando le dimensioni di 10-20 (o più) appesi gocce dimensione media e il confronto tra i gruppi di trattamento. Noi di solito analizzare le immagini utilizzando il software ImageJ dalla soglia ogni immagine poi la conversione di immagini in modalità binaria, dopo di che applicare l&#39;analisi delle particelle per rivelare la superficie totale dell&#39;immagine in pixel. Questo può essere facilmente convertito in micron quadrati. La figura 2 rappresenta un confronto dimensioni per le cellule non trattate e Meki MLL-trattati. Come notato, il trattamento Meki riduce significativamente formato del foglio rispetto da parte di Student t-test (p &lt;0,0001). Per questo confronto, 10 aggregati per ciascuna delle cellule non trattate e trattate sono state misurate. Figura 3 rappresenta un embrionale sferoide fegato pulcino formato dopo 24 ore di sospensione cultura goccia e 2 giorni in un bagno shaker. Il dosaggio goccia può anche essere modificato per includere più di un tipo di cellule per rivelare il posizionamento spaziale adottato. Per esempio, le cellule del fegato pulcino embrionali possono essere isolate, tinto con un fluorescente membrana intercalando colorante e mescolate con cellule del cuore pulcino embrionali che sono stati colorati con un marcatore fluorescente diversa. Mostra la figura 4A che piuttosto che rimanere mescolati, le cellule del fegato e il cuore tendono a &quot;sort-out&quot; da un altro e adottare una sfera-in-a-sfera di configurazione in cui le cellule del cuore sono avvolti dalle cellule del fegato. Questa configurazione può essere spiegata da differenze di adesione intercellulare tra fegato e cellule del cuore. Questo concetto è codificato nella ipotesi Adesione differenziale che è stato usato per spiegare il comportamento delle cellule di smistamento tra il tessuto embrionale foglietto embrionale per anfibi teleostei 3 e 4 embrioni, per l&#39;organizzazione delle cellule delle isole pancreatiche 5 e per l&#39;ordinamento delle cellule tra due popolazioni di cellule identiche in ogni punto di vista diverso per i livelli di espressione dello stesso tipo di caderina (6 e Figura 4B). Questo risultato è particolarmente importante per dimostrare come ingegneria una semplice differenza di adesione tra popolazioni di cellule può portare alla generazione di struttura degli organi, per esempio, un isole pancreatiche 5. <img src="/files/ftp_upload/2720/2720fig1.jpg" alt="Figura 1" /> Figura 1. Immagini di cellule incubate in sospeso cultura goccia per 18 ore sia in assenza (A) o presenza (B) del 25 micron della inibitore MEK PD98059. Le immagini sono state catturate con una Nikon Eclipse TE 300 e un microscopio a epifluorescenza fotometrici CoolSnap ES fotocamera digitale. <img src="/files/ftp_upload/2720/2720fig2.jpg" alt="Figura 2" /> Figura 2. Quantificazione di Meki indotta compattazione delle cellule tumorali della prostata di ratto MLL. Culture goccia di cellule non trattate e MLL Meki trattati sono stati incubati per 18 ore dopo di che dimensioni aggregato è stato determinato come sopra descritto. L&#39;analisi statistica della dimensione aggregata era di Student t-test. L&#39;asterisco rappresenta una differenza statisticamente significativa nelle dimensioni aggregate (P &lt;0,0001) tra le cellule non trattate e trattate con Meki e suggerisce che Meki-trattamento si traduce in aggregati molto più piccoli e compatti. <img src="/files/ftp_upload/2720/2720fig3.jpg" alt="Figura 3" /> Figura 3. Formata da uno sferoide incubando cellule del fegato embrionale pulcino in sospeso cultura goccia per 18 ore poi in un incubatore / agitazione bagno per 48 ore. Questa immagine è stata catturata usando un microscopio stereo SMZ800 Nikon e Sony Nero camera CCD. <img src="/files/ftp_upload/2720/2720fig4.jpg" alt="Figura 4" /> Figura 4. Hanging cultura goccia rivela ordinamento-out comportamento tra diversi tipi di cellule. Nel pannello A, le cellule del fegato pulcino embrionali sono state colorate con PKH-2 membrana colorante intercalante e miscelati in proporzioni uguali con PKH-26 macchiato le cellule del cuore pulcino embrionali. Nel pannello B, due N-caderina transfettate cloni cellulari L (LN4 e LN2a), esprimendo N-cad a loro superficie nel rapporto di 2.4:1, sono stati colorati con le tinte fluorescenti membrana intercalando PKH-2 e PKH-26 , (Sigma-Aldrich), miscelati in proporzioni uguali e colto come appendere gocce 7. Dopo 18 ore di cultura, fogli cellulari sono stati trasferiti in fiasche agitazione e incubate per altre 48 ore. Aggregati sono stati fissati nel 2% paraformaldeide in PBS e visualizzati con un BioRad MRC600 sistema di scansione laser confocale microscopio collegato a un Optiphot-2 microscopio Nikon. Sezioni ottiche da entrambi i canali verde e rosso sono stati raccolti e una Silicon Graphix Crimson VGX postazione di lavoro dotata di Vital Images Voxel Ultra View software di imaging è stato utilizzato per assegnare i falsi colori per entrambi i canali e di unire le immagini, rivelando le configurazioni anatomiche generato. (A) confocale sezione ottica attraverso un avvolgimento aggregato dimostrazione delle cellule del cuore (qui in uno pseudo-colore giallo) da parte delle cellule del fegato in pseudocolori blu (da 8). (B) sezione confocale ottico attraverso un avvolgimento aggregato dimostrando di alta N-caderina cellule che esprimono (verde) da parte di coloro che esprimono bassi livelli di N-caderina (rosso) (da 6).

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Nessun conflitto di interessi dichiarati.

Gli studi hanno dimostrato che le cellule in coltura tridimensionale contesto (3D) produce distinte morfologia cellulare e di segnalazione rispetto ad un rigido bidimensionale (2D) sistema cultura 9. Per esempio, fibroblasti popolate gel collagene dimostrare che la morfologia dei fibroblasti in 3D è ben distinta da quella osservata in 2D 10,11. Allo stesso modo, la cultura 3D può indurre tessuto-specifica differenziazione delle cellule epiteliali mammarie. Sistemi di coltura 3D sono stati utilizz...

<ul><li> cellule contatore automatico (BioRad TC10) </li><li> scuotendo bagnomaria con CO 2 Modello timpano di copertura 3540 (Lab-line) </li></ul>

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

Studi di cellula-cellula di coesione e adesione cellula-substrato sono storicamente stati effettuati su colture monostrato aderente a substrati rigidi. Cellule all&#39;interno di un tessuto, però, sono in genere racchiusi all&#39;interno di una massa di tessuto stipati in cui le cellule stabilire connessioni intime con molti quasi vicini di casa e con componenti della matrice extracellulare. Di conseguenza, l&#39;ambiente chimico e le forze fisiche sperimentato da cellule all&#39;interno di un tessuto 3D sono fondamentalmente diversi da quelli vissuti da cellule cresciute in coltura monostrato. Questo è stato dimostrato che l&#39;impatto notevolmente la morfologia cellulare e di segnalazione. Diversi metodi sono stati studiati per generare colture di cellule 3D tra cui l&#39;incapsulamento di cellule in gel di collagene 1 o in biomateriale impalcature 2. Tali metodi sono utili, ma non ricapitolare l&#39;intimo diretto cellula-cellula architettura di adesione si trovano in tessuti normali. Piuttosto, più da vicino i sistemi di coltura approssimativi in ​​cui le cellule singole sono liberamente disperso all&#39;interno di un reticolo 3D di prodotti ECM. Qui, descriviamo un semplice metodo in cui sono inserite cellule in sospensione cultura goccia e incubate in condizioni fisiologiche fino a formare veri sferoidi 3D in cui le cellule sono in contatto diretto con loro e con i componenti della matrice extracellulare. Il metodo non richiede attrezzature specializzate e può essere adattato per includere l&#39;aggiunta di alcun agente biologico in quantità molto piccole che possono essere di interesse per chiarire gli effetti sulla cellula-cellula o cellula-ECM interazione. Il metodo può essere utilizzato anche per co-coltura due (o più) diverse popolazioni cellulari in modo da chiarire il ruolo di cellula-cellula o cellula-ECM interazioni nello specificare le relazioni spaziali tra le celle. Cellula-cellula e cellula-coesione ECM adesione sono i cardini di studi di sviluppo embrionale, tumore stromale interazione cellula maligna invasione, la guarigione delle ferite, e per applicazioni di ingegneria dei tessuti. Questo semplice metodo fornirà un mezzo per generare tessuti come aggregati cellulari per la misurazione di proprietà biomeccaniche o per l&#39;analisi molecolari e biochimici in un modello fisiologicamente rilevanti.

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

Descriviamo un metodo semplice, rapido di generare 3D tessuti come sferoidi e la loro potenziale applicazione di quantificare le differenze nelle interazioni cellula-cellula.

Un semplice Hanging goccia Cultura protocollo cellulare per la generazione di sferoidi 3D

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

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Bioengineering

56.9K Views.  UMDNJ-Robert Wood Johnson Medical School. Descriviamo un metodo semplice, rapido di generare 3D tessuti come sferoidi e la loro potenziale applicazione di quantificare le differenze nelle interazioni cellula-cellula.

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

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.

Generazione e recupero dei β-cellule Spheroids dal passaggio di crescita PEG-peptide idrogel

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

Ricerca

Bioingegneria

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 Ponteggi per articolare condrociti, Cultura e Cartilagine 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.

Sintonizzabile idrogel da polmonare matrice extracellulare per colture cellulari 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 ...

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.

Un protocollo per il monitoraggio in tempo reale 3D singola particella

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.

Costruzione di un foglio di multistrato delle cellule staminali mesenchimali con un sistema di cultura dinamica 3D

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.

Un modello di coltura a matrice extracellulare extracellulare -adipocyte umano per lo studio del crosstalk metabolico a matrice

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.

Produzione automatizzata su larga scala di sferoidi a cellule staminali di derivazione adiposa per la bioprinting 3D

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.

Generazione di microambienti cellulari multicue mediante fotopatterning UV di substrati tridimensionali di colture cellulari

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.

Generazione, screening ad alto rendimento e biobanca di sferoidi cardiaci derivati da cellule staminali pluripotenti indotte dall'uomo

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

Saggio di co-coltura 3D: nicchie osteogeniche vascolarizzate per lo screening di farmaci antitumorali

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

Autore in primo piano: Semplice creazione di una nicchia di midollo osseo osteogenico vascolarizzato utilizzando idrogel prefabbricati di poli(glicole etilenico) (PEG) in una micropiastra di imaging  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

Semplice creazione di una nicchia vascolarizzata del midollo osseo osteogenico utilizzando idrogel prefabbricati di poli(glicole etilenico) (PEG) in una micropiastra di imaging

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...