Name: preparation of chitosan-based injectable hydrogels and its application in 3d cell culture
Uploaded: 2017-09-29T14:00:00.000+00:00
Duration: 8 min 5 s
Description: Watch this Scientific Journal Video about Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture at JoVE.com

Questa ricerca è stata sostenuta dal National Science Foundation cinese (21474057 e 21604076).

Attenzione: si prega di consultare tutte le schede di dati di sicurezza (MSDS) prima dell'uso. Si prega di utilizzare pratiche adeguate di sicurezza durante gli esperimenti di chimica, compreso l'uso di una cappa aspirante e dispositivi di protezione individuale (occhiali protettivi, guanti protettivi, camice da laboratorio, ecc.). Il protocollo richiede cella standard gestione tecniche (sterilizzazione, recupero delle cellule, cellule passaggio, congelamento delle cellule, macchiatura delle cellule, ecc.).
 1. preparazione di idrogeli <ol><li>sintesi della benzaldeide terminato funzionalizzati di polietilene glicole (spina DF) <ol><li>pre-essiccazione di PEG polimero <ol><li>pesare 4,00 g PEG (4.000 MW, 1.00 mmol), trasferirla in un pallone a fondo tondo (250 mL) e aggiungere toluene (100 mL). 
			Nota: Altre pioli con diversi pesi molecolari può lavorare per la formazione di idrogel ma porterà a diversa rigidezza.</li>
			<li>Riscaldare la soluzione da una pistola di calore (o un piatto caldo intorno ai 40 ° C) leggermente per aiutare a sciogliere tutti i polimeri. Dopo tutti i polimeri si dissolvono, è possibile utilizzare un evaporatore per rimuovere tutti i solventi. Ripetere questo processo secco e sciogliere due volte per rendere il PEG secche polimeri. 
			Attenzione: Questa operazione deve essere eseguita in una cappa con estrema cura. Il toluene è infiammabile.</li>
		</ol></li> <li>Reazione di terminazione di benzaldeide di PEG <ol><li>aggiungere un agitatore magnetico e tetraidrofurano (THF, 100 mL) al pallone e sciogliere PEG con un agitatore. Aggiungere 4-carboxybenzaldehyde (0,90 g, 6,0 mmol) e 4-dimetilaminopiridina (0,07 g, 0,6 mmol) in sequenza per le soluzioni di sciogliere tutti i solidi completamente.</li>
			<li>Aggiungi N, N &#39;-dicyclohexylcarbodiimide (DCC, 1,25 g, 6,0 mmol) alla soluzione e aggiungere un tubo di essiccazione che viene riempito con anidro CaCl 2 al pallone. Mantenere la reazione sotto agitazione a temperatura ambiente (~ 20 ° C) per circa 12 h.</li>
		</ol></li> <li>Processo di post-reazione <ol><li>filtrare i solidi bianchi generati nella soluzione di vuoto dopo la reazione è completa. Versare la soluzione nel freddo etere dietilico (500 mL) sotto agitazione per precipitare i solidi bianchi. Raccogliere tutti i solidi bianchi dal filtro e la sostanza secca in una cappa aspirante.</li>
			<li>Dissolversi ancora i solidi bianchi in THF, filtrare qualsiasi solidi bianchi insolubili, versare la soluzione nel fresco freddo etere etilico per precipitare i solidi bianchi e asciugarlo. Ripetere questo processo due o tre volte e quindi posizionare i solidi bianchi in un forno di essiccazione sotto vuoto (20 ° C, 0,1 mbar) per asciugarle completamente. Raccogliere la polvere bianca come il prodotto finale: benzaldeide terminato DF PEG.</li>
		</ol></li></ol></li> <li>Preparazione di idrogeli <ol><li>pesare quantità diverse di DF PEG (0,11 g, 0,028 mmol; 0,22 g, 0.055 mmol; 0,44 g, 0.110 mmol) in un tubo (10 mL), aggiungere acqua deionizzata (5,0 mL) e utilizzare un vortice o agitatore magnetico per aiutare sciogliere il polimero.</li>
		<li>Sciogliere glicole chitosano (0,495 g, 6,18 x 10 -3 mmol) in acqua deionizzata (15,0 mL) in un tubo (50 mL) e utilizzare un vortice per pochi minuti per omogeneizzare la soluzione di chitosano (3% in peso).</li>
		<li>Aggiungere la soluzione di chitosano di glicole (0,2 mL, 3% in peso) e soluzioni di PEG DF (0,2 mL) in sequenza in un tubo (2,0 mL). Utilizzare un vortice per mescolare le soluzioni in modo omogeneo a forma idrogel in alcuni minuti. Seguire i rapporti nella tabella 1 per rendere idrogeli di resistenze meccaniche differenti. 
		Nota: Utilizzare il metodo invertente tubo per determinare se l'idrogel è già formato.</li>
	</ol></li> <li>Analisi di reologia <ol><li>effettuare analisi di reologia su un reometro rotazionale con una piastra di acciaio parallela (diametro: 20 mm). Per la prova di gelificazione, pubblicizza chitosano glicolata (0,2 mL, 3% in peso) su una piastra inferiore. Aggiungere soluzioni acquose DF PEG (0,2 mL, 2% in peso) uniformemente goccia a goccia sulla superficie della soluzione del chitosano, quindi mescolare rapidamente con una pipetta. Più basso giù la piastra superiore e cominciano a test.</li>
		<li>Per idrogel &#39; prova di rigidezza s, tagliare un idrogel in un cerchio (diametro: 20 mm) e misurare il modulo elastico (G &#39;) valori contro analisi di frequenza al 1% di deformazione. Tipico G &#39; valori a 6,3 rad s &#8722; 1 sono elencati nella tabella 1. 
		Nota: Effettuare le analisi di reologia di idrogel 0,5 h dopo la formazione di idrogel per garantire la stabilizzazione dinamica legame dell'idrogel.</li>
		<li>Per l'idrogel &#39; s self-sanabile proprietà testare, tagliare un idrogel a pezzi e raccogliere i pezzi insieme a Self-Heal a un pezzo integrale. Quindi tagliare il pezzo di idrogel per fare un cerchio (diametro: 20 mm) e metterlo sul reometro per eseguire l'analisi di reologia.</li>
	</ol></li></ol> 2. colture cellulari 3D in idrogel <ol><li>preparazione delle soluzioni di gelificazione di idrogel <ol><li>pesare DF PEG (0,44 g, 0,11 mmol) in una provetta sterile (4,0 mL), aggiungere in terreni di coltura delle cellule (RPMI-1640, 2.0 mL) e utilizzare un vortice o agitatore per aiutare a sciogliere il polimero per ottenere la soluzione di polimero (20% in peso). Caricare la soluzione in una siringa (10,0 mL) e poi sterilizzarlo facendolo passare attraverso un filtro batteri ritentiva micron (0,22 µm).</li>
		<li>Pesare il chitosano di glicole (0,165 g, 2.06 x 10 -3 mmol) in una provetta sterile (15,0 mL), aggiungere in terreni di coltura delle cellule (RPMI-1640, 4,0 mL) e vortex per aiutare a sciogliere il polimero per ottenere la soluzione di glicole chitosano (4,0% in peso). Caricare la soluzione in una siringa (10,0 mL) e filtrare con un filtro di batteri a rimanenza da 0,22 µm.</li>
	</ol></li> <li>Colture cellulari in idrogel Attenzione: eseguire tutto cella procedure correlate in una cappa di coltura del tessuto. Conoscenza di base della tecnica sterile è previsto.
	<ol><li>Preparazione della sospensione di cellule <ol><li>cultura L929 cellule in RPMI-1640 supplementato con 10% FBS, 5% penicillina (10 mL) in un Petri dish (diametro 10 cm) e incubare a 37 ° C, 5% CO 2. Modificare il mezzo ogni giorno prima dell'uso.</li>
			<li>Raccogliere le celle L929 con PBS contenente tripsina (0,025% w/v) ed EDTA (0,01% w/v), poi centrifugare (5 min a 70 x g) e risospendere le cellule nel medium RPMI-1640 (1,0 mL). Eseguire la conta cellulare utilizzando standard di funzionamento del Consiglio di sangue-conteggio. Risospendere le cellule per regolare la concentrazione cellulare di ~ 3,75 × 10 6 cellule/mL.</li>
		</ol></li> <li>Incapsulamento di cellule in idrogel <ol><li>mescolare le sospensioni di cellula L929 (0,4 mL, 3.75 x 10 6 cellule mL -1) con chitosano glicolata (0,4 mL) in un tubo (4,0 mL) Vortex. Dispensare la soluzione di chitosano L929/glicole (0,8 mL) in al centro di un piatto di Petri confocale (diametro 2,0 cm). Pipettare le soluzioni DF PEG (0,2 mL) nello stesso piatto e pipettare delicatamente per mescolare la soluzione e inducono la formazione di idrogel. 
			Nota: Valutare la formazione di idrogel inclinando il piatto petri.</li>
			<li>Per la coltura cellulare diretto, aggiungere ulteriori quantità di terreni di coltura RPMI-1640 (1,0 mL) in cima l'idrogel. Mettere l'idrogel di cella incorporato (1,0 mL, 1.5 wt % glicole chitosano, 4,0 wt % DF PEG, 1,5 × 10 6 cellule mL -1) in un incubatore (37 ° C, 5% CO 2) e cambiare il mezzo ogni giorno. Preparare per l'imaging delle cellule il giorno 1, 3, 5 e 7 dopo incapsulamento cella.</li>
		</ol></li> <li>Della cultura post-cella 3D in idrogel dopo l'iniezione, preparare cella caricata idrogel (1,0 mL, vedi punto 2.2.2) in una siringa (10,0 mL, ago 48 G). Dopo le forme di idrogel, iniettare lentamente l'idrogel in una capsula Petri per imaging confocale. Aggiungere un importo supplementare di terreni di coltura (1,0 mL) in cima l'idrogel e cambiare ogni giorno. Mettere la capsula di Petri in un incubatore (37 ° C, 5% CO 2) e preparare per l'imaging in seguito. 
		Attenzione: Si prega di controllare e seguire il protocollo di funzionamento di sicurezza di una siringa.</li>
	</ol></li> <li>Analisi di attuabilità delle cellule <ol><li>confocale osservazione <ol>
			<li>Sciacquare l'idrogel con PBS (1,0 mL) per due volte. Macchia nel caso degli idrogeli con diacetato di fluoresceina (FDA, 0,5 mL, 0,05 mg/mL) e soluzioni di propidio ioduro (PI, 0,5 mL, 0,08 mg/mL) per 15 min. Dopo la colorazione, rimuovere tutti i solventi.</li>
			<li>Osservare nel caso degli idrogeli utilizzando un microscopio confocale in lunghezze d'onda di eccitazione di 488 nm e 543 nm di visualizzare dal vivo e morto cellule, rispettivamente. Prendere z-stack attraverso ogni profondità di 2 µm di idrogeli per convalidare una distribuzione uniforme delle cellule in tutto. 
			Nota: FDA macchie cellule vive mentre le cellule morte di macchie PI.</li>
		</ol></li> <li>Degradare l'idrogel (1,0 mL) con acido acetico (HAc, 3% v, 1,0 mL) per 5 min e pipettare in una provetta (4,0 mL). Raccogliere le cellule di centrifuga (70 x g, 5 min) e risospendere le cellule in terreno di coltura cellulare di RPMI-1640 (1,0 mL). Eseguire la conta cellulare utilizzando un sangue conteggio pensione.</li>
	</ol>
	</li>
</ol>

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L'idrogel presentato in questo protocollo (Figura 1) ha due componenti principali: il chitosano di glicole di polimero naturale e un sintetico benzaldeide terminato gelator polimero DF PEG, che sono entrambi materiali biocompatibili. Sintesi di DF PEG sono presentata utilizzando una reazione di modificazione di uno stadio. PEG del peso molecolare 4.000 fu scelto nel presente protocollo per preoccupazioni di solubilità, efficienza di modificazione, come pure la rigidità di idrogel. Una seri...

Idrogeli sono materiali polimerici reticolati con grandi quantità di acqua e morbido proprietà meccaniche, e sono stati utilizzati in molte applicazioni bio-medicali1,2. Idrogeli possono offrire un ambiente morbido e bagnato, che è molto simile all'ambiente fisiologico per cellule in vivo. Di conseguenza, idrogeli sono diventati uno dei più popolari impalcature per cella 3D cultura3,4. Rispetto alla coltura delle cellule 2D di Petri, coltura cellulare 3D ha avanzato velocemente per offrire che una matrice extracellulare (ECM) ha imitato il microambiente per celle da contattare e montare per proliferazione e differenziazione di scopi5. Inoltre, idrogeli contenenti polimeri naturali potrebbero offrire ambienti bio-compatibili e promozione per le cellule di proliferare e differenziare3. Idrogeli derivati da polimeri sintetici sono preferibili per i loro componenti semplici e chiare, che escludono le influenze complesse come proteine di origine animale o virus. Tra tutti i candidati di idrogel per colture cellulari 3D, idrogeli che facilmente sono preparati e hanno una proprietà coerente sono sempre preferiti. La possibilità di regolare le proprietà di idrogel per soddisfare i requisiti di ricerca differenti è importante come ben6.
Qui vi presentiamo una facile preparazione di un idrogel a base di chitosano glicole usando immina dinamica chimica, che diventa una piattaforma versatile idrogel per cella 3D cultura7. In questo metodo, noto bio-compatibili glicole chitosano vengono utilizzati per stabilire cornici delle reti di idrogel. Suoi gruppi amminici sono ha reagiti con un glicole di polietilene benzaldeide terminato come il gelator di polimeri per formare legami immina dinamico come crosslinkages di idrogeli8. Immina dinamico obbligazioni possono formare e si decompongono reversibilmente e responsabilmente ai dintorni, dotando l'idrogel con reticolati regolabile meccanicamente reti9,10,11. A causa del suo contenuto di acqua alta, materiali bio-compatibili e resistenze meccaniche regolabili, l'idrogel è applicato con successo come impalcatura per celle L929 in cella 3D cultura12,13. Il protocollo qui i dettagli delle procedure, tra cui la sintesi del polimero gelator, idrogel preparazione, l'incorporamento delle cellule e coltura delle cellule 3D.
L'idrogel Mostra anche diverse altre caratteristiche grazie alla sua dinamica immina crosslinkages, tra cui il multi-sensible a reagire ai vari bio-stimoli (acido/pH, piradossale derivato di vitamina B6, papaina proteine, ecc.), che indica che potrebbe essere l'idrogel indotto a decomporsi sotto condizioni fisiologiche8. L'idrogel è anche self-sanabile e iniettabili, che significa l'idrogel potrebbe essere amministrato per mezzo di un metodo di iniezione invasiva minimale e ottenere un vantaggio in droga e cella consegne14,15. Con l'aggiunta di additivi funzionali o gelators polimero specifico predefiniti, l'idrogel è compatibile per ottenere proprietà specifiche come magnetico, temperatura, pH sensibile, ecc.16,17, che potrebbe compiere un vasta gamma di esigenze di ricerca. Tali proprietà rivelano la capacità potenziale di idrogel per essere un iniettabile più vettori per farmaci e cellule sia in vitro che in vivo ricerca bio-medica e applicazioni.

<table><tbody><tr><td>Glycol chitosan</td><td>Wako Pure Chemical Industries</td><td>39280-86-9</td><td>90% degree of deacetylation</td></tr><tr><td>4-Carboxybenzaldehyde</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>619-66-9</td><td>99%</td></tr><tr><td>N, N&#039;-dicyclohexylcarbodiimide</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>538-75-0</td><td>99%</td></tr><tr><td>Calcium chloride anhydrous</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>10043-52-4</td><td>96%</td></tr><tr><td>4-dimethylamiopryidine</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>1122-58</td><td>99%</td></tr><tr><td>Polyethyleneglycol</td><td>Sino-pharm Chemical Reagent</td><td>5254-43-7</td><td>99%</td></tr><tr><td>Tetrahydrofuran</td><td>Sino-pharm Chemical Reagent</td><td>109-99-9</td><td>99%</td></tr><tr><td>Toluene</td><td>Sino-pharm Chemical Reagent</td><td>108-88-3</td><td>99%</td></tr><tr><td>Ethyl ether</td><td>Sino-pharm Chemical Reagent</td><td>60-29-7</td><td>99%</td></tr><tr><td>Acetic acid</td><td>Sino-pharm Chemical Reagent</td><td>64-19-7</td><td>99%</td></tr><tr><td>Anhydrous CaCl2</td><td>Sino-pharm Chemical Reagent</td><td>10043-52-4</td><td>99%</td></tr><tr><td>Fluorescein diacetate</td><td>Sigma</td><td>596-09-8</td><td>99%</td></tr><tr><td>Propidium iodide&amp;nbsp;</td><td>Sigma</td><td>25535-16-4</td><td>94%</td></tr><tr><td>RPMI-1640 culture media</td><td>Gibco</td><td></td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td></td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td></td><td>0.25%</td></tr><tr><td>PBS</td><td>Solarbio</td><td></td><td>0.01 M</td></tr><tr><td>Penicillin streptomycin solution</td><td>Hyclone</td><td></td><td>10,000 U/mL</td></tr><tr><td>Rheometer</td><td>TA Instrument</td><td></td><td>AR-G2</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td></td><td>710-3channel</td></tr><tr><td>L929 Cells</td><td>ATCC</td><td></td><td>NCTC clone 929; L cell, L929, derivative of Strain L</td></tr><tr><td>Evaporator</td><td>EYELA</td><td></td><td>N-1100</td></tr><tr><td>48 guage needle</td><td>ShanghaiZhiyu Medical Material Co., LTD</td><td></td><td>48-guage</td></tr><tr><td>Microscope</td><td>Leica</td><td></td><td>DM3000 B</td></tr><tr><td>Microscope software</td><td>Imaris</td><td></td><td></td></tr><tr><td>Heat gun</td><td>Confu</td><td></td><td>KF-5843&amp;nbsp;</td></tr><tr><td>Petri dish</td><td>NEST</td><td></td><td></td></tr></tbody></table>

preparation of chitosan-based injectable hydrogels and its application in 3d cell culture

Il protocollo presenta un metodo facile, efficiente e versatile per preparare idrogel a base di chitosano usando immina dinamica chimica. L'idrogel è preparata mescolando soluzioni di glicole chitosano con un gelator di polimeri sintetizzati benzaldeide terminato e idrogeli sono ottenuti in modo efficiente in diversi minuti a temperatura ambiente. Dai vari rapporti tra glicole chitosano, gelator di polimero e contenuto di acqua, idrogel versatile con tempi di gelificazione differenti e rigidità sono ottenuti. Quando danneggiato, l'idrogel può ritrovare apparenze e modulo elastico, dovuto la reversibilità delle obbligazioni immina dinamico come crosslinkages. Questa proprietà auto-sanabile consente l'idrogel di essere iniettabili, poiché esso può essere auto-guarito da pezzi spremuti da un idrogel di massa integrale dopo il processo di iniezione. L'idrogel è anche multi-sensible a reagire a molti stimoli di bio-attivi a causa di Stati di equilibrazione differenti delle obbligazioni immina dinamico. Questo idrogel è stata confermata come bio-compatibili e cellule di fibroblasti di topo L929 erano incorporate seguenti procedure standard e la proliferazione delle cellule è stata valutata facilmente tramite un processo di coltivazione di cella 3D. L'idrogelo in grado di offrire una piattaforma regolabile per diverse ricerche dove è profittato di un mimo fisiologico di un ambiente 3D per le cellule. Insieme alle sue proprietà multi-reattivo, self-sanabile e iniettabili, nel caso degli idrogeli potenzialmente può essere applicato come elementi portanti multipli per farmaci e cellule in future applicazioni bio-medicali.

Una presentazione schematica di questo protocollo su idrogel preparazione e il suo uso come coltura cellulare 3D è offerto nella Figura 1. Informazioni di idrogel Sommario e rapporti preparati con diversi punti di forza meccaniche sono riassunta nella tabella 1. L'idrogel di self-sanabile e proprietà di reologia presenta rigidità di idrogel di modulo elastico contro frequenza test nella Figura 2. Le immagini confocal cellulare e numeri di cel...

Watch this Scientific Journal Video about Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture at JoVE.com

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

Qui descriviamo una facile preparazione di idrogeli iniettabili a base di chitosano usando immina dinamica chimica. Metodi per regolare la resistenza meccanica di idrogel e la sua applicazione nella coltura cellulare 3D sono presentati.

Preparazione di idrogeli iniettabili a base di chitosano e la sua applicazione nella coltura cellulare 3D

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

preparation-chitosan-based-injectable-hydrogels-its-application-3d

Universidad San Sebastian

Research

JoVE Journal

Chemistry

19.0K Views.  Tsinghua University. Qui descriviamo una facile preparazione di idrogeli iniettabili a base di chitosano usando immina dinamica chimica. Metodi per regolare la resistenza meccanica di idrogel e la sua applicazione nella coltura cellulare 3D sono presentati.

Video: Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Facile manipolazione delle architetture in idrogel a base di proteine per applicazioni della coltura delle cellule

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Ricerca

Biochimica

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Costruire un idrogel collagene per la fornitura di Stem Cell-loaded Chitosan Microsfere

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

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 ...

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

Formazione di spheroidcellule a celle di legamento parontale umano su Chitosan Films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

Biologia dello sviluppo

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 ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D per la terapia rigenerativa iniettabile e droga di High-throughput Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

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.

Produzione di elastina-come proteina idrogeli per incapsulamento e Immunostaining delle cellule in 3D

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

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable alginato/gelatina idrogel 3D In Vitro sistemi modello inducono la formazione di sferoide di cella

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Protocolli di biostampa 3D di Gelatin Abasecryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Fabbricazione di microgel di chitosano-genipina a grandezza controllata e priva di emulsioni per applicazioni di ingegneria tissutale

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...