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

Denne forskning blev støttet af National Science Foundation of China (21474057 og 21604076).

forsigtighed: Rådfør dig med alle relevante materiale sikkerhedsdatablade (MSDS) før brug. Skal du bruge passende sikkerhedspraksis ved udførelse af kemi eksperimenter, herunder brug af et stinkskab og personlige værnemidler (sikkerhedsbriller, beskyttende handsker, laboratoriekittel, osv.). Protokollen kræver standard celle håndtering teknikker (sterilisering, celle opsving, celle passaging, celle frysning, celle farvning, etc.).
 1. forberedelse af Hydrogels <ol><...

<ol>
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Rev. 43 (23), 8114-8131 (2014).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulus-regulated+3D-cell+proliferation+in+an+injectable+self-healing+hydrogel.">Modulus-regulated 3D-cell proliferation in an injectable self-healing hydrogel.</a> Colloid. Surface. B. 149, 168-173 (2017).</li><li>Tseng, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Injectable,+Self&#8208;Healing+Hydrogel+to+Repair+the+Central+Nervous+System.">An Injectable, Self&#8208;Healing Hydrogel to Repair the Central Nervous System.</a> Adv. Mater. 27 (23), 3518-3524 (2015).</li><li>Yu, L., Ding, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+hydrogels+as+unique+biomedical+materials.">Injectable hydrogels as unique biomedical materials.</a> Chem. Soc. Rev. 37 (8), 1473-1481 (2008).</li><li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+Tumor+Chemotherapy+Effect+by+Using+an+Injectable+Self-healing+Hydrogel+as+Drug+Carrier.">Improving Tumor Chemotherapy Effect by Using an Injectable Self-healing Hydrogel as Drug Carrier.</a> Polym. Chem. , (2017).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+magnetic+self-healing+hydrogel.">A magnetic self-healing hydrogel.</a> Chem. 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Hydrogel præsenteret i denne protokol (figur 1) har to hovedkomponenter: naturlige polymer glycol chitosan og en syntetisk benzaldehyd opsagt polymer gelator DF PIND, som er både biokompatible materialer. Syntese af DF PIND er præsenteret ved hjælp af en et-trins ændring reaktion. PEG med molekylevægt 4.000 blev valgt i denne protokol i bekymringer af opløselighed, modifikation effektivitet samt hydrogel stivhed. En serie af hydrogels med forskellige mekaniske styrker blev udarbejdet ...

Hydrogels er krydsbundet polymer materialer med store mængder vand og bløde mekaniske egenskaber, og de har været brugt i mange bio-medicinske programmer1,2. Hydrogels kan tilbyde en blød og våde miljø, som er meget lig de fysiologiske omgivelser for celler i vivo. Derfor er hydrogels blevet en af de mest populære stilladser for 3D celle kultur3,4. I forhold til 2D petriskål cellekultur, har 3D cellekultur avancerede hurtigt for at tilbyde en ekstracellulære matrix (ECM) efterlignede mikromiljø for cellerne til at kontakte og samle for spredning og differentiering formål5. Derudover kunne hydrogels indeholdende naturlige polymerer tilbyde bio-kompatible og fremme miljøer for celler til at formere sig og differentiere3. Hydrogels stammer fra syntetiske polymerer er at foretrække for deres enkle og klare komponenter, som udelukker komplekse påvirkninger som animalsk oprindelse proteiner eller virus. Blandt alle hydrogel kandidater til 3D cellekultur er hydrogels, der er let at tilberede og har en konsekvent ejendom altid foretrukket. Mulighed for at justere den hydrogel egenskaber til at passe forskellige forskningsbehov er vigtigt som godt6.
Her vil vi præsentere en letkøbt forberedelse af en glykol chitosan-baserede hydrogel ved hjælp af dynamiske imine kemi, som bliver en alsidig hydrogel platform for 3D celle kultur7. I denne metode anvendes velkendte bio-kompatible glycol chitosan at etablere rammer for hydrogel's netværk. Dens amino grupper er reageret med et benzaldehyd opsagt polyethylenglycol som polymer gelator at danne dynamisk imine obligationer som crosslinkages af hydrogels8. Dynamisk imine obligationer kan danne og nedbrydes reversibelt og lydhørt til omgivelserne, tilføre hydrogels med mekanisk indstillelig crosslinked netværk9,10,11. På grund af sin høje vand indhold, bio-kompatible materialer og justerbar mekaniske styrker er hydrogel anvendt med succes som et stillads til L929 celler i 3D celle kultur12,13. Protokol her beskriver de procedurer, herunder polymer gelator syntese, hydrogel forberedelse, integrering af celle og 3D celle dyrkning.
Hydrogel viser også flere andre funktioner på grund af dens dynamiske imine crosslinkages, herunder dens multi lydhør over for forskellige bio-stimuli (syre/pH, vitamin B6 afledte pyridoxal, protein papain, etc.), der angiver, at hydrogel kunne være induceret for at nedbrydes under fysiologiske forhold8. Hydrogel er også selv healable og injicerbar, hvilket betyder hydrogel kunne administreres via en minimal invasiv injektion metode og få en fordel i stof og celle leverancer14,15. Ved at tilføje funktionelle tilsætningsstoffer eller specifikke foruddesignede polymer gelators, hydrogel er kompatible hen til opnå specifikke egenskaber ligesom magnetisk, temperatur, pH lydhør, etc.16,17, som kunne opfylde et bred vifte af forskningsbehov. Disse egenskaber afsløre den hydrogel potentielle kapacitet til at være en injicerbar flere luftfartsselskaber for lægemidler og celler i både in vitro og i vivo bio-medicinske forskning og anvendelser.

<table border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Glycol chitosan</td>
			<td style="width:360px;">Wako Pure Chemical Industries</td>
			<td style="width:131px;">39280-86-9</td>
			<td style="width:385px;">90% degree of deacetylation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Carboxybenzaldehyde</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>619-66-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N, N&#39;-dicyclohexylcarbodiimide</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>538-75-0</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride anhydrous</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>10043-52-4</td>
			<td>96%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-dimethylamiopryidine</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>1122-58</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethyleneglycol</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>5254-43-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetrahydrofuran</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>109-99-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Toluene</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>108-88-3</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethyl ether</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>60-29-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>64-19-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anhydrous CaCl2</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>10043-52-4</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein diacetate</td>
			<td>Sigma</td>
			<td>596-09-8</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium iodide&#160;</td>
			<td>Sigma</td>
			<td>25535-16-4</td>
			<td>94%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI-1640 culture media</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA</td>
			<td>Gibco</td>
			<td></td>
			<td>0.25%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Solarbio</td>
			<td></td>
			<td>0.01 M</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin streptomycin solution</td>
			<td>Hyclone</td>
			<td></td>
			<td>10,000 U/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rheometer</td>
			<td>TA Instrument</td>
			<td></td>
			<td>AR-G2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>710-3channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L929 Cells</td>
			<td>ATCC</td>
			<td></td>
			<td>NCTC clone 929; L cell, L929, derivative of Strain L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Evaporator</td>
			<td>EYELA</td>
			<td></td>
			<td>N-1100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48 guage needle</td>
			<td>ShanghaiZhiyu Medical Material Co., LTD</td>
			<td></td>
			<td>48-guage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DM3000 B</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope software</td>
			<td>Imaris</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat gun</td>
			<td>Confu</td>
			<td></td>
			<td>KF-5843&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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

Protokollen præsenterer en letkøbt, effektiv og alsidig metode for at forberede chitosan-baserede hydrogels ved hjælp af dynamiske imine kemi. Hydrogel er fremstilles ved at blande løsninger af glycol chitosan med en syntetiseret benzaldehyd opsagt polymer gelator, og hydrogels fremstilles effektivt i flere minutter ved stuetemperatur. Af varierende forhold mellem glycol chitosan, polymer gelator og vand indhold, er alsidig hydrogels med forskellige gellation gange og stivhed opnået. Når beskadiget, kan hydrogel inddrive sine optrædener og modulus, på grund af reversibilitet af de dynamiske imine obligationer som crosslinkages. Denne selvstændige healable egenskab aktiverer hydrogel skal injicerbar, da det kan være selvstændige helbredt fra klemt stykker til en integreret bulk hydrogel efter injektion proces. Hydrogel er også flere modtagelig hen til mange bio-aktive stimuli på grund af forskellige ækvilibrering statusser for dynamisk imine obligationer. Denne hydrogel blev bekræftet som bio-kompatible, og L929 mus fibroblastceller blev integreret følgende standardprocedurer og celleproliferation var let vurderet af en 3D celle dyrkning proces. Hydrogel kan tilbyde en justerbar platform for forskellige forskning hvor en fysiologisk efterligner af et 3D miljø for celler er nydt. Sammen med sin multi lydhør, selv healable og injicerbar egenskaber, kan hydrogels potentielt kunne anvendes som flere luftfartsselskaber for lægemidler og celler i fremtidige bio-medicinske anvendelser.

En skematisk præsentation af denne protokol på hydrogel forberedelse og dens brug som 3D cellekultur tilbydes i figur 1. Oplysninger af hydrogel indholdet og nøgletal tilberedt med forskellige mekaniske styrker er opsummeret i tabel 1. Hydrogel's egen healable og reologi egenskab præsenterer den hydrogel stivhed af opbevaring modulus kontra frekvens test i figur 2. Celle Konfokal billeder og celle numre med dage af kultur i hydrogels er præ...

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

Her beskriver vi en letkøbt forberedelse af chitosan-baserede injicerbare hydrogels ved hjælp af dynamiske imine kemi. Metoder til at justere den hydrogel mekanisk styrke og dens anvendelse i 3D cellekultur præsenteres.

Forberedelse af Chitosan-baserede injicerbare Hydrogels og dens anvendelse i 3D cellekultur

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

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Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Bio-inspirerede Polydopamine overflade ændring af Nanodiamonds og dets begrænsning af sølv nanopartikler

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

Photogeneration of N-Heterocyclic Carbenes: Application in Photoinduced Ring-Opening Metathesis Polymerization

Photogeneration af N-Heterocycliske Carbenes: ansøgning i lysinducerede Ring-åbning Metathesis polymerisering

We report a method to generate the N-heterocyclic carbene (NHC) 1,3-dimesitylimidazol-2-ylidene (IMes) under UV-irradiation at 365 nm to characterize IMes ...

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

Syntese af blød Polysiloxane-urea elastomerer intraokulær linse anvendelse

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses ...

Preparation and Application of a New Bacterial Biosensor for the Presumptive Detection of Gunshot Residue

Klargøring og anvendelse af en ny bakteriel biosensor til formodet påvisning af skud rester

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, ...