Protokoller af 3D Bioprinting af gelatine Methacryloyl hydrogel baserede Bioinks
Summary
Præsenteret her er en metode til 3D bioprinting af gelatine methacryloyl.
Abstract
Gelatine methacryloyl (GelMA) er blevet en populær biomaterialet i området for bioprinting. Afledningen af dette materiale er gelatine, som hydrolyseres fra pattedyr kollagen. Således arginin-glycin-aspartinsyre (RGD) sekvenser og målmotiver af matrix metalloproteinase (MMP) forbliver på de molekylære kæder, som hjælper med at opnå celle fastgørelse og nedbrydning. Desuden er formation egenskaber af GelMA alsidige. Methacrylamid-grupperne tillader, at et materiale hurtigt bliver tværbundet under let bestråling i nærværelse af en fotoinitiator. Derfor, det giver god mening at etablere egnede metoder til syntetisere tredimensionelle (3D) strukturer med dette lovende materiale. Men dens lave viskositet begrænser Gelma’s print barhed. Præsenteret her er metoder til at udføre 3D bioprinting af gelma hydrogels, nemlig fabrikation af gelma mikrokugler, gelma fibre, gelma komplekse strukturer, og gelma-baserede mikrofluidisk chips. De resulterende strukturer og biokompatibilitet af materialer samt trykning metoder diskuteres. Det menes, at denne protokol kan fungere som en bro mellem tidligere anvendte biomaterialer og GelMA samt bidrage til etableringen af GelMA-baserede 3D-arkitekturer til biomedicinske applikationer.
Introduction
Hydrogels menes at være et egnet materiale inden for biofabrikation1,2,3,4. Blandt dem, gelatine methacryloyl (GelMA) er blevet en af de mest alsidige biomaterialer, oprindeligt foreslået i 2000 af van den Bulcke et al.5. GelMA syntetiseres ved direkte reaktion af gelatine med methacrylanhydrid (MA). Gelatine, som hydrolyseres af pattedyr kollagen, består af målmotiver af matrix metalloproteinase (MMP). Således, in vitro tredimensionale (3D) væv modeller etableret af GelMA kan ideelt efterligne samspillet mellem celler og ekstracellulære matrix (ECM) in vivo. Endvidere, arginin-glycin-aspartinsyre (rgd) sekvenser, som er fraværende i nogle andre silicagelrogeler såsom alginates, forblive på de molekylære kæder af gelma. Dette gør det muligt at realisere fastgørelse af indkapslede celler inde hydrogel netværk6. Desuden er dannelsen evne GelMA lovende. Methacrylamid-grupperne på GelMA-molekyle kæderne reagerer med photoinitiator under milde reaktionsbetingelser og danner kovalente bindinger ved udsættelse for lysbestråling. Derfor kan de trykte strukturer hurtigt tværbundet for at opretholde de designede figurer på en enkel måde.
Baseret på disse egenskaber, en række felter udnytte GelMA til at udføre forskellige anvendelser, såsom vævsteknik, grundlæggende cytologi analyse, Drug screening, og biosensing. Derfor er forskellige fabrikations strategier også blevet påvist7,8,9,10,11,12,13,14. Men, det er stadig udfordrende at udføre 3D bioprinting baseret på GelMA, som er på grund af sine grundlæggende egenskaber. GelMA er et temperaturfølsomt materiale. Under trykningen skal temperaturen i udskrivnings atmosfæren kontrolleres strengt for at opretholde biotrykens fysiske tilstand. Desuden er viskositeten af gelma generelt lavere end andre almindeligt silicagelrogeler (dvs. alginat, chitosan, hyaluronsyre osv.). Men andre forhindringer står over for, når du opbygger 3D-arkitekturer med dette materiale15.
Denne artikel opsummerer flere tilgange til 3D bioprinting af gelma foreslået af vores laboratorium og beskriver de trykte prøver (dvs. syntesen af gelma mikrokugler, gelma fibre, gelma komplekse strukturer, og gelma-baserede mikrofluidisk chips). Hver metode har specialiserede funktioner og kan vedtages i forskellige situationer med forskellige krav. GelMA mikrokugler er genereret af en elektro assisteret modul, som danner ekstra ekstern elektrisk kraft til at krympe Dråbestørrelse. Med hensyn til GelMA fibre, de er ekstruderet af en koaksial bioprinting dyse ved hjælp af viskøs natriumalginat. Desuden er etableringen af komplekse 3D-strukturer opnås med en digital Light Processing (DLP) bioprinter. Endelig foreslås en dobbelt Cross Linking-strategi for at bygge gelma-baserede mikrofluidisk-chips, der kombinerer gelma-hydrogel og traditionelle mikrofluidisk-chips. Det menes, at denne protokol er et væsentligt Resumé af GelMA bioprinting strategier, der anvendes i vores laboratorium og kan inspirere andre forskere i relative områder.
Protocol
Representative Results
Discussion
Denne artikel beskriver flere strategier til at fabrikere gelma 3D strukturer, nemlig gelma mikrokugler, gelma fibre, gelma komplekse strukturer, og gelma-baserede mikrofluidisk chips. GelMA har lovende biokompatibilitet og dannelse kapacitet og er meget udbredt i området for biofabrikering. Mikrosphere strukturer er velegnede til kontrolleret frigivelse af stoffer, vævsdyrkning, og injektion i organismer til yderligere behandling21,22,<sup class="xr…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev sponsoreret af det nationale centrale forsknings-og udviklings program i Kina (2018YFA0703000), national Nature Science Foundation i Kina (no. U1609207, 81827804), videnskabs fonden for kreative forskningsgrupper af National Natural Science Grundlæggelsen af Kina (nr. 51821093).
Materials
| 0.22 μm filter membrane | Millipore | ||
| 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) | Yeasen Biological Technology Co., Ltd., Shanghai, China | ||
| 3D bioprinter | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| 405nm wavelength light | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| co-axial nozzle | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| confocal fluorescence microscope | OLYMPUS FV3000 | ||
| digital light processing (DLP) bioprinter | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| DLP printer | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| Dulbecco's Phosphate Buffered Saline (DPBS) | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| Dulbecco's Modified Eagle Medium (DMEM) | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| Dulbecco's Modified Eagle Medium with L-glutamine (DMEM/F-12) | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| EFL Software | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| fetal bovine serum (FBS) | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| gelatin | Sigma-Aldrich, Shanghai, China | ||
| gelatin methacryloyl (GelMA) | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| high voltage power | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP) | SuZhou Intelligent Manufacturing Research Institute, SuZhou, China | ||
| paraformaldehyde | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| penicillin/streptomycin | Tangpu Biological Technology Co., Ltd., Hangzhou, China | ||
| sodium alginate (Na-Alg) | Sigma-Aldrich, Shanghai, China | ||
| TRITC phalloidin | Yeasen Biological Technology Co., Ltd., Shanghai, China | ||
| Triton X-100 | Solarbio Co., Ltd., Shanghai, China |
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