3D Bioprinting af Murine Kortikale Astrocytter til Engineering Neural-lignende Væv
Summary
Her rapporterer vi en metode til 3D bioprinting murine kortikale astrocytter til biofabricating neural-lignende væv til at studere funktionaliteten af astrocytter i centralnervesystemet og de mekanismer, der involverer gliaceller i neurologiske sygdomme og behandlinger.
Abstract
Astrocytter er gliaceller med en væsentlig rolle i centralnervesystemet (CNS), herunder neuronal støtte og funktionalitet. Disse celler reagerer også på neurale skader og virker for at beskytte vævet mod degenerative hændelser. In vitro-undersøgelser af astrocytters funktionalitet er vigtige for at belyse de mekanismer, der er involveret i sådanne hændelser, og bidrage til at udvikle behandlinger til behandling af neurologiske lidelser. Denne protokol beskriver en metode til biofabricate en neural-lignende væv struktur rig på astrocytter ved 3D bioprinting astrocytter-laden bioink. En ekstruderingsbaseret 3D-bioprinter blev brugt i dette arbejde, og astrocytter blev udvundet fra C57Bl/6 mus hvalpe hjerne cortices. Bioinken blev fremstillet ved at blande kortikale astrocytter fra op til passage 3 til en biomaterialeopløsning bestående af gelatine, gelatine-methacryloyl (GelMA) og fibrinogen, suppleret med laminin, som præsenterede optimale bioprinting betingelser. 3D-bioprintforholdene minimerede cellestress og bidrog til astrocytternes høje levedygtighed under processen, hvor 74,08% ± 1,33% af cellerne var levedygtige lige efter bioprinting. Efter 1 uges inkubation steg astrocytternes levedygtighed betydeligt til 83,54% ± 3,00%, hvilket indikerer, at 3D-konstruktionen repræsenterer et passende mikromiljø til cellevækst. Den biomateriale sammensætning tilladt celle vedhæftet fil og stimuleret astrocytisk adfærd, med celler, der udtrykker de specifikke astrocytter markør glial fibrillary surt protein (GFAP) og besidder typisk astrocytisk morfologi. Denne reproducerbare protokol giver en værdifuld metode til biofabricate 3D neural-lignende væv rig på astrocytter, der ligner cellernes indfødte mikromiljø, nyttigt for forskere, der har til formål at forstå astrocytter funktionalitet og deres forhold til de mekanismer, der er involveret i neurologiske sygdomme.
Introduction
Astrocytter er den mest rigelige celletype i centralnervesystemet (CNS) og spiller en central rolle i hjernen homøostase. Ud over vedvarende neuronal støtte, astrocytter er ansvarlige for modulerende neurotransmittere optagelse, opretholde blod – hjerne barriere integritet, og regulere neuronal synaptogenese1,2. Astrocytter har også en væsentlig rolle i CNS-inflammation, der reagerer på skader på hjernen i en proces, der fører til astrocitær reaktivitet eller reaktiv astrogliose3,4, danner et glimt ar, der forhindrer sund vævsudposition til degenerative stoffer5. Denne hændelse resulterer i ændringer i astrocytters genekspression, morfologi og funktion6,7. Derfor er undersøgelser, der involverer astrocytter funktionalitet, nyttige til udvikling af behandlinger til behandling af neurologiske lidelser.
In vitro-modeller er afgørende for at studere mekanismer relateret til neurologiske skader, og selvom vellykket isolation og todimensionel (2D) kultur af kortikale astrocytter er blevet etableret8, giver denne model ikke et realistisk miljø, der efterligner indfødte celleadfærd og reproducerer kompleksiteten af hjernen9 . I 2D-tilstand påvirker den dårlige mekaniske og biokemiske støtte, lav cellecelle- og cellematrixinteraktioner og celleudfladning, der fører til fravær af basal-apikal polaritet, cellesignaldynamik og eksperimentelle resultater, der fører til ændret cellemorfologi og genekspression, hvilket kompromitterer reaktionen på behandlinger10. Derfor er det afgørende at udvikle alternativer, der giver et mere realistisk neuralt miljø, der sigter mod at oversætte resultaterne til klinikken.
Tredimensionel (3D) cellekultur repræsenterer en mere avanceret model, der opsummerer med øgede troskabsfunktioner i organer og væv, herunder CNS11. Med hensyn til glialkultur bidrager 3D-modeller til vedligeholdelse af astrocyttermorfologi, celle basal-apikal polaritet og cellesignalering12,13. 3D bioprinting-teknologien opstod som et kraftfuldt værktøj til at biofabricate 3D levende væv på en kontrolleret måde ved at bruge celler og biomaterialer til at genskabe strukturen og egenskaberne af indfødte væv. Brugen af denne teknologi har ført til en betydelig forbedring af resultaterne forudsigelse og har bidraget til regenerativ medicin anvendes til CNS14,15,16.
Protokollen beskrevet her beskriver isolation og kultur kortikale astrocytter. Protokollen beskriver også en reproducerbar metode til bioprint astrocytter indlejret i gelatine / gelatine methacryloyl (GelMA) / fibrinogen, suppleret med laminin. I dette værk blev en ekstruderingsbaseret bioprinter brugt til at udskrive biomaterialesammensætningen, der indeholder kortikale astrocytter med en massefylde på 1 x 106 celler/mL. Bioprinting forskydningsstress blev minimeret ved at kontrollere udskrivningshastigheden, og astrocytter viste høj levedygtighed efter processen. Bioprintede konstruktioner blev kultiveret i 1 uge, og astrocytter var i stand til at sprede, vedhæfte og overleve inden for hydrogelen, opretholde den astrocytiske morfologi og udtrykke en bestemt markør glial fibrillary surt protein (GFAP)4.
Denne procedure er kompatibel med stempeldrevne ekstruderingsbaserede bioprintere og kan bruges til bioprint astrocytter afledt af forskellige kilder. Den 3D bioprintede model, der foreslås her, er velegnet til en bred vifte af neurale tekniske applikationer, såsom undersøgelser af de mekanismer, der er involveret i astrocytterfunktionalitet i sundt væv og forståelse af udviklingen af neurologiske patologier og behandlingsudvikling.
Protocol
Representative Results
Discussion
3D bioprinting-teknologien har vist sig som et biofabricationalternativ, der gør det muligt at konstruere raffinerede konstruktioner, der strukturelt og fysiologisk ligner indfødte væv22, herunder hjernen23. Biofabrication af neurale-lignende væv giver mulighed for in vitro native mikromiljø modellering, er et vigtigt redskab til at forstå de cellulære og molekylære mekanismer forbundet med udvikling og behandling af mange sygdomme, der påvirker CNS<sup cl…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af The São Paulo Research Foundation (FAPESP), bevillingsnumrene 2018/23039-3 og 2018/12605-8; Det Nationale Råd for Videnskabelig og Teknologisk Udvikling (CNPq), tilskudsnumre 465656/2014-5 og 309679/2018-4; koordinering til forbedring af personale på de videregående uddannelser (CAPES), finansiel kode 001.
Materials
| 3D Bioprinter | 3D Biotechnology Solutions | Extrusion-based bioprinter | |
| Blunt-tip forceps | Integra Miltex | 6–30 | Forceps for brain dissection previously sterilized |
| Bovine serum albumin | Sigma-Aldrich | 9048-46-8 | Protease free, fatty acid free, essentially globulin free |
| CaCl2 | Sigma-Aldrich | 10043-52-4 | |
| Cell culture flask | Fisher Scientific | 156340 | Culture flask T25 |
| Cell strainer | Corning Incorporated | 352340 | Cell strainer 40 µm |
| Confocal microscope | Leica | Confocal TCS SP8 microscopy coupled with an Olympus FluoView 300 confocal system | |
| Conical tubes | Thermo Scientific | 339651, 339652 | Sterile tubes of 15 mL and 50 mL |
| DAPI | Abcam | ab224589 | DAPI staining solution |
| DMEM/F12 | Gibco; Life Technologies Corporation | 12500062 | DMEM/F-12 50/50, 1X (Dulbecco's Mod. Of Eagle's Medium/Ham's F12 50/50 Mix) with L-glutamine |
| Dyalisis tubing | Sigma-Aldrich | D9527 | Molecular weight cut-off = 14 kDa |
| Ethanol | Fisher Scientific | 64-15-5 | Reagent grade |
| Fetal Bovine Serum | Gibco; Life Technologies Corporation | 12657011 | Research Grade |
| Fibrinogen | Sigma-Aldrich | 9001-32-5 | Fibrinogen cristalline powder from bovine plasma |
| Gelatin | Sigma-Aldrich | 9000-70-8 | Gelatin powder from porcine skin |
| Glycine | Sigma-Aldrich | 56-40-6 | Glycine powder |
| Hanks Buffered Salt Solution (HBSS) | Gibco; Life Technologies Corporation | 14175095 | No calcium, no magnesium, no phenol red |
| L-Glutamine | Sigma-Aldrich | 56-85-9 | L-Glutamine crystalline powder |
| Laminin | Sigma-Aldrich | 114956-81-9 | Laminin 1-2 mg/mL L in 50 mM Tris-HCl |
| Live dead kit cell imaging kit | Thermo Scientific | R37601 | Green fluorescence in live cells (ex/em 488 nm/515 nm). Red fluorescence in dead cells (ex/em 570 nm/602 nm) |
| Methacrylic anhydride | Sigma-Aldrich | 760-93-0 | For GelMA preparation |
| Microtubes | Corning Incorporated | MCT-150-C | Microtubes of 1,5 mL |
| NaCl | Sigma-Aldrich | 7647-14-5 | |
| Needle 22G | Fisher Scientific | NC1362045 | Sterile blunt needle |
| Operating scissor | Integra Miltex | 05–02 | Sharp scissor for brain dissection previously sterilized |
| Paraformaldehyde | Sigma-Aldrich | 30525-89-4 | Paraformaldehyde powder |
| Penicillin/Streptomycin | Gibco; Life Technologies Corporation | 15070063 | Pen Strep (5,000 Units/ mL Penicillin; 5,000 ug/mL Streptomycin) |
| Petri dish | Corning Incorporated | 430591, 430588 | Sterile petri dishes of 35 and 100 mm |
| Phalloidin | Abcam | ab176753 | iFluor 488 reagent |
| Photoinitiator | Sigma-Aldrich | 106797-53-9 | 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone |
| Phosphate buffer saline (PBS) | Gibco; Life Technologies Corporation | 10010023 | PBS 1 x, culture grade, no calcium, no magnesium |
| Poly-L-lysine | Sigma-Aldrich | 25988-63-0 | Poly-L-lysine hydrobromide mol wt 30,000-70,000 |
| Primary antobody | Abcam | ab4674 | Chicken polyclonal to GFAP |
| Secondary antibody | Abcam | ab150176 | Alexa fluor 594 anti-chicken |
| Spatula | Miltex | V973-70 | Number 24 cement spatula previously sterilized |
| Stereomicroscope | Fisherbrand | 3000038 | Microscope for brain dissection |
| Syringe 5 mL | BD | 1222C84 | Sterile syringe |
| Syringe filter 2 µm | Fisher Scientific | 09-740-105 | Polypropylene filter for sterilization |
| Thrombin | Sigma-Aldrich | 9002–04-4 | Thrombin cristalline powder from bovine plasma |
| Triton X-100 | Sigma-Aldrich | 9002-93-1 | Laboratory grade |
| Trypsin-EDTA | Gibco; Life Technologies Corporation | 15400054 | Trypsin no phenol red 1 x diluted in PBS |
| Versene solution | Gibco; Life Technologies Corporation | 15040066 | Versene Solution (0.48 mM) formulated as 0.2 g EDTA(Na4) per liter of PBS |
| Well plate | Thermo Scientific | 144530 | Sterile 24-well plate |
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