A Method for 3D Bioprinting Murine Cortical Astrocytes

Abstract

Source: de Melo, B. A. G., et al., 3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue. J. Vis. Exp. (2021)

The video demonstrates the process of creating a 3D bioprinted construct using astrocytes suspended in a bioink solution. The bioprinted construct, after crosslinking, facilitates astrocyte adhesion and their spreading, forming neural-like tissue.

Protocol

1. Synthesis of gelatin methacryloyl (GelMA)

1. Weigh 10 g of gelatin obtained from porcine skin and dissolve in 100 mL of PBS by letting the solution stir on a heating plate at 240 rpm and 50 °C until complete dissolution.
2. Under a hood, add 2 mL of methacrylic anhydride (MA) for a low degree of functionalization, and let the gelatin emulsion stir at 240 rpm and 50 °C for 2 h.
   CAUTION: MA hazard statement: H302 + H332 (harmful if swallowed or inhaled), H311 (toxic in contact with skin), 314 (causes severe skin burns and eye damage), 315 (causes skin irritation), H317 (may cause an allergic skin reac....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
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 crystalline powder from bovine plasma
Gelatin	Sigma-Aldrich	9000-70-8	Gelatin powder from porcine skin
Hanks Buffered Salt Solution (HBSS)	Gibco; Life Technologies Corporation	14175095	No calcium, no magnesium, no phenol red
Laminin	Sigma-Aldrich	114956-81-9	Laminin 1-2 mg/mL L in 50 mM Tris-HCl
Methacrylic anhydride	Sigma-Aldrich	760-93-0	For GelMA preparation
Needle 22G	Fisher Scientific	NC1362045	Sterile blunt needle
Petri dish	Corning Incorporated	430591, 430588	Sterile petri dishes of 35 and 100 mm
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
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
Well plate	Thermo Scientific	144530	Sterile 24-well plate