3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Summary

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Abstract

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes' functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups' brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% ± 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% ± 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells' native microenvironment, useful to researchers that aim to understand astrocytes' functionality and their relation to the mechanisms involved in neurological diseases.

Introduction

Astrocytes are the most abundant cell type in the Central Nervous System (CNS) and play a key role in brain homeostasis. In addition to enduring neuronal support, astrocytes are responsible for modulating neurotransmitters uptake, maintaining the blood-brain barrier integrity, and regulating neuronal synaptogenesis^1,2. Astrocytes also have an essential role in CNS inflammation, responding to injuries to the brain in a process that leads to astrocitary reactivity or reactive astrogliosis^3,4, forming a glial scar that prevents healthy tissue exposition t....

Protocol

All the procedures involving animals followed international guidelines for animal use in research (http://www.iclas.org) and were approved by the Committee for Ethics in Research of Universidade Federal de São Paulo (CEUA 2019/ 9292090519).

1. Mice brain dissection

1. Transfer 10 mL of cold Hanks Buffered Salt Solution (HBSS) to a 100 mm culture dish and 1 mL to a 1.5 mL microtube. Prepare one microtube per animal.
   NOTE: Both the culture dish and the microtube need to be kept on ice.
2. Prepare astrocytes culture medium using DMEM F12 + 10% Fetal Bovine Serum (FBS), 2% glutamine, and 1% Penicillin-Streptomycin (P....

Results

This work aimed to develop a neural-like tissue using the 3D bioprinting technology to deposit layer-by-layer primary astrocytes-laden gelatin/GelMA/fibrinogen bioink. Astrocytes were extracted and isolated from the cerebral cortex of mice pups (Figure 1), added to a biomaterial composition, allowing the biofabrication of a living 3D construct.

The computer-aided-design (CAD) was developed using the G-code (Supplemental file) as an interconnected .......

Discussion

The 3D bioprinting technology has emerged as a biofabrication alternative that allows the engineering of refined constructs that structurally and physiologically resemble native tissues²², including the brain²³. The biofabrication of neural-like tissues allows for in vitro native microenvironment modeling, being an important tool for understanding the cellular and molecular mechanisms associated with the development and treatment of many diseases that affect the CN.......

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
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