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.
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.
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 synaptogenesis1,2. 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 astrogliosis3,4, forming a glial scar that prevents healthy tissue exposition to degenerative agents5. This event results in changes in astrocytes' gene expression, morphology, and function6,7. Therefore, studies involving astrocytes' functionality are helpful for the development of therapies to treat neurologic disorders.
In vitro models are crucial for studying mechanisms related to neurological injuries, and although successful isolation and two-dimensional (2D) culture of cortical astrocytes have been established8, this model fails to provide a realistic environment that mimics native cell behavior and to reproduce the complexity of the brain9. In 2D condition, the poor mechanical and biochemical support, low cell-cell and cell-matrix interactions, and cell flattening leading to the absence of basal-apical polarity, affect cell signaling dynamics and experimental outcomes leading to altered cell morphology and gene expression, which compromise response to treatments10. Therefore, it is crucial to develop alternatives that provide a more realistic neural environment, aiming to translate the results to the clinic.
Three-dimensional (3D) cell culture represents a more advanced model that recapitulates with increased fidelity features of organs and tissues, including the CNS11. Regarding glial culture, 3D models contribute to the maintenance of astrocytes morphology, cell basal-apical polarity, and cell signaling12,13. The 3D bioprinting technology emerged as a powerful tool to biofabricate 3D living tissues in a controlled manner by using cells and biomaterials to recreate the structure and properties of native tissues. The use of this technology has led to a substantial improvement of results prediction and has contributed to regenerative medicine applied to the CNS14,15,16.
The protocol described here details the isolation and culture of cortical astrocytes. The protocol also details a reproducible method to bioprint astrocytes embedded in gelatin/gelatin methacryloyl (GelMA)/fibrinogen, supplemented with laminin. In this work, an extrusion-based bioprinter was used to print the biomaterial composition containing cortical astrocytes at a density of 1 x 106 cells/mL. Bioprinting shear stress was minimized by controlling the printing speed, and astrocytes showed high viability after the process. Bioprinted constructs were cultured for 1 week, and astrocytes were able to spread, attach, and survive within the hydrogel, maintaining the astrocytic morphology and expressing a specific marker glial fibrillary acidic protein (GFAP)4.
This procedure is compatible with piston-driven extrusion-based bioprinters and can be used to bioprint astrocytes derived from different sources. The 3D bioprinted model proposed here is suitable for a wide range of neural engineering applications, such as studies of the mechanisms involved in astrocytes functionality in healthy tissues and understanding the progression of neurological pathologies and treatment development.
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
2. Astrocytes isolation and culture
3. Synthesis of gelatin methacryloyl (GelMA)
4. Bioink preparation
NOTE: In order to obtain 1 mL of bioink, it is recommended to fabricate at least 3 mL of biomaterial solution, as there may be losses during filtration.
5. Preparation of the crosslinker solution
6. Bioprinting astrocytes-laden bioink using an extrusion-based bioprinter
7. Assessment of astrocytes viability
8. Immunostaining of astrocytes
9. Confocal imaging
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 frame of square shape (0.6 x 0.6 mm), with pores of 1 mm, aiming to facilitate the diffusion of nutrients and oxygen. The frame was composed of 6 layers placed on top of each other, changing at an angle of 90° in every layer (Figure 2A i). The designed structure possessed approximately 5 mm of high (Figure 2A ii), allowing tissue manipulation. The bioink composition also allowed the fabrication of constructs of different shapes (Figure 2B).
The preparation of the bioink composed of gelatin, GelMA, and fibrinogen comprised two crosslinking steps. First, GelMA was crosslinked under UV light, in which inter and intramolecular covalent bonds are formed, followed by fibrin crosslinking. In this step, thrombin enzymatically cleaves fibrinogen chains, resulting in the formation of fibrin fibers17, a reaction stabilized by Ca2+ ions18. Then, GelMA and fibrin fibers form a stable interpenetrated polymer network (IPN) supplemented with laminin, suitable support for cell attachment and spread19,20 (Figure 2C).
Prior to bioprinting, after 12 days of isolation, astrocytes were characterized by the presence of GFAP, a protein constituent of astrocyte intermediate filaments4 (Figure 3A). Then, trypsinized astrocytes were mixed with the gelatin/GelMA/fibrinogen solution at a density of 1 x 106 cells/mL, generating an astrocytes-laden bioink. The bioink was transferred to a 5 mL syringe connected to a 22 G blunt needle, composing the bioprinter printhead (Figure 3B i). The needle allowed the bioink extrusion without clogging and preventing high shear stress to the cells.
Due to the viscoelastic properties of gelatin, which behaves as a fluid at higher temperatures and as a gel at lower temperatures21, the bioprinted construct retained shape fidelity (Figure 3B ii). After the bioprinting of two consecutive layers of bioink, the formation of a well-defined structure was observed (Figure 3C), with cells entrapped within the biomaterial.
After bioprinting and crosslinking processes, the construct was incubated with astrocyte medium, and after 1 day of bioprinting, most of the cells still presented a round morphology (Figure 3D). Bioprinted scaffolds maintained integrity after 7 days of incubation, and although some round cells were observed, a large number of astrocytes spread throughout the construct, presenting astrocytic morphology and interconnection (Figure 3E).
Because the parameters of bioprinting, such as speed, could directly affect cell viability, different bioprinting speeds (400, 600, and 800 mm/min) were tested and astrocytes survival evaluated using the Live/Dead assay with calcein-AM (live cells, green fluorescence) and ethidium homodimer-III (EthD-II) (dead cells, red fluorescence). The percentage of viable cells was quantified using a computational software, by calculating the number of living and dead cells. Cell viability was evaluated at time 0 (right after bioprinting), and results showed that at the lower speed, 400 mm/min, viable cells represented 74.08% ± 1.33% of total cells, being significantly higher than cells bioprinted at 600 and 800 mm/min (60.25% ± 1.93% and 62.94 ± 6.18%, respectively) (Figure 4A). Therefore, the speed of 400 mm/min was used in this work.
Prior to bioprinting, 2D cultured astrocytes were characterized as the percentage of viable cells, and viability of bioprinted astrocytes was normalized to this condition. Results showed that 2D culture presented 90.98% ± 0.94% of viable cells. Viability of bioprinted astrocytes (day 7) was 83.54% ± 3.00%, representing 0.92 ± 0.03 of the 2D value, which was significantly higher than that of day 0 (0.81 ± 0.01) (Figure 4B). Images of astrocytes stained with the live/dead reagent are presented in Figure 4C, and show that after bioprinting, cells possessed a round morphology (Figure 4C i). After 1 week of incubation, astrocytes spread throughout the construct (Figure 4C ii), showing a distinct morphology of cells from 2D culture (Figure 4C iii).
Bioprinted astrocytes were stained to show cell density and cell morphology within the construct. Figure 5A shows a bioprinted construct after 7 days of incubation, with high density of astrocytes stained with phalloidin, an F-actin cytoskeleton dye. Although few round cells were observed, astrocytes presented mainly a star-like morphology. Bioprinted astrocytes showed to be GFAP positive when stained after 7 days of bioprinting, indicating that cells maintained their astrocytic phenotype (Figure 5B i). Figure 5B ii and 5B iii show the Z-stacked images of the bioprinted GFAP+ astrocytes within the construct. These results indicate that the bioink composition provided a biocompatible microenvironment to promote astrocytes' adhesion, spread, and growth.
Figure 1: Extraction of brain and cortex separation for primary astrocytes culture. Primary astrocytes were isolated from the cortex of C57Bl/6 mice pups' (post-natal day 1) brain. After extracting the brain from the animal, the meninges were removed under a microscope, and the cortex separated followed by tissue digestion and astrocytes culture. Please click here to view a larger version of this figure.
Figure 2: Schematic illustration of 3D bioprinting process. (A) CAD file designed for the 3D bioprinting of neural tissue showing (i) 2 layers, top view, and (ii) 6 layers, side view, of the construct. (B) Images showing the capacity of the bioink to print structures of different shapes (i) square, (ii) capillary, and (iii) star. (C) Scheme of biomaterials crosslinking after 3D bioprinting showing, where GelMA is crosslinked under UV light followed by fibrin crosslinking in a thrombin:Ca2+ bath. Please click here to view a larger version of this figure.
Figure 3: 3D boprinting of astrocytes-laden gelatin/GelMA/fibrinogen bioink. (A) Characterization of astrocytes after 12 days of isolation and culture, stained for GFAP, green and DAPI, blue. (B) (i) Printhead setup and (ii) 3D bioprinted construct right after bioprinting. (B) Two-layered construct showing the bioprinted frame. Magnification of 4x. (C) Image of bioprinted astrocytes 1 day after bioprinting, showing cells in a round morphology. (D) Image of bioprinted astrocytes 7 days after the bioprinting process, showing cells with astrocytic morphology with few round cells, indicating their affinity with the mimetic tissue. Magnification of 10x. Please click here to view a larger version of this figure.
Figure 4: Assessment of bioprinted astrocytes viability. (A) Viability of astrocytes bioprinted at different speeds. (B) Viability of bioprinted astrocytes on (i) day 0 (right after bioprinting) and (ii) after 7 days of bioprinting, normalized to viability of astrocytes in 2D culture. Statistical analysis by means of One-Way Anova with Tukey's test, n = 3, *p < 0.05. (C) Fluorescent images of astrocytes stained with Live/Dead reagent. Bioprinted astrocytes on (i) day 0 (right after bioprinting), (ii) day 7, and (iii) 2D culture of astrocytes. Magnification of 10x. Please click here to view a larger version of this figure.
Figure 5: Characterization of 3D bioprinted astrocytes. Immunofluorescence of 3D bioprinted astrocytes after 7 days of incubation stained for (A) F-actin (phalloidin, red) and nuclei (DAPI, blue) with magnification of 10x and 40x and for (B) GFAP, green (i) with magnification of 10x, (ii) Z-stacked showing the X-Y-Z axis of the bioprinted construct, and (iii) image showing the X-Z axis of GFAP+ astrocytes. Please click here to view a larger version of this figure.
Supplemental File. Please click here to download this File.
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 CNS11. Due to the important role of glial cells in neural functionality, cortical astrocytes have been used in many studies, such as brain development24, biomolecules transport25, neurite outgrowth26, and in brain-like tissue biofabrication14.
Methods for engineering 3D culture of astrocytes were reported previously, using different biomaterials and scaffolding techniques27,28,29. Similarly, a method for 3D bioprinting human induced pluripotent stem cells (hiPSCs)-derived neural aggregates was also reported, and showed the capacity of these bioprinted cells to differentiate and mature in vitro30. However, there are no reports of methods for biofabrication of 3D brioprinted astrocytes constructs in the literature. Then, this protocol aimed to describe a reproducible method for 3D bioprinting cortical astrocytes.
In this protocol, astrocytes were isolated from the cerebral cortices of C57Bl/6 mice pups, an animal model widely used in research31,32, which can be replaced by astrocytes derived from other sources, such as hiPSCs and spinal cord. After isolation, culture, and subculture, astrocytes remain in a proliferative state up to passage 3, which is the maximal number of splitting recommended due to their intrinsic limitation of proliferation8. It was verified that proliferation of astrocytes from passage 4 was limited, being difficult to achieve the desired amount of cells for 3D bioprinting.
In the described method, a concentration of 1.0 x 106 cells/mL of biomaterial solution was used, as a proof of concept to evaluate the capacity of cells to survive the bioprinting process and to maintain their viability and intrinsic properties during culture within the hydrogel. In the previous work, a 3D bioprinted neural-like tissue was biofabricated by co-culturing astrocytes and neurons, and to increase cellular interaction, astrocytes concentration was 8.0 x 106 cells/mL14. Then, concentration of cells may be optimized for specific studies.
A bioink for 3D bioprinting is composed of cells and a biomaterial or a combination of biomaterials33. In this protocol, a combination of gelatin/GelMA/fibrinogen was used, which showed to be favorable for both bioprinting and maintainance of astrocytes in culture. The production of a bioprintable bioink is challenging when using extrusion-based bioprinting technique. The biomaterial composition must possess viscoelastic properties that, at the same time, allow the extrusion of the bioink, while retaining the 3D shape after the printing process34. In addition, it should be able to maintain cell viability during the process, which depending on the bioprinting conditions, can cause shear stress to the cells leading to death35.
Bioprintability was assured by gelatin, a biomaterial that possesses optimal viscoelastic properties due to its sol-gel transition capacity36. This allows gelatin macromolecules to rearrange and behave as a fluid during extrusion, and as a gel after bioprinting, maintaining the 3D structure. In addition, as a collagen derivate, gelatin is composed of glycine-amino acid peptide triplet repetitions, which assure cell-specificity21. However, intra and intermolecular bonds of gelatin are weak, and due to its thermoreversibility, gelatin has no stability at 37 °C, being released from the construct during cell culture. Therefore, GelMA became an alternative as a stable hydrogel, due to the covalent bonds formed after UV light exposition, maintaining cell-specificity properties19. The physical properties of GelMA, such as porosity, degradation, and elastic modulus can be tuned to suit different tissue engineering applications19. The stiffness of GelMA scaffolds can be controlled by varying the methacryloyl substitution37, allowing to achieve a stiffness similar to that of the tissue to be modeled. That is, by decreasing the degree of functionalization, a lower stiffness can be achieved37. Therefore, due to the softness of the mouse brain38,39 and the direct effect of extracellular matrix (ECM) stiffness on cell behavior40, in this protocol a low degree of gelatin functionalization was proposed. In the previous works, the capability of GelMA hydrogels to mimic CNS physical features, showing high suitability for culturing astrocytes, neurons, and neural stem cells14,41,42 was reported.
Besides GelMA, fibrinogen, a native biopolymer that forms fibrin fibers through enzymatic reaction, has also been widely used to biofabricate neural-like tissue, showing high specificity and offering a suitable microenvironment for neural cells to attach and grow30,43,44. Other biomaterials, such as alginate and chitosan have been used to bioprint neural-like tissues, mixed to gelatin, GelMA, and/or fibrinogen, aiming to improve printability and physical properties of the 3D scaffolds14,30. In the present method, an optimal bioprintability, physical stability, and cell-specificity was achieved using gelatin, GelMA, and fibrinogen as components of the bioink. In order to increase astrocytes' recognition to the microenvironment, laminin-a component of the brain ECM-was used to supplement the bioink.
In this protocol, gelatin was used at a concentration of 4% (w/v), which allowed the sol-gel transition of the bioink at 25 °C within approximately 10 min. A faster gelation can be achieved by increasing the concentration of gelatin. However, sterilization by filtration using 0.2 µm filter can be compromised. The bioink filtration is a critical step, verifying that higher concentrations of gelatin (>5% w/v) may prevent filtration. An alternative to achieve a faster gelation point during bioprinting is to leave the syringe containing the bioink at 4 °C for 2 min before connecting it at the bioprinter printhead. After bioprinting, it is crucial to maintain the construct at 25 °C to avoid destabilizing the 3D structure and maintain a gelled condition prior to UV exposition. Notably, the construct should be solid when the fibrinogen crosslinker solution is added to the plate. For GelMA crosslinking, it is important to flip the sample (2 x 60 s each side) to ensure UV exposition throughout the construct. For fibrinogen crosslinking, the crosslinker solution should cover the construct completely. Therefore, if using a larger dish or plate, the volume should be adjusted. After crosslinking, the hydrogel should look homogeneous under phase contrast microscopy and cells should be homogeneously distributed throughout the construct, possessing a round morphology.
This protocol allowed the gelatin/GelMA/fibrinogen bioink to print structures of different shapes, maintaining the integrity and the 3D shape of the constructs. Due to the limitations of the temperature to reach 25 °C inside the laminar flow, the bioprinting was performed in the culture room outside the laminar flow. Bioprinting time was approximately 1 min for each sample, and no contamination was observed during cell culture.
Cell integrity can be affected by bioprinting, due to the shear stress caused in the process35. This can be controlled by optimizing printing parameters, such as printing speed. In this work, different bioprinting speeds were tested, 400, 600, and 800 mm/min, observing a significant decrease in cell viability when speed was increased from 400 to 600 mm/min. At 400 mm/min, around 74% of the cells remained viable after bioprinting, and this value increased significantly after 7 days of incubation (>80%). Therefore, lower speeds were not used in order to avoid the excessive exposition of cells to the environment. The 2D culture of astrocytes showed higher viability (~90%) as compared to the bioprinted cells. However, as shown by fluorescent images, morphology is affected by the type of culture. While 2D cells were flat, astrocytes in 3D environment possessed a star-like shape, interconnecting with each other by cellular processes.
Notably, the mimetic microenvironment was favorable for cortical astrocytes culture, as cell viability significantly increased after 1 week, suggesting cell proliferation within the construct. Laminin, a glycoprotein present in the brain ECM, was added to the bioink aiming to achieve a higher adherence of the astrocytes to the hydrogel14. Cytoskeleton F-actin staining showed that bioprinted astrocytes were in high density within the construct, indicating that the cell concentration used in this protocol allowed astrocytes interconnection. Immunohistochemistry for GFAP localization, a marker correlated with astrocytes' extensive arborization and cell hypertrophy45, corroborated by F-actin staining, showed that cells presented typical astrocytic morphology, indicating that the mimetic 3D system was favorable for cells to attach and behave as in their native environment.
The protocol presented here describes an efficient and reproducible procedure for 3D bioprinting cortical astrocytes. Due to the importance of astrocytes in neuroinflammation response to injury, as well as in regulating neuronal functionality, studies involving this glial cell could contribute to many aspects in the understanding of diseases that affect the CNS. Therefore, the 3D in vitro model presented here is useful in future applications that aim to study astrocyte-neuron interactions, the functionality of astrocytes in brain pathologies, and the potential of astrocytes as therapeutic targets.
The authors have nothing to disclose.
This work was supported by The São Paulo Research Foundation (FAPESP), grant numbers 2018/23039-3 and 2018/12605-8; National Council for Scientific and Technological Development (CNPq), grant numbers 465656/2014-5 and 309679/2018-4; and Coordination for the Improvement of Higher Education Personnel (CAPES), financial code 001.
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 |