Protocol for 3D Matrigel embedding of primary murine astrocytes for a physiologically relevant culture model

Summary

Primary astrocyte 2D cultures often exhibit a phenotype that differs significantly from astrocytes in the brain. Here, we present a detailed protocol for culturing primary murine astrocytes within a 3D Matrigel environment, which better mimics the in vivo conditions. We describe a step-by-step guide for the isolation and preparation of primary striatal astrocytes, their embedding and cultivation in Matrigel, and the functional characterization of astrocytes grown in this 3D system.

Graphical abstract

Highlights

• •Guided differentiation protocol of murine primary astrocytes
• •Steps for embedding primary murine astrocytes in Matrigel
• •Procedures for functional characterization of astrocytic uptake by live imaging

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Primary astrocyte 2D cultures often exhibit a phenotype that differs significantly from astrocytes in the brain. Here, we present a detailed protocol for culturing primary murine astrocytes within a 3D Matrigel environment, which better mimics the in vivo conditions. We describe a step-by-step guide for the isolation and preparation of primary striatal astrocytes, their embedding and cultivation in Matrigel, and the functional characterization of astrocytes grown in this 3D system.

Before you begin

We describe the specific steps for generating 3D primary murine astrocytic cultures starting from newborn pups. Specifically, starting from postnatal day 0–2 (P0–P2) mice, we detail how to obtain 2D primary astrocyte cultures, expand them, embed them in a 3D Matrigel matrix, and subsequently characterize them via immunofluorescence and functional assays, including assessment of their internalization activity. We describe a protocol that can be used for any transgenic mice, opening to the opportunity to use a more advanced in vitro model for the experiments.

Innovation

Our 3D culture loses the classical polygonal morphology that primary astrocytes typically acquire on 2D surface. Moreover, they stop proliferating in the 3D system and acquire a sessile profile typical of the in vivo condition. Compared to 2D cultures, 3D astrocytes display enhanced morphological complexity reflecting a more homeostatic, physiologically relevant phenotype. These features make 3D astrocyte cultures a more suitable system to gain mechanistic insights into astrocyte biology.

Institutional permissions

Housing and handling of mice were done in compliance with national guidelines. All procedures performed with mice were approved by the Ethical Committee of the University of Padova and the Italian Ministry of Health (license 200/2019).

Primary striatal astrocyte isolation and culturing in 2D

Timing: 1.5 h

This section provides a step-by-step protocol for culturing primary murine striatal astrocytes which we used as a starting point to generate 3D cultures.

Note: The focus of our lab is directed toward the study of molecular mechanisms underlying Parkinson’s Disease (PD). Therefore, striatum is a region of interest for us. However, this protocol can be used for other brain regions (e.g., cortex).

• 1.
  Striatum dissection:

  • a.
    Sacrifice postnatal pups (P0-P2) via decapitation.
  • b.
    Cut the skin and skull using scissors. Use forceps to expose the brain.
  • c.
    Remove the brain with a spoon, lifting from the cerebellum to the olfactory bulbs. Place the brain in cold Dulbecco’s Phosphate-Buffered Saline (DPBS).
  • d.
    Under a stereomicroscope, dissect the cortices to expose the striata.
  • e.
    Collect three striata together to achieve a higher yield and place them in a new dish containing cold DPBS.

CRITICAL: During the dissection work quickly and use cold DPBS to maximize cells viability and yield.

• 2.
  Tissue dissociation and primary astrocytes isolation:

  • a.
    Work under a biological hood. Carefully remove DPBS with a pipette, avoiding tissue aspiration.
  • b.
    Add 1 mL of DMEM supplemented with 1% Penicillin-Streptomycin (PenStrep). Mechanically homogenize the tissue by thorough pipetting.
  • c.
    Place a 70 μm cell strainer on top of a 50 mL tube.
  • d.
    Transfer the homogenized tissue onto the strainer.
  • e.
    Use 1 mL of supplemented DMEM to rinse the dish containing the striata, collect the remaining cells, and transfer them onto the strainer.
  • f.
    Use the plunger of a 20 mL syringe to facilitate the passage of cells through the strainer.
  • g.
    Add 25 mL of supplemented DMEM to the tube.
  • h.
    Centrifuge at 0.3 RCF for 15 min at room temperature (RT). Discard the supernatant.
  • i.
    Resuspend the pellet in 25 mL of supplemented DMEM and centrifuge again.
  • j.
    Repeat the centrifugation and resuspension process two more times (three washes in total).
  • k.
    After three washes, resuspend the pellet in Basal Medium Eagle (BME) supplemented with 1% PenStrep and 10% Fetal Bovine Serum (FBS).
  • l.
    Seed the cells in a T75 flask and incubate at 37°C with 5% CO₂.
  • m.
    After 7 days, discard the medium completely and replace it with fresh supplemented BME.
  • n.
    After 14 days in vitro (DIV), primary astrocytes reach confluency and are ready for experiments.

Note: Avoid keeping the cells in over confluency for a long period of time. Primary cultures have inhibited proliferation due to contact-contact inhibition which can lead to the appearance of senescence markers.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Gfap (dilution 1/200)	Proteintech	60190-1-Ig
Aldhl1 (dilution 1/200)	Abcam	Ab190298
Phalloidin-iFluor 647 reagent (dilution 1/1,000)	Abcam	Ab176759
Hoechst (1:10,000)	Invitrogen	H3569
Alexa 568 rabbit (1/200)	Invitrogen	A11036
Alexa 488 rabbit (1/200)	Invitrogen	A11034
Alexa 568 mouse (1/200)	Invitrogen	A11004
Alexa 488 mouse (1/200)	Invitrogen	A11029
Chemicals, peptides, and recombinant proteins
DPBS	Biowest	L0615
BME	Biowest	L0042
DMEM	Biowest	L0103
FBS	S.I.A.L. Group	yourSIAL-FBS-SA
PenStrep	Biowest	L0022
Matrigel, phenol red-free, LDEV-free	Corning	356237, lot: 3039003
Trypsin 10X	Gibco	15090046
EDTA	Carlo Erba	6381926
Trypan blue	Sigma-Aldrich	T8154
Triton X-100	Sigma-Aldrich	9002931
Sodium azide	Sigma-Aldrich	S2002
Paraformaldehyde 4% (PFA)	Thermo Scientific	J19943-K2
α-synuclein fibrils - pHrodo	Giusti et al.1	N/A
Opti-MEM	Gibco	31985047
CellMask Green plasma membrane stain (1/1,000)	Thermo Scientific	C37608
Experimental models: Organisms/strains
C57BL/6 J wild-type (WT) mice	Jackson Laboratory	N/A
Other
μ-Slide 8 well	ibidi	80806
μClear 96-well (Greiner)	Greiner	655090
Operetta CLS	PerkinElmer	N/A

Materials and equipment

Trypsin + EDTA

Reagent	Final concentration	Amount
Trypsin 10X	1X	5 mL
DPBS	N/A	44.95 mL
EDTA (0.5 M)	0.53 mM	53 μL
Total	N/A	50 mL

Store at +4°C up to 6 months.

Permeabilization solution

Reagent	Final concentration	Amount
Triton X-100	0.1%	1 μL
DPBS	N/A	999 μL
Total	N/A	1 mL

Prepare fresh.

Blocking solution

Reagent	Final concentration	Amount
FBS	5%	50 μL
DPBS	N/A	950 μL
Total	N/A	1 mL

Prepare fresh.

Alternatives: As blocking solution, 5% BSA or other serums (e.g., Donkey serum) can be used.

Step-by-step method details

Embedding in Matrigel of primary murine astrocytes

Timing: 1 h

This section provides a step-by-step protocol for the generation of 3D astrocyte cultures starting from 2D primary astrocytes.

Note: One hour before starting the protocol, thaw Matrigel on ice and keep it on ice during the whole procedure.

• 1.Aspirate the medium from the flask and wash gently with warm DPBS.
• 2.Add 2 mL of Trypsin 1X supplemented with 50 mM EDTA and incubate for 2 min at 37°C.
• 3.Add 5 mL of supplemented BME to inactivate the trypsin and transfer to a 15 mL tube.
• 4.
  Count cells as follows:

  • a.
    Resuspend the cell suspension thoroughly.
  • b.
    Mix 20 μl of cell suspension with 180 μL of Trypan blue in a 0.5 mL tube and mix gently by pipetting.
  • c.
    Count the cells using the Burker chamber.
• 5.The proportion of cells is 5.000 cells for each μl of Matrigel. Transfer the appropriate amount of cells suspension to a new tube. For imaging purposes, form a 20 μl Matrigel drop in each well.

CRITICAL: The number of cells must be precise otherwise the embedding will not be successful.

• 6.Centrifuge the cells at 0.3 RCF for 5 min at RT and discard the supernatant.
• 7.Place the tube containing the cell pellet on ice and add the proper amount of Matrigel to the cell pellet (e.g., for 500.000 cells add 100 μl of Matrigel).
• 8.Mix gently but quickly the cell pellet with the Matrigel by maintaining the tube in the ice (troubleshooting 1).

CRITICAL: The cells re-suspension must be quick to avoid the polymerization of the Matrigel and long exposure of the cells to a cold environment.

• 9.Pipette 20 μl of cell-Matrigel suspension in the μ-Slide 8 Well or a μClear 96-well plate (troubleshooting 2).

Note: Keep the μ-Slide 8 Well | Chambered Coverslip for Cell Imaging on a flat surface and do not tilt to avoid obtaining a deformed drop. For the same reason, dispense the Matrigel drop while holding the pipette completely perpendicular to the well (Figure 1A).

Note: We recommend using a μ-Slide 8 Well or μClear 96-well plate as it is compatible with 3D culture and imaging.

• 10.Incubate the μ-Slide 8 Well for 10 min at 37°C – 5% CO₂ to allow the polymerization of the Matrigel (Figure 1B).
• 11.Add 300 μl of supplemented BME to each well.
• 12.Incubate the μ-Slide 8 Well for 7 days at 37°C – 5% CO₂.

Note: After plating, 3D primary astrocytes can be subjected to different treatments (e.g., transfection, inhibitors). The incubation time might be longer to allow the proper diffusion of the molecules into the Matrigel drop.

Figure 1.

Matrigel embedding of primary murine astrocytes

(A) Proper pipette positioning: the pipette should be held perpendicular to the ibidi surface to obtain a perfectly round drop in the center of the chamber well.

(B) Representative image of the Matrigel drop after polymerization.

Fixation and immunostaining

Timing: 2 days

This section provides a step-by-step protocol for the fixation and staining of the 3D embedded astrocytes using astrocytic markers.

• 13.Aspirate gently the medium tilting the μ-Slide 8 Well chamber to avoid touching the Matrigel drop.
• 14.Wash with warm PBS for 5 min in agitation to allow the complete removal of the medium also inside the Matrigel drop.
• 15.Add PFA 4% and incubate in agitation at RT for 1 h or o/n at 4°C.
• 16.Wash three times with PBS for 5 min in agitation.
• 17.Add PBS + 0.5% Triton-X 100 to permeabilize the cells.
• 18.Incubate for 20 min in agitation at RT.
• 19.Remove the permeabilization solutions and add the blocking solution for 1 h.
• 20.Incubate the embedded astrocytes with the primary antibodies diluted in the blocking solution o/n at 4°C in agitation.
• 21.Wash three times with PBS for 5 min each at RT.
• 22.Incubate the embedded astrocytes with the secondary antibodies diluted in blocking solution for 1 h at RT.
• 23.Wash three times with PBS for 5 min each at RT.
• 24.Incubate the embedded astrocytes for 30 min with Phalloidin-iFluor 647 Reagent to visualize the actin cytoskeleton.
• 25.Wash three times with PBS for 5 min each at RT.
• 26.Incubate the embedded astrocytes for 5 min with Hoechst for nuclei staining.
• 27.Wash two times with PBS for 5 min each at RT.
• 28.Add PBS containing 0.03% sodium azide for long-term storage.

Note: To analyze the retaining of astrocytic identity in the 3D embedded cultures we recommend the use of specific astrocytic markers such as, Gfap or Aldhl1 in combination with a generic cellular marker (e.g., actin cytoskeleton [phalloidin]) (Figure 2A). The 3D cultured astrocytes display a higher morphological complexity compared to the traditional 2D cultures (Figures 2A–2E).

Note: Due to the absence of the mounting agent, the confocal imaging must be performed optimally within a week from the staining. Longer storage time of the fixed embedded astrocytes results in the dehydration of the Matrigel drop.

Figure 2.

Comparison between 2D and 3D cultures of astrocytes

(A) Representative confocal z-stacks (40× magnification) of astrocyte cultures (2D vs. 3D) stained for Aldh1l1 (green), F-actin (red), and nuclei (blue). Scale bar: 50 μm.

(B) Quantification of the maximum branch length per cell.

(C) Quantification of the number of junctions per cell.

(B and C) The analysis was performed using the AnalyzeSkeleton plugin (version 3.4.2) after cell skeletonization with the Skeletonize plugin. Statistical analysis was performed using an unpaired Mann–Whitney test (n = 3). p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

(D) Quantification of the longest shortest path.

(E) Quantification of the number of branches per cell using the AnalyzeSkeleton plugin (version 3.4.2) after cell skeletonization with the Skeletonize plugin (n = 3).

Live imaging for functional assays with 3D embedded primary astrocytes

Timing: 7 days

This section presents an assay to evaluate whether the embedded primary astrocytes maintain their functional capacity. Given the well-documented ability of astrocytic cultures to internalize extracellular materials, including protein aggregates,² we adapted a previously established protocol for 2D cultures to a 3D environment.

Note: We recommend the use of pHrodo conjugated fibrils since the fluorescence in dependent on the acidic pH of the lysosomes – hence when the fibrils are internalized by the astrocytes. This allows to minimize the background fluorescence that would arise if non-pH sensitive fluorophore were to be used. Moreover, the use of the pHrodo conjugated aggregates allows a live assay.¹

• 29.Treat the embedded astrocytes at DIV 0 (from embedding) with 0.5 μM of α-synuclein (α-syn). fibrils conjugated with the pHrodo dye diluted in supplemented BME for 90 min at 37°C.
• 30.Wash the cells twice with warm DPBS in agitation for 5 min.
• 31.Incubate the cells for 30 min with Hoechst and CellMask resuspended in Optimem.
• 32.Wash the cells twice with warm DPBS in agitation for 5 min.
• 33.Add fresh supplemented BME supplemented.
• 34.Acquire the images using Operetta CLS High-Content Analysis System after 1 h, 6 h, 24 h, 48 h, 72 h, and 7 days (Figures 3A and 3B).

CRITICAL: Add the medium or DPBS carefully and close to the wall of each well to avoid the disruption of the Matrigel drop.

Figure 3.

Kinetic analysis of α-synuclein internalization by 3D-embedded astrocytes

(A) Representative images from a live-cell assay performed with the high-throughput Operetta (PerkinElmer) at 6 h, 24 h, 72 h, and 7 days after co-polymerization of α-syn fibrils tagged with pHrodo (red) and 3D astrocytes transfected with eGFP (green) prior to embedding. Scale bar: 20 μm.

(B) Quantification of the number of α-syn spots per field in untreated and treated astrocytes.

Expected outcomes

We describe a protocol for the generation of 3D embedded astrocytes that acquire a more complex phenotype compared to the traditional primary polygonal astrocytes cultured on 2D surfaces (Figures 2B–2E). Initially, the embedded astrocytes exhibit a rounded morphology without processes, which is expected following detachment. However, after seven days, these cells demonstrate the ability to self-organize into a network, protruding and extending their processes within the Matrigel droplet. This process is likely facilitated by the secretion of metalloproteases, which degrade the proteins comprising the Matrigel. Moreover, the culture period can likely be adjusted within the established viability window (up to 21 DIV) without requiring major protocol modifications. Co-culturing these astrocytes with other cell types also appears feasible, particularly with other glial populations (e.g., microglia) and potentially with neurons. In fact, primary neurons embedded in Matrigel have been reported to form networks by 7 DIV and to reach electrophysiological maturity by 21 DIV.³ While optimization of the relative cell ratios would be necessary, such co-cultures are theoretically possible based on available literature.

Limitations

Primary cells are significantly more sensitive than immortalized cell lines; therefore, it is crucial to handle them quickly and gently during resuspension in Matrigel. Improper resuspension can lead to cell clustering, preventing them from acquiring a ramified morphology and instead causing them to remain rounded. Additionally, seeding density plays a critical role in cell distribution and viability.

Overall, this protocol provides a valuable in vitro model for better capturing astrocyte heterogeneity. However, to ensure reproducibility and minimize variability across experiments, strict standardization of handling techniques is essential.

Troubleshooting

Problem 1

Cells do not form a network in the Matrigel drop but form a cluster (Figure 4B).

Figure 4.

Proper cell seeding density

(A) Representative optical microscope images (EVOS M5000, Invitrogen) of primary astrocytes immediately after embedding in the Matrigel drop at cell densities lower, equal to, or higher than 5,000 cells/μL Matrigel.

(B) Representative confocal z-stack images (10×) of primary astrocytes embedded in Matrigel at a density higher than 5,000 cells/μL, stained for Gfap (green), F-actin (pseudocolored red), and nuclei (blue). Scale bar: 50 μm.

Potential solution

If the cell pellet is not properly resuspended in Matrigel, the cells remain clustered together during drop formation, preventing their uniform distribution. This aggregation hinders the establishment of a proper astrocytic network, as the cells remain confined to the same space rather than spreading appropriately (Figure 4B).

Problem 2

Bubble formation during cells resuspension.

Potential solution

• •Matrigel is a highly viscous solution that tends to form bubbles during pipetting, which can interfere with imaging. To minimize bubble formation, we recommend the following:
• •Use pre-cut filter tips to reduce resistance and shearing.
• •Employ a gentle resuspension technique to avoid introducing air.
• •Pre-cool pipette tips at −20°C before use, which helps keep the Matrigel cold and in a liquid state during handling.
• •These precautions will improve pipetting accuracy and maintain sample integrity for imaging.

Problem 3

Cell density is suboptimal during 3D embedding.

Potential solution

When the seeding density exceeds 5000 cells/μL Matrigel, cells may not distribute evenly, leading to aggregation. Conversely, when the seeding density falls below the recommended threshold, cells may undergo cell death, likely due to insufficient contact with neighboring cells, which is necessary for survival (Figure 4A). To address this, cells should be accurately counted before embedding in Matrigel, and the density should be adjusted to ensure sufficient cell–cell contact without overcrowding, promoting optimal network formation.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Laura Civiero (laura.civiero@unipd.it).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Gurkirat Kaur (gurkirat.kaur@phd.unipd.it).

Materials availability

The cell line used above was obtained from C57BL/6 J wild-type (WT) mice purchased from the Jackson Laboratory.

Data and code availability

This study did not generate datasets or analyze code.

Acknowledgments

We acknowledge and respect the University of Padova for support of L.C. as an associate professor and the IRCCS San Camillo Hospital in Venice, Italy, for scientific collaboration. We also thank Federico Soria, PhD (Achucarro Basque Center for Neuroscience, Leioa, Spain), for his valuable advice. This work was supported by the Italian Ministry of Health (GR-2021-12372494) and by the Ministry of Education, University and Research (MIUR) with the Progetti di rilevante interesse nazionale 2022 (PRIN – 20229RTWSZ).

Author contributions

G.K. and L.C. conceived and designed the experiments, supervised the entire project, and wrote the manuscript. G.K., V.G., and M.E.S. performed the experiments. G.K. and L.C. contributed to data analysis and manuscript preparation.

Declaration of interests

The authors declare no competing interests.