Isolation and Culture of Mouse Brain Microvascular Endothelial Cells

Summary

A protocol is presented for the isolation and primary culture of brain microvascular endothelial cells (BMECs) from neonatal murine brain tissue to generate a robust in vitro model for studying blood-brain barrier function under physiological and pathological conditions.

Abstract

Brain microvascular endothelial cells (BMECs) are the main cellular component of the blood-brain barrier (BBB) and play a key role in regulating molecular transport and maintaining central nervous system homeostasis. Reliable in vitro models of brain endothelium are essential for studying BBB physiology and dysfunction in neurological and systemic diseases. A reproducible protocol for the isolation and primary culture of BMECs from neonatal murine cerebral cortex is described. The method includes cortical tissue dissection, mechanical and enzymatic dissociation, removal of myelin debris using a bovine serum albumin (BSA) density gradient, and enrichment of endothelial cells using CD31-based immunoselection. Isolated cells are subsequently cultured on collagen-coated plates and characterized by morphological assessment, immunofluorescence detection of endothelial markers, angiogenic tube formation assays, and transendothelial electrical resistance (TEER) measurements to evaluate barrier properties. This protocol provides a reliable approach for obtaining primary BMEC cultures suitable for investigating BBB biology, endothelial signaling, and neurovascular interactions under physiological and pathological conditions.

Introduction

The blood-brain barrier (BBB) is a highly specialized structure that strictly regulates the exchange of substances between the central nervous system (CNS) and the systemic circulation¹. The BBB is primarily formed by brain microvascular endothelial cells connected by tight junctions. These endothelial cells function within a multicellular system known as the neurovascular unit (NVU), which also includes astrocytes, pericytes, neurons, glial cells, and the basement membrane². Among these components, brain microvascular endothelial cells (BMECs) play a central role by forming a continuous layer with tight junctions, thousands of selective transporter systems, limited vesicular trafficking, and by restricting immune cell infiltration through low expression of adhesion molecules that limit the passage of solutes and cells into the brain parenchyma^3,4.

In addition to their role as a physical and selective barrier, BMECs are actively involved in essential processes, including neurovascular signaling, cerebral blood flow regulation, local immune responses, and, particularly, cerebral angiogenesis⁵. The latter process is key during embryonic development, but it is also reactivated in pathological conditions such as stroke, ischemia, or inflammation. BMECs respond to proangiogenic signals such as vascular endothelial growth factor (VEGF) and placental growth factor (PLGF), coordinating the formation and maturation of new vessels^6,7. Thus, a brain endothelial dysfunction would severely compromise the integrity of the BBB and the brain homeostasis.

Studying BMECs in vitro allows us to model both the physiology of the BBB and the mechanisms that regulate brain angiogenesis. However, isolating these cells from brain tissue poses significant technical challenges due to the complexity of neural tissue, the abundance of myelin, and the need to obtain cultures with high endothelial purity. Although immortalized cell lines are available, they have significant limitations in terms of their phenotypic and functional representativeness⁸.

In this context, the protocols for primary culture of BMECs from neonatal murine models constitute a powerful experimental tool, as they preserve many of the morphological, molecular, and functional characteristics of the brain endothelium in vivo. However, several established protocols for murine BMEC isolation rely on mechanical dissociation combined with density gradients (dextran or Percoll), often yielding variable endothelial enrichment and residual contamination by pericytes, astrocytes, and myelin debris⁹. Antibiotic-based purification using puromycin improves endothelial purity (up to ~95%) but may introduce cellular stress due to its inhibition of protein synthesis and potential effects on endothelial phenotype¹⁰. Alternatively, Fluorescence-Activated Cell Sorting (FACS)-based approaches provide highly purified populations but require specialized equipment and extensive processing, which can reduce viability and scalability¹¹. More recent neonatal protocols avoid density gradients and reduce the number of centrifugation steps, improving yield while limiting contamination¹².

Building on this background, this work describes an optimized protocol for isolating and culturing endothelial cells from neonatal mouse brain microvasculature. This method combines precise anatomical dissection, efficient enzymatic digestion, myelin removal using a Bovine Serum Albumin (BSA) gradient, and enrichment by immunoselection with magnetic microspheres conjugated to the CD31 marker. The protocol enables the obtaining of viable, reproducible, and enriched primary cultures, suitable for studies of cerebral angiogenesis, neurovascular interaction, endothelial signaling, and BBB dysfunction under both physiological and pathological conditions.

Protocol

The Bioethics and Biosafety Committee of the University of Bío-Bío approved this project (Fondecyt grant 1240925). The work with animals was carried out in accordance with the cardinal principles of the three R's in the use of experimental animals¹³ and in accordance with the recommendations of the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health of the United States¹⁴. The animals were kept in appropriate environments at the Bioterium of the University of Bío-Bío under standard housing conditions (12 h light/dark cycle, controlled temperature of 22 ± 2 °C, with ad libitum. access to feed and water).

To obtain the cells, wild-type (WT) pregnant mice (C57BL/6), aged between 4 and 6 months, were used. After delivery, the pups remained with the mother until postnatal day 5 (P5). At this point, the offspring were deeply anesthetized with isoflurane until loss of reflexes prior to euthanasia. Hypothermia was used solely as an adjunct to minimize movement and was immediately followed by decapitation. All procedures were performed in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020)¹⁵ and international regulations for the care and use of laboratory animals (NIH)¹⁴. The cerebral cortex was isolated and used to develop this experimental protocol (see Protocol Step 3). The Table of Materials lists the materials and antibodies used in this protocol.

1. Preparing the endothelial culture medium

1. To prepare 500 mL of culture medium for murine brain endothelial cells, in a sterile container, mix 250 mL of DMEM supplemented with 10% fetal bovine serum (FBS), 250 mL of endothelial cell culture medium supplemented with 10% FBS.
   NOTE: The culture medium provides essential endothelial growth factors and supports cell proliferation. If the referenced product (see the Table of Materials) is not available, equivalent endothelial cell supplements (e.g., ECGS, hydrocortisone, and L-glutamine) can be added to DMEM + FBS to achieve similar growth conditions¹⁶.
2. Sterile-filter the entire medium (10% FBS) through a 0.22 µm filter to ensure sterility.
3. Store the prepared medium at 4 °C until use. Prepare fresh medium every 2–4 weeks to maintain growth factor activity.

2. Coating the culture plates

NOTE: To promote cell adherence and maintain the endothelial phenotype, culture plates must be precoated with type I collagen. The steps to this process are as follows:

1. Prepare the precoating solution by mixing Type I collagen (0.1 mg/mL), M of acetic acid to solubilize the collagen, and sterile basal medium to reach a total volume of 15 mL. Mix gently until the collagen is fully dissolved and adjust the solution to pH 4.0 if necessary. Sterile-filter the solution through a 0.22 µm filter and keep on ice until use to preserve collagen stability.
   NOTE: Type I collagen mimics the basement membrane, supports endothelial cell attachment, and helps to maintain cell phenotype.
2. Homogenize the solution under sterile conditions. Apply the solution evenly to cover the entire surface of the culture plates.
   NOTE: Only the volume necessary to cover the surface is used.
3. Incubate the plates at 37 °C overnight (12–16 h) to allow the collagen to adsorb properly. Before seeding cells, remove excess coating solution and wash the plates 2x with sterile 1x phosphate-buffered saline (1x PBS, pH 7.4) to remove residual acid and unbound collagen.

3. Isolation of brain mixed cell culture

NOTE: This protocol builds on previous reports^9,17 that aimed to isolate BMECs from the cerebral cortex of P5 mice.

1. Sacrifice five P5 pups using deep anesthesia with isoflurane; use hypothermia only as an adjunct, followed by cervical decapitation.
   NOTE: From this step onward, all procedures must be performed in a laminar-flow biological safety cabinet.
2. Collect the brains (Figure 1A) and place them in sterile cold PBS (1x) in a 50 mL conical tube until all brains are processed.
3. Separate the meninges by carefully rolling each brain on a sterilized nitrocellulose membrane. Transfer the brains to a 100 mm plate containing cold PBS (1x). Using a sterile scalpel, remove the brainstem, cerebellum, thalamus, and olfactory bulb (Figure 1B,C).
4. Collect the resulting meninges-free cerebral cortices and store them in cold PBS (1x). Immediately transfer all brain cortex to a 50 mL conical tube containing 5 mL of PBS 1x on ice (Figure 1D).
5. Mechanically dissociate the tissue using sterile scissors, followed by a 1 mL pipette until no pieces are observed, and the suspension is homogeneous (Figure 1E).
   NOTE: The pipette tip can be cut to reduce mechanical stress during trituration. Tissue dissociation is achieved by controlled suction and gentle pipetting, while a wider tip opening minimizes shear forces and mechanical cell lysis, thereby preserving cell viability.
6. Collect all tissues and place them in 15 mL tubes.
7. To digest the tissue, transfer the sample to a sterile 15 mL conical tube containing 2 mL of collagenase solution (2 mg/mL in DMEM) and incubate for 2 h at 37 °C on an orbital shaker at 180 rpm.
8. After digestion, add 5 mL of DMEM supplemented with 10% FBS to the suspension and centrifuge at 3,000 × g. for 10 min (see Figure 1F). Discard the supernatant.
9. To remove myelin, resuspend the pellet in 4 mL of 20% BSA in PBS (w/v) and centrifuge at 3,000 × g. for 20 min (Figure 1G). After centrifugation, carefully remove the top milky layer containing myelin using a Pasteur pipette and discard any remaining supernatant (Figure 1H).
10. Resuspend the pellet in 2 mL of DMEM containing 10% FBS, then centrifuge again at 3,000 × g. for 10 min. Discard the supernatant, then gently resuspend the final pellet in 1 mL of DMEM containing 10% FBS.
    NOTE: At this stage, the final resuspension contains a heterogeneous population of brain-derived cells resulting from tissue dissociation. This mixture may include brain microvascular endothelial cells, astrocytes, pericytes, and other neural cells. Subsequent purification and culture steps are required to enrich BMECs selectively.
11. Before plating the isolated cells, remove the coating solution from the prepared culture plates (see Step 2.3) and rinse each well twice with sterile PBS (1x).
12. Seed the resuspended cells (step 3.10) onto the coated plates (recommended: 60 mm dishes).
    NOTE: Typically, the entire cell suspension obtained from one isolation is distributed into one or two coated dishes, each containing 5 mL of endothelial growth medium.
13. Maintain the cultures in endothelial cell medium at 37 °C in a humidified incubator with 5% CO₂. Replace the medium every 2–3 days. When cultures reach 90–100% confluence, proceed with immunoselection using CD31-coated beads to enrich for endothelial cells.
    NOTE: Following isolation and initial culture establishment, the procedure typically yields approximately 5 × 10⁶ cells per preparation, which adhere to collagen-coated plates and develop the characteristic cobblestone morphology of endothelial monolayers.

4. Isolation of brain microvascular endothelial cells (BMECs) using CD31 magnetic beads

1. Preparation and detachment of cells
   1. Once cultures reach 90–100% confluence, wash the cells 3x with PBS (1x) to remove residual medium.
   2. Add 1 mL of trypsin (0.5% 1x) to the plate and incubate for 3–5 min at 37 °C to detach the cells.
   3. Neutralize the trypsin by adding 1 mL of endothelial culture medium with 10% FBS. Transfer the cell suspension (~2 mL) to a sterile microcentrifuge tube and centrifuge at 220 × g. for 10 min at 4 °C. Discard the supernatant.
2. Preparation of CD31 magnetic beads
   1. Place 8 µL of CD31 magnetic beads in a new 15 mL tube. Wash the beads with 1 mL of 1% Hank’s solution, gently resuspending them by moving the tube.
   2. Place the tube on a magnet for 2 min to precipitate the beads. Carefully remove the supernatant without disturbing the beads. Repeat the wash step 3x.
      NOTE: Always work close to the magnet to avoid losing the beads, and perform this step under sterile conditions to prevent contamination of the primary cell culture.
3. Incubation of cells with beads
   1. Resuspend the cell pellet in 1 mL of 1% Hank’s solution containing FBS (to reduce non-specific binding).
   2. Transfer the cells into the tube containing the prewashed beads using a 1,000 µL pipette. Incubate the mixture on ice with gentle shaking for 20 min.
4. Magnetic separation of endothelial cells
   1. Place the tube on a magnet for 3 min to precipitate bead-bound endothelial cells.
   2. Remove the supernatant carefully.
      NOTE: At this stage, two isolated cell types can be purified—one endothelial cell (see step 4.5), and a mix of different types of brain cells. At this point, two options are available.
      1. Remove and discard the supernatant (i.e., the mixture of brain cells) while keeping the tube on the magnet. Wash the bead-bound cells (i.e., CD31⁺ cells) with 2 mL of Hank's solution and repeat 3x.
      2. Use the supernatant containing cellular debris to generate a new culture containing unselected cells (e.g., fibroblasts, astrocytes, microglia, neurons, pericytes, among others).
5. Culturing purified BMECs
   1. Resuspend the bead-bound endothelial cells in 7 mL of complete endothelial medium (10% FBS).
   2. Seed the cells onto a 60 mm plate previously coated with collagen overnight (see section 2). Incubate under standard conditions (37 °C, 5% CO₂).
      NOTE: The endothelial phenotype is preserved up to passage (p) 3 under the described culture conditions. To maintain blood–brain barrier–related properties, experimental assays are recommended to be performed using early passages (p0–p3).

5. Cell characterization

1. Microscopic observation of bead-captured cells
   NOTE: Under the culture conditions described in this protocol, primary BMECs can typically be maintained for up to three passages (p3) while preserving their characteristic endothelial morphology and phenotype. Depending on seeding density and growth rate, cultures can generally be maintained for up to approximately 2–3 weeks in vitro before significant phenotypic changes may occur.
   1. After immunoselection with CD31 magnetic beads, observe the attached cells under a phase-contrast microscope (Figure 2A).
      1. Look for CD31-positive endothelial cells exhibiting a typical cobblestone morphology and forming homogeneous monolayers within 5–7 days.
         ​NOTE: During immunoselection, CD31-coated magnetic beads bind specifically to endothelial cells, thereby enriching the endothelial population. After the selection process, the beads (approximately 4.5 µm in diameter) may still be observed associated with or internalized by endothelial cells during the early passages. These typically decrease with subsequent washes and culture maintenance and do not interfere with BMEC viability or cell growth (Figure 2B).
      2. Use the uniform morphology and absence of elongated or astrocyte-like cells to estimate the purity of the endothelial population.
2. Immunofluorescence for CD31 and GLUT1
   1. Seed 2 × 10³ BMECs on collagen-coated coverslips and culture until reaching 80–90% confluence.
   2. Fix the cells with 4% paraformaldehyde for 10 min at room temperature (22–25 °C) and wash with PBS.
   3. Permeabilize with 0.1% Triton X-100 for 5 min and block non-specific binding with 5% BSA for 30 min.
   4. Incubate overnight at 4 °C with primary antibodies against CD31 and GLUT1 (glucose transporter specific for brain endothelium).
   5. Wash with PBS and incubate for 1 h with the appropriate fluorescent secondary antibodies.
   6. Counterstain nuclei with 4′,6-diamidino-2-phenylindole (DAPI) and mount the coverslips with antifade mounting medium.
   7. Visualize under a fluorescence or confocal microscope. Look for CD31 localized at cell borders (Figure 3A), while GLUT1 shows cytoplasmic and membrane distribution, confirming endothelial identity and brain endothelium phenotype (Figure 3B).
      NOTE: Representative images from independent BMEC isolations demonstrate the typical morphology and marker expression of cultured BMECs. The presence of tight junction proteins, such as claudin-5, can also be used to characterize the brain endothelium. To further assess the purity of the endothelial cultures, additional immunofluorescence staining can be performed using negative markers for other neural cell types. For example, Glial fibrillary acidic protein (GFAP) may be used to detect astrocytes, and Ionized calcium-binding adapter molecule 1 (Iba1) to identify microglial cells. The absence of staining for these markers confirms the lack of contamination from these non-endothelial cell populations (Figure 3C,D).
3. Tube formation assay
   NOTE: Endothelial cells exhibit proangiogenic capacity in the Matrigel assays¹⁸, and endothelial identity can be confirmed using this assay.
   1. Precoat a 96-well plate with Matrigel and allow it to polymerize for 30 min at 37 °C.
   2. Seed 1 × 10⁴ BMECs per well in 100 µL of complete endothelial medium (10% FBS). Incubate at 37 °C and monitor tube formation over 4–8 h using a phase-contrast microscope (Figure 4A).
   3. Quantify total tube length, number of junctions, and meshes using ImageJ (Angiogenesis Analyzer plugin)¹⁹. (Figure 4B).
      NOTE: The ability of BMECs to form interconnected tubular structures confirms their angiogenic potential and endothelial functionality.
4. Measurement of transendothelial electrical resistance (TEER)
   NOTE: Brain endothelial cells must form a tight barrier, as they are a critical component of the BBB²⁰. The endothelial capacity to generate an electrical barrier needs to be confirmed. 
   1. Seed 20,000 BMECs on collagen-coated Transwell inserts (pore size 0.4 µm) in the upper compartment and culture until they reach confluence.
   2. Measure TEER using an epithelial voltohmmeter to assess monolayer integrity. Record TEER values daily until stabilization; an increase in resistance indicates the formation of tight intercellular junctions, characteristic of functional endothelial barriers (Figure 4C).
      NOTE: Remnants of the non-selected cell mixture, including astrocytes, can be used to generate a coculture system. This configuration requires careful optimization of the cell ratio between the upper (BMECs) and lower (non-selected cell mix) compartments, which should be standardized in each laboratory. This coculture configuration was adapted from previously described BBB models²¹.
   3. Seed the non-selected cell mix (step 4.4.2.2) in the bottom well of the culture plate and BMECs on the upper side of the Transwell insert membrane.
      NOTE: A representative experiment comparing monoculture and coculture conditions was included to evaluate barrier tightness by TEER measurements (Figure 4C).

6. Expansion and maintenance of purified BMECs

1. Culture conditions
   1. Maintain the purified BMECs in complete endothelial culture medium (10% FBS) at 37 °C in a humidified incubator (95% humidity) with 5% CO₂. Change the culture medium every 2–3 days to provide fresh nutrients and maintain cell viability.
2. Monitoring cell growth and morphology
   1. Observe the cultures daily under a phase-contrast microscope to monitor cell attachment, growth, and morphology. Look for the characteristic cobblestone morphology of BMECs, which form a confluent monolayer within 5–7 days.
3. Subculturing and passage
   1. When cultures reach 80–90% confluence, detach cells using 0.05–0.1% trypsin for 3–5 min at 37 °C.
   2. Neutralize the trypsin with an equal volume of complete medium, centrifuge at 220 × g. for 10 min at 4 °C, and resuspend the pellet in fresh endothelial medium.
   3. Seed the cells onto collagen-coated plates at the desired density for continued expansion.
      NOTE: Limit BMEC passages to 3–5 to maintain endothelial phenotype and functional integrity.
4. Optional cryopreservation
   1. For long-term storage, resuspend BMECs in freezing medium (10% DMSO in complete endothelial medium). Freeze gradually to –80 °C in a controlled-rate freezer, then transfer to liquid nitrogen for long-term storage.

Results

The protocol described here enables the isolation and culture of primary BMECs from neonatal mice (P5). These cells are the main cellular component of the BBB and are therefore a valuable experimental tool for studying cerebrovascular physiology and pathophysiology. Successful isolation and culture of brain microvascular endothelial cells (BMECs) were demonstrated through morphological, phenotypic, and functional assessments.

The isolation workflow resulted in a cell suspension derived from di...

Discussion

The isolation of BMECs is an instrumental tool for studying the mechanisms that regulate the physiology of the BBB and its dysfunction in various central nervous system (CNS) pathologies^4,9, including stroke. Unlike immortalized cell lines, primary BMEC cultures preserve key phenotypic and functional features of the brain endothelium, including the expression of tightly bound proteins, and an angiogenic capacity representative of the in vivo. context

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
60 mm culture plates	Corning	430166
96 Well culture plates	Corning	41122107
Acetic acid	Sigma-Aldrich	A6283
Antibiotic-Antimycotic (optional)	Gibco	15240062
Anti-CD31	Santa Cruz	sc-46694	1:100
Anti-GFAP	Thermo Fisher Scientific	13-0300	1:500
Anti-GLUT1	Abcam	ab115730	1:200
Anti-Iba1	Santa Cruz	sc-32725	1:500
Anti-mouse IgG Alexa Fluor (594 Dye)	Thermo Fisher Scientific	A-11005	1:500
Anti-rabbit IgG Alexa Fluor  (488 Dye)	Thermo Fisher Scientific	A-11008	1:500
Automatic and serological pipettes	Oxford LP	OXL.OXL.OSP-5C
Basal medium (for collagen solution)	Gibco (DMEM)	11965092
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A9647
Collagenase type II	Sigma-Aldrich	C6885
DAPI	Thermo Fisher Scientific	62248	1 µg/mL
DMEM (high glucose)	Thermo Fisher Scientific	11965092
EndoGRO-MV Complete Medium	Merck Millipore	SCME004	Endothelial cell growth medium
Falcon tubes (15 & 50 mL)	Corning®	352096 / 352070
Fetal Bovine Serum (FBS)	Gibco (Thermo Fisher)	16000044
Fluorescence Microscope	Motic	BA410
Gloves, masks, lab consumables	Various suppliers	—
Heparin (100 U/mL)	Sigma-Aldrich	H3393
Inverted Microscope	Olympus	CKX41
Magnetic separation magnet	EasySep	NC9284020
Orbital Shaker (37 °C)	Thermo Fisher Scientific	SK-0180-S
Parafilm	Bemis	PM996
PBS (1×)	Thermo Fisher Scientific	10010023
Refrigerated Centrifuge	HITACHI	CT5RE
Scalpel	Fine Science Tools	Various
Scissors and fine tweezers	Fine Science Tools	Various
Sterile nitrocellulose membrane	BIO-RAD	1620094
Type I collagen	Corning	30394