Plasticity of primary microglia on micropatterned geometries and spontaneous long-distance migration in microfluidic channels
- Susanna Amadio†1,
- Adele De Ninno†2, 3,
- Cinzia Montilli1,
- Luca Businaro2,
- Annamaria Gerardino2 and
- Cinzia Volonté1Email author
© Amadio et al.; licensee BioMed Central Ltd. 2013
Received: 25 March 2013
Accepted: 3 October 2013
Published: 13 October 2013
Microglia possess an elevated grade of plasticity, undergoing several structural changes based on their location and state of activation. The first step towards the comprehension of microglia’s biology and functional responses to an extremely mutable extracellular milieu, consists in discriminating the morphological features acquired by cells maintained in vitro under diverse environmental conditions. Previous work described neither primary microglia grown on artificially patterned environments which impose physical cues and constraints, nor long distance migration of microglia in vitro. To this aim, the present work exploits artificial bio-mimetic microstructured substrates with pillar-shaped or line-grating geometries fabricated on poly(dimethylsiloxane) by soft lithography, in addition to microfluidic devices, and highlights some morphological/functional characteristics of microglia which were underestimated or unknown so far.
We report that primary microglia selectively adapt to diverse microstructured substrates modifying accordingly their morphological features and behavior. On micropatterned pillar-shaped geometries, microglia appear multipolar, extend several protrusions in all directions and form distinct pseudopodia. On both micropatterned line-grating geometries and microfluidic channels, microglia extend the cytoplasm from a roundish to a stretched, flattened morphology and assume a filopodia-bearing bipolar structure. Finally, we show that in the absence of any applied chemical gradient, primary microglia spontaneously moves through microfluidic channels for a distance of up to 500 μm in approximately 12 hours, with an average speed of 0.66 μm/min.
We demonstrate an elevated grade of microglia plasticity in response to a mutable extracellular environment, thus making these cells an appealing population to be further exploited for lab on chip technologies. The development of microglia-based microstructured substrates opens the road to novel hybrid platforms for testing drugs for neuroinflammatory diseases.
KeywordsConfocal analysis Long distance migration Microglia plasticity Microfabrication Time-lapse microscopy
Microglia are part of the immune system being the resident macrophages of the brain and spinal cord, and were first discovered and defined by Pio Del Rio Hortega  as an independent cellular phenotype abundantly present in the central nervous system (CNS). Microglia are of mesodermal origin and possess an elevated grade of plasticity, undergoing a variety of structural changes based on their location and particular state of activation. Unlike other cells in the brain, they are extremely versatile and dynamic . They have the capacity to sense and adjust to the microenvironment, to migrate, proliferate and phagocytose. During early development, microglia enter the brain using vessels and white matter tracts as guiding structure for migration, and then disperse throughout the CNS occupying a defined territory . Late in development, they transform from an amoeboid morphology into a branched, ramified phenotype composed of long, constantly extending, shrinking and re-growing processes with small, fairly motionless cell bodies, known today as surveilling microglia. These shape-shifting cells constitute about 20% of the total glial cell population within the adult brain and act as the first and main form of active immune defense in the CNS, by frequently scavenging for pathogens, damaged or dead cells and misfolded proteins, but also trimming away weak or damaged synapses between neurons [4–6]. Indeed, after a pathological event, microglia respond to the environment by undergoing profound transition in morphological appearance, motility and state of activation, and reacquiring an amoeboid shape similar to the one observed early in development. This high level of plasticity is required to fulfill the vast variety of immunological functions that microglia perform, as well as for maintaining homeostasis within the brain [2, 7–12].
The first step towards the comprehension of microglia’s biology consists in discriminating if different morphological features can be acquired in vitro when microglia are cultured on diverse surface topography that are mimetic of an in vivo resembling three-dimensional (3D) environment. To this aim, we employed microstructured pillar-shaped and line-grating geometries produced on poly(dimethylsiloxane) (PDMS) by standard soft lithography techniques, and two-layer microfabrication photolithography. While the use of artificial bio-mimetic microtextured substrates and microfluidic devices  was mostly exploited for cancer cells [14–16] and neurons [17, 18], only few studies were performed so far on microglia [19, 20].
Microglia spontaneously adjust to micropatterned structures
Morphometric analysis of microglia on micropatterned structures
Morphological transition of microglia in microfluidic devices
Morphometric analysis of microglia in microfluidic devices
Primary microglia microchannels
Primary microglia microchambers
N9 cells microchannels
N9 cells microchambers
523 ± 52
1807 ± 545
487 ± 176
680 ± 141
20 ± 9
1.36 ± 0.19
1.56 ± 0.38
1.51 ± 0.34
0.08 ± 0.03
0.36 ± 0.12
0.25 ± 0.14
0.39 ± 0.2
Primary microglia spontaneously migrate in microfluidic channels
Additional file 1: The video shows baseline motility of primary microglia for long distances on artificial bio-mimetic microfluidic substrates with controlled physical/chemical characteristics. We performed regular 30 min time-lapse recording (JuLI, Digital Bio) over a 20 h period. Primary microglia spontaneously travel through fibronectin-coated PDMS microfluidic channels (width 12 μm and length 500 μm) for a distance of up to 500 μm in approximately 12 h, with an average speed of 0.66 μm/min. (WMV 3 MB)
Morphometric analysis of primary microglia migrating in microfluidic channels
Microglia consist of at least two subpopulations that coexist in the adult CNS and derive from different sources: one that originates from bone marrow-derived cells and migrates to the CNS during embryonic development for colonization of the nervous system parenchyma, the second that develops from myeloid progenitor cells and enters the brain after birth , becoming fundamental for microglial maintenance of the CNS homeostasis [6, 33]. Since adult microglia can play a twofold role, either amplifying the effects of inflammation and mediating cell degeneration, or protecting the nervous system from pathological insults , efficient chemotaxis can acquire either neuroprotective or inflammatory and detrimental roles. Whereas we are currently capable of distinguishing amoeboid-phagocytic from branched-surveilling microglia [3, 5], we are unaware of how these morphological shapes can denote a neuroprotective or neurotoxic role. In other words, it is still an open matter how the vast morphological heterogeneity existing within the mixed microglia population might relate to functional diversification.
Cell behavior strictly depends on the stiffness and shape of the microenvironment, and the response to surface topography is a very critical determinant of cell morphogenesis. Microglia respond to micro- nano-structured pits, protrusions and grooves with altered morphology, adhesion, and directional growth . In our work, we establish that primary microglia retain in vitro the intrinsic competence of modifying their structure in response to contact guidance cues. When grown on pillar-shaped substrates, microglia acquire a center-stage multi-polar morphology and develop abundant button-like pseudopodia emerging from the cell body with a nearly radial orientation. This situation might very well depict the in vivo condition of microglia not committed to migrate, but exploring the environment with short forward, backward and sideways steps, with the final purpose to orient their migration. When placed on a line-grating substrate in the presence of parallel and symmetrical grooves, microglia elicit a longitudinally flattened appearance with elongated bipolar shape. This in vitro condition might favor a polarized extension of filopodia at the leading edge of the cell, in preparation of a forward translocation of the cell body, with retraction of the rear of the cell . The adjustment of microglia to a line-grating geometry is thus highly suggestive of commitment to follow a straight path in vivo. Thus, surface topography can induce a synchronous and homogeneous metamorphosis of microglia in vitro, distinguishing the center-stage multipolar (on micropatterned pillars) from the elongated bipolar (on line-grating micropatterns) cells. With the use of specific markers and cytoskeleton-perturbing drugs, future work will aim to establish if these subpopulations can be correlated to: a) microglia generated from bone marrow rather than from myeloid progenitor cells; b) microglia colonizing the CNS during development, rather than maintaining CNS homeostasis in adulthood; c) more importantly, “beneficial” rather than “detrimental” microglia. Moreover, future analysis of parameters such as cell adhesion, distribution of actin cytoskeleton and microtubules, phagocytosis, antigen presenting power, beneficial or detrimental chemokine/cytokine expression and release, will allow to determine the functional identity of multipolar versus bipolar microglia.
Moreover, by the use of bio-mimetic 3D microfluidic substrates, our work demonstrates that primary microglia appear intrinsically different from immortalized N9 cells. During cell polarization inside the microchannels, primary microglia tend to align longitudinally and individually, whereas immortalized N9 microglia assume a more compact morphology with the propensity to establish cell contacts. This might be reminiscent of the immortalization program that likely confers to N9 cells a compact morphology optimizing cell division and contact formation, rather than the aptitude of primary cells to explore the environment with a direction-oriented polarization. In both cases, specific receptors and structural molecules are found enriched respectively at the cell-to-cell surface (N9 cells) or leading edges (primary microglia), as evinced by increased phalloidin and P2Y12 receptor signals at these sites.
Cell adhesion and morphology are dynamically mutable during cell migration . The last issue that we addressed in our work is thus the free motility of microglia in culture. While numerous publications cite motility and short migration of microglia in vivo, only few works describe microglia moving for long distances. This is the case for example of the work by Carbonell and coauthors , demonstrating by intracerebroventricular injection of rhodamine and time-lapse confocal microscopy that subventricular microglia at the interface of the cerebrospinal fluid and brain parenchyma, exhibit the “in situ” ability to migrate for several hundred microns into the parenchyma, towards a deafferentation injury of the hippocampus. Conversely, Nimmerjahn and colleagues indicate only static movement of microglia, branch motility, but not cell bodies migration . Using transcranial two photon microscopy, Davalos and coauthors  confirm that only microglial processes are highly dynamic in intact brain, reaching up to several micrometers in length, or retracting until they completely disappear. This baseline dynamism of microglial processes is thus in sharp contrast to the stability of the microglial cell bodies and surrounding neuronal processes .
In vitro studies on this subject have instead shown only transwell device infiltration directed by a chemical gradient. Lee and Chung  describe that ADP stimulates chemotaxis of immortalized BV2 microglia from the upper to the lower side of the transwell membrane. Karlstetter and co-authors  present evidence that curcumin inhibits basal and LPS-induced relocation of BV2 microglia from the upper to the lower transwell membrane surface . However this passage across a physical barrier limits the possibility of investigating long distance migrations and, moreover, provides only indirect evaluation of kinetic parameters. Honda and collaborators  using the Dunn chemotaxis chambers report that primary rat microglia have weak motility in the absence of ligands, but perform a mean displacement of about 50 μm in the presence of ATP or ADP. Nasu-Tada et al.  confirm that primary microglia are almost static in the absence of stimulants, but show chemotactic responsiveness to ADP with a maximal displacement of about 140 μm. Finally, Haynes and coauthors  prove that the leading edge of mouse primary microglia moves for a maximal displacement of 50 and 120 μm after 30 minutes, in a gradient of ATP or ADP, respectively. However, the average distance spontaneously migrated in the absence of any provided stimulus is only 0.8 μm.
All considered, spontaneous long distance migration of microglia is described in vivo with conflicting results, but never in vitro until now. Here, we demonstrate that biocompatible polimeric channels constitute a more suited environment than transwell devices or Dunn chemotaxis chambers for permitting and measuring spontaneous long distance migration in vitro. Indeed, the 500 μm traveled by primary microglia inside the microfluidic channels in the absence of stimuli are much above the known average distance so far reported and this achievement might be perhaps improved.
In summary, by the use of interdisciplinary techniques, our data indicate that microfluidic technologies and microstructured patterns with control over the presentation of adhesion sites, are able to sort out different morphological structures within a mixed microglia population and allow free motility in vitro. By reproducing a specialized niche for the cells, these technologies can help to understand the role of structural determinants in priming morphogenesis and free motility of microglia and may be exploited for translational research on functional tissue engineering and implantable device design.
With our work we have shown that a strict control over biomaterial surface topography by soft lithography techniques can highly impact on microglia morphology and greatly improve spontaneous motility in vitro. The use of microstructured 3D devices to manipulate microglia is thus of interest to scientists working on the mechanisms of cell substrate/matrix interactions, morphogenesis and migration, and particularly on microglia responses and functions also during neurodevelopmental and neuroinflammatory disorders. The advantage of using primary microglia in microfluidics and micropatterning opens the road to the use of these devices for testing drug candidates for CNS neuroinflammatory diseases.
All reagents for cell culture are obtained from Sigma-Aldrich, unless otherwise stated. The culture media DMEM and DMEM-F12 are acquired from Invitrogen. Fetal bovine serum (FBS) is obtained from Gibco.
Fabrication and preparation of microstructured PDMS substrates
Microtopographic silicon masters are realized to replica-mold substrates on PDMS (Sylgard 184, Dow Corning) with line-grating and pillar-shaped geometries. The micropatterns dimensions in both cases are width: 1500 nm, pitch: 3 μm, height: 550 nm. The fabrication process is started with the patterning of PMMA by 100 kV e-beam lithography on a Si substrate. A 20 nm Cr film is evaporated by electron gun followed by lift-off process in acetone at 50°C and sonication. Samples are then etched up to 550 nm by Reactive Ion Etching using CHF3, O2, SF6 and Ar gas mixtures. After Cr wet etching, Si substrates are thermally oxidized at 950°C for 2 h in O2 atmosphere to reduce surface roughness and then immersed in the hydrofluoric acid buffer to remove the thin oxide layer. After cleaning in Piranha solution (H2SO4/H2O2, 3:1), microstructured Si wafers are silanized with 10% trimethylchlorosilane in toluene in N2 environment to generate a low-energy surface and facilitate the subsequent mold-PDMS separation. The micropatterns are reproduced on PDMS by standard soft lithographic techniques. A 10:1 (v/v) mixture of monomer and curing agent is prepared and diluted in a 5% (v/v) solution in n-heptane, in order to lower the viscosity. The mixture spin-coated onto the Si master mold is left undisturbed for 2–3 h to allow the solution to penetrate into the voids of the master and for solvent evaporation. Then, it is cured for 30 min at 60°C. Onto this thin PDMS layer, a liquid prepolymer of Sylgard 184 PDMS 10:1 (v/v) is poured and degassed. Then, it is reticulated on a hotplate for 1 h at 60°C and peeled off from the mold. Micropatterned PDMS samples are plasma oxidized and kept in water before cell culture, in order to produce and maintain a hydrophilic surface.
Fabrication and design of microfluidic devices for real-time cell analysis
Standard soft lithography procedures are performed to fabricate all microdevices in PDMS, a biocompatible thermo-curable elastomer [40, 41]. The microfluidic device features two cell culture compartments (1 mm wide, 7 mm in length and 100 μm high) connected via a set of micron-size channels each with dimensions: width = 12 or 18 μm (depending on the experiment), length = 500 μm, height = 10 μm (Figure 4). The circular wells (8 mm in diameter) serve as loading inlets and cell medium reservoirs for nutrient and gas exchange. The design configuration was adapted from Hosmane and collaborators , optimizing microchannel dimensions to the microglia physical scale. The master molds are realized by two-layer microfabrication process using the negative photoresist SU-8 (MicroChem Corp, Newton, MA). Briefly, patterns for standard photolithography are designed with CAD software and transferred on two chrome masks by electron-beam lithography. Silicon wafers are spin coated with a layer of SU-8 3005 at a rate of 1000 rpm (resist thickness 10 μm), pre-baked at 95°C for 3 min, exposed to a i-line (365 nm) UV light source for 16 s through a photo mask (with the microchannel pattern and alignment marks) and post-baked (1 min at 65°C, 3 min at 95°C). Next, the second photolithography step on Su-8 3050 (100 μm thick) transfers the chamber areas and reservoirs aligned to the first pattern (pre-bake: 45 min at 95°C, exposure time: 18 seconds, post-bake: 1 min at 65°C, 5 min at 95°C). PDMS (Sylgard 184, Dow Corning) chips are obtained by replica molding, casting the prepolimer base and cross-linker at the volume ratio of 10:1, over the patterned master template. After degassing for 30 minutes in a vacuum chamber the PDMS is allowed to polymerize at 120°C on a hotplate for 1 h. Once cross-linked, it is carefully released from the mold and then fluidic access ports are created using a suite of 8 mm dermal biopsy punch tools (Kai Medical). To form an irreversible bonding, the surfaces of PDMS replica and microscope glass slides are O2 plasma-activated (Oxford Plasma Lab 80 plus, RF Power: 20 W, Flux: 60 sccm, Pressure: 700 mtorr, Time: 30 s) and put in contact immediately after exposure. Assembled devices are post-baked at 70°C for 2 h to complete and enhance adhesion strength. Before plasma treatment bonding, microscope glass slide (52 mm × 76 mm, Menzel-Glaser) are cleaned in Piranha solution (H2SO4/H2O2 3:1), rinsed in DI water and dried with N2 gun to remove debris and other surface contaminants.
Coating of micropatterned structures with fibronectin
After UV sterilization for 20 min, the micropatterned devices are kept immersed in a solution of 10 μg/ml fibronectin (Sigma-Aldrich) for 1 h at room temperature, followed by three washes with sterile water.
Coating of microfluidic devices with fibronectin
After UV sterilization for 20 min, the coating of microfluidic devices is performed according to Park and coauthors . Each reservoir is loaded with 100 μl of 10 μg/ml fibronectin (Sigma-Aldrich) and the entire device is allowed to be filled. The coating solution is kept for 1 h at room temperature and three washes are performed after removal of the fibronectin solution. Homogeneity of fibronectin coating is verified by indirect immunofluorescence, in the presence of anti-fibronectin (Calbiochem) used at a dilution of 1:100 in phosphate buffer saline (PBS, 24 h at 4°C), followed by rabbit anti-goat rhodamine conjugated antiserum (3 h at 24°C). Confocal analysis is performed (as described below), and the profile of fluorescence intensity is obtained with the Zen software of Zeiss LSM 700 microscope.
Primary cortical microglia
All animal procedures have been performed according to the European Guidelines for the use of animals in research (86/609/CEE) and the requirements of Italian laws (D.L. 116/92). The ethical procedure has been approved by the Animal Welfare Office, Department of Public Health and Veterinary, Nutrition and Food Safety, General Management of Animal Care and Veterinary Drugs of the Italian Ministry of Health. All efforts were made to minimize animal suffering and to use the number of animals only necessary to produce reliable results. Primary microglial cultures are prepared from 1 to 2 day-old rat and mouse, as previously described by Chen and collaborators . In brief, after removing the meninges, cortices are minced and digested with 0.01% trypsin and 10 μg/ml DNase I. After dissociation and passage through 70-μm nylon cell strainer (BD Biosciences Europe), cells are resuspended in DMEM medium supplemented with 20% heat-inactivated FBS, 4 mM glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml gentamicin and plated in T75 poly-D-lysine-coated flasks, at about 10 million cells/flask. The cultures are kept at 37°C in a 5% CO2 and 95% air atmosphere. Every 2–3 days, the medium is entirely changed for the next 12-days. At about 14 days after plating, mixed glial cultures are shaken at 200 rpm at 37°C for 1 hour. The microglial cells are collected from each flask and plated at different density on microfluidic devices or micropatterned supports coated with 10 μg/ml fibronectin. A population 99% pure of microglial cells is obtained as verified by immunofluorescence with GFAP (for astrocytes), NeuN (for neurons), NG2 (for oligodendrocytes) and CD11b clone OX42 (for microglia).
Microglial N9 cell line
The murine N9 microglia cell line is grown in DMEM-F12 medium supplemented with 10% heat-inactivated FBS, 4 mM glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin and 100 μg/ml gentamicin. The N9 microglia is kept at 37°C in a 5% CO2 and 95% air atmosphere.
Loading of the cells in the microfluidic devices is performed mainly according to Park and coauthors . Briefly, the cell device is maintained filled with 200 μl of culture medium for 1 h in a humidified incubator, before plating the cells. The medium is then removed from the reservoirs and 1×104 cells are immediately seeded. The device is kept in the incubator for 15–20 min to allow cell adhesion and, after replacement of fresh media in the reservoirs, the device is then maintained at 37°C in a 5% CO2 and 95% air atmosphere, until used. Plating density is set in the range of 125 cells/mm2.
Immunofluorescence using micropatterned structures
Microglia are washed three times with PBS, fixed with 4% paraformaldehyde for 20 min, washed, permeabilized with 0.05-0.1% Triton X-100 for 10 min, rinsed, blocked for 30 min in 1% PBS/BSA (bovine serum albumin), and stained with 5 μg/ml Cy2-phalloidin (Sigma-Aldrich) alone, or in combination with the primary antiserum against P2Y12 receptor (Anaspec) used at 1:100 diluition, in 1% PBS/BSA, for about 3 h at 37°C, or stained with anti-paxillin (BD Biosciences) used at 1:1000. The secondary antibodies used for double labeling are Cy3-conjugated donkey anti-rabbit IgG (1:100, Jackson Immunoresearch) or Cy2-conjugated donkey anti-mouse IgG (1:100, Jackson Immunoresearch). The cells are then extensively washed and stained with the nucleic acid blue dye, Hoechst 33342 (1:1000). After rinsing, the cells are covered with gel/mount™ anti-fading medium (Biomeda Corporation) and a coverslip. Incorporated fluorescence is analyzed by means of a fluorescence microscope (Olympus BX51). Brightness and contrast of digital images are adjusted using Microsoft Office PowerPoint 2007.
Immunofluorescence using microfluidic devices
Microglia is washed three times by loading each reservoir with 200 μl PBS. Cells are then fixed with 4% paraformaldehyde for 20 min, washed, permeabilized with 0.05-0.1% Triton X-100 for 15 min, rinsed, blocked for 30 min in 1% PBS/BSA, and stained with 5 μg/ml Cy2-phalloidin (Sigma-Aldrich), alone or in combination with the primary antiserum against P2Y12 receptor (Anaspec) used at 1:100 dilution, or with IBA1 (Wako Chemicals GmbH) at 1:200, in 1% PBS/BSA, for 24 h at 4°C. The secondary antibody used for double immunofluorescence is Cy3-conjugated donkey anti-rabbit IgG (1:100, Jackson Immunoresearch). Finally, the cells labeled with IBA1 are also extensively washed and allowed to incorporate the nucleic acid blue dye, Hoechst 33342 (1:1000).
After rinsing of the cells, double or triple incorporated immunofluorescence is analyzed by means of a confocal laser scanning microscope (LSM 700, Zeiss) equipped with four laser lines: 405 nm, 488 nm, 561 nm and 639 nm. The brightness and contrast of the digital images are adjusted using Microsoft Office PowerPoint 2007.
Primary microglia are recorded with objective 4× at regular time-lapse (30 min) over a 20 h period, with a bright-field microscope equipped by a time-lapse system (JuLI, Digital Bio). The images are captured with CMOS 1.3 M pixels camera and exported as TIFF files.
Fluorescence microscopy images are imported into Matlab, converted into binary 8-bit images and processed by a code developed according to Kozlowsky and Weimer . The images are then subjected to quantitative analysis of morphometric parameters. Tracking analysis of time-lapse microphotographs is performed by Image J manual tracking plugin. The generated tracking data are used to calculate microglia motility parameters. Audio Video Interleave (AVI) files are generated by using the entire time-lapse image sequence with Image J software (Additional file 1).
The Mann–Whitney test is used for non-parametric analysis of differences between groups. P <0.05 is considered statistically significant.
Bovine serum albumin
Central nervous system
Fetal bovine serum
Cell perimeter length
Phosphate buffer saline
We thank Dr. N. D’Ambrosi and Dr. S. Apolloni for useful advice and help with primary cultures preparation. This study was supported by grant from Ministero della Salute (GR-2009-1523273).
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