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In this study, we hypothesized that the sensor system could be used for non-invasive real-time monitoring of microglial metabolism to further elucidate the level of polarization of specific phenotypes in dynamic microenvironments
.
We designed and fabricated a microfluidic chip for culturing microglia under continuous flow and correlated changes in oxygen and pH with polarization states upon activation
.
Inflammation is a complex series of events triggered by stimuli such as foreign antigens, pathogens, and chemicals
.
It plays a key role in a variety of diseases, such as autoimmune diseases, cancer and wound healing processes [1]
.
Macrophages and microglia play important roles in inflammation and neuroinflammation, respectively, due to their antigen presentation, phagocytosis, and immunomodulatory properties [2]
.
Microglia, as a subtype of macrophages, also function like macrophages in neuroimmune responses [3]
.
Depending on the signals they are exposed to in the physiological microenvironment, microglia can transform into two distinct phenotypes, termed M1 and M2 [4]
.
Pro-inflammatory M1 microglia, also known as "classically activated", can be induced by lipopolysaccharide (LPS), while IL-4 and IL-13 induce an "alternately activated" anti-inflammatory M2 phenotype [5,6]
.
Metabolic changes have been shown to occur in macrophages and microglia in response to stimulation of the M1 phenotype
.
During LPS stimulation, the production of nitric oxide (NO) inhibits oxidative phosphorylation, causing the cell to switch to glycolysis
.
Due to this reaction, lactate production increases, which in turn increases the extracellular acidification rate (ECAR)
.
Monitoring pH changes can provide insight into the physiological and metabolic states of cells and tissues [7]
.
The polarized state of microglia can often be detected by gene expression, cell surface markers, and proteins released into the culture medium or accumulated in the extracellular matrix
.
PCR, flow cytometry, ELISA and Western Blotting are among the most common methods in this regard, which are invasive, laborious and time-consuming
.
These techniques also cannot provide insight and real-time monitoring of changes in polarization states
.
In vitro experiments
.
Microfluidic organ-on-chip systems provide an excellent in vitro platform to simulate complex organs/tissues by providing heterogeneous cell populations with a 3D layered architecture with dynamic culture models
.
Neuroinflammation models of diseases with complex etiologies have been reported previously, further highlighting the advantages of bioengineered microenvironments [8]
.
Materials and Methods The microfluidic chip was made of polydimethylsiloxane (PDMS) because of its flexibility, facilitating insertion of sensor plugs and sealing leaks
.
To fabricate the PDMS components of the chip, PDMS was cast on polymethyl methacrylate (PMMA) molds with a 1:10 mixture of curing agent and PDMS, respectively
.
After degassing under vacuum, bake the mold at 80 ºC for one hour, then cut pieces from the mold
.
Chip A is a hybrid chip that includes both PMMA and PDMS components
.
The bottom of the chip contains a channel for 3D culture and fluid flow with a PDMS layer on top of these layers with inlet, outlet and sensor plug ports
.
An additional layer of PMMA surrounds the chip, providing structural support for the cable, facilitating compression of the bolt
.
All these layers are held together with the help of 6 bolts (Fig.
1C)
.
Chip B has two layers
.
After soft lithography, the fabricated PDMS components were plasma bonded to microscope slides
.
After bonding, the chips were placed on a hot plate at 120°C for 2 hours to enhance bond strength
.
Figure 1: Images of two differently fabricated microfluidic chips for 3D and 2D microglia polarization
.
Design of chip A for 3D cell culture: A) AutoCAD drawing, dimensions in [mm], B) layers of chip A from top to bottom, C) chip with blue dye and sensor plug dummy Image of A, Chip B designed for 2D cell culture: D) Dimensions of Chip B, E) Number of layers of Chip B, F) Image of Chip B with red dye and sensor plug dummy
.
The N9 mouse microglia cell line was used for macrophage polarization experiments
.
Cell expansion was performed using complete RPMI (phenol-free) medium 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Pen/Strep)
.
Cells were grown to 80% confluency and harvested with 0.
25% trypsin-EDTA
.
Ethanol-sterilized chips were seeded with high cell density (4.
5 x 10 5 cells/cm 2 ) and incubated overnight at 37 °C and 5% CO 2 under static conditions
.
Once the microglia adhered and formed a monolayer culture, they were administered using a syringe pump (Harvard Instruments) at a continuous flow of 0.
5 µl/min (Figure 2)
.
Microglia were polarized to an M1-like phenotype with 300 ng/ml lipopolysaccharide (LPS) for 24 hours
.
Unstimulated cells were used as a control throughout the study
.
Place the microfluidic chip in the cell incubator and connect the sensor plug to the fiber optic cable
.
Collect oxygen and pH sensor measurements for 24 hours
.
During data acquisition, the incubator was not opened to avoid any gas and temperature fluctuations
.
Figure 2: Experimental setup: A) image of sensor connected to fiber optic instrument and computer, B) image of syringe pump and chip, C) chip inside incubator and connected fiber optic cable
.
Figure 2: Experimental setup: A) image of sensor connected to fiber optic instrument and computer, B) image of syringe pump and chip, C) chip inside incubator and connected fiber optic cable
.
After 24 hours of dynamic culture in the microfluidic chip, lysis buffer containing 15 µM dithiothreitol (DTT) was pipetted into the channel to lyse cells
.
Lysates were collected and stored at -80°C for RNA isolation
.
RNA was isolated using the Macherey-Nagel RNA isolation kit
.
The control and M1 groups were compared based on their TNFα, IL-6 and iNOS gene expression levels by qPCR analysis
.
The supernatant collected from the outlet of the microfluidic chip during the 24 h incubation was used for ELISA
.
The Affymetrix eBioscience Mouse TNFα Kit was used according to the manufacturer's instructions
.
To validate our sensor readings and confirm M1 polarization, we determined the levels of TNFα released in the control and M1 groups
.
Results Dissolved oxygen showed a decrease in LPS-stimulated microglia and unstimulated controls
.
Although there were similar trends in both conditions, oxygen levels in M1-polarized microglia were lower than in controls during the 24-h stimulation period
.
For LPS-stimulated cells, the dissolved oxygen levels of the control were reduced by 22.
2% and 29.
5% (Fig.
3A)
.
In the pH monitoring chip, pH levels dropped over 24 hours with a final value of 6.
8 for the control and 6.
5 for the LPS-stimulated cells
.
Figure 3: Sensor data of O2 and pH profiles during 24 h stimulation
.
A) O 2 % change in control and LPS-stimulated cells, B) pH change in control and LPS-stimulated cells
.
Gene expression analysis of TNFα, IL-6, and iNOS at the end of 24 hours showed significant fold changes, confirming M1 polarization of LPS-stimulated microglia under dynamic microfluidic culture
.
ELISA assay showed a significant increase in TNFα cytokine release from LPS-stimulated cells (M1)
.
TNFα concentrations after 24 hours under dynamic conditions were measured as 102 pg/ml for unstimulated controls and 470 pg/ml for LPS-stimulated microglia
.
Figure 4: mRNA expression in control and LPS stimulated cells
.
A) TNFα mRNA expression in control and LPS-stimulated cells, B) IL-6 mRNA expression in control and LPS-stimulated cells, C) iNOS mRNA expression in control and LPS-stimulated cells
.
*P≤0.
05, **P≤0.
01, ***P≤0.
001
.
P-values were calculated from a two-tailed Student's t-test
.
Figure 5: Concentration levels (pg/ml) of TNFα in media collected from control and LPS-stimulated chip outlets
.
*P≤0.
05, **P≤0.
01, ***P≤0.
001
.
P-values were calculated from a two-tailed Student's t-test
.
Discussion The original purpose of this study was to observe the metabolic changes that occur in microglia during polarization in 3D culture
.
To this end, we designed and fabricated Chip A for 3D dynamic culture and stimulated cells to develop towards the M1 phenotype
.
We observed that cells in 3D cultures were not efficiently polarized compared to 2D cultures
.
We conclude that a lower number of successfully polarized cells may not be sufficient to alter overall metabolic measures
.
To demonstrate the advantages of novel sensing systems in microfluidic research, we focus on more traditional 2D cultures and discuss the results with the literature
.
Therefore, we decided to perform a two-dimensional dynamic monolayer culture within a microfluidic chip
.
These results also prompted us to change the microfluidic chip A to chip
B.
After 24 hours of stimulation, we ensured the success of M1 polarization by qPCR analysis
.
The mRNA expression levels of typical M1 status markers TNFα, IL-6 and iNOS were significantly higher than those in the control group
.
Next, we show that stimulated cells actually complete the translation process from the mRNA to the protein level
.
To this end, we measured the level of secreted TNFα protein at the outlet of the microfluidic chip by performing an ELISA
.
The pH changes are consistent with the literature: in several previous studies, M1 polarization has been shown to lead to lower pH levels because stimulated cells have higher ECAR
.
In our study, we were able to observe a much lower pH within the LPS stimulated chip compared to the control chip
.
But when we look at the initial pH of the chip, they are different
.
This may be due to bubble formation within the microchannels
.
Air bubbles affect the dissolved gas concentration within the chip medium, and dissolved gas changes the pH of the medium
.
CO 2 affects the medium by lowering the pH, on the other hand, if the medium is exposed to an oxygen-enriched gas, the pH of the medium will increase
.
This could explain the higher pH if the control medium was exposed to O2-rich bubbles
.
There is another possibility for this difference
.
It takes approximately 40 minutes to set up the experiment and start measuring
.
Cells were exposed to stimulatory factors prior to measurement initiation
.
This may be due to the fact that LPS-stimulated cells reduce the pH of the medium through the formation of iNOS after stimulation [7]
.
O levels did not match the literature: LPS stimulation of N9 microglia reduced proliferation rates and altered cellular metabolism, making it glycolysis-dependent
.
These changes in cellular metabolism result in decreased oxygen consumption, and due to cell cycle arrest [9] combined with relatively low cell numbers, measurements of dissolved oxygen levels in LPS-stimulated cells are expected to be higher than in controls and must have a tendency to increase 24 hours stimulation
.
Our results showed a 7% reduction in oxygen levels in LPS-stimulated cells
.
More experiments must be performed to better understand why this divergence from the literature occurs
.
.
We designed and fabricated a microfluidic chip for culturing microglia under continuous flow and correlated changes in oxygen and pH with polarization states upon activation
.
Inflammation is a complex series of events triggered by stimuli such as foreign antigens, pathogens, and chemicals
.
It plays a key role in a variety of diseases, such as autoimmune diseases, cancer and wound healing processes [1]
.
Macrophages and microglia play important roles in inflammation and neuroinflammation, respectively, due to their antigen presentation, phagocytosis, and immunomodulatory properties [2]
.
Microglia, as a subtype of macrophages, also function like macrophages in neuroimmune responses [3]
.
Depending on the signals they are exposed to in the physiological microenvironment, microglia can transform into two distinct phenotypes, termed M1 and M2 [4]
.
Pro-inflammatory M1 microglia, also known as "classically activated", can be induced by lipopolysaccharide (LPS), while IL-4 and IL-13 induce an "alternately activated" anti-inflammatory M2 phenotype [5,6]
.
Metabolic changes have been shown to occur in macrophages and microglia in response to stimulation of the M1 phenotype
.
During LPS stimulation, the production of nitric oxide (NO) inhibits oxidative phosphorylation, causing the cell to switch to glycolysis
.
Due to this reaction, lactate production increases, which in turn increases the extracellular acidification rate (ECAR)
.
Monitoring pH changes can provide insight into the physiological and metabolic states of cells and tissues [7]
.
The polarized state of microglia can often be detected by gene expression, cell surface markers, and proteins released into the culture medium or accumulated in the extracellular matrix
.
PCR, flow cytometry, ELISA and Western Blotting are among the most common methods in this regard, which are invasive, laborious and time-consuming
.
These techniques also cannot provide insight and real-time monitoring of changes in polarization states
.
In vitro experiments
.
Microfluidic organ-on-chip systems provide an excellent in vitro platform to simulate complex organs/tissues by providing heterogeneous cell populations with a 3D layered architecture with dynamic culture models
.
Neuroinflammation models of diseases with complex etiologies have been reported previously, further highlighting the advantages of bioengineered microenvironments [8]
.
Materials and Methods The microfluidic chip was made of polydimethylsiloxane (PDMS) because of its flexibility, facilitating insertion of sensor plugs and sealing leaks
.
To fabricate the PDMS components of the chip, PDMS was cast on polymethyl methacrylate (PMMA) molds with a 1:10 mixture of curing agent and PDMS, respectively
.
After degassing under vacuum, bake the mold at 80 ºC for one hour, then cut pieces from the mold
.
Chip A is a hybrid chip that includes both PMMA and PDMS components
.
The bottom of the chip contains a channel for 3D culture and fluid flow with a PDMS layer on top of these layers with inlet, outlet and sensor plug ports
.
An additional layer of PMMA surrounds the chip, providing structural support for the cable, facilitating compression of the bolt
.
All these layers are held together with the help of 6 bolts (Fig.
1C)
.
Chip B has two layers
.
After soft lithography, the fabricated PDMS components were plasma bonded to microscope slides
.
After bonding, the chips were placed on a hot plate at 120°C for 2 hours to enhance bond strength
.
Figure 1: Images of two differently fabricated microfluidic chips for 3D and 2D microglia polarization
.
Design of chip A for 3D cell culture: A) AutoCAD drawing, dimensions in [mm], B) layers of chip A from top to bottom, C) chip with blue dye and sensor plug dummy Image of A, Chip B designed for 2D cell culture: D) Dimensions of Chip B, E) Number of layers of Chip B, F) Image of Chip B with red dye and sensor plug dummy
.
The N9 mouse microglia cell line was used for macrophage polarization experiments
.
Cell expansion was performed using complete RPMI (phenol-free) medium 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Pen/Strep)
.
Cells were grown to 80% confluency and harvested with 0.
25% trypsin-EDTA
.
Ethanol-sterilized chips were seeded with high cell density (4.
5 x 10 5 cells/cm 2 ) and incubated overnight at 37 °C and 5% CO 2 under static conditions
.
Once the microglia adhered and formed a monolayer culture, they were administered using a syringe pump (Harvard Instruments) at a continuous flow of 0.
5 µl/min (Figure 2)
.
Microglia were polarized to an M1-like phenotype with 300 ng/ml lipopolysaccharide (LPS) for 24 hours
.
Unstimulated cells were used as a control throughout the study
.
Place the microfluidic chip in the cell incubator and connect the sensor plug to the fiber optic cable
.
Collect oxygen and pH sensor measurements for 24 hours
.
During data acquisition, the incubator was not opened to avoid any gas and temperature fluctuations
.
Figure 2: Experimental setup: A) image of sensor connected to fiber optic instrument and computer, B) image of syringe pump and chip, C) chip inside incubator and connected fiber optic cable
.
Figure 2: Experimental setup: A) image of sensor connected to fiber optic instrument and computer, B) image of syringe pump and chip, C) chip inside incubator and connected fiber optic cable
.
After 24 hours of dynamic culture in the microfluidic chip, lysis buffer containing 15 µM dithiothreitol (DTT) was pipetted into the channel to lyse cells
.
Lysates were collected and stored at -80°C for RNA isolation
.
RNA was isolated using the Macherey-Nagel RNA isolation kit
.
The control and M1 groups were compared based on their TNFα, IL-6 and iNOS gene expression levels by qPCR analysis
.
The supernatant collected from the outlet of the microfluidic chip during the 24 h incubation was used for ELISA
.
The Affymetrix eBioscience Mouse TNFα Kit was used according to the manufacturer's instructions
.
To validate our sensor readings and confirm M1 polarization, we determined the levels of TNFα released in the control and M1 groups
.
Results Dissolved oxygen showed a decrease in LPS-stimulated microglia and unstimulated controls
.
Although there were similar trends in both conditions, oxygen levels in M1-polarized microglia were lower than in controls during the 24-h stimulation period
.
For LPS-stimulated cells, the dissolved oxygen levels of the control were reduced by 22.
2% and 29.
5% (Fig.
3A)
.
In the pH monitoring chip, pH levels dropped over 24 hours with a final value of 6.
8 for the control and 6.
5 for the LPS-stimulated cells
.
Figure 3: Sensor data of O2 and pH profiles during 24 h stimulation
.
A) O 2 % change in control and LPS-stimulated cells, B) pH change in control and LPS-stimulated cells
.
Gene expression analysis of TNFα, IL-6, and iNOS at the end of 24 hours showed significant fold changes, confirming M1 polarization of LPS-stimulated microglia under dynamic microfluidic culture
.
ELISA assay showed a significant increase in TNFα cytokine release from LPS-stimulated cells (M1)
.
TNFα concentrations after 24 hours under dynamic conditions were measured as 102 pg/ml for unstimulated controls and 470 pg/ml for LPS-stimulated microglia
.
Figure 4: mRNA expression in control and LPS stimulated cells
.
A) TNFα mRNA expression in control and LPS-stimulated cells, B) IL-6 mRNA expression in control and LPS-stimulated cells, C) iNOS mRNA expression in control and LPS-stimulated cells
.
*P≤0.
05, **P≤0.
01, ***P≤0.
001
.
P-values were calculated from a two-tailed Student's t-test
.
Figure 5: Concentration levels (pg/ml) of TNFα in media collected from control and LPS-stimulated chip outlets
.
*P≤0.
05, **P≤0.
01, ***P≤0.
001
.
P-values were calculated from a two-tailed Student's t-test
.
Discussion The original purpose of this study was to observe the metabolic changes that occur in microglia during polarization in 3D culture
.
To this end, we designed and fabricated Chip A for 3D dynamic culture and stimulated cells to develop towards the M1 phenotype
.
We observed that cells in 3D cultures were not efficiently polarized compared to 2D cultures
.
We conclude that a lower number of successfully polarized cells may not be sufficient to alter overall metabolic measures
.
To demonstrate the advantages of novel sensing systems in microfluidic research, we focus on more traditional 2D cultures and discuss the results with the literature
.
Therefore, we decided to perform a two-dimensional dynamic monolayer culture within a microfluidic chip
.
These results also prompted us to change the microfluidic chip A to chip
B.
After 24 hours of stimulation, we ensured the success of M1 polarization by qPCR analysis
.
The mRNA expression levels of typical M1 status markers TNFα, IL-6 and iNOS were significantly higher than those in the control group
.
Next, we show that stimulated cells actually complete the translation process from the mRNA to the protein level
.
To this end, we measured the level of secreted TNFα protein at the outlet of the microfluidic chip by performing an ELISA
.
The pH changes are consistent with the literature: in several previous studies, M1 polarization has been shown to lead to lower pH levels because stimulated cells have higher ECAR
.
In our study, we were able to observe a much lower pH within the LPS stimulated chip compared to the control chip
.
But when we look at the initial pH of the chip, they are different
.
This may be due to bubble formation within the microchannels
.
Air bubbles affect the dissolved gas concentration within the chip medium, and dissolved gas changes the pH of the medium
.
CO 2 affects the medium by lowering the pH, on the other hand, if the medium is exposed to an oxygen-enriched gas, the pH of the medium will increase
.
This could explain the higher pH if the control medium was exposed to O2-rich bubbles
.
There is another possibility for this difference
.
It takes approximately 40 minutes to set up the experiment and start measuring
.
Cells were exposed to stimulatory factors prior to measurement initiation
.
This may be due to the fact that LPS-stimulated cells reduce the pH of the medium through the formation of iNOS after stimulation [7]
.
O levels did not match the literature: LPS stimulation of N9 microglia reduced proliferation rates and altered cellular metabolism, making it glycolysis-dependent
.
These changes in cellular metabolism result in decreased oxygen consumption, and due to cell cycle arrest [9] combined with relatively low cell numbers, measurements of dissolved oxygen levels in LPS-stimulated cells are expected to be higher than in controls and must have a tendency to increase 24 hours stimulation
.
Our results showed a 7% reduction in oxygen levels in LPS-stimulated cells
.
More experiments must be performed to better understand why this divergence from the literature occurs
.
