Neutrophil Transmigration through Collagen Gels

Intro

For the past couple months, I have been working with Tejas to recreate his previous experiments using a more complex system. The goal of the research is to study the transmigration of neutrophils and represent how they behave during chronic inflammation in vitro. To do so, we planned to use a setup that included collagen gels above and below the membrane, endothelial cells, and neutrophils. The experiments described below use a setup with gels and no endothelial cells. We have been working to identify why the neutrophils can transmigrate through the endothelial cells (as seen in the video highlighting specific cells) and not through the collagen gel.

Materials and Methods

• 2 mg/ml (old) collagen 1 gel: 10 ul 10X PBS, 1.33 ul NaOH, 30.67 ul H2O, 58 ul collagen
• 2 mg/ml (new) collagen 1 gel: 20 ul collagen, 5 ul 10X PBS, 5 ul 1N NaOH, 20 ul sterile H2O
• buffer: 238 mg HEPES, 500 mg of 5 mg/ml BSA, 100 ml HBSS
• making fMLP: 5 mg fMLP powder + 1 ml DMSO to make 10 mM stock, then 1 ul 10 mM stock = 999 ul DMSO to make 10 uM fMLP
  • The 10 uM fMLP was aliquoted into small vials of 10 ul and stored in the freezer.
• diluting fMLP: 38 uM to 100 nM –> 2.6316 ul fMLP, 997.37 ul media
• fixing: use 2.5% glutaraldehyde

Experiments

Gel concentration and chip porosity (6/1/17)

The motivation behind conducting this experiment was to see whether chip porosity and collagen gel concentration hindered neutrophil migration. The idea was that with a higher porosity chip or a lower concentration of collagen gel, the neutrophils would more easily be able to migrate through the membrane.

• neutrophil count: 11.4 million cells/ml
• 4 devices: 5% porosity with 1 mg/ml collagen (D1), 5% porosity with 2 mg/ml collagen (D2), 20% porosity with 1 mg/ml collagen (D3), and 20% porosity with 2 mg/ml collagen (D4)

Everything was previously warmed to 37 degC, including the 100 nM fMLP used. For D1, the fMLP was added to the device and placed in the incubator for 40 minutes. After that time, neutrophils were added at 10X dilution; the cells were not concentrated enough and another bolus was added. For D2, D3, and D4, fMLP was also added 40 minutes prior to the neutrophils, which were added at 5X dilution. We found that none of the neutrophils migrated through the gels.

 

Gel configuration (6/6/17-6/7/17)

Since the previous experiment showed us that chip porosity was not the factor limiting the neutrophil migration, we decided to test if the possible cross-linking between top and bottom gel was hindering the migration. In order to do so we set up multiple devices with different gel configurations – top gel, bottom gel, top and bottom gelled simultaneously, and top gel one hour before bottom gel.

• neutrophil count: 7.525 million cells/ml
• 4 devices: top and bottom gel separately (D1), top and bottom gel simultaneously (D2), bottom gel only (D3), top gel only (D4)

For D1, we used 4X dilution of neutrophils along with 100 nM fMLP that was not pre-warmed. The neutrophils were activated, but no migration was seen after 35 minutes. For D2, 3X dilution of neutrophils was used, along with an added bolus since the concentration was still low. We added 2X dilution of neutrophils to both D3 and D4 along with the 100 nM fMLP. In the end, we found only a few neutrophils on the plane of the membrane of D2; otherwise, none of the devices showed signs of neutrophil migration.

 

Gel vs. no gel (6/14)

No migration was seen during the last experiment using different gel configurations, so we decided to try using no gel on two devices to see if the membrane itself was the problem. Two devices were gelled on the top and two devices were not gelled at all. The goal was to look for neutrophil migration through the membrane and possibly through the gel.

• neutrophil count: 8.625 million cells/ml
• 4 devices: no gel and fMLP added after one hour (D1), no gel and fMLP added with neutrophils (D2), 2 mg/ml collagen gel (D3), 2 mg/ml collagen gel (D4)

D1 had neutrophils added one hour prior to the fMLP. The neutrophils were checked 10 minutes before the fMLP was added and they were not yet activated. When the 100 nM fMLP was added and incubated for 40 minutes, the neutrophils were still rounded but there were a few on the coverslip level. For D2, the neutrophils and fMLP were added at the same time and incubated.  D3 and D4 both had 4X dilution of neutrophil added along with old 100 nM fMLP. D3 was taken apart and reassembled using a new coverslip and PDMS since cells appeared to be below the coverslip layer. The old fMLP was again used and the cells appeared rounded after 30 minutes of incubation. All in all, the was no major neutrophil migration observed in the gelled devices.

 

fMLP concentration (6/16)

After multiple days of testing if the gelling was what was hindering the neutrophil migration, we decided to test if the fMLP used was activating the neutrophils and creating a gradient to induce migration. To do so, we used two different concentrations of fMLP. We mixed the fMLP in with the blood sample and smeared a drop onto a slide. The neutrophils were then observed under the microscope.

• 2 devices: 100 nM fMLP, 10 nM fMLP

The blood used was obtained from finger pricks and neutrophil counts were lower than other experiments previously conducted due to this. The fMLP was found to activate the neutrophils at both 100 nM and 10 nM.

Note: We can use the 10 nM fMLP in future work.

 

6/20/17-6/22/17

2 mg/ml gel used, top gel only for devices 1 and 2, endothelial cells added to devices and once they were confluent the flow began for one device

• neutrophil count: 11.525 million cells/ml
• 4 devices: 2 mg/ml top gel, 2 mg/ml top gel with fMLP added after one hour of incubation with neutrophils, 20% porosity with 2 mg/ml bottom gel, 2 mg/ml top gel under flow

The fMLP and media were pre-warmed to 37 degC. For device 1, neutrophils and fMLP were added simultaneously and incubated. Pictures were taken after 30 minutes and cells were found at an intermediate level that appeared to be floating. The device was flipped and the cells settled slightly above the membrane. The PDMS block was removed and we concluded that some neutrophils were on the underside (in the trench) and that the fMLP was definitely working. Device 2 was incubated for an hour with only neutrophils before any fMLP was added. Many of the neutrophils seemed slightly activated but none had migrated through the top gel. After an hour of incubation with the fMLP, the neutrophils were more highly concentrated in one corner. It was concluded that no neutrophils migrated in device 2. Device 3, using a 20% porosity chip, was gelled on the bottom. The neutrophils and fMLP were added simultaneously. Neutrophils were seen on the coverslip level after one hour of incubation. After another 30 minutes, the neutrophils were still activated, meaning the fMLP was working. There were no signs of any major neutrophil migration since there were no cells at any intermediate levels. Device 4 was put under flow and had only a top gel. The neutrophils and fMLP were added simultaneously. After both 20 minutes and 40 minutes, there were no signs of neutrophil migration. Devices 2, 3, and 4 were fixed and imaged. No neutrophil migration was observed in any of the four devices.

Note: fMLP is definitely working.

 

Collagen gel concentration (6/26/17)

After many tests involving gel configurations, we decided to further test whether the collagen gels had too high of a concentration. If the concentration was too high, the cross-linking could be too dense for neutrophils to migrate through efficiently. Tejas made gels of varying concentrations to test the lowest we could use that would still gel but allow for neutrophil migration.

• neutrophil count: 7.8 million cells/ml
• 5 devices: no gel (D0), 1 mg/ml with 100 nM fMLP (D1), 1 mg/ml with 10 nM fMLP (D2), 0.7 mg/ml (D3), 0.35 mg/ml (D4)
  • all of the devices with gel were only gelled on the top

For D0, the neutrophils and fMLP were added simultaneously. The neutrophils were observed to be activated after only five minutes. After 30 minutes of incubation, the cells were seen at the coverslip level as expected with no gel. D1, D3, and D4 were all assembled and 100 nM fMLP was added along with neutrophils. The neutrophils were checked after an hour of incubation and no sign of neutrophil migration was seen. D2, which had 10 nM fMLP added along with the neutrophils, was incubated and after an hour, no cells were found to have migrated (some were still activated though). All in all, there were no signs of neutrophil migration.

 

Gel type and media (7/19)

In the past, Tejas had used a different collagen 1 gel and it had worked for his experiments. He decided to try this gel again, as well as geltrex of an unknown concentration. The hope was that this gel, used only on the bottom side, would allow for neutrophil migration.

• neutrophil count: 3.275 million cells/ml (pellet was sticking together)
  • reconcentrated neutrophils to 8 million cells/ml
• 4 devices: two with 2 mg/ml (old) collagen gel, two with unknown concentration of geltrex (could range from 12-18 mg/ml, gels at 10 mg/ml)

The neutrophils and 20 nM fMLP were added to collagen 1 and geltrex 1 simultaneously. In col 1, after five minutes no cells were seen in the bottom channel, but the neutrophils were activated. After 40 minutes of incubation, cells were seen at intermediate levels of col 1, but not in geltrex 1. A video was taken of the intermediate cells of col 1 after two hours of incubation. Neutrophils and 20 nM fMLP were also added to collagen 2 and geltrex 2 simultaneously. After two hours of col 2 incubation, there were a few neutrophils at intermediate levels, but not close to the numbers seen in col 1. At this point, col 1 has many deactivated neutrophils, but still at many intermediate levels. In conclusion, the old 2 mg/ml collagen gel supported neutrophil migration, as seen in col 1.

 

Next step

• Build back up to using the whole system – top and bottom collagen gels, endothelial cells, and neutrophils.