µSiM Sampling Method for Small Molecule Permeability

Some notes for researchers prior to beginning experimentation: The goal of this experiment is to determine the concentration of a fluorescent small molecule that passes a cell layer and enters the bottom channel of the µSiM. This enables us to determine permeability of the cells. To sample consistent volumes and sufficiently clear all dye from the channel, we add a 50 µl reservoir to one port, and draw a 50 µl sample from the opposite port. See this post and this post for details on optimising and validating this process. See this post for theoretical underpinnings of our measurements and calculations.

When we add the reservoir to one port, fluid will often passively pump across the channel and begin to come out of the opposite port. To contain this sample and prevent from spilling, use a hydrophobic pen or tape around the sampling port.

Method

1. Prior to experiment, take pictures of all the membrane windows for measurements of surface area.
2. Seed cells in mod-µSiMs and grow to desired confluency. Prepare a coated-control without cells to isolate endothelial permeability from system permeability.
3. Prepare fluorescent small molecule solution.
4. Add 20 µl fresh media to the bottom channel of all devices.
5. For those using a hydrophobic pen (eg. Millipore Sigma PAP pen for immunostaining, Ref: Z672548, 2 mm tip, or Dako Pen, Ref: S2002, 5 mm tip), draw a half circle around the sampling port prior to dye addition. This will give the hydrophobic ink time to dry. Make sure the device is dry around the port first. If media is pooled around the port, pipet it off and dab with a kimwipe or let it air dry a few minutes. Avoid drawing over the port. Leave excess space between the pen and port. 
6. Start experiment: Add 100 µl of desired fluorescent small molecule at the desired concentration in medium to the top well of mod-µSiM devices. Include a cell-free, coated control.
7. Incubate at 37°C, 5% CO₂ for 1 hr.
   1. For those using hydrophobic tape, a few minutes prior to the 1 hr timepoint, add tape to the sampling port. We recommend 3M tape (467MP 200MP) and a biopsy punch to make 2 mm holes. You can see the media gathering in the tape with the addition of the reservoir (right). 
8. Following incubation, sample media from the bottom channel and transfer to a black, flat bottomed 96-well plate. To sample from the channel, perform the following steps:
   1. Remove dye from the top well of all devices to stop the diffusive process.
   2. Draw 50 µl media into a pipet tip and lodge into the reservoir port of one µSiM.
   3. Add 50 µl media back into the top well of the same device to prevent cell loss (only if performing downstream analysis).
   4. Fit an empty pipet tip into the sampling port and draw out 50 µl from of the channel. Add to a black, flat bottomed 96-well plate.
      1. If passive pumping occurs and there is media coming out of the sampling port, be sure to pipet that sample as you are fitting the tip into the port, as this is where most of the dye will be. Try to avoid pipetting air, to keep the volume added to the plate at 50 µl. See videos of Sampling Method below (bullet 9) for technique.
      2. There will likely be remnant media in the reservoir tip. This does not mean you did not sample 50 µl, but is rather media displacement from the space the tip takes up in the port.
   5. You may need to stagger dye addition, to prevent cells from drying after dye removal. We recommend working in groups of three.
9. Sampling Method Movie (Hydrophobic Pen)
10. Prepare a reference ladder with the fluorescent small molecule. Make from same mix used for µSiMs, to ensure measurements fall on standard curve. Transfer reference samples in 50 μl aliquots in triplicate to the 96-well plate.
11. Measure fluorescence intensity at the appropriate Ex/Em for dye used in a microplate reader.
12. Calculate total permeability using the following equation:where [A]A is the concentration of fluorescent small molecule in the abluminal chamber (bottom channel, determined by matching to linear reference ladder), V is the volume sampled and added to plate (0.050 ml), [A]L is the concentration of fluorescent small molecule added to the luminal chamber (top well), t is time of incubation (in either seconds or min depending on desired units), and S is the membrane surface area (usually 0.014 cm2 but this should be measured using ImageJ, as variability in membrane area has been seen and significantly affects calculated permeabilities).
    1. [A]A is calculated from the a linear fit to the reference ladder. Make sure to only use the portion of the standard curve that is linear. For experimental samples, use the equation to solve for concentration (x), from the sample’s fluorescence intensity (y).
13. Calculate endothelial permeability (P_e) using the following equation:where PS is the system permeability as calculated above and PC is system permeability calculated across the coated-control device.