Matthew Usevitch and Alonzo Cook, Department of Chemical Engineering
The purpose of this project is to further understand the function of extruded polytetrafluoroethylene (ePTFE) membranes in the pursuit of creating an implantable, removable pancreas device. This device would be filled with pancreas cells and automatically regulate blood sugar for patients with diabetes. Dr. Alonzo Cook’s lab is currently pursuing understanding the process of taking a patient’s own cells, reprogramming them into stem cells, and then differentiating them into pancreas cells. The device that is the end goal of this project is the main foreseen use of these differentiated pancreas cells. This project focuses on studying how essential nutrients diffuse through the ePTFE membrane as well as whether the cells themselves can migrate across.
The initial idea to conduct the diffusion analysis was to use a Boyden Chamber design, as given in the application report. This consists of a membrane-separated well as the figure shows. The membrane (“Filter” in Figure 1) separates the top and bottom chambers and diffusion can be conducted and measured. This idea was modified to work better with the tube shape of the membranes used. In this design (Figure 2), the membrane’s initial shape and the diffusion design of having two chambers separated by a membrane is retained. Small plastic caps are used to seal the ends of the tubes. The inside of the tube is filled with a concentrated solution and then allowed to diffuse out.
Initially the ePTFE membranes are water-impermeable and must be chemically modified to allow diffusion. To do this, a process used by Gore Technologies1 was adopted and modified as follows:
- Immerse in 100% isopropyl alcohol for 1 min
- Transfer to hot (boiling) 2% polyvinyl alcohol / de-ionized (DI) water for 30 min
- Rinse in DI water for 10 min
- Place in 2% glutaraldehyde / 1% hydrochloric acid – DI water solution. Leave overnight
- Rinse in DI water for 15 min
- Air dry.
For the glucose diffusion testing, test solutions were created by dissolving D-glucose into DI water. After producing a calibration curve to account for the difference between measuring a test solution concentration rather than glucose, it was determined that concentrations of 500 and 1500 mg/dL should be used to mimic typical high and low blood glucose concentrations. The tubes were filled with .3 μL of solution and the surrounding microcentrifuge tube was filled with 1.2 mL of DI water to mimic a 1:5 dilution. After waiting 15 minutes, the glucose concentration of the surrounding solution was measured.
The initial glucose diffusion testing yielded the following results with the three different densities of ePTFE membranes. A general increasing trend is shown on the low concentration test. However, the errors experienced during the high concentration test indicate that more testing should be performed to validate these results.
This data confirms the hypothesis that the membrane is permeable to glucose. In the low glucose test, the data seem to show a correlation between the membrane density and glucose diffused, however the errors experienced in the high concentration test indicate that more testing needs to be performed to validate these results.
Due to difficulties in finding a way to measure insulin concentrations, this part of the testing has not been accomplished at this time. The protocol will mimic that of the glucose testing, with the exception of using Essential 8 media in replacement of the DI water as a solvent. It is anticipated that the concentrations will be measured by radioimmunoassay (RIA) with the assistance of Dr. Jeffery Tessem in the Nutrition department who routinely does these measurements. In addition, the cell diffusion test will be accomplished using the assistance of a cell culture chamber slide system. With the assistance of Dr. Tessem, pancreatic β-cells will be grown and then loaded with media into the tube devices used for the diffusion assay. These will then be laid on cell culture chamber slides, left to allow time for migration, then stained and analyzed under a microscope to ascertain if migration happened.
This project has been an important first step to understanding the nature of ePTFE membranes. The first part of the hypothesis that the membranes will be glucose has been confirmed, and later work will help to show whether this holds true for insulin and whether cells will be able to migrate through the membrane. To further this research, the migration analysis tests could be carried out with cells that are known to be migratory to confirm the results.
1US Patent 5,843,069 – Implantable Containment Apparatus for a Therapeutical Device and Method for Loading and Reloading the Device Therein