Fabrication of Porous PDMS Thin Films as a Microfluidic Blood Brain Barrier!Nathaniel M. Braman1, Lucas H. Hofmeister1,2, Virginia Pensabene3, David K. Schaffer3, Lino Costa4,5, Christina Marasco1, John Wikswo1,3.!1Systems Biology and Bioengineering Undergraduate Research Experience, Vanderbilt University. 2Combinatorial Biomaterials and Biointerface Laboratory, Vanderbilt University. 3Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University. 4Center for Laser Applications, University of Tennessee Space Institute, Tullahoma, TN. 5 Department of Materials Science and Engineering, University of Tennessee, Knoxville, TN.!

Testing Hexane Dilution  

References & Acknowledgements  

Porous PDMS Membranes using PDMS Compression Molds  

Porous PDMS Membranes Spun Onto Sacrificial Needle Substrates  

Porous H2O-in-PDMS Emulsion Membranes   Conclusions and Future Directions  Introduction  

The development of a microfluidic Brain-on-a-Chip (BoC) device that accurately models the environment of the human brain would significantly improve design of central nervous system drugs and enhance understanding of brain physiology. A major challenge presented by the BoC is recreating the blood-brain barrier (BBB) in a microfluidic environment. In this study, we explore the fabrication of porous spin-coated PDMS membranes suitable for use as a microfluidic blood-brain barrier (μBBB). !  

Optimizing Thickness  

The BoC (F+ig. 1) consists of 2 compartments: a section seeded with glial cells that models the brain (a,b), and a vascular layer seeded with endothelial cells (ECs) simulating the brain’s capillaries (d). A microfluidic BBB membrane (μBBB) (c), a porous interface only a few μm thick, sits at the junction of the two and is responsible for facilitating the formation and operation of a BBB within the BoC. !!Polydimethylsiloxane (PDMS) possesses numerous advantages for μBBB fabrication. Most significantly, the membrane can be bonded directly into the PDMS BoC, making a PDMS μBBB easier to handle and tightly seal between layers of the device.!!

This study focused on the manipulation of two crucial μBBB attributes in PDMS thin films:!!

Thickness. A μBBB membrane must be thick enough to support the cells of the BBB, yet still allow the signalling between layers necessary for barrier formation. PDMS alone cannot be spun sufficiently thin due to its high viscosity. Dilution of PDMS pre-polymer with hexane is a well-established and effective method of thinning PDMS thin films.1,2 The relationship between hexane dilution and film thickness was investigated to optimize films to a target thickness of 1-3 μm, the ideal thickness of the μBBB. !"

Porosity. The size of the μBBB’s pores must be finely tuned to to allow cell communication while still providing support and promoting the high selectivity of the BBB. Three distinct methods of generating pores within spun PDMS-hexane thin films were explored in hopes of creating membranes with consistent pores at a target size of 2-3 microns. !

Nonporous PDMS thin films were produced at a variety of hexane dilutions to characterize the relationship of hexane dilution and film thickness. (Fig. 2, a) Poly-vinyl alcohol (PVA) (green) and hexane-diluted PDMS (red) were spun onto silicon. PDMS-coated wafers were cured on a hot plate, evaporating hexane and thinning the PDMS layer. (b) Samples were bonded to PDMS blocks for ease of handling. (c) Samples were cut along the border of PDMS blocks. (d) Samples were placed in deionized water to dissolve the underlying PVA layer and separate membranes ]from silicon. Thickness was measured using a profilometer.!

Fig. 2. Nonporous thin film production. !

•  Thickness measurements were averaged and used to construct a hexane dilution curve (Fig. 3). !

•  Two dilutions tested, 80% (2.56 ± 0.40 μm) and 90% hexane (1.11 ± 0.30 μm), yielded thicknesses in the target range of 1-3 μm.!

•  A dilution of 80% hexane was chosen for porous membrane production, as at 90% hexane membranes tore easily.!

Fig. 3. Film thickness at various hexane dilutions. All films were spun at 4000 RPM for one minute. "

Fig. 4. (a&b) Membrane with a H20-SDS:PDMS mass ratio of .25, diluted to 50% hexane. (c,d) Membrane with a H20-SDS:PDMS ratio of .10, diluted to 80% hexane. Scale bars indicate (a) 1.00 mm, (b) 150 μm, (c) 231 μm, and (d) 300 μm. !

•  1:100 H2O:sodium dodecyl sulfate (SDS) solution was vigorously mixed into PDMS-hexane in various quantities and spun. !

•  This technique proved to be ineffective. !

•  At a dilution of 50% hexane (Fig. 4, a&b), Pores were overly large and c lustered near membrane edges. !

•  A t 8 0 % h e x a n e ( c & d ) , membranes possessed zero pores. "

Fig. 4. (a-c) SU-8 post arrays were patterned onto silicon using s tandard pho to l i t hog raphy technique. (d) PDMS was cast over post arrays. (e) PDMS was separated from master, inverted, and silanized to form a secondary mold. (f) PDMS was cast over secondary mold. Compression molds bearing original post array were cut f rom PDMS and silanized. "

Fig. 5. (a) PDMS-hexane was spun onto blocks of silanized PDMS. (b) PDMS compression molds were placed atop uncured PDMS-hexane. (c) PDMS-hexane and molds were compressed. Post arrays penetrated membranes as they cured. (d) Two different compression methods were investigated. Top: two membranes and molds were placed between large glass slides and compressed at each end with binder clips. Bottom: 6-8 membranes and molds were placed between steel plates. A screw was tightened, compressing membranes. (e) PDMS compression molds were lifted from the membrane, leaving (f) cured, porous PDMS membranes. (g) Membranes were bonded to PDMS then (h) pried from silanized PDMS. !

Fig. 6. (a-f): Porous membranes created by compression with PDMS post arrays. (a-c): Membranes compressed with binder clips. (d-f): Membranes compressed with a screw clamp. (g): Pore sizes of membranes compressed with binder clips (n=2) and screw clamps (n=3).! !

Fig. 7. (a) Glass substrates were patterned with micropores using a femtosecond laser. (b) PVA was spun over substrates. (c) PVA was attached to a cover slip and inverted, forming PVA needle array substrates. (d) PDMS-hexane was spun atop PVA needles and cured. (e) Samples were bonded to the vascular layer of the BoC. (f) Membranes were soaked in water, dissolving PVA and separating membranes and cover slips. !

Fig. 8. BoC devices assembled with PDMS membranes spun onto needle substrates. !

•  Compression with a PDMS c o m p r e s s i o n m o l d successfully created pore a r rays w i t h an ove ra l l average pore size of 4.06 ± 0.80 μm (Fig. 6). !

!•  Membranes compressed

between glass slides using binder clips (Fig. 6, a-c) had a r r a y s o f w e l l - d e fi n e d circular pores, but were not compressed equally across the entire membrane. As a result, some areas were left without pores (c). !

!•  Screw clamped membranes

( d - f ) w e r e o f t e n o v e r -compressed with oval pores (d,e). Other membranes received less force and were only dimpled (f). Screw clamp pores were overall larger than pores compressed with binder clips (g). !

(g)!

0  1  2  3  4  5  6  

Binder  Clip   Screw  Clamp  

Pore  Size  (μm)  

Compression  Method  

Pore  Size  of  PDMS  Membranes  Following  Various  Methods  of  Compression    

•  Membranes were bonded into BoCs for future testing of diffusive properties!

•  Membranes transferred well f rom covers l ip to BoC, separa t ing eas i l y when soaked and forming a tight seal with the vascular layer that was free of bubbles or tears. !

•  Hexane dilution was effective in reaching optimal thickness of the μBBB. !

•  A dilution of 80% hexane was chosen for porous membrane production. !

•  Membrane production using an H2O-in-PDMS emulsion was shown to be an ineffective method of pore generation. !

•  PDMS post compression demonstrated promise as a method of μBBB fabrication. It produced pore arrays of consistent size, shape, and spacing. !

•  Neither of the compression methods used delivered optimal results and further investigation into new compression methods is required.!

•  Exploration into tougher polymers for compression mold fabrication may help reduce post deformation under high compression.!

•  The fabr icat ion of compression molds using photomasks with smaller slits will be investigated in hopes of achieving target pore size.!

•  Membranes spun onto sacrificial needle substrates were easily bonded from PVA substrates and incorporated into the BoC for future testing. !

•  Diffusive properties must be tested in order to compare fabrication methods, as well as determine baseline properties in an acellular BoC. !

•  Future studies will measure the permeability and molecular weight cutoff of porous membranes by measuring the diffusion of fluorescent-albumin and fluorescent-dextran. "

(a)!

(b)!

(c)!

(d)!

Fig. 1. Diagram   of   BoC  layers,   created   from   CAD  d r aw ing s   p rov i ded   b y  Virginia  Pensabene   !

Compression Mold Fabrication"

Fabrication of porous PDMS membranes using post compression"

Needle substrate fabrication and membrane production"

Nonporous membrane production"

References:!1.  A. L. Thangawng, R. S. Ruoff, M. A. Swartz, and M. R.

Glucksberg, Biomed. Microdevices, 2007, 9, 587–595.!2.  S. Wang, A. Kallur, and A. Goshu, 2011, vol. 7935, p.

79350M–79350M–6."

Acknowledgements: The project is made possible through the support of the Searle Systems Biology and Bioengineering Undergraduate Research Experience. Additionally, the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) is thanked for the use of its facilities in this study. !!Dr. Anthony Hmelo and the Vanderbilt Institute of Nanoscale Science and Engineering are thanked for providing SEM access and necessary training. Jake Brady is thanked for providing training in photolithography and soft lithography. !!Lucas Hofmesiter and Dr. Chrissy Marasco are thanked for their assistance editing this poster. The presenter specially thanks Lucas Hofmeister for his excellent mentorship throughout the course of the project. !