Our most important design criteria, based on ideal aspects of current models perfusion flow chamber models:

1. Support Cell Culture

– Because perfusion flow chambers are used to study the shear stress and/or the tensile strain placed on cells in the human cardiovascular system, our design must be able to support cell culture. We will focus on vascular endothelial cells, and incorporate controls on temperature, pH, and humidity to simulate ideal conditions. (2)

-In order to properly support our cell cultures, we must consider what type of cell media will be used in our culture plates,  as well as what specific type of cell we will be growing in our culture

-In our consideration of cell cultures, we looked at RPMI medium and DMEM which is a minimal essential medium for culturing cells. Ultimately we chose to use DMEM with 10% Fetal Bovine Serum because it is the most common choice used to culture endothelial cells, it is the same density as water, and Fetal Bovine Serum is a low-cost supplement to promote cell growth (1).

-In our consideration of cell types,  we looked at three types: Bovine Arterial Endothelial Cells (6), Human Arterial Endothelial Cells (6), and Human Umbilical Vascular Endothelial Cells. After reviewing these options, it became clear that Human Umbilical Vascular Endothelial Cells (7) were the best choice because they are commonly used in endothelial cell cultures (8), they were the least expensive option at $350/flask, and they would still accurately replicate in vivo conditions.

 

2. Achieve Shear Stress & Tensile Strain 

– In vivo, vascular endothelial cells are subject to both shear stress from blood flow and tensile strain from conditions such as blood pressure. It is important that this perfusion flow chamber mimics both at once since it more closely mimics in vivo conditions. The device will ideally simulate consistent distributions of these forces on the cultured cells.(4)

-To achieve shear stress, the flow of culture media through the chamber must have a laminar flow profile (5). This can be determined by calculating the velocity of the media being disbursed by the pump, and later the Reynolds number of the fluid.

-To achieve tensile strain the PDMS flexible membrane will be pulled uniaxially by magnets that will be bonded on both sides of the membrane. The optimal range in percent strain change of the sample is 2-15% (4) and the amount of force that would be needed to achieve the extremes of this range can be calculated using the Young’s modulus of PDMS.

3. New Approach: An Open System

– In an open system, fluid flows through the chamber once and new fluid flows in from a source.

– Many current models are either open systems without flexible membranes, or closed systems with flexible membranes. Both successfully induce cells to similar stresses and strains endured in the human body, but a model has yet to include a flexible membrane and be an open system.

-Our device is compatible with a peristaltic pump, which will circulate the culture media through the system.

 

Testing: There are some important computational and mechanical tests that can be run on our system to verify its function.

– Computation Modeling of Flow Distribution: 

– In order to verify that the flow through our device subjects cell culture to adequate shear stress, we could computationally model the flow distribution to verify numerical predictions through programs such as ADINA or a 3D FSI model (4). These techniques are rather common in areas such as microfluidics.

– Mechanical Testing: Percent Deformation of PDMS

– Another important aspect is to test the strength of the PDMS membrane, so as to not rip it. Our team has decided that we could do so by first curing the PDMS at a thickness of 50 micrometers, marking it with a dot of ink and then stretching it to just before its breaking point. The width of the distorted dot can then be measured just before this  point, thereby quantifying the attainable percent deformation of the membrane.

– Mechanical Testing: Magnetic Strength

– Even though we have a known value of neodymium magnet strength, it is important to mechanically test the strength of the magnetic adherence to the structure attached to the PDMS membrane to provide a  tensile strain. To do so, we can simply attach the stacked magnets to the PDMS by depositing a layer of chromium, the target metal of the neodymium magnets, SU-8, and finally uncured PDMS onto a glass substrate (9). After baking the layers together, the PDMS could then be carefully peeled off the glass substrate to be put into the device.

 

– Mechanical Testing: Vibration from closure

– We plan to utilizing clasps to seal our chamber closed. The ‘clicking’ of the clasps into the necessary divots on the chambers base may cause a slight vibration of the chamber. However, current studies indicate that only significant vibratory motions may disrupt the culture. (10)

 

 

 

 

 

 

References:

 

  1. Arora, M. (2013). Cell culture media: A review. Mater methods, 3(175), 24.
  2. Bacabac, Rommel. Et al. “Dynamic Shear Stress in Parallel-Plate Flow Chambers.” Journal of Biomechanics. (2005): 159-167. Web.<https://www.academia.edu/17864064/Dynamic_shear_stress_in_parallel-plate_flow_chambers>
  3. Burkhart, Collin, et al. (2015) . A Dynamic Cell Culturing System. Rochester Institute of Technology.
  4. Meza, Daphne, et al. (2016, March 1). A Shearing-Stretching Device That Can Apply Physiological Fluid Shear Stress and Cyclic Stretch Concurrently to Endothelial Cells. Retrieved from https://asmedigitalcollection.asme.org/biomechanical/article-abstract/138/3/031007/370473/A-Shearing-Stretching-Device-That-Can-Apply?redirectedFrom=fulltext
  5. Ruel, J., Lemay, J., Dumas, G., Doillon, C., & Charara, J. (1995). Development of a parallel plate flow chamber for studying cell behavior under pulsatile flow. ASAIO Journal , 41(4), 876–883.
  6. Grabowski, E. F., & McDonnell, S. L. (1993). Human versus bovine endothelial cell culture on glass and tissue-culture plastic. Journal of tissue culture methods, 15(4), 190-198.
  7. Wang, Y. X., Xiang, C., Liu, B., Zhu, Y., Liu, S. T., & Qin, K. R. (2016). A multi-component parallel-plate flow chamber system for studying the effect of exercise-induced wall shear stress on endothelial cells. Biomedical engineering online, 15(2), 154.
  8. Jutila, M. A., Walcheck, B., Bargatze, R., & Palecanda, A. (2007). Measurement of neutrophil adhesion under conditions mimicking blood flow. In Neutrophil Methods and Protocols (pp.239-256). Humana Press.
  9. Koh, D., Wang, A., Schneider, P., Bosinski, B., & Oh, K. W. (2017). Introduction of a Chemical-Free Metal PDMS Thermal Bonding for Fabrication of Flexible Electrode by Metal Transfer onto PDMS. Micromachines, 8(9). https://doi.org/10.3390/mi8090280

  10. Wang, I. J. and F.-R. Hu (1997). “Effect of shaking on corneal endothelial preservation.” Current Eye Research 16(11): 1111-1118.