Design of an integrated microfluidic system for culturing patterned tissue

Allyson Fry1, Bryan Gorman1, Jonathan Lin2, William Wong1

Advisors: John Wikswo1, Dmitry Markov1, Lisa McCawley3, Phil Samson1

Departments of 1Biomedical Engineering, 2Electrical Engineering, and 3Cancer Biology at Vanderbilt University.

Introduction Characterization of tissue culture model

AcknowledgementsJason Greene contributed suggestions for device design and experimental testing. This project is a continuation of a senior design project started in 2004-2005 by Barnett, Garrett, Harvill, Mayer, and McClintock. This project was made possible by use of resources provided by the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) directed by Dr. John Wikswo. Dr. Paul King is the course instructor for BME 272.

A Two-layer SU-8 photolithography mold

Detect pHCompare pH value with desired pH

range

Outside of range?

Process error using PID equation

C(t) = P +I + D

Change pumping rate of high pH

pump

Do not change pumping rates

YES

Find magnitude of error e(t)

Change pumping rate of low pH

pump by equal and opposite amount

Microfluidic pH control in LabVIEW

Changes in pH are detected by a pH sensor and processed by a computer. Appropriate commands are then sent to the syringe pumps to compensate for these changes by altering pumping rates. Overall flow rates are kept constant by close coordination between the changes in pumping rates for each pump.

Bioreactors are commonly used biotechnology equipment in which small organisms and other biological components are grown. In this project, a bioreactor is being used to grow a three dimensional organotypic cell culture comprised of primary endothelial cells and fibroblasts embedded in a collagen matrix. We have shown that this tissue culture model produces a capillary network. One of the main goals of this project is to show that the growth of this capillary network can be guided using a Matrix Enabled Capillary Scaffold (MECS). Perfusion is then introduced using a microfluidic network so that the cultured cells can receive nutrients and be rid of wastes in a controlled environment. The flow of this perfusion network is monitored for pH content with a LabView control system.

Overall Bioreactor DesignThe inspiration for the scaffold design is the fractal pattern of vasculature occurring in nature. Cells grown in fractal flows are exposed to similar nutrient, waste, and shear stress levels. The purpose of the bioreactor is to add another spatial dimension to transwell plate cocultures. In this design, a scaffold is seeded with endothelial cells and placed between two layers of collagen seeded with fibroblasts. Parallel supply networks above and below perfuse the tissue culture with nutrients via diffusion through supporting nano-pore filters.

Polycarbonate Filter

Lower collagen and fibroblast layer

Upper collagen and fibroblast layer

Arrows point to endothelial tubes. For reference, the scaffold is 10 um in thickness.

The Proportional, Integral, Differential (PID) control theory equation is used to control the pumps. All three terms contribute to the resulting control signal using different pieces of information. The proportional term (P) takes into account the immediate presence of error. The integral term (I) helps compensate for persistent, steady-state error. The differential term (D) compensates for trends in error.

Cells Media needed (ul/cell/hr)

Flow rates (ul/hr) Volume of bioreactor

(ul)

Desired pH range

45,000 4e-4 5-20 1-10 7.2-7.6

An image of the PDMS scaffold in cross-section

An organotypic culture is any cell culture that mimics the in vivo tissue in form and function. The organotypic culture in use in this project is adapted from a model used by Manuela Martins-Green at University of California-Riverside1. Our cultures contain two layers of human dermal fibroblasts embedded in a collagen matrix and separated by a layer of human microvascular endothelial cells. This culture is constructed in a transwell chamber in a normal 12-well culture plate. The culture is fed by placing media below the chamber, allowing it to perfuse upward to the culture through a polycarbonate membrane, as well as being placed on top of the entire culture and seeping downward.

In order to study the functions and mechanisms of a system, scientists commonly use animal models such as mice, rats, and rabbits. However this practice has several shortcomings. First, the use of animals for research is fraught with ethical implications concerning the worth of animal life. Second, although animal studies can provide a reasonable basis to answer vital questions, results can never full predict the response of human tissue to treatment. Finally, an animal system is relatively difficult to manipulate, and often much of the control must be done on the molecular level by reconstructing the genome. The manipulations frequently cannot carry over from one study to another, leading to a redesigning of the entire model. Our project overcomes these shortcomings by creating a completely human model, with many levels of control, and easy adaptability.

Social Impact

• Model a complete microvascular network• Support cellular processes• Allow evaluation of molecular mechanisms• Provide environment for endothelial cell differentiation• Facilitate formation of stable tubular structures• Support a flow of perfusate

Project Goals:

Perfusion of the bioreactor occurs by a fluidics system that both injects media to support the cells, and also maintains stable pH and oxygen levels. Two syringe pumps inject low- and high-pH RPMI media into separate flow lines that unite and feed fluid through an oxygenator. After passing through the bioreactor, the flow line is split and fed into two dedicated output syringes mounted onto the same pumps as the input syringes, so as not to re-use any fluid. Changes in pH are detected by a pH sensor and processed by computer. Appropriate commands are then sent to the syringe pumps to raise or lower pH while keeping flow rate constant.

Fabrication of scaffolds

Physical constraints for pH control:

The tissue culture model used as the basis for the design of the bioreactor and for assessing the

angioconductive ability of scaffold designs

Endothelial tubes as seen using H&E staining on a developed tissue culture with no scaffold added.

Cost and market analysisThe appeal of this product will be the high level of microenvironment sophistication and control, relative to current products.  The cost will also be relatively low.  Although the pump, sensors, and computer hardware will likely cost over $10000 alone, they represent a one-time purchase.  However, the plastic microfluidic chips are disposable and can be batch-produced off a single mold for less than several dollars each. 

Dimensions of the PDMS egg-crate scaffold

Integration of scaffolds into tissue cultureEndothelial cells were seeded into the three different tissue culture scaffolds, which were then placed between two layers of collagen interspersed with fibroblasts using the organ culture model characterized above. Three trials using each type scaffold were conducted. The results are forthcoming. Based on these results the ability of each type of scaffold to induce endothelial alignment and vascular tube formation will be assessed with iimmunofluorescence and confocal imaging.

We have demonstrated the fabrication of three different types of tissue culture scaffold:

1. The PDMS ‘Egg-crate’ scaffold1. Advantage: supports arterial and venous

branches in rigid network2. Disadvantages: difficult to construct, not

biodegradable2. The patterned Matrigel scaffold

1. Advantage: biodegradable, easy fabrication

2. Disadvtantage: poor mechanical features3. The SU-8 Lift-off scaffold

1. Advantage: good mechanical features, easy fabrication

2. Disadvantage: poor biocompatibility

In conclusion, we have characterized an angiogenic tissue culture model, fabricated three types of capillary scaffolds, and tested those scaffolds directly into the tissue culture model. Additionally, we have written pH control software in LabVIEW and set up a system for controlling the pH of a microenvironmnent. Now, we must make experimental measurements of pH control and analyze the results of the scaffold-patterned tissue cultures.

Conclusion

1. Martins-Green M, Li QJ, and Yao M. FASEB 2005; 19:222-224.

An 80 um PDMS scaffold resting on a glass slide

NO