Chapter 4: Combinatorial Cell Assay Device on Glass with Membrane-Based Cell
4.2 Experimental
4.2.1 Device Design
of Parylene C (poly(chloro-p-xylylene)) and eliminated the need for the bonding steps as in many other multilayer microfluidic devices. The device presented in this chapter is fabricated based on the extension of such procedure. We have characterized concentration distribution of each output from the combinatorial mixer by using a fluorescence method. Two quantitative cell-based assays were performed using this device. We cultured rat neuronal cells, B-35, on this device and screened for the ability of three chemicals to attenuate cell death caused by cytotoxic hydrogen peroxide. We also assayed for the combinatorial effects of three chemotherapeutic compound exposures on human breast cancer carcinoma, MDA-MB-231.
4.2 Experimental
Figure 4-1. Schematics of the microfluidic device. (Left) 3-D rendition of the device before assembly. (Right) Side view of the device.
Figure 4-2 shows the layout of the 1.7 cm × 1.7 cm 3-D microfluidic Parylene- based chip with integrated combinatorial mixer. The combinatorial mixer has three inputs, which are recombined into the seven possible outputs. An unmixed control channel is also included. Like the chip described in the previous chapter, the overpass structure allows one microfluidic stream to cross over other microchannels, and a control channel that receives none of the three inputs is included. Unlike the previous chip, glass wafer will be used as the substrate instead of silicon. The chamber diameter is 1 millimeter. The 3-D microfluidic Parylene chip is consisted of two different layers of microfluidic network, with the bottom 14 µm height channels making up the basic combinatorial mixer channels and the top 25 µm height channels making up the overpasses and the outlet channels.
Figure 4-2. Layout of the 3-D microfluidic Parylene-based chip.
Compounds A, B, and C are delivered into eight chambers with different combinations and concentrations, and the concentration of each compound inside the chamber is determined by the fluidic resistance of the channels. As the channel width is much larger than the channel height, the resistance can be modeled using the simplified formula for rectangular channel:
3
12 wh R L
. (4.2)
R is the resistance, µ is the fluid viscosity, and L, w, and h are the length, width and height of the channel, respectively. Chambers receiving multiple compounds are designed to have equal dilution of the input compounds. For example, chamber 2 is designed to be 50% of input A and 50% of input B so the channel connecting input A and chamber 2 (channel AII) and the channel connecting input B and chamber 2 (channel BI) would have the same fluid resistance. As the channel heights are the same for all the channels in the combinatorial mixer, the channel widths and lengths are tuned to achieve
the desired flow resistance. The fluidic channels can be represented as network of fluidic resistors and the schematic is shown in Figure 4-3.
Figure 4-3. Equivalent fluidic resistance network of the chip.
In addition, the fluidic networks are designed so the flow rate into each chamber is the same to prevent variation in shear stress that can affect the cells. This is done by making the combined fluid resistances leading up to each chamber to be the same. Thus, the fluidic resistances of channels, AI, BII, CIV, and N, which lead to chambers with single compounds, would be the same. The fluidic resistances of channels leading to chambers with two compounds are twice of the fluid resistances of channels leading to chambers with single compounds. For example, channels AII and BI, which connect input A to chamber 2 and input B to chamber 2, respectively, would have twice the fluidic resistance of channel AI. The fluidic resistances of channels leading to chambers with three compounds are three times of the fluid resistances of channels leading to chambers with
single compounds. The flow rate, Q is related to the pressure drop, ∆P, and fluidic resistance, R, by the following equation,
R
Q P. (4.3)
With a constant flow rate source (syringe pump) delivering a flow rate, QO, into each input, Q1, the flow rate into chamber 1, which receives a single compound, would be
] 1 [
] 1 [
1
TOTAL I O
A R
A Q R
Q . (4.4)
R[ATOTAL] is defined by the following equation:
] [
1 ] [
1 ] [
1 ] [
1 ] [
1
IV III
II I
TOTAL R A R A R A R A
A
R . (4.5)
R represents the resistance and the name inside the square bracket indicates the specific channel. For example, R[AI] is the fluidic resistance for channel AI. The expressions for
] [BTOTAL
R and R[CTOTAL] follow same expression as equation 4.5.
Q2, the flow rate into chamber 2, would be the combined flow rate of flow through channel AII and channel BI:
] 1 [
] 1 [ ]
1 [
] 1 [
2
TOTAL I O
TOTAL II O
B R
B Q R
A R
A Q R
Q . (4.6)
As stated above,
] 3 [
] 1 2 [
] 1 2 [ ] 1 2 [
] 1
[AI R AII R BI R AIV R AIII
R
] [ ] [ ] [ ]
[A R B R C R N
R I II IV
] [ ] [ ] [ ] [ ] [ ]
[AII R AIV R BI R BIV RCII RCIII
R
] [ ] [ ]
[AIII R BIII RCI
R
] [
1 ]
[ 1 ]
[ 1
TOTAL TOTAL
TOTAL R B R C
A
R
Thus, the two chambers, chamber 1 and chamber 2 would receive the same flow rate of 3/7QO. The designs for all the other channels follow the same principle so the flow rates into all the chambers would be equal and the chambers receiving two or more inputs would have equal flow rate from each of the connecting channels.
Figure 4-4 shows the layout of the 2.5 cm × 2.5 cm PDMS microfluidic chip with 100 µm height channels. It has punched holes that match the locations of the ports on the Parylene microfluidic chip. The PDMS piece also has a cell loading inlet and a one-to- eight manifold that distributes cells or reagents into eight chambers simultaneously. In addition, the chamber locations of the PDMS chip align to the locations of the culture chambers of the Parylene microfluidic chip. At the cell culture chamber area, a 0.5 cm × 1.5 cm transparent PET (polyethylene terephthalate) membrane with 0.4 or 1.0 µm diameter pores is sandwiched in between those two chips, and the membrane separates the culture chamber into two levels. During the device operation, the cells will be loaded via the PDMS channels, and the membrane allows cells to be efficiently collected, eliminating the need to coat the microfluidic channels with cell attachment factors.
During the cell assay experiments, cells will be maintained at the cell culture level, on top of the membrane and treatment solution will be delivered via the fluid delivery level, at the bottom of the membrane. To expose all chambers to a common fluid solution such as during washing or staining the cells, fluid can be injected via the cell loading inlet or the outlet ports. For treating cells under combinatorial conditions, the fluids will be injected into combinatorial mixer inlets. The chamber for cell culture on the PDMS chip is 100
µm height and 600 µm in diameter and the chamber on the Parylene chip is 90 µm height and 1,000 µm in diameter. This results in a total volume of 100 nL. As typical cell culture has cell density range from 104 to 107 cells/mL, this would translate to about 1 to 1,000 cells per chamber. Typical cell culture procedure calls for changing of media every two to three days, so cells within the chambers under that density range should have sufficient nutrients to grow in two to three days without culture media change.
Figure 4-4. Layout of the PDMS microfluidic chip.