BASIC Integration: Cell Culture and Electrochemical Lysis
Cultured cells are used in a variety of contexts ranging from high throughput screening of drugs to systems biology. Microfluidic devices for cell culture studies offer numerous advantages over plate-based cell culture, and because of this, are being increasingly used in laboratory settings. Microfluidic devices can provide physiologically relevant microenvironments by allowing for constant perfusion and 3D tissue-like structure. Additionally, time and costs are reduced due to decreased reagent volumes and automated handling. Higher surface-to-volume ratios can also offer improved and novel detection schemes.
Fig 1: Integrated microfluidic cell culture and lysis on a chip. (a) A top view of the chip with six separate devices (filled with dye for visualization). (b) A magnified image of one chamber showing the trapping region structure which consists of an array of four cell traps separated by spacers. Electrodes are on either side of the trapping region, which is preceded by a high resistance, or pinched, section. Scale bars are 1.3 mm.
Existing microfluidic systems for cell-based lysate studies require the addition of lysis buffers and subsequent washing steps, increasing the complexity of such devices and reducing their ease of use. We have developed an integrated microfluidic cell analysis system that allows for continuous perfusion cell culture with on-demand cell lysis. Lysis is achieved by applying a DC voltage to electrochemically generate hydroxide inside the device. This lysis method differs from other electrical lysis techniques. Rather than relying on high electric fields to irreversibly electroporate the cells, electrochemically generated hydroxide ions permanently disrupt the cellular membrane by cleaving fatty acid groups, thereby releasing intracellular material.
Fig 2: (a) Schematic of how the chip is loaded using a four way valve connected to a syringe with cells and a syringe with media or buffer. (b) Schematic of electrochemical lysis via hydroxide generation at the cathode upstream of the cell traps.
By combining two BASICs (Biological Application Specific Integrated Circuits) previously developed in our lab, we introduce an integrated cell analysis package that minimizes the need for external reagents and manual procedures. Here we demonstrate the practical use of this device by examining the culture and lysis of 4 different cell lines (HeLa, Jurkat, CHO-K1, and MCF-7). Additionally, we investigate the effects of this lysis technique on two biological molecules, horseradish peroxidase (HRP) and p53. HRP is an enzyme (derived from the plant of the same name) that is oft-used in molecular biology. P53, the 1993 molecule of the year, is a transcription factor that plays a central role in many cancer mechanisms. We show that the immunodetection of p53 is not compromised by the lysis procedure within the device. The enzymatic activity of HRP is diminished as applied DC voltage increases. However, we also show that it is possible to lyse cells using a voltage with minimal effect on HRP enzymatic activity. Given the many applications that require a combination of cell culture and lysis, we believe this integration of microfluidic devices is a valuable advancement in the field of biological research and diagnostics.
Fig 3: (a) Enzymatic activity of HRP. HRP solution exposed to 0, 1, 1.5, 2, 2.5, 3, 4, 5, and 6 V. Error bars show standard deviation for triplicate experiments. (b) Immunological capture activity of p53: p53 solution exposed to 0, 1, 2, 3, and 6 V. The "Off" refers to a negative control that was never passed through the chip. Inset image is a magnified data point, with error bars showing standard deviation from triplicate experiments. The significance of these two plots is that it may be possible to perform highly efficient immunodetection on chip because the lysis conditions can produce immunodetectable proteins while simultaneously reducing the activity of enzymes. The reduction of protease activity could greatly enhance the quality of results.
Video 1 HeLa cells loading into a single trap. This video is shown in real time, and illustrates that the traps can be completely filled with cells to form a tight three dimensional pack.
Video 2 CHO-K1 cells loading into a single trap. This video is shown in real time. Notice that the cells initially fill in the edges of the trap before filling in completely.
Video 3 Time lapse video of MCF-7 cells cultured over 72 hours. The cells form a spheroid within the cell chamber which is indicative of good cell viability. The device was placed on a heated stage (WIS1, Carel) and the video was recorded using a small inverted microscope (MIC-D, Olympus).
Video 4 A video of HeLa cells being lysed. The lysing electrodes are the large black structures at the left and right edges of the image. A voltage of 2.6V was used for lysis.
Video 5 A video of CHO-K1 cells being lysed under a voltage of 2.6V.
Video 6 Another video of CHO-K1 cells being lysed under a voltage of 2.6V, where these cells are sandwiched underneath the thin section of the cell trap. These cells are confined underneath this 2 µm gap between the glass and PDMS. The blebbing of the cells during lysis is especially apparent because of this confinement.
Video 7 A video of HeLa cells being lysed. These cells were transfected to produce intracellular GFP. The cells that express this protein show up as green spots in these fluorescent images. The GFP can be seen to disappear as the cells are lysed and the membrane is compromised.
Video 8 A video of CHO-K1 cells being lysed. These cells have a membrane protein that is attached to GFP (green), and the nuclear material is stained with Hoescht (blue). This lysis video demonstrates that the cellular material leaks out of the cell and moves downstream (flow is from left to right) upon lysis. The fluorescently labelled material can be seen as streaks moving downstream, indicating that cellular proteins and genetic material can be found in the lysate.
JT Nevill, R Cooper, M Dueck, DN Breslauer, and LP Lee. "Integrated microfluidic cell culture and lysis on a chip," Lab Chip, 2007, 7, 1689-1695.