UROP Proposal Spring 2014 (CTC)

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UROP Proposal Spring 2014 (CTC)

  1. 1. Microfluidic Platforms for Enrichment and Capture of Circulating Tumor Cells in Blood Student Researcher: Anthony Han Faculty Mentor: William C. Tang, Biomedical Engineering Department Background There is a plethora of different types of cancer, each with its own distinct causes and characteristics. However, cancers are generally categorized in two main subgroups: benign and malignant. The primary difference between the two groups is that malignant cancers have the ability to spread to other parts of the body during metastasis via blood vessels while benign cancers do not. If not promptly discovered and removed, most cancers are deadly due to their potential to metastasize, as cells detach from a primary tumor site and travel to other parts of the body to form new tumors. These circulating tumor cells (CTCs) can be detected in the bloodstream. Metastasis is one of the most dangerous aspects of cancer, as it is often difficult to predict where the cancer will spread. As such, successful isolation and capture of the CTCs could potentially aid in the endeavor to promote understanding and knowledge of metastasis. Furthermore, isolation of CTCs is associated with a wide range of medical and clinical applications, including monitoring of cancer in patients and medical prognosis. However, CTC isolation is no simple task as the relative scarcity of CTCs in the bloodstream compared to other normal hematologic cells makes it difficult to detect and isolate them; there is approximately 1 CTC for every 1 billion hematologic cells in 1 mL of blood [1]. Objective There are several approaches possible to achieve CTC isolation. Some of the common approaches often employ focus on the biochemical properties of CTCs. For example, one approach is to functionalize the channels of microfluidic devices with antibodies coated against EpCAM (Epithelial Cell Adhesion Molecule), a protein often overexpressed on the surfaces of CTCs. However, our research team’s approach is to exploit the mechanical properties and blood rheology characteristics of CTCs in designing our device. In essence, we are utilizing shear-modulated inertial microfluidics to separate the CTCs out from whole blood and to collect them in one of our device’s outlets. This is possible due to their relatively larger sizes in comparison to normal hematologic cells: as a comparison, normal cells are ~8-14 μm in diameter while CTCs are ~16-20 μm [2]. We opt to not use a biochemical approach because we believe that our approach is more beneficial and advantageous as it allows us to save on the costs of antibodies and reagents necessary for channel functionalization, thus proving to be more cost-efficient. Another advantage of our approach is that the CTCs that we collect are viable samples for further research analysis and studies since they will be preserved in their pristine states, unbound to and unreacted with any additional reagents nor substrates.
  2. 2. Approaches Blood Debulking Prior to utilizing inertial microfluidics in our spiral device to isolate CTCs in blood, we will pass our samples through a preliminary blood debulking stage where we would essentially filter out the bigger nucleated cells such as CTCs and white blood cells (WBCs), from the rest of the smaller cellular and protein components of blood. Not only will this ensure better separation and isolation of CTCs in the later stages, but also further enriches our sample before it enters the spiral device and increases the purity of the final output. The blood debulking method that we will be employing is continuous flow deterministic lateral displacement (DLD). This stage of the device will be composed of an array of microposts varying in pillar size and array offset so as to achieve an arrangement of posts designed to deflect particles above a certain size away from the primary suspension. A section of the micropost array is shown in Figure 1. In our case, we will be trying to deflect CTCs and WBCs away from the rest of the blood components, which should not be deflected by the microposts and would simply pass straight through the array. Thus we can create two separate outlets for this stage, one designated for the collection of the non- deflected particles in the primary fluid suspension on the left, and another for the deflected particles of interest on the right. The deflected particles would then be led to the inlet of our spiral device where they will undergo the final stages of separation and collection. As can be seen in the schematic of the array’s functionality in Figure 2, The WBCs and CTCs will be deflected off to the right while the non-deflected blood components will pass straight through on the left. [3] Figure 1. A section of the micropost array. Figure 2. A schematic of the functionality of the micropost array.
  3. 3. Inertial Microfluidics Inertial microfluidics govern the flow profiles for particles in a solution flowing through curved microchannels. A combination of both an inertial lift force generated by the channel wall and the Dean Drag Force generated by the Dean vortices arising from centrifugal acceleration could achieve size-based separation of particles in a solution after it has reached equilibrium [4]. Thus, CTCs could potentially be separated from other hematologic cells in blood due to their larger sizes. The inertial lift force ( ) that a particle of diameter experiences from the channel walls cause the particle to move away from the walls. It is quantified as follows [5]: where is the density of the fluid medium, is the fluid shear rate and is proportional to the maximum fluid velocity within the channel, , such that , with being the hydraulic diameter of the channel. Also, is the lift coefficient, and is a function of the position of the particle within the channel regardless of the particle size, rising from zero at the channel center to a maximum value before falling back to zero again at a distance of . An illustration of the lift forces acting on a particle subjected to a parabolic flow through a channel due to the shear gradient and the channel walls is shown below in Figure 3. [5] Figure 3. Illustration of lift forces. Shear induced lift forces are dominant at the center of the microchannel while wall induced lift forces are dominant near the walls. The magnitude of the Dean vortices formed due to centrifugal acceleration of the fluid can be quantified by a dimensionless number known as the Dean number ( ) [5]: where is the average fluid velocity, is the fluid viscosity, and is the radius of curvature.
  4. 4. The particles in a solution experience Dean Drag Forces ( ) that induce the particles to move laterally as they travel through the curved channels. This force can be expressed as [5]: A graphical representation of the net forces that a particle experiences while flowing through a curved channel is represented below in Figure 4. [5] Figure 4. The net lift forces vertically separate the flowing particles in the microchannel while the Dean vortices exert a drag force that laterally isolates them. Both forces contribute to each particle’s unique flow profile and equilibrium position. If whole blood were to be run through curved channels such as those in a spiral, the inertial lift force and Dean drag forces would balance out and cause large cells such as CTCs to reach an equilibrium position that is close to the proximal end of the curve, while smaller hematologic cells would equilibrate somewhere near the distal end of the curve. For this project, we introduce branches on the proximal end of the primary curved channels to initiate earlier collection of the CTCs. The branches would then divert flow into another stream that terminates at one of the collection outlets. Double Spiral Design Ultimately, our microfluidic device aims to exploit the different flow profiles for particles of different sizes that are governed by the mechanics of inertial microfluidics, to achieve size based separation. The main channel consists of two segments linked in series, each section composing of a spiral moving in a specific direction (first one counterclockwise and second clockwise). The main channel is then fixed with 3 collections branches that stem from the inner walls of the channel and divert flow away from it. The branches on the inner walls are intended to capture the CTCs/big beads as the flow in these collection branches eventually congregate and merge into another channel that leads to the CTC collection outlet. The rest of the solution not collected by the inner branches continues along the primary
  5. 5. channel in the device until they reach the other outlet reserved for hematologic cell collection. Our device ensures that there will be adequate distance for the solution to run initially after injection so that the particles in the solution are able to separate and equilibrate upon reaching the first branch. Illustrated below, is a mask of our device that we used in fabricating the SU-8 master mold. Figure 5 illustrates the design for our device which is composed of two spirals each consisting of 3 whole turns (3 turns counterclockwise followed by 3 turns clockwise). Figure 5. Mask for our device The main channel in our device is initially 500 μms in diameter and subsequently becomes thinner as it passes by the 3 collections branches located on the second spiral (clockwise) as the solution starts it exit out of the device. Specifically, as the main channel passes the first branch its diameter is attenuated to 400 μms; 300 μms at the second branch; 210 μms at the third branch. On the other hand, the collection channel that the flows diverted away from the main channel by the branches merge into is initially 100 μms in diameter at the first branch, and expands to 200 μms upon the second branch and 290 μms upon the third. Experiment Before we actually move on to test our devices using whole blood, we want to first test our devices using microbead solutions. The microbead solutions are intended to simulate whole blood as they are composed of a mixture of fluorescently dyed beads. The solutions contain green, large beads (~20-27μm) and white, small beads (~7.3μm). The microbeads solution is supposed to represent whole blood as the large beads are to represent CTCs while the small beads represent normal hematologic cells. Running this solution through our device and conducting data analysis on the bead composition of the outlet streams will allow us to assess the effectiveness and accuracy of our device. After testing with beads, we hope to move to testing with whole blood, using a spiked number of cancer cells to test the device. Fabrication
  6. 6. Our microfluidic devices will be fabricated through soft lithography using polydimethylsiloxane (PDMS). We will employ CAD softwares such as L-Edit, Solidworks, and AutoCAD to design the masks that will ultimately be used to pattern the SU-8 molds of our devices through photolithography techniques; the SU-8 will be situated on top of silicon wafers. The first step after the molds have been created, is to hard bake the SU-8 molds so that they retain on the silicon wafers better. Next, we have the molds undergo a silanization process to prevent the PDMS from sticking to the mold. After the molds have been preprocessed, we pour the PDMS that we made using a 10:1 ratio of base to curing agent, on top of the molds and allow it to cure at 65°C. We then extract the device by cutting it out and separating the PDMS from the molds, and then puncture holes in the device where the inlet and outlets are located. We then clean the device and prepare it to be plasma treated so that it may irreversibly bond to a glass slide. The final step is to inspect the devices under an optical microscope to look for defects, abnormalities, and cleanliness. [7] Student Responsibilities As an undergraduate researcher working on this project in collaboration with my graduate student mentor, my primary responsibilities involve fabrication of the microfluidic devices, data acquisition and analysis, and reporting findings to both the research team and the rest of the lab. Once I have the device, I can start running the microbead experiment as simulations for CTC capture. After the bead solution has run through the device, I analyze the results by using a hemocytometer to count the beads collected at both outlets to assess the accuracy and success of the device’s ability to capture ‘CTCs’. Under the guidance of my PI, Professor William Tang, I will be conducting my experiments in the Microbiomechanics Laboratory located on the third floor of Engineering Hall. My lab encourages and emphasizes teamwork and I will communicate my findings and results to the entire lab at the general lab meetings and be open to all suggestions and feedback. Timeline Month Objectives 1st Review and analyze the results of past research projects and journal papers to brainstorm and integrate improvements in our new design. Brainstorm designs for masks for the new double spiral devices on CAD software. 2nd Conduct experiments on old devices to acquire quantitative data for future comparison to results of new devices. Establish experiment protocol. Continue designing masks. 3rd Finish up any experiments and data acquisition for former devices.
  7. 7. Finalize experiment protocol. Finish designing masks for the new devices. 4th & 5th Fabricate new double spiral devices using PDMS and the new SU-8 molds. Run microbeads experiments on the new devices. Assess accuracy and effectiveness of new designs and if necessary, formulate design modifications and improvements. 6th Extract and congregate all data, results, and findings in preparation for symposium. Itemized Budget: Clean room access fee $650 Fabrication chemicals and supplies $200 Masks $100 Laboratory Consumables $50 Total $1000 References [1] Ross, A. et al. “Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques,” Blood, 82(9), 2605–2610, November 1993 [2] Lee, W. et al., “Highthroughput cell cycle synchronization using inertial forces in spiral microchannels,” Lab on a Chip, 11, 13591367, January 2011. [3] Karabacak, N. et al., “Microfluidic, Marker-Free Isolation of Circulating Tumor Cells from Blood Samples,” Nature, 27, February 2014. [4] Zhou, J. Papautsky, I. “Fundamentals of inertial focusing in microchannels,” Lab Chip, 13, 1121, January 2013 [5] Chatterjee, A. et al, “Inertial microfluidics for continuous separation of cells and particles,” SPIE, 7929, 2011. [6] Russom, A. et al., “Differential inertial focusing of particles in curved low-aspect- ratio microchannels,” New Journal of Physics, 11, July 2009.
  8. 8. [7] Friend, J. R. and Yeo, L., “Fabrication of microfluidic devices using polydimethylsiloxane,” Biomicrofluidics 4, 026502, (2010)