Embedded Files
University of California, San Diego
Mechanical and Aerospace Engineering
MAE 156B: Senior Design Project (Spring 2024)
Sponsored by Dr. Ben Croker Ph.D. , Croker Lab
Primary Objective:
Primary Objective:
Objective:
Objective:
Design a high-throughput, high-efficiency microfluidic chip capable of isolating single-cell interactions and is compatible with super resolution instruments.
Project Description:
Project Description:
Microfluidics is a rapidly growing field enabling high-throughput studies through exploiting fluid and chemical properties at the micron scale. Life sciences divisions have been increasingly employing microfluidics devices as a means to study cells in a manner that is cost effective and resource friendly while maintaining a small working footprint. In this work we design, fabricate, and image microfluidics chips capable of sorting and trapping coccidioidomycosis (cocci) fungal cells and neutrophils to facilitate investigation of single cell-cell interactions at a large scale.
Leveraging computational fluid dynamics (CFD), empirical research and soft-photolithography fabrication techniques two polydimethylsiloxane (PDMS) chips were fabricated, one capable of sorting cells via deterministic lateral displacement (DLD) and the other capable of retaining paired cells in hydrodynamic trap arrays. In considering the health implications cocci poses to the general public, it is hoped this work will aid in the development of a vaccine and greater understanding of potential treatment methods.
Final Design
Final Design
The final design utilizes two polydimethylsiloxane (PDMS) microfluidic chips: a trapping array consisting of 1336 traps and a filter chip which uses deterministic lateral displacement (DLD) to passively separate cells based on a chosen critical diameter of 10 μm. Used in series, these chips serve to filter the cocci cells from a size range of 10 - 100 μm to a range of 10 - 20 μm and retain pairs of cells in an array, allowing for imaging.
The DLD filter design incorporates a wide array of pillars to manipulate the streamlines of the flow. As the cells flow along the streamlines, smaller cells zig-zag through the array while larger cells collide with the pillars, resulting in lateral displacement. Given a chosen critical diameter of 10 μm, the inserted cells separate into groups, with 10 μm as the dividing size. This simple, passive, and robust design has a high rate of success and can be easily modified to filter any size range required.
The trap array utilizes a similar passive hydrodynamic mechanism to flow cells into the traps. The traps consist of two pockets, a large pocket in the forward direction and a smaller pocket in the reverse direction. Initially, neutrophil cells are inserted into the system in the reverse direction, with one cell per trap. Then, the flow direction is reversed and the cocci spores are introduced, flowing the neutrophils and cocci cells into the forward-facing trap. Through this procedure, it is possible to ensure only two cells occupy a particular trap. With an array of more than a thousand traps, this chip provides the capability to collect a large amount of data from a large sample size simultaneously.
The chips are designed to interface seamlessly with an inverted confocal microscope to image the flow and the behavior of the cells within the traps. The chips are connected to a syringe pump which drives the flow and is the mechanism which injects the cells into the chips. The whole system is connected by 23G microbore tubing, with steel tubes acting at the interface of the syringe and the chips.
Summary of Performance Results
Summary of Performance Results
Scaled Prototype Testing
Scaled Prototype Testing
To facilitate rapid prototyping and testing, the hydrodynamic trap array and the DLD Filter chips were 3D printed using the Stratasys Objet350 Connex printer at an 80x scale. At this scale, glass microbeads, Delrin beads, and tobiko fish eggs could be used as models for the cells to be flown through the to-scale prototype. Tobiko fish eggs were chosen for their deformability, which makes them ideal candidates to test the effect the deformability of the cells has on the trapping efficiency of the prototype. In this test, it was found that 116 of the total 350 traps were occupied, occupying 47% of the total traps. Additionally, it is clear 11 traps contained 2-3 eggs and approximately 11 eggs were within the array, but not contained within any particular trap. 47% is a more than adequate success rate, when considering the to-scale array has over a thousand traps, predicting approximately 500 traps successfully filled.
Figure 1: A) ImageJ processed image of the results of the caviar flow experiment, showing the cells residing in the array as white dots across a black background. B) The pre-processed results of the caviar flow experiment, showing clearly the amount of eggs trapped within the array in individual traps.
Confocal Microscope Imaging
Confocal Microscope Imaging
The experiments at the microfluidic scale were conducted on the Zeiss LSM T-PMT inverted confocal microscope in a temperature controlled environment. The trap array was placed in the enclosure, tubing routed from the chip to the syringe pump and a drain, with PDMS-fabricated Y-connectors splitting the flow. Neutrophil cells, lab grown to a diameter of 10 μm, and cocci cells, cultured and filtered to a diameter of 16-20 μm, were flown into the chip from the syringe pump. The bicarbonate-free media RPMI-1640 Medium acted as the working fluid, eliminating the need to introduce CO2 incubation chambers into the working system. With a constant fluid flow, changes in pH due to cell metabolism would not be an issue as it would be for long-term static liquid cultures.
From initial imaging, the prototype design was validated and results showed the trap array functioned as intended. As seen in Figure 2, the first stage of the loading process proved to be very successful, with a 78% trapping efficiency from the sampled population of neutrophils. Results show most traps contain a single neutrophil, as intended, with six traps containing two or three cells and a wide variety of cells floating throughout the array.
Figure 2: A) Sample imaging result from neutrophil injection in the reverse flow direction, with the neutrophil cells dyed red. B) ImageJ processed image of the the sampled results, contrasting the white cells over the black background.
Figure 3: Imaging results from the first attempt to obtain single cell interactions. Left: zoomed out view of many cocci and neutrophil cells interacting, some in undesirable locations outside of traps. Right: zoomed in view of a single cell cocci-neutrophil interaction. Characteristic clustering of neutrophils is present.
Final Presentation
Presentation for Croker 6-7 MAE 156B.pdfExecutive Summary
Executive Summary - MAE 156B SP24 Team 8.pdfPoster
MAE 156B Poster Team 8.pdfVideo
Team8Vid2.mp4Final Report
Final Report - MAE 156B SP24 Team 8.pdfPage updated
Report abuse