Microfluidic chips for cellular heterogeneity studies.

Abstract

In recent years several studies have found significant heterogeneity among even purified cells that were previously treated as if all cells were essentially identical. But since many measurements have been reported as averages over large numbers of cells, assuming that all cells are same when they might really be a mixture of different cell subtypes, this can lead to incorrect or at least imprecise and harder to replicate results. There is a macroscopic technique for single cell analysis known as dilution method: diluting and depositing cells in a conventional platform such as a 96 well plate. However, it is very labor-intensive, low-throughput, low single-cell loading efficiency, and poor reproducibility and thus is rarely performed. We have designed a high-throughput microfluidic chip which solves these problems and is superior to existing devices in allowing heterogeneous cell studies at scales and accuracies not previously possible and with minimum labor and cost. This chip performs 1) single-cell capture and culture to generate their colonies, and 2) sorting multiple specific target cells. Hydrodynamic force and magnetic force were studied to position or to sort cells in pre-determined locations precisely. We designed a novel hydrodynamic guiding structure which can automatically capture and position single cells into each microwell with high capturing efficiency, using only gravity flow, with which we could capture 80% of cells at a single cell resolution from all the injected cells (~ 2 orders of magnitude improvement, comparing to the state-of-art and widely adopted passive weir structures for single cell trapping). This hydrodynamic guiding scheme was applied to a high-throughput microfluidic array chip, which is the first chip capable of culturing single cells into their clonal colonies inside individual microwells and introducing test-reagents to their clones. To track cells carefully over time and ensure clonal outgrowth from single cells, we should prevent cell migration between neighbouring microwells, which can generally happen in other reported single cell culture chips. We integrated a surface patterning technique into a microwell, which effectively confines cells' movement inside each microwell. In addition, we utilized a gravity flow caused by pressure difference between inlet and out reservoir, which allows us a simple and easy operation without the necessity of external equipments. Using this chip we can detect heterogeneity of cells identify subtypes of clones and monitor drug responsiveness. We observed three different subtypes grown from a prostate cancer cell line, PC3 cells. These distinctive subtypes have different morphologies and proliferation rate, as well as different drug responsiveness. This single cell clonal chip can be extended to a larger array as well as used for multiple reagents by integrating pneumatic valves. An alternative method of cell sorting for heterogeneity studies has been explored where the objective cells have well-known identifiable surface markers. It is also desirable to collect cells at a specific target size especially for effective drug screening purpose. A macro magnetic sorter can separate target cells; however, the screened cells are not 100% pure and their sizes vary in a wide range. We devised a novel magnetic sorter which can separate multiple target cells into corresponding microwells. We have designed a magnetic sorter based on the phenomenon that magnetic particles move towards the minima of field and flowing electrical current generates controllable magnetic field. The magnetic sorter could separate different-sized cells by generating a local magnetic field gradient with the integrated current-carrying lines. We successfully demonstrated three different sizes of magnetic beads can be sorted under the different electrical current through the embedded current-carrying lines in three successive sorting units. More unit stages can be added and the number of stages can be determined to meet the sorting purpose.