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A Single Cell AnaLysis and Sorting Platform based on Lensfree digital imaging techniques applied to Rapid Detection of Cancer

Final Report Summary - SCALPEL (A Single Cell AnaLysis and Sorting Platform based on Lensfree digital imaging techniques applied to Rapid Detection of Cancer)

Metastasis is responsible for > 90% of cancer-related deaths. Billions of dollars have been spent trying to cure primary tumors but very little was spent in trying to detect or kill the highly aggressive tumor cells that cause disease spreading. One of the reasons is that single cell studies of rare cells in blood still present a large challenge. Single cell analysis remains tedious with many different instruments and protocols, typically taking a few days of hands-on work. This slows down research, but also hinders the translation to application in future clinical practice. In SCALPEL, we envisage a high-content, high-throughput cell imaging and sorting platform, more compact and easier to use than any existing single cell analyzer. The high content results from lens free digital imaging of single cells on a high-speed CMOS active optical pixel matrix to analyze the morphology of cells. The high throughput results from a highly parallelized fluidic matrix that steers cells at high speed over the CMOS imaging blocks. Lens free cell sorters can be realized in a cheap and compact platform, as all optomechanical components (lenses, detectors, nozzles,...) are replaced by nanoelectronics, advanced imaging and signal processing technology.

In the course of the SCALPEL grant, we were able to demonstrate the capabilities of the different modules as well as translate to different clinical applications based on engineering prototypes that became available during the grant. .

First, we successfully built a compact lens-free in-line holographic microscope and used it to image cancer cell lines and blood cells flowing in a microfluidic chip. We used the lensfree microscope for characterizing migration behaviour of metastatic cell lines (Lab on a chip 10.1039/c6lc00860g): . We employed numerical reconstruction of the captured holograms to classify unlabeled leukocytes into three main subtypes: lymphocytes, monocytes, and granulocytes. This classification was done using an in house developed scale-space recognition analysis. Results obtained were benchmarked with a conventional hematological analyzer and found to be in good agreement with it. These results were published in the journal Lab on a chip (doi: 10.1039/c41c01131g). Furthermore, we implemented additional improvements which allowed for acquisition of much higher data accuracy and development of a new processing algorithm and classification method. This led to another publication in Plos One (Doi: 10.1016/j.compbiomed.2018.03.008). Finally, we have improved the processing algorithm further to allow computation at high throughput using a simple GPU. This improvement was published in Optics express (10.1364/OE.26.014329).

Secondly, we fabricated a microfluidic cell sorter allowing high rate and fully enclosed cell sorting. The sorter chip consists of an array of micro heating hotspots. Pulsed resistive heating in the hotspots produces numerous micro vapor bubbles with short duration, giving rise to a rapid jet flow for cell sorting. We have demonstrated that this bubble jet sorter is capable of high-rate sorting and could be further improved to achieve a throughput comparable to commercial FACS. We have validated a significant enrichment of rare cells (such as cancer cells) from PBMC thereby positioning the cell sorter as a powerful tool for contamination-free and affordable clinical diagnostic cell sorting. These results are detailed in our manuscript published in the journal lab on chip (doi: 10.1039/c61c01560c).
Secondly, we validated the performance of the chip for gentle isolation of T cells, a key step in the manufacturing process of CAR T cell therapy. We have shown that the functional properties of the sorted cells related to viability, cell growth and activation are equal to the currently used method based on magnetic bead isolation. In contradiction to the current magnetic isolation, our celsorter platform can be scaled, automated, and integrated easily in line, which will really change and improve the full manufacturing process. These findings are gathered in a manuscript that will be submitted to the journal of Cytotherapy.
Further, the capability of the chip to perform sensitive and clinically relevant flow cytometry was validated in collaboration with the research group of professor Coosemans at KU Leuven focusing on immune profiling in ovarian cancer patients. They have found that an immune read out of immune cells in the blood of ovarian patients can discriminate between a benign or malignant tumor without the need for a biopsy. Further, this immune readout can be followed up during primary treatment and provide information on the success of the current treatment. We have validated the performance of our platform to perform a limited immune readout, based on 4 markers against a conventional flow cytometer. We found a good correlation between the two measurements and results will be gather for submission to Cytometry A.

In the last part of the grant, we have investigated and developed the concepts needed to multiplex the sorter chip to enable high throughput without compromising on the quality of the sorted product. First, we assessed how the fluidics could be multiplexed in a robust way while minimizing overall chip complexity. Hydrodynamic focusing was identified as the most robust method that can be integrated rather easily on chip with a small form factor. The only drawback is the need for a high number of in/outlet holes per channel. To solve this, further development is needed to efficiently split channels on chip, thereby sharing the same inlet/outlet holes over multiple channels. We envision that this could be done by implementing two-layer fluidics. Besides the fluidics, also the optics needs to be parallelized. Given the requirement for fluorescent detection, optics parallelization is a complex challenge. First, a paper study was performed to develop a scalable concept for multicolor excitation and detection. This concept was based on waveguides that guide light of different wavelength to different channels in the chip. Upon excitation, cells would emit fluorescence that would be captured with lenses and guided to a pixel in an APD array. As a first step, robust excitation waveguides were designed and fabricated that produce a uniform light beam orthogonal to the flow direction. These waveguides were validated with QC calibration beads and the detection efficiency was found to be less dependent to the position of the bead in the flow channel compared to conventional waveguide platforms.

The ERC grant has given us the opportunity to de-risk some modules, perform proof of concepts and early prototype. Moreover, the translation to clinical applications has been initiated with already a number of successes on the way.