Deterministic Lateral Displacement: The Next-Generation CAR T-Cell Processing?

Reliable cell recovery and expansion are fundamental to the successful scale-up of chimeric antigen receptor (CAR) T cells or any therapeutic cell-manufacturing process. Here, we extend our previous work in whole blood by manufacturing a highly parallel deterministic lateral displacement (DLD) device incorporating diamond microposts and moving into processing, for the first time, apheresis blood products. This study demonstrates key metrics of cell recovery (80%) and platelet depletion (87%), and it shows that DLD T-cell preparations have high conversion to the T-central memory phenotype and expand well in culture, resulting in twofold greater central memory cells compared to Ficoll-Hypaque (Ficoll) and direct magnetic approaches. In addition, all samples processed by DLD converted to a majority T-central memory phenotype and did so with less variation, in stark contrast to Ficoll and direct magnetic prepared samples, which had partial conversion among all donors (<50%). This initial comparison of T-cell function infers that cells prepared via DLD may have a desirable bias, generating significant potential benefits for downstream cell processing. DLD processing provides a path to develop a simple closed system that can be automated while simultaneously addressing multiple steps when there is potential for human error, microbial contamination, and other current technical challenges associated with the manufacture of therapeutic cells.

Maximizing particle concentration in deterministic lateral displacement arrays.

We present an improvement to deterministic lateral displacement arrays, which allows higher particle concentration enhancement. We correct and extend previous equations to a mirror-symmetric boundary. This approach allows particles to be concentrated into a central channel, no wider than the surrounding gaps, thereby maximizing the particle enrichment. The resulting flow patterns were, for the first time, experimentally measured. The performance of the device with hard micro-spheres and cells was investigated. The observed flow patterns show important differences from our model and from an ideal pattern. The 18 µm gap device showed 11-fold enrichment of 7 µm particles and nearly perfect enrichment-of more than 50-fold-for 10 µm particles and Jurkat cells. This work shows a clear path to achieve higher-than-ever particle concentration enhancement in a deterministic microfluidic separation system.

Erratum: Publisher's Note: "Maximizing particle concentration in deterministic lateral displacement arrays" [Biomicrofluidics 11, 024121 (2017)].

[This corrects the article DOI: 10.1063/1.4981014.].

Automated leukocyte processing by microfluidic deterministic lateral displacement.

We previously developed a Deterministic Lateral Displacement (DLD) microfluidic method in silicon to separate cells of various sizes from blood (Davis et al., Proc Natl Acad Sci 2006;103:14779-14784; Huang et al., Science 2004;304:987-990). Here, we present the reduction-to-practice of this technology with a commercially produced, high precision plastic microfluidic chip-based device designed for automated preparation of human leukocytes (white blood cells; WBCs) for flow cytometry, without centrifugation or manual handling of samples. After a human blood sample was incubated with fluorochrome-conjugated monoclonal antibodies (mAbs), the mixture was input to a DLD microfluidic chip (microchip) where it was driven through a micropost array designed to deflect WBCs via DLD on the basis of cell size from the Input flow stream into a buffer stream, thus separating WBCs and any larger cells from smaller cells and particles and washing them simultaneously. We developed a microfluidic cell processing protocol that recovered 88% (average) of input WBCs and removed 99.985% (average) of Input erythrocytes (red blood cells) and >99% of unbound mAb in 18 min (average). Flow cytometric evaluation of the microchip Product, with no further processing, lysis or centrifugation, revealed excellent forward and side light scattering and fluorescence characteristics of immunolabeled WBCs. These results indicate that cost-effective plastic DLD microchips can speed and automate leukocyte processing for high quality flow cytometry analysis, and suggest their utility for multiple other research and clinical applications involving enrichment or depletion of common or rare cell types from blood or tissue samples. © 2016 International Society for Advancement of Cytometry.

Microfluidics and circulating tumor cells.

Circulating tumor cells (CTCs) are shed from cancerous tumors, enter the circulatory system, and migrate to distant organs to form metastases that ultimately lead to the death of most patients with cancer. Identification and characterization of CTCs provides a means to study, monitor, and potentially interfere with the metastatic process. Isolation of CTCs from blood is challenging because CTCs are rare and possess characteristics that reflect the heterogeneity of cancers. Various methods have been developed to enrich CTCs from many millions of normal blood cells. Microfluidics offers an opportunity to create a next generation of superior CTC enrichment devices. This review focuses on various microfluidic approaches that have been applied to date to capture CTCs from the blood of patients with cancer.

Microfluidic control of cell pairing and fusion.

Cell fusion has been used for many different purposes, including generation of hybridomas and reprogramming of somatic cells. The fusion step is the key event in initiation of these procedures. Standard fusion techniques, however, provide poor and random cell contact, leading to low yields. We present here a microfluidic device to trap and properly pair thousands of cells. Using this device, we paired different cell types, including fibroblasts, mouse embryonic stem cells and myeloma cells, achieving pairing efficiencies up to 70%. The device is compatible with both chemical and electrical fusion protocols. We observed that electrical fusion was more efficient than chemical fusion, with membrane reorganization efficiencies of up to 89%. We achieved greater than 50% properly paired and fused cells over the entire device, fivefold greater than with a commercial electrofusion chamber and observed reprogramming in hybrids between mouse embryonic stem cells and mouse embryonic fibroblasts.

An active bubble trap and debubbler for microfluidic systems.

We present a novel, fully integrated microfluidic bubble trap and debubbler. The 2-layer structure, based on a PDMS valve design, utilizes a featured membrane to stop bubble progression through the device. A pneumatic chamber directly above the trap is evacuated, and the bubble is pulled out through the gas-permeable PDMS membrane. Normal device operation, including continuous flow at atmospheric pressure, is maintained during the entire trapping and debubbling process. We present a range of trap sizes, from 2 to 10 mm diameter, and can trap and remove bubbles up to 25 microL in under 3 h.

The Urey instrument: an advanced in situ organic and oxidant detector for Mars exploration.

The Urey organic and oxidant detector consists of a suite of instruments designed to search for several classes of organic molecules in the martian regolith and ascertain whether these compounds were produced by biotic or abiotic processes using chirality measurements. These experiments will also determine the chemical stability of organic molecules within the host regolith based on the presence and chemical reactivity of surface and atmospheric oxidants. Urey has been selected for the Pasteur payload on the European Space Agency's (ESA's) upcoming 2013 ExoMars rover mission. The diverse and effective capabilities of Urey make it an integral part of the payload and will help to achieve a large portion of the mission's primary scientific objective: "to search for signs of past and present life on Mars." This instrument is named in honor of Harold Urey for his seminal contributions to the fields of cosmochemistry and the origin of life.

Analysis of neuroactive amines in fermented beverages using a portable microchip capillary electrophoresis system.

A portable microfabricated capillary electrophoresis (CE) instrument is used for the determination of neurologically active biogenic amines, especially tyramine and histamine, in fermented beverages. The target molecules are labeled on their primary amino groups with fluorescamine in a 10-min reaction, and the samples analyzed directly, producing a detailed electropherogram in only 120 s on a microfabricated glass CE device containing 21.4-cm-long separation channels. Tyramine was found mainly in red wines at <1-3.4 mg/L, while the histamine content of these samples ranged from 1.8 to 19 mg/L. The highest levels of histamine (20-40 mg/L) were found in sake. The analysis of samples drawn from grape crush through malolactic fermentation in four varieties of zinfandel red wines revealed that histamine and tyramine are produced during yeast and malolactic fermentation, respectively. Following malolactic fermentation, the histamine content in these samples ranged from 3.3 to 30 mg/L, and the tyramine content ranged from 1.0 to 3.0 mg/L. This highly sensitive and rapid lab-on-a-chip analysis method establishes the feasibility of monitoring neurologically active amine content and potentially other chemically and allergenically important molecules in our food supply.

Application of the Mars Organic Analyzer to nucleobase and amine biomarker detection.

The Mars Organic Analyzer (MOA), a portable microfabricated capillary electrophoresis instrument being developed for planetary exploration, is used to analyze a wide variety of fluorescamine-labeled amine-containing biomarker compounds, including amino acids, mono and diaminoalkanes, amino sugars, nucleobases, and nucleobase degradation products. The nucleobases cytosine and adenine, which contain an exocyclic primary amine, were effectively labeled, separated, and detected at concentrations <500 nM. To test the general applicability of the MOA for biomarker detection, amino acids and mono- and diamines were extracted from bacterial cells using both hydrolysis and sublimation followed by analysis. The extrapolated limit of detection provided by the valine biomarker was approximately 4 x 10(3) cells per sample. Products of an NH(4)CN polymerization that simulate a prebiotic synthesis were also successfully isolated via sublimation and analyzed. Adenine and alanine/serine were detected with no additional sample cleanup at 120 +/- 13 microM and 4.1 +/- 1 microM, respectively, corresponding to a reaction yield of 0.04% and 0.0003%, respectively. This study demonstrates that the MOA provides sensitive detection and analysis of low levels of a wide variety of amine-containing organic compounds from both biological and abiotic sources.

Microfluidic serial dilution circuit.

In vitro evolution of RNA molecules requires a method for executing many consecutive serial dilutions. To solve this problem, a microfluidic circuit has been fabricated in a three-layer glass-PDMS-glass device. The 400-nL serial dilution circuit contains five integrated membrane valves: three two-way valves arranged in a loop to drive cyclic mixing of the diluent and carryover, and two bus valves to control fluidic access to the circuit through input and output channels. By varying the valve placement in the circuit, carryover fractions from 0.04 to 0.2 were obtained. Each dilution process, which is composed of a diluent flush cycle followed by a mixing cycle, is carried out with no pipeting, and a sample volume of 400 nL is sufficient for conducting an arbitrary number of serial dilutions. Mixing is precisely controlled by changing the cyclic pumping rate, with a minimum mixing time of 22 s. This microfluidic circuit is generally applicable for integrating automated serial dilution and sample preparation in almost any microfluidic architecture.

Rapid on-column analysis of glucosamine and its mutarotation by microchip capillary electrophoresis.

A novel electrophoretic microchip method for analyzing alpha- and beta-d-glucosamine and their interconversion in solution is presented. d-Glucosamine is labeled with fluorescamine and analyzed by capillary electrophoresis in under 2 min revealing its pH-dependent mutarotation between the alpha- and beta-anomers. The forward interconversion rates for the labeled sugars, based on an iterative analysis of the plateau heights between the peaks, are 0.72+/-0.09, 1.3+/-0.1, and 2.2+/-0.3 x 10(-3)s(-1) at pH 8.99, 9.51 and 10.01, respectively. In a separate experiment, the mutarotation of the unlabeled alpha-d-anomer was followed; the relative intensities of the alpha- and beta-peaks as a function of reaction time at pH 9.51 give a forward rate constant of 0.6+/-0.1 x 10(-3)s(-1). These results demonstrate that fast microchip separations, previously exploited for amine, amino acid, and nucleobase analysis, can also be used to analyze amino sugars and their mutarotation.

Development and evaluation of a microdevice for amino acid biomarker detection and analysis on Mars.

The Mars Organic Analyzer (MOA), a microfabricated capillary electrophoresis (CE) instrument for sensitive amino acid biomarker analysis, has been developed and evaluated. The microdevice consists of a four-wafer sandwich combining glass CE separation channels, microfabricated pneumatic membrane valves and pumps, and a nanoliter fluidic network. The portable MOA instrument integrates high voltage CE power supplies, pneumatic controls, and fluorescence detection optics necessary for field operation. The amino acid concentration sensitivities range from micromolar to 0.1 nM, corresponding to part-per-trillion sensitivity. The MOA was first used in the lab to analyze soil extracts from the Atacama Desert, Chile, detecting amino acids ranging from 10-600 parts per billion. Field tests of the MOA in the Panoche Valley, CA, successfully detected amino acids at 70 parts per trillion to 100 parts per billion in jarosite, a sulfate-rich mineral associated with liquid water that was recently detected on Mars. These results demonstrate the feasibility of using the MOA to perform sensitive in situ amino acid biomarker analysis on soil samples representative of a Mars-like environment.

Integrated hydrogenated amorphous Si photodiode detector for microfluidic bioanalytical devices.

Hydrogenated amorphous silicon (a-Si:H) PIN photodiodes have been developed and characterized as fluorescence detectors for microfluidic analysis devices. A discrete a-Si:H photodiode is first fabricated on a glass substrate and used to detect fluorescent dye standards using conventional confocal microscopy. In this format, the limit of detection for fluorescein flowing in a 50-microm deep channel is 680 pM (S/N = 3). A hybrid integrated detection system consisting of a half-ball lens, a ZnS/YF3 multilayer optical interference filter with a pinhole, and an annular a-Si:H photodiode is also developed that allows the laser excitation to pass up through the central aperture in the detector. Using this integrated detection device, the limit of detection for fluorescein is 17 nM, and DNA fragment sizing and chiral analysis of glutamic acid are successfully performed. The a-Si:H detector exhibits high sensitivity at the emission wavelengths of commonly used fluorescent dyes and is readily microfabricated and integrated at low cost making it ideal for portable microfluidic bioanalyzers and emerging large scale integrated microfluidic technologies.

Chiral separation of fluorescamine-labeled amino acids using microfabricated capillary electrophoresis devices for extraterrestrial exploration.

Chiral separations of fluorescamine-labeled amino acids are characterized and optimized on a microfabricated capillary electrophoresis (CE) device. A standard mixture of acidic and neutral amino acids is labeled with fluorescamine in less than 5 min and the hydroxypropyl-beta-cyclodextrin (HPbetaCD) concentration, temperature, and pH are optimized (15 mM HPbetaCD, 6 degrees C, pH < 9) to achieve high-quality and low background chiral separations in less than 200 s. All four stereoisomers formed in the labeling reaction of the chiral dye with the chiral amino acids are typically resolved. At pH > 9, isomerization of the dye chiral center is observed that occurs on the time scale of the chip separation. Typical limits of detection are approximately 50 nM. These results demonstrate the feasibility of combining fluorescamine labeling of amino acids with microfabricated CE devices to develop low-volume, high-sensitivity apparatus and methods for extraterrestrial exploration.