Mass measurements during lymphocytic leukemia cell polyploidization decouple cell cycle- and cell size-dependent growth.

Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e., growth efficiency) than intermediate-sized cells in the same population. Size-dependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size- and cell cycle-dependent growth is challenging. To address this, we monitored growth efficiency of pseudodiploid mouse lymphocytic leukemia cells during normal proliferation and polyploidization. This was enabled by the development of large-channel suspended microchannel resonators that allow us to monitor buoyant mass of single cells ranging from 40 pg (small pseudodiploid cell) to over 4,000 pg, with a resolution ranging from ∼1% to ∼0.05%. We find that cell growth efficiency increases, plateaus, and then decreases as cell cycle proceeds. This growth behavior repeats with every endomitotic cycle as cells grow into polyploidy. Overall, growth efficiency changes 33% throughout the cell cycle. In contrast, increasing cell mass by over 100-fold during polyploidization did not change growth efficiency, indicating exponential growth. Consistently, growth efficiency remained constant when cell cycle was arrested in G2 Thus, cell cycle is a primary determinant of growth efficiency. As growth remains exponential over large size scales, our work finds no evidence for transport limitations that would decrease growth efficiency.

Rapid and high-precision sizing of single particles using parallel suspended microchannel resonator arrays and deconvolution.

Measuring the size of micron-scale particles plays a central role in the biological sciences and in a wide range of industrial processes. A variety of size parameters, such as particle diameter, volume, and mass, can be measured using electrical and optical techniques. Suspended microchannel resonators (SMRs) are microfluidic devices that directly measure particle mass by detecting a shift in resonance frequency as particles flow through a resonating microcantilever beam. While these devices offer high precision for sizing particles by mass, throughput is fundamentally limited by the small dimensions of the resonator and the limited bandwidth with which changes in resonance frequency can be tracked. Here, we introduce two complementary technical advancements that vastly increase the throughput of SMRs. First, we describe a deconvolution-based approach for extracting mass measurements from resonance frequency data, which allows an SMR to accurately measure a particle's mass approximately 16-fold faster than previously possible, increasing throughput from 120 particles/min to 2000 particles/min for our devices. Second, we describe the design and operation of new devices containing up to 16 SMRs connected fluidically in parallel and operated simultaneously on the same chip, increasing throughput to approximately 6800 particles/min without significantly degrading precision. Finally, we estimate that future systems designed to combine both of these techniques could increase throughput by nearly 200-fold compared to previously described SMR devices, with throughput potentially as high as 24 000 particles/min. We envision that increasing the throughput of SMRs will broaden the range of applications for which mass-based particle sizing can be employed.

Microfluidic platform for characterizing TCR-pMHC interactions.

The physical characteristics of the T cell receptor (TCR)-peptide-major histocompatibility complex (pMHC) interaction are known to play a central role in determining T cell function in the initial stages of the adaptive immune response. State-of-the-art assays can probe the kinetics of this interaction with single-molecular-bond resolution, but this precision typically comes at the cost of low throughput, since the complexity of these measurements largely precludes "scaling up." Here, we explore the feasibility of detecting specific TCR-pMHC interactions by flowing T cells past immobilized pMHC and measuring the reduction in cell speed due to the mechanical force of the receptor-ligand interaction. To test this new fluidic measurement modality, we fabricated a microfluidic device in which pMHC-coated beads are immobilized in hydrodynamic traps along the length of a serpentine channel. As T cells flow past the immobilized beads, their change in speed is tracked via microscopy. We validated this approach using two model systems: primary CD8+ T cells from an OT-1 TCR transgenic mouse with beads conjugated with H-2Kb:SIINFEKL, and Jurkat T cells with beads conjugated with anti-CD3 and anti-CD28 antibodies.

High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays.

Methods to rapidly assess cell growth would be useful for many applications, including drug susceptibility testing, but current technologies have limited sensitivity or throughput. Here we present an approach to precisely and rapidly measure growth rates of many individual cells simultaneously. We flow cells in suspension through a microfluidic channel with 10-12 resonant mass sensors distributed along its length, weighing each cell repeatedly over the 4-20 min it spends in the channel. Because multiple cells traverse the channel at the same time, we obtain growth rates for >60 cells/h with a resolution of 0.2 pg/h for mammalian cells and 0.02 pg/h for bacteria. We measure the growth of single lymphocytic cells, mouse and human T cells, primary human leukemia cells, yeast, Escherichia coli and Enterococcus faecalis. Our system reveals subpopulations of cells with divergent growth kinetics and enables assessment of cellular responses to antibiotics and antimicrobial peptides within minutes.

A microfluidic platform enabling single-cell RNA-seq of multigenerational lineages.

We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function--including Granzyme B--are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology.

Measuring single cell mass, volume, and density with dual suspended microchannel resonators.

Cell size, measured as either volume or mass, is a fundamental indicator of cell state. Far more tightly regulated than size is density, the ratio between mass and volume, which can be used to distinguish between cell populations even when volume and mass appear to remain constant. Here we expand upon a previous method for measuring cell density involving a suspended microchannel resonator (SMR). We introduce a new device, the dual SMR, as a high-precision instrument for measuring single-cell mass, volume, and density using two resonators connected by a serpentine fluidic channel. The dual SMR designs considered herein demonstrate the critical role of channel geometry in ensuring proper mixing and damping of pressure fluctuations in microfluidic systems designed for precision measurement. We use the dual SMR to compare the physical properties of two well-known cancer cell lines: human lung cancer cell H1650 and mouse lymphoblastic leukemia cell line L1210.

A microfluidic "baby machine" for cell synchronization.

Common techniques used to synchronize eukaryotic cells in the cell cycle often impose metabolic stress on the cells or physically select for size rather than age. To address these deficiencies, a minimally perturbing method known as the "baby machine" was developed previously. In the technique, suspension cells are attached to a membrane, and as the cells divide, the newborn cells are eluted to produce a synchronous population of cells in the G1 phase of the cell cycle. However, the existing "baby machine" is only suitable for cells which can be chemically attached to a surface. Here, we present a microfluidic "baby machine" in which cells are held onto a surface by pressure differences rather than chemical attachment. As a result, our method can in principle be used to synchronize a variety of cell types, including cells which may have weak or unknown surface attachment chemistries. We validate our microfluidic "baby machine" by using it to produce a synchronous population of newborn L1210 mouse lymphocytic leukemia cells in G1 phase.

Integrated microelectronic device for label-free nucleic acid amplification and detection.

We present an integrated microelectronic device for amplification and label-free detection of nucleic acids. Amplification by polymerase chain reaction (PCR) is achieved with on-chip metal resistive heaters, temperature sensors, and microfluidic valves. We demonstrate a rapid thermocycling with rates of up to 50 degrees C s(-1) and a PCR product yield equivalent to that of a bench-top system. Amplicons within the PCR product are detected by their intrinsic charge with a silicon field-effect sensor. Similar to existing optical approaches with intercalators such as SYBR Green, our sensing approach can directly detect standard double-stranded PCR product, while in contrast, our sensor does not require labeling reagents. By combining amplification and detection on the same device, we show that the presence or absence of a particular DNA sequence can be determined by converting the analog surface potential output of the field-effect sensor to a simple digital true/false readout.

Monitoring of heparin and its low-molecular-weight analogs by silicon field effect.

Heparin is a highly sulfated glycosaminoglycan that is used as an important clinical anticoagulant. Monitoring and control of the heparin level in a patient's blood during and after surgery is essential, but current clinical methods are limited to indirect and off-line assays. We have developed a silicon field-effect sensor for direct detection of heparin by its intrinsic negative charge. The sensor consists of a simple microfabricated electrolyte-insulator-silicon structure encapsulated within microfluidic channels. As heparin-specific surface probes the clinical heparin antagonist protamine or the physiological partner antithrombin III were used. The dose-response curves in 10% PBS revealed a detection limit of 0.001 units/ml, which is orders of magnitude lower than clinically relevant concentrations. We also detected heparin-based drugs such as the low-molecular-weight heparin enoxaparin (Lovenox) and the synthetic pentasaccharide heparin analog fondaparinux (Arixtra), which cannot be monitored by the existing near-patient clinical methods. We demonstrated the specificity of the antithrombin III functionalized sensor for the physiologically active pentasaccharide sequence. As a validation, we showed correlation of our measurements to those from a colorimetric assay for heparin-mediated anti-Xa activity. These results demonstrate that silicon field-effect sensors could be used in the clinic for routine monitoring and maintenance of therapeutic levels of heparin and heparin-based drugs and in the laboratory for quantitation of total amount and specific epitopes of heparin and other glycosaminoglycans.