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© 2020 National Academy of Sciences. All rights reserved. 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. Sizedependent 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.

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. Keywords: Particle size analysis; Signal processing; Microfluidic devices; Phase lock loop; Cantilever; Resonator device; MEMS devices; Mass measurement, Cancer Systems Biology Consortium (Grant R33 CA191143), National Cancer Institute (Grant U54 CA217377), National Cancer Institute. Koch Institute Support (Grant P30-CA14051)

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017., Cataloged from PDF version of thesis., Includes bibliographical references (pages 139-142)., The development of single-cell RNA-sequencing has led to a new degree of resolution in the characterization of complex, heterogeneous biological systems. However, existing methods are often limited in their ability to link these whole-transcriptome profiles with complimentary measurements of single-cell phenotype. In this thesis we present a microfluidic toolset which allows us to link a panel of single-cell phenotypic measurements - including lineage history, cell cycle stage, cell size, and growth rate - with their corresponding transcriptional profiles. Using a microfluidic platform that employs an array of hydrodynamic traps to capture and culture single cells for multiple generations we measured single-cell growth kinetics, lineage hierarchies and cell cycle stage. By subsequently releasing individual cells from this device for downstream scRNA-seq we were able to generate whole-transcriptome profiles of primary, activated murine CD8+ T cell and lymphocytic leukemia cell line lineages. For both cell types we found distinct transcriptional patterns associated with single-cell lineage relationships as well as cell cycle progression. In order to link single-cell size and growth rate measurements with gene expression, we have also developed a system that relies on an array of suspended microchannel resonators (SMR) - high resolution single-cell buoyant mass sensors - in combination with an automated method of isolating single cells to conduct scRNA-seq downstream. Using this platform, we were able to collect linked transcriptional and biophysical measurements for a murine leukemia cell line, primary murine CD8+ T cells, and a patient-derived glioblastoma multiforme (GBM) cell line. For all cell models measured, we found that single-cell buoyant mass showed a strong correlation with the expression of cell cycle genes. Furthermore, we found that single-cell growth rate and buoyant mass measurements can be used to characterize the degree to which GBM cells respond to, by Robert J. Kimmerling., Ph. D.