Chunxiong Luo

A fast cell loading and high‐throughput microfluidic system for long‐term cell culture in zero‐flow environments

We present a simple technique for cell loading, culturing, and phenotypic study in a multi-chamber microfluidic device made of polydimethylsiloxane (PDMS). This technique is based on the use of degassing induced aspiration of PDMS which allows loading cells into micro-cavities within 1 min. A large number of triangle cavities are patterned aside main flow channels with narrow connections so that cells can be loaded by aspirating into each cavity. In our device, high throughput and long-term monitoring can be done with minimum shear force of the flow. As a demonstration, we show a controlled loading at single cell level and the phenotypic variation of gene expression of the yeast strain w303 as a function of copper ion concentration of the medium.

Single Cell Analysis of Yeast Replicative Aging Using a New Generation of Microfluidic Device

A major limitation to yeast aging study has been the inability to track mother cells and observe molecular markers during the aging process. The traditional lifespan assay relies on manual micro-manipulation to remove daughter cells from the mother, which is laborious, time consuming, and does not allow long term tracking with high resolution microscopy. Recently, we have developed a microfluidic system capable of retaining mother cells in the microfluidic chambers while removing daughter cells automatically, making it possible to observe fluorescent reporters in single cells throughout their lifespan. Here we report the development of a new generation of microfluidic device that overcomes several limitations of the previous system, making it easier to fabricate and operate, and allowing functions not possible with the previous design. The basic unit of the device consists of microfluidic channels with pensile columns that can physically trap the mother cells while allowing the removal of daughter cells automatically by the flow of the fresh media. The whole microfluidic device contains multiple independent units operating in parallel, allowing simultaneous analysis of multiple strains. Using this system, we have reproduced the lifespan curves for the known long and short-lived mutants, demonstrating the power of the device for automated lifespan measurement. Following fluorescent reporters in single mother cells throughout their lifespan, we discovered a surprising change of expression of the translation elongation factor TEF2 during aging, suggesting altered translational control in aged mother cells. Utilizing the capability of the new device to trap mother-daughter pairs, we analyzed mother-daughter inheritance and found age dependent asymmetric partitioning of a general stress response reporter between mother and daughter cells.

PDMS microfludic device for optical detection of protein immunoassay using gold nanoparticles

A simple but highly specific immunoassay system for goat anti-human IgG has been developed using gold nanoparticles and microfluidic techniques. The assay is based on the deposition of gold nanoparticles that are coated with protein antigens in the presence of their corresponding antibodies to microfluidic channel surface. The effects of time accumulation, the flow velocity, and the concentration of antibodies to the red light absorption percentage (RAP) of deposition were investigated with an ordinary optical microscope. By controlling the reaction time and flow velocity, a dynamic range of 3 orders of magnitude and a detection sensitivity of 10 ng ml(-1) of goat anti-human IgG were achieved. Because of its simplicity and flexibility, this new technique should be useful for fast, highthroughput screening of antibodies in clinical diagnostic applications.

Heteroresistance at the Single-Cell Level: Adapting to Antibiotic Stress through a Population-Based Strategy and Growth-Controlled Interphenotypic Coordination

ABSTRACT Heteroresistance refers to phenotypic heterogeneity of microbial clonal populations under antibiotic stress, and it has been thought to be an allocation of a subset of “resistant” cells for surviving in higher concentrations of antibiotic. The assumption fits the so-called bet-hedging strategy, where a bacterial population “hedges” its “bet” on different phenotypes to be selected by unpredicted environment stresses. To test this hypothesis, we constructed a heteroresistance model by introducing a blaCTX-M-14 gene (coding for a cephalosporin hydrolase) into a sensitive Escherichia coli strain. We confirmed heteroresistance in this clone and that a subset of the cells expressed more hydrolase and formed more colonies in the presence of ceftriaxone (exhibited stronger “resistance”). However, subsequent single-cell-level investigation by using a microfluidic device showed that a subset of cells with a distinguishable phenotype of slowed growth and intensified hydrolase expression emerged, and they were not positively selected but increased their proportion in the population with ascending antibiotic concentrations. Therefore, heteroresistance—the gradually decreased colony-forming capability in the presence of antibiotic—was a result of a decreased growth rate rather than of selection for resistant cells. Using a mock strain without the resistance gene, we further demonstrated the existence of two nested growth-centric feedback loops that control the expression of the hydrolase and maximize population growth in various antibiotic concentrations. In conclusion, phenotypic heterogeneity is a population-based strategy beneficial for bacterial survival and propagation through task allocation and interphenotypic collaboration, and the growth rate provides a critical control for the expression of stress-related genes and an essential mechanism in responding to environmental stresses. IMPORTANCE Heteroresistance is essentially phenotypic heterogeneity, where a population-based strategy is thought to be at work, being assumed to be variable cell-to-cell resistance to be selected under antibiotic stress. Exact mechanisms of heteroresistance and its roles in adaptation to antibiotic stress have yet to be fully understood at the molecular and single-cell levels. In our study, we have not been able to detect any apparent subset of “resistant” cells selected by antibiotics; on the contrary, cell populations differentiate into phenotypic subsets with variable growth statuses and hydrolase expression. The growth rate appears to be sensitive to stress intensity and plays a key role in controlling hydrolase expression at both the bulk population and single-cell levels. We have shown here, for the first time, that phenotypic heterogeneity can be beneficial to a growing bacterial population through task allocation and interphenotypic collaboration other than partitioning cells into different categories of selective advantage. Heteroresistance is essentially phenotypic heterogeneity, where a population-based strategy is thought to be at work, being assumed to be variable cell-to-cell resistance to be selected under antibiotic stress. Exact mechanisms of heteroresistance and its roles in adaptation to antibiotic stress have yet to be fully understood at the molecular and single-cell levels. In our study, we have not been able to detect any apparent subset of “resistant” cells selected by antibiotics; on the contrary, cell populations differentiate into phenotypic subsets with variable growth statuses and hydrolase expression. The growth rate appears to be sensitive to stress intensity and plays a key role in controlling hydrolase expression at both the bulk population and single-cell levels. We have shown here, for the first time, that phenotypic heterogeneity can be beneficial to a growing bacterial population through task allocation and interphenotypic collaboration other than partitioning cells into different categories of selective advantage.

Discovery of novel chemoeffectors and rational design of Escherichia coli chemoreceptor specificity

Significance Chemotaxis is a universal phenomenon whereby motile cells, like motile bacteria, navigate by following chemical gradients in their environment. Bacterial chemoreceptors can bind with specific chemoeffectors and transfer environmental signals to the cell. However, the molecular mechanisms for chemoeffector binding and signaling are not fully understood, and rational design of bacteria to respond to new chemicals has been challenging. In this study, by using a combined experimental and computational approach, we discovered novel antagonists and attractants for the Escherichia coli chemoreceptor Tar. The interaction differences of the novel antagonists and attractants with Tar provide clues to alter Tar–ligand specificity. Based on these understandings, E. coli strain was successfully engineered to sense l-arginine, a ligand unrecognized before. Bacterial chemoreceptors mediate chemotactic responses to diverse stimuli. Here, by using an integrated in silico, in vitro, and in vivo approach, we screened a large compound library and found eight novel chemoeffectors for the Escherichia coli chemoreceptor Tar. Six of the eight new Tar binding compounds induce attractant responses, and two of them function as antagonists that can bind Tar without inducing downstream signaling. Comparison between the antagonist and attractant binding patterns suggests that the key interactions for chemotaxis signaling are mediated by the hydrogen bonds formed between a donor group in the attractant and the main-chain carbonyls (Y149 and/or Q152) on the α4 helix of Tar. This molecular insight for signaling is verified by converting an antagonist to an attractant when introducing an N-H group into the antagonist to restore the hydrogen bond. Similar signal triggering effect by an O-H group is also confirmed. Our study suggests that the Tar chemoeffector binding pocket may be separated into two functional regions: region I mainly contributes to binding and region II contributes to both binding and signaling. This scenario of binding and signaling suggests that Tar may be rationally designed to respond to a nonnative ligand by altering key residues in region I to strengthen binding with the novel ligand while maintaining the key interactions in region II for signaling. Following this strategy, we have successfully redesigned Tar to respond to l-arginine, a basic amino acid that does not have chemotactic effect for WT Tar, by two site-specific mutations (R69′E and R73′E).

Dynamics between cancer cell subpopulations reveals a model coordinating with both hierarchical and stochastic concepts.

Tumors are often heterogeneous in which tumor cells of different phenotypes have distinct properties. For scientific and clinical interests, it is of fundamental importance to understand their properties and the dynamic variations among different phenotypes, specifically under radio- and/or chemo-therapy. Currently there are two controversial models describing tumor heterogeneity, the cancer stem cell (CSC) model and the stochastic model. To clarify the controversy, we measured probabilities of different division types and transitions of cells via in situ immunofluorescence. Based on the experiment data, we constructed a model that combines the CSC with the stochastic concepts, showing the existence of both distinctive CSC subpopulations and the stochastic transitions from NSCCs to CSCs. The results showed that the dynamic variations between CSCs and non-stem cancer cells (NSCCs) can be simulated with the model. Further studies also showed that the model can be used to describe the dynamics of the two subpopulations after radiation treatment. More importantly, analysis demonstrated that the experimental detectable equilibrium CSC proportion can be achieved only when the stochastic transitions from NSCCs to CSCs occur, indicating that tumor heterogeneity may exist in a model coordinating with both the CSC and the stochastic concepts. The mathematic model based on experimental parameters may contribute to a better understanding of the tumor heterogeneity, and provide references on the dynamics of CSC subpopulation during radiotherapy.

A micro-channel-well system for culture and differentiation of embryonic stem cells on different types of substrate

We have developed a combined micro-channel and micro-well system for easy cell loading, culture and post-culture operation on a chip. To demonstrate the reliability of the system, on chip cell culture and differentiation were performed with different types of substrates made of culture dish, glass cover slide and polydimethylsiloaxe (PDMS). As expected, mouse embryo fibroblasts (MEF) showed different adhesion and growth rate on different substrates. When embryonic stem (ES) cells were co-cultured with MEFs, the formation of ES colonies is efficient on both glass and Petri dish, although PDMS could also be used. Finally, ES cell differentiation with neuron growth factors was performed on different substrates, showing clear advantages of using culture Petri dish over both glass and PDMS.

Angle sensing in magnetotaxis of Magnetospirillum magneticum AMB-1

The mechanism of how magnetotactic bacteria navigate along the magnetic field has been a puzzle. Two main models disagree on whether the magnetotactic behavior results from passive alignment with the magnetic field or active sensing of the magnetic force. Here, we quantitatively studied the swimming patterns of Magnetospirillum magneticum AMB-1 cells to understand the origin of their magnetotactic behaviors. Single-cell tracking and swimming pattern analysis showed that the cells follow a mixed run-reverse-tumble pattern. The average run time decreased with the angle between the cells moving velocity and the external magnetic field. For mutant cells without the methyl-accepting chemotaxis protein (MCP) Amb0994, such dependence disappeared and bacteria failed to align with magnetic field lines. This dysfunction was recovered by complementary Amb0994 on a plasmid. At high magnetic field (>5 mT), all strains with intact magnetosome chains (including the Δamb0994-0995 strains) showed alignment with the external magnetic field. These results suggested that the mechanism for magnetotaxis is magnetic field dependent. Due to the magnetic dipole moment of the cell, the external magnetic field exerts a torque on the cell. In high magnetic fields, this torque is large enough to overcome the random re-orientation of the cell, and the cells align passively with the external magnetic field, much like a compass. In smaller (and biologically more relevant) external fields, the external force alone is not strong enough to align the cell mechanically. However, magnetotactic behaviors persist due to an active sensing mechanism in which the cell senses the torque by Amb0994 and actively regulates the flagella bias accordingly to align its orientation with the external magnetic field. Our results reconciled the two putative models for magnetotaxis and revealed a key molecular component in the underlying magneto-sensing pathway.

Studies of Antibiotic Resistance of Beta-Lactamase Bacteria under Different Nutrition Limitations at the Single-Cell Level

Drug resistance involves many biological processes, including cell growth, cell communication, and cell cooperation. In the last few decades, bacterial drug resistance studies have made substantial progress. However, a major limitation of the traditional resistance study still exists: most of the studies have concentrated on the average behavior of enormous amounts of cells rather than surveying single cells with different phenotypes or genotypes. Here, we report our study of beta-lactamase bacterial drug resistance in a well-designed microfluidic device, which allows us to conduct more controllable experiments, such as controlling the nutrient concentration, switching the culture media, performing parallel experiments, observing single cells, and acquiring time-lapse images. By using GFP as a beta-lactamase indicator and acquiring time-lapse images at the single-cell level, we observed correlations between the bacterial heterogeneous phenotypes and their behavior in different culture media. The feedback loop between the growth rate and the beta-lactamase production suggests that the beta-lactamase bacteria are more resistant in a rich medium than in a relatively poor medium. In the poorest medium, the proportion of dormant cells may increase, which causes a lower death rate in the same generation. Our work may contribute to assaying the antibiotic resistance of pathogenic bacteria in heterogeneous complex media.

Degassing-assisted patterning of cell culture surfaces.

We developed an alternative patterning technique which is capable of producing both topographic and biochemical features for cell culture studies. This technique is based on microaspiration induced with a degassed poly (dimethylsiloxane) (PDMS) mold. After degassing in a rough vacuum chamber and placed on a sample surface, liquid solution can be aspired through channels and cavities created in the PDMS mold. Depending on the composition of the solution and the associated drying or incubation processes, a variety of surface patterns can be produced without applying external pressure. For demonstration, we fabricated agarose gel microwells and biomolecule patterns either on a glass plate or in a cell culture Petri dish, both applicable for cell culture studies.