Microfluidic Immunoassays for Time-Resolved Measurement of Protein Secretion from Single Cells

Measurement of humoral factors secreted from cells has served as an indispensable method to monitor the states of a cell ensemble because humoral factors play crucial roles in cell–cell interaction and aptly reflect the states of individual cells. Although a cell ensemble consisting of a large number of cells has conventionally been the object of such measurements, recent advances in microfluidic technology together with highly sensitive immunoassays have enabled us to quantify secreted humoral factors even from individual cells in either a population or a temporal context. Many groups have reported various miniaturized platforms for immunoassays of proteins secreted from single cells. This review focuses on the current status of time-resolved assay platforms for protein secretion with single-cell resolution. We also discuss future perspectives of time-resolved immunoassays from the viewpoint of systems biology.

Extracellular matrix stiffness regulates force transmission pathways in multicellular ensembles of human airway smooth muscle cells

For an airway or a blood vessel to narrow, there must be a connected path that links the smooth muscle (SM) cells with each other, and transmits forces around the organ, causing it to constrict. Currently, we know very little about the mechanisms that regulate force transmission pathways in a multicellular SM ensemble. Here, we used extracellular matrix (ECM) micropatterning to study force transmission in a two-cell ensemble of SM cells. Using the two-SM cell ensemble, we demonstrate (a) that ECM stiffness acts as a switch that regulates whether SM force is transmitted through the ECM or through cell-cell connections. (b) Fluorescent imaging for adherens junctions and focal adhesions show the progressive loss of cell-cell borders and the appearance of focal adhesions with the increase in ECM stiffness (confirming our mechanical measurements). (c) At the same ECM stiffness, we show that the presence of a cell-cell border substantially decreases the overall contractility of the SM cell ensemble. Our results demonstrate that connectivity among SM cells is a critical factor to consider in the development of diseases such as asthma and hypertension.

Synapse-specific representation of the identity of overlapping memory engrams

Memories are integrated into interconnected networks; nevertheless, each memory has its own identity. How the brain defines specific memory identity out of intermingled memories stored in a shared cell ensemble has remained elusive. We found that after complete retrograde amnesia of auditory fear conditioning in mice, optogenetic stimulation of the auditory inputs to the lateral amygdala failed to induce memory recall, implying that the memory engram no longer existed in that circuit. Complete amnesia of a given fear memory did not affect another linked fear memory encoded in the shared ensemble. Optogenetic potentiation or depotentiation of the plasticity at synapses specific to one memory affected the recall of only that memory. Thus, the sharing of engram cells underlies the linkage between memories, whereas synapse-specific plasticity guarantees the identity and storage of individual memories.

AFM-based bivariate morphological discrimination of apoptosis induced by photodynamic therapy using photosensitizer-functionalized gold nanoparticles

Typical examples of the morphology of one viable and one apoptotic cell together with the statistical analysis of a larger cell ensemble subsequent to photodynamic treatment.

Enhancing Range of Transport in Optical Tweezers Assisted Microfluidic Chambers Using Automated Stage Motion

In this paper, we present a planning approach for automated high-speed transport of cells over large distances inside an Optical Tweezers (OT) assisted microfluidic chamber. The transport is performed in three steps that combine the optical trap and motorized stage motions. This includes optical trapping and transporting the cells to form a desired cell-ensemble that is suitable for a long distance transport, automatically moving the motorized stage to transport the cell-ensemble over a large distance while avoiding static obstacles, and distributing the cells from the ensemble to the desired locations using OT. The speeds of optical traps and the motorized stage are determined by modeling the motion of the particle under the influence of optical trap. The desired cell-ensemble is automatically determined based on the geometry of the microfluidic chamber. We have developed a greedy heuristic method for optimal selection of the initial and the final location of the cell-ensemble to minimize the overall transport time while satisfying the constraints of the OT workspace. We have discussed the computational complexity of the developed method and compared it with exhaustive combinatorial search. The approach is particularly useful in applications where cells are needed to be rapidly distributed inside a microfluidic chamber. We show the capability of our planning approach using physical experiments.