Dynamic single cell screening – A short review

Abstract: Dynamic single cell screening

Single cell screening helps us discover mechanisms which are not necessarily evident in bulk cell populations. Isolation and inoculation of single cells into separate culture chambers is a key requirement for such studies. Several highly sophisticated methods like fluorescence-activated cell sorting (FACS), Raman tweezers and laser capture are currently being used for single cell isolation. The authors present an elegant system which can be used for single cell screening and printing (inoculating) them into separate cell culture wells. The proposed microfluidic chip uses a dual microvalve system to screen cells based on their size. Two adjacent pneumatic microvalves connected to a programmable pressure controller were used to achieve size-based screening of cells. The upper and lower limits for the cell screening can be dynamically controlled based on the pressure employed at each of the valves. Screening of single beads, endothelial cells and their subsequent printing into well plates was demonstrated with 100% efficiency.

Introduction: Dynamic single cell screening

A high level of heterogeneity exists among individual cells, in terms of their morphology, proliferation and response to external stimuli. Experimental studies of heterogeneity at the single-cell level help us understand mechanisms, which cannot be elucidated from population-based studies. It is essential to extract individual cells from a mixed population to perform any single cell study in the fields of clonal expansion, monoclonality development or genomic studies. The most widely used methods for acquiring single cells are fluorescence-activated cell sorting (FACS)(1) and limiting dilution(2). FACS is not always accessible as it requires sophisticated equipment and trained professionals for operating it. Limiting dilution is highly cumbersome to implement as only a mere 30% of the wells can be seeded with single cells. 

On the other hand, microfluidics-based cell sorting has emerged as a highly efficient and easily accessible tool. The precise manipulation of fluids enabled by these platforms allows for implementation of a wide variety of cell sorting architectures. Microfluidic techniques for cell sorting include microfluidic filters (3), hydrodynamic sorting (4), deterministic lateral displacement, inertial microfluidic sorting, electrophoresis and acoustophoresis. Size-based sorting methods are the most optimal for single-cell studies. With no requirement for labelling, the cells remain viable with minimal damage for longer periods of time. In most scenarios, cell size is indicative of its type, as well as differences in cell function and metabolic changes. Size-based cell screening has been employed to perform a large number of studies on circulating tumor cells (5), white blood cells (6), platelets (7) and others. Among these, micro-filter membranes, hydrodynamic traps and microwells have been extensively studied for isolating a large number of single-cells for drug screening (8) and clonal culture studies (9). Microfluidic techniques have been proven to be highly efficient, with most techniques with more than 70% efficiency.

Video from “Dynamic screening and printing of single cells using a microfluidic chip with dual microvalves” by Chang chen, Dong Xu, Siwei Bai, Zhihang Yu, Yonggang Zhu, Xiao Xing and Huaying chen, published in Lab on Chip Journal.

Aim & Objectives

• To sort cells on a microfluidic chip based on their size
• To enable a sorting system, with dynamic control over lower and upper limits for size of cells to be sorted
• To assess the screening efficiency of the system and perform viability studies on the screened cells

Key findings: Dynamic single cell screening

The authors have employed a three-layer microfluidic device with two pneumatic microvalves to achieve size-based cell screening and subsequent printing into a standard well plate. The gas and fluid layers are separated by a membrane layer. The gas layer consists of two pneumatic channels, a 10 µm thick membrane layer with holes and a fluid layer with flow channels (as shown in Figure 1). The device was fabricated with PDMS using multi-layer soft-lithography.

Upon application of pressure in the gas channel, the membrane gets deformed. The difference between the overall channel height and membrane deflection will act as the valve clearance. With the application of the required pressure, the valve clearance at both the front and rear valves can be dynamically controlled. This enables an on-the-fly control over the lower and upper limits of cells that can be sorted. 

A numerical study was performed to determine the deformation of the PDMS membrane. The deformation of the membrane was experimentally characterized using confocal microscopy. Diluted fluorescent dye was flown through the fluid layer while the pressure acting at the valves was controlled using Elveflow OB1 Mk3 pressure controller. The flow channel was imaged to obtain the deflection curve determined by the dye boundary at various loading conditions. 

When the bead size (d) was less than clearance of the front valve (C1), then the cell does not get trapped at either of the valves. It flows freely into the waste channel due to the negative pressure applied at the waste outlet. When the size (d) is higher than the clearance of rear valve, the cell gets captured first at the front valve and is then released to be captured again at the rear valve. After this it flows through to the waste channel due to negative pressure. When d is within the range of both clearances, it gets trapped at the front valve and then gets released. Here, positive pressure is applied at the waste channel to make sure the cell/bead flows in the culture well.

In summary, the authors employed a pressure driven microfluidics with dual microvalve system to dynamically control the size limits of cells to be screened. The efficiency of printing single endothelial cells was found to be 100%. Further, the demonstrated viability studies prove that the damage to the cells was very low.

References