How to dynamically screen and print single cells using microfluidics

This short review article is based on the research paper titled “The method to dynamically screen and print single cells using microfluidics with pneumatic microvalves”, authored by Chang Chen, Yonggang Zhu, Joshua W.K. Ho and Huaying Chen.

The research paper was published in The MethodsX. Journal in December, 2020. The study explores a method to dynamically screen and print single cells using microfluidics with pneumatic microvalves. The authors employed a microfluidic chip with dual microvalves to dynamically control the size limits of cells to be screened and printed into standard well plates. A pressure-driven flow controller was used to precisely regulate the negative and positive pressure to either activate the pneumatic microvalves or drive the fluid flow for printing.

Abstract: how to dynamically screen and print single cells

Single-cell clonal expansion and sequencing using traditional equipment, like other single-cell analysis, have the crucial need to print single cells into individual chambers. Types, functions, and even cell cycle phases influence their size. Thus, to perform single-cell analysis, there is a need to print individual cells within the desired size range.

This paper presents a method for the development of a microfluidic chip integrating pneumatic microvalves to dynamically screen and print single cells. The reported method provided essential guidelines for single cell screening and printing (inoculating) into separate cell culture wells. The proposed microfluidic chip uses a dual microvalve system to screen cells based on their size.

Watch the webinar by our researcher Lisa Muiznieks about dynamic cell culture!

 You can also read our application note about medium recirculation for dynamic cell culture

Introduction

Single-cell printing describes the capability of isolating a large amount of desired individual cells from a mixed population. Inoculation of single cells into separate culture chambers is one of the key requirements in single-cell analysis [1]. 

Microfluidic technologies have been exploited for:

• on-chip analysis (cell culture [2] and biomarker analysis [4]
• pre-treatment on chip before further analysis using traditional equipment, e.g., cell inoculation and CRISPR-Cas9 delivery [5]
• automatisation and high-speed isolation of single cells by inkjet-like printing [6] 

A microfluidic chip using a dual microvalve system was developed by Chen et al. [1] to screen cells based on their size. 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, human umbilical vein endothelial cells (HUVECs) and their subsequent printing into well plates was demonstrated with 100% efficiency.

The aim of this paper is to deliver a method on how to dynamically screen and print single cells, using microfluidics with pneumatic microvalves. It presents the fabrication details of the multi-layer microfluidic chip, control of the membrane deflection to screen cell size, and printing of single cells.

Aim & objectives

 In short, this paper is about :

• the manufacturing of the chip using standard soft lithography  
• the protocol to set up a sorting system, with control over lower and upper limits for size of cells
• the screening efficiency of the system and how to perform viability studies on suspended human umbilical vein endothelial cells (HUVECs) into 384-well plates with high viability.

Key findings

1. Manufacturing of the chip using standard soft lithography

a- Design of the microfluidic chip

The planar design of the chip was depicted in Fig. 2A. The chip is composed of three layers: the gas layer, the membrane layer, and the fluid layer. The microfluidic network layouts were created using AutoCAD2018 (Educational version).

b- Device manufacture

The masters of the flow and pneumatic layers were manufactured by standard photolithography in the clean room. The flow and pneumatic layers were fabricated by casting uncured PDMS against the maters. The PDMS membrane was spin-coated on the silicon water. The membrane thickness at various spin speeds was reported elsewhere [1,3]. Three PDMS layers were irreversibly bonded together (Fig. 2C) following oxygen plasma oxidiation.

c- Platform construction

The microfluidic chip was connected to airtight tubes and the ELVEFLOWⓇ OB1 Mk3 pressure controller was used to regulate the pressure (both negative and positive, with precision) which permitted to either drive the fluid flow for printing or activate the pneumatic microvalves.

For more information about the working principle of pressure-driven flow controlled microfluidics, please refer to this video!

2. The protocol to screen and print single cells with desired sizes

Channel clearance defined by the deflected valve membrane allows control over lower and upper limits for size of cells.

The valve membrane was precisely regulated by the positive pressure from the pressure controller. The pressure applied on both microvalves to screen cells was determined according to the empirical equation below [1]:

P=2.12-0.096 D cell

Where P (atm) was the applied pressure and D cell (µm) was either the upper or the lower diameter limit of the cell for screening.

3. Screening efficiency of the system

The dual pneumatic microvalves were designed for screening suspended cells. The front microvalve activated using a bigger pressure (P1) determined the lower limit (d1) of the cells, whereas the back one applied with a smaller pressure (P2) adjusted the upper limit (d2) of the cells [1]. During the cell screening experiments, the microvalves were controlled manually using the supporting software called ElveflowⓇ Smart Interface.

Finally, HUVECs were employed as a model to demonstrate the screening and printing capabilities of the microfluidic chip. After trypsinizing and resuspending of HUVECs in the medium, the cell suspension was stored in the airtight tube (Fig. 3) for cell printing for less than 1.5 h to avoid the influence of the cell viability by suspension state.

The microfluidic chip with dual valves is capable of dynamic size selection and printing of single cells. It will be of broad application potential in the field of single-cell study.

Conclusion

The authors employed a pressure-driven flow controlled microfluidics with a 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%. This study shows the great potential of the microfluidic chip with dual microvalves in the field of single-cell inoculation for subsequent analyses.

References