Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers

Abstract

Microfluidic cultivation, with its high level of environmental control and spatio-temporal resolution of cellular behavior, is a well-established tool in today's microfluidics. Yet, reliable retention of (randomly) motile cells inside designated cultivation compartments still represents a limitation, which prohibits systematic single-cell growth studies. To overcome this obstacle, current approaches rely on complex multilayer chips or on-chip valves, which makes their application for a broad community of users infeasible. Here, we present an easy-to-implement cell retention concept to withhold cells inside microfluidic cultivation chambers. By introducing a blocking structure into a cultivation chamber's entrance and nearly closing it, cells can be manually pushed into the chamber during loading procedures but are unable to leave it autonomously in subsequent long-term cultivation. CFD simulations as well as trace substance experiments confirm sufficient nutrient supply within the chamber. Through preventing recurring cell loss, growth data obtained from Chinese hamster ovary cultivation on colony level perfectly match data determined from single-cell data, which eventually allows reliable high throughput studies of single-cell growth. Due to its transferability to other chamber-based approaches, we strongly believe that our concept is also applicable for a broad range of cellular taxis studies or analyses of directed migration in basic or biomedical research.

Figure 1

Structure and cell retention concept of the MSCC device with enhanced cell retention for CHO suspension cell lines. (a) Microfluidic PDMS-glass-based cultivation device. (b) Scanning electron microscopy image of the microfluidic structure illustrating the devices dimensions and trapping barrier. (c) Schematic drawing of the loading procedure and cell retention concept based on a PDMS barrier that is traversable by applying pressure during cell loading but non-traversable by random cellular movement during cultivation. (d) Scanning electron microscopy image of the PDMS barrier located in the cultivation chamber’s entrance.

Figure 2

Microfluidic characterization of the MSCC designs. (a) Individual cultivation array that contains 60 cultivation chambers with either our previous chamber design (Design 1) or the chamber design from this work (Design 2). (b) Image sequence of trace substance experiments to quantify diffusive mass exchange for the MSCC device with Design 2. (c) Medium exchange duration until full equilibrium between channel and chamber is achieved based on rel. fluorescein signal for both designs. (d) Glucose concentration profile during MSCC cultivation assuming a steady state with 181 cells inside the chamber with a constant glucose uptake rate of 3800 nmol per 10⁶ cells and day for both designs.

Figure 3

MSCC of CHO cells and cell retention assessment of the MSCC designs. (a) Growth of three CHO-K1 microcolonies from three individual cultivation arrays cultivated applying Design 1. (b) Semi-logarithmically plotted growth profile of the microcolonies cultivated applying Design 1. (c) Growth of three CHO-K1 microcolonies from two individual cultivation arrays cultivated applying Design 2. (d) Semi-logarithmically plotted growth profile of the microcolonies cultivated applying Design 2.

Figure 4

Comparison of single-cell division behavior between the two microfluidic cell retention concepts. Depicted are the single-cell doubling times t_D of cells cultivated in chambers with Design 1 (n = 29, 25, 24) and Design 2 (n = 25, 27, 30). The colored segment marks the interquartile range from 25 to 75%, the horizontal lines show the median. The whiskers represent the 10% and 90% percentile and the tilted squares mark rare cellular events outside the predefined percentiles.