The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation

Abstract

There is a recognized and growing need for rapid and efficient cell assays, where the size of microfluidic devices lend themselves to the manipulation of cellular populations down to the single cell level. An exceptional way to analyze cells independently is to encapsulate them within aqueous droplets surrounded by an immiscible fluid, so that reagents and reaction products are contained within a controlled microenvironment. Most cell encapsulation work has focused on the development and use of passive methods, where droplets are produced continuously at high rates by pumping fluids from external pressure-driven reservoirs through defined microfluidic geometries. With limited exceptions, the number of cells encapsulated per droplet in these systems is dictated by Poisson statistics, reducing the proportion of droplets that contain the desired number of cells and thus the effective rate at which single cells can be encapsulated. Nevertheless, a number of recently developed actively-controlled droplet production methods present an alternative route to the production of droplets at similar rates and with the potential to improve the efficiency of single-cell encapsulation. In this critical review, we examine both passive and active methods for droplet production and explore how these can be used to deterministically and non-deterministically encapsulate cells.

Droplet microfluidics for synthetic biology

“…To improve these statistics, various active and more controlled encapsulation methods are being developed. 61 An interesting approach is to use droplet microfluidics and a simple motorized system to streak droplets with a single cell on agar plates (Fig. 3b).…”

Dissecting the stem cell niche with organoid models: an engineering-based approach

“…An advantage of this technique over microwells is the speed of droplet generation and the ease of automation, which allows high-throughput assembly of cell aggregates (Allazetta and Lutolf, 2015;Chan et al, 2013;Tumarkin et al, 2011). In contrast to microwells, which rely on probabilistic cell loading, recent advances in droplet-based microfluidics can achieve singlecell droplet loading and can load droplets with combinations of cell types with a precision that exceeds Poisson limitations (Collins et al, 2015;Edd et al, 2008;Schoeman et al, 2014). These devices can also be used to capture precise cell pairs, facilitating the dynamic dissection of cell-cell interactions from the initiation of contact (Dura et al, 2016).…”

Parallel droplet microfluidics for high throughput cell encapsulation and synthetic microgel generation

“…Furthermore, cells suspended at high concentrations (410 6 cells per mL) tend to aggregate, which can lead to incomplete cell encapsulation as well as increased microgel diameters. Therefore, low concentration cell suspensions (10 4 -10 5 cells per mL) are typically used, often resulting in a high percentage of empty microgels according to the Poisson distribution 25 , which is undesirable for in vivo efficacy and limits applications of encapsulation. Microgels containing cells can be purified by sorting or can be selectively crosslinked 7,24,26,27 , but encapsulating clinically relevant numbers of cells in these very small microgels is highly challenging, since several tens of millions of cells may be required for therapeutic efficacy 28,29 .…”