E. COLI CULTURE IN DROPLETS USING dSURF FLUOROSURFACTANT
In vitro cell culture is a fundamental component of biological production systems and biotechnological research. The ability to grow cells outside of their natural environment offers many possibilities ranging from high quantity production of enzymes to cell toxicity studies.
In this application note, discover how using microfluidics allows one to encapsulate single or multiple cells into tiny droplets of pL volume which are generated at a rate of approximately one thousand per second.
Traditionally, in vitro cell culture methods use petri dishes or culture flasks for laboratory cell culture. In culture flasks, cells are grown in a homogenous liquid medium, while petri dishes allow the growth of colonies on a solid or semi-solid substrate surface. Nevertheless, these two methods have limitations including the difficulty to compartmentalize clones and single cells.Compartmentalization allow single cells or clones to be individually assayed or exposed to different culture conditions or stimuli for versatile (or multiparametric) analysis of cells heterogeneity.
Long term cell culture experiments have been successfully performed with water-in-perfluorinated oil droplets. Moreover, fluorinated oils show lower organic molecule transfer drop to drop. Finally, while mineral oils swell PDMS chips, fluorinated oils show a better compatibility. Though perfluorinated oils offer benefits for applications such as cell cultivation or dPCR, to find an adequate and efficient surfactant for droplet stabilization in this oil remains a challenge. To date, most commercially available surfactants present limitations. The objective of this study is to highlight the biocompatibility of the new surfactant dSURF by culturing microorganisms in picoliter droplets.
MATERIALS & METHODS
Continuous phase Reagents:
Novec HFE-7500 (Sigma Aldrich) containing 0.5% or 3% dSURF (Fluigent, P/N DR-RE-SU-A1)
Dispersed phase Reagents:
500µl suspension of E. coli ECJW922 in TB (Terrific broth) medium with OD600 (optical density) of 0.005 (5 x 106 CFU/ml) or 0.01 (10 x 107 CFU/ml), which means cell concentration in the first suspension was 5 x 106 CFU/ml and in second suspension 10 x 107 CFU/ml. CFU – colony forming unit.
Droplet generation was performed with customer designed PDMS chip but we would recommend to use the EZ drop chip (Fluigent, P/N DropKit01)
To generate droplets, fluid handling devices such as pressure controller, syringe pump or peristaltic pump can be used with a flow focusing PDMS chip. Pressure controller as Fluigent LineUPTM series or MFCSTM-EZ are best suited to optimize droplet generation performance such as Flow rate stability and droplet monodispersity. In this case, E Coli suspension and dOIL with dSURF are loaded into vials. A pressure is applied to the reservoir to ensure a continuous and pulseless injection of both phases into the chip. The determined flow rate is monitored and controled by using the A-i-O software or local control on the Flow EZTM to achieve the desired droplet size and frequency.
Test with E. coli cells at OD= 0.005 and 0.5% surfactant concentration, incubation at 37 °C:
Legend: (A) Images of droplets under bright-field after 4 h incubation (B) Image of droplets under bright-field after 20h incubation
C) Images of droplets under dark-field after 4 h incubation (D) Images of droplets under dark-field after 20 h incubation
Stable, monodispersed droplet emulsions were observed on both images. High numbers of E. coli cells were observed in almost half of the droplets, particularly in 46±0.8 % of droplets, which is in a good agreement with the theoretical number of 54% calculated from the Poisson distribution of cells over 33.6 µm droplets (160 pL) at a given concentration of 5 x 106 CFU/ml.
The variability between theoretical and experimental numbers could be explained by the choice to use a low E. coli concentration and fractional droplet occupation with cells as well as by small variation in the experimental procedure, such as general pipetting imprecision.
Growth of E. coli after 20 h of incubation in all above described cases has highlighted that dSURF is biocompatible at concentrations ranging from 0.5% to 3%. For routine applications and cost reduction, 0.5% concentration of dSURF can be used. It is also suitable for experiments starting with single and multiple microbial cells per droplet.
The emulsion stability after 20 h shows the good droplet stabilization which will be of use for longer duration experiments.
This study has been made in collaboration with Dr Oksana Shvydkiv and her lab from Leibniz Institute for Natural Product Research and Infection Biology.
This application note was made in collaboration with Oksana Shvydkiv and her lab. For more information on the team visit :https://www.leibniz-hki.de/en/droplet-based-microfluidics.html