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Microfluidic Technology

Dolomite Bio systems employ the principle of microfluidic flow technology focussing to rapidly encapsulate single cells in millions of aqueous droplets in oil. The droplets are identically sized and, depending on the application, may be 10 – 100 µm in diameter (optionally with beads and reagents).

Benefits of encapsulating cells in microfluidic droplets

• Enables analysis of millions of single cells
               o e.g. more than 105 single-cell libraries/hour
• Single-cell reactions become efficient and robust
               o Droplets are small (often 10s to 100s of picolitres), so e.g., effective mRNA concentration is high
• Reliable and reproducible performance
               o Precisely-controlled micro-reactor volumes and avoidance of cross-contamination
• Droplets can be used as micro-compartments or micro-reactors
• Can capture quantitative data from rare cells

MicrofluidictechnologyFlow focusing

Droplet production is achieved using two immiscible fluids (aqueous droplets in a fluorocarbon oil carrier phase with bio-compatible surfactant). A flow-focusing method, coupled with extremely smooth pressure driven pumps is used to make monodisperse i.e. <5% CV (coefficient of variation) droplets. Typically, immediately prior to dropletization, two independent aqueous streams (e.g. cells and beads) are combined.

Use of fluorinated oils and surfactants

Fluorocarbon oil (rather than for example mineral oil) is used as the carrier phase for droplet production in Dolomite Bio Systems. This is because it is stable, inert, biocompatible and allows gas exchange. As a result, if required, cell viability can be maintained.
A biocompatible surfactant (Pico-SurfTM) is added to the fluorinated oil to improve droplet stability, i.e. to ensure that droplets do not coalesce after formation.

Introduction of cells and beads into droplets and Poisson distribution

Cells are loaded into the µEncapsulator System as a 100µl suspension with a pipette. In the RNA Seq System, cells are loaded into a microcentrifuge tube which is externally agitated. Beads may either be introduced in a similar way to cells or loaded via a sample loop (in the case of dense or fragile beads).

A suspension of cells and beads will be delivered into droplets with a Poisson distribution. e.g. when aiming to achieve 1 cell per droplet, the actual number of cells per droplet, over 10 droplets, may be 1, 1, 1, 2, 0, 1, 1, 0, 1, 2.
It is frequently desired to have not more than one cell per droplet to avoid for example mRNA from two cells being captured on one barcoded bead. This is generally achieved by increasing the dilution, e.g. to 1 cell per 10 droplets, thus minimizing the frequency of two cells in one droplet, e.g. when aiming to achieve 1 cell per 10 droplets, the number of cells per droplets, over 10 droplets, should be 0, 0, 1, 0, 0, 0, 0, 0, 0, 0.

Double emulsions

Where an oil shell is required around the droplet (e.g. for FACS sorting), the aqueous droplets are first formed in oil, as if regular single emulsions were required. The resulting aqueous droplets in fluorinated oil are then re-introduced into another flow-focusing droplet junction with aqueous carrier phase. More information on applications for the technology can be found at Droplets for FACS-sorting libraries.

Intellectual property for droplet generation

Dolomite Bio is a licensee of Japan Science and Technology Agency (“JST”) under JST’s microdroplet generation technology. We are authorised to directly grant sub-licenses under JST’s microdroplet generation technologies for R&D purposes, please contact us for other usage.
The JST family of patents is core to much of micro-droplet technology. By purchasing a JST sub-license from Dolomite Bio, you will be gaining access to:
WO2002/068104 Patent Family for process for producing emulsion and microcapsules and apparatus thereof, including patents 7268167, 7772287, 7717615, 7375140
WO2005/089921 Patent Family for method and device for producing microdroplets, including patent 0196397