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Microfluidic Technology

Dolomite Bio systems, Nadia Instrument and Nadia Innovate, employ the principle of microfluidic flow technology focusing to rapidly encapsulate single cells in millions of aqueous droplets in oil. The droplets are identically sized and, depending on the application, may be 60 – 100 µm in diameter.

Microfluidic Technology- How it works

On a microfluidic chip droplet production is achieved using two immiscible fluids. Aqueous droplets are formed in a fluorocarbon oil carrier phase containing 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.Nadia junction - Microfluidic Technology

Use of fluorinated oils and surfactants

Fluorocarbon oil (rather than for example mineral oil) is used as the carrier phase for droplet production in the Dolomite Bio platform. 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 is added to the fluorinated oil to improve droplet stability, i.e. to ensure that droplets do not coalesce after formation.

Benefits of encapsulating cells in microfluidic droplets

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

Introduction of cells and beads into droplets at Poisson distribution

Cells are loaded into the Nadia Instrument with a pipette in a 250 to 100 µl suspension. Dolomite Bio’s patented stirring technology enbales low doublet rates and reduced clumbing of cells and beads during a scRNA-Seq run.

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.

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

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