Methods and apparatus for transplantation of nucleic acid molecules
Inventors
Mershin, Andreas • Pelletier, James • Gershenfeld, Neil • Glass, John • Strychalski, Elizabeth
Assignees
Massachusetts Institute of Technology • National Institute of Standards and Technology NIST
Publication Number
US-9834747-B2
Publication Date
2017-12-05
Expiration Date
2034-07-31
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Abstract
In exemplary implementations, transplantation of nucleic acids into cells occurs in microfluidic chambers. The nucleic acids may be large nucleic acid molecules with more than 100 kbp. In some cases, the microfluidic chambers have only one orifice that opens to a flow channel. In some cases, flow through a microfluidic chamber temporarily ceases due to closing one or more valves. Transplantation occurs during a period in which the contents of the chambers are shielded from shear forces. Diffusion, centrifugation, suction from a vacuum channel, or dead-end loading may be used to move cells or buffers into the chambers.
Core Innovation
The invention addresses the problem of damage to nucleic acids during conventional methods of transferring them into biological cells, particularly large nucleic acids greater than 100,000 base pairs, which are especially fragile and susceptible to shear forces generated during fluid flow. Such damage reduces the success likelihood of transplantation into recipient cells.
The invention provides a method where interaction between large nucleic acids and recipient cells occurs within microfluidic chambers shielded from shear forces. These chambers have controlled chemical environments through fluidic methods, allow parallelized processing for high-throughput and multiplexed screening, and enable high spatiotemporal visualization via microscopy during the transfer operations. Cells and buffers are introduced gently into the chambers, for example, by diffusion, centrifugation, suction from vacuum channels, or dead-end loading, thus avoiding damaging shear forces.
In this method, the microfluidic chamber may have only one orifice opening to a flow channel, with flow temporarily ceased when valves close, ensuring the contents are free from shear forces during transplantation. Buffers flow gently into the chamber mainly by diffusion or slow induced flow, maintaining low Reynolds numbers and very slow fluid velocities, which protect the nucleic acids and cells. Lysis agents can diffuse into the chamber to lyse donor sources, followed by recipient cells and transplantation agents diffusing in to trigger transplantation within the chamber, all under conditions preventing attachment of cells or nucleic acids to chamber walls.
Claims Coverage
The patent includes one independent claim defining a method involving microfluidic chambers, moving donor sources, lysis agents, recipient cells, and transplantation agents to achieve transplantation inside the chamber with limited cell portals.
Method of transplantation within microfluidic chambers with single cell portal
A method providing a microfluidic chamber having no more than one cell portal, moving donor sources containing nucleic acids into the chamber, introducing lysis agents to lyse donor sources inside the chamber, removing lysis agents, moving recipient cells into the chamber, introducing transplantation agents, and triggering transplantation of nucleic acids into recipient cells within the chamber.
Use of large nucleic acids during transplantation
Specifying that the nucleic acids involved in transplantation within the chamber are large nucleic acids.
Microfluidic chamber size constraints
The chamber includes a cavity with volume less than one nanoliter.
Loading steps by dead-end loading or diffusion
Moving lysis agents, removing them, moving recipient cells, and moving transplantation agents into the chamber is by dead-end loading or diffusion.
Prevention of attachment to chamber walls
Recipient cells and nucleic acids are not attached to chamber walls before or during transplantation.
Geometrical constraints on chamber and portal
The cell portal's maximum inner rim-to-rim distance is less than 0.8 times the chamber's maximum inner wall-to-wall distance perpendicular to the elongated chamber axis.
Orientation between chamber axis and channel axis
The chamber's longitudinal axis is at least 45 degrees relative to the longitudinal axis of the external channel to which the cell portal opens.
Steradian subtending of the cell portal
At least one sphere exists with its center inside the chamber such that the cell portal subtends less than 3.14 steradians from the sphere's center.
The claims collectively cover a microfluidic method and apparatus for transplantation of nucleic acids into recipient cells inside chambers specifically designed to shield contents from shear forces, with controlled loading, lysis, and transplantation steps under fluidic and geometrical conditions ensuring protection and efficiency, especially for large nucleic acids.
Stated Advantages
The microfluidic method protects fragile large nucleic acids from damaging shear forces during transplantation by confining cells and nucleic acids in chambers with controlled fluid flow.
Parallelized microfluidic processing enables high-throughput and multiplexed screening of transplantation conditions.
Controlled chemical and physical environments allow fast and precise modification of conditions to optimize transplantation.
Use of automated microscopy and sensors enables real-time, high-resolution monitoring of transplantation processes.
Gentle loading methods, such as diffusion, centrifugation, and vacuum-assisted loading, reduce damage to nucleic acids and cells.
Microfluidic confinement increases transplantation yield and reproducibility by preserving nucleic acid integrity and controlling physical interactions.
Documented Applications
Use of microfluidic platforms to transplant large nucleic acids, including entire genomes or chromosomes, into biological recipient cells.
Determining optimal growth and transplantation conditions for difficult-to-culture microorganisms using microfluidic screening of gradients.
Generation of cloned or recoded bacteria resistant to bacteriophages for industrial or medical biomanufacturing.
Microfluidic isolation of yeast centromeric plasmids and human artificial chromosomes and their transplantation into suitable recipient cells.
High-throughput screening and optimization of physical and chemical factors influencing nucleic acid transplantation and genetic modification.
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