Available Technology

METHODS AND APPARATUS FOR TRANSPLANTATION OF NUCLEIC ACID MOLECULES

This invention mitigates a problem that occurs during conventional transfer of nucleic acids into a biological cell:  Nucleic acids outside cells, extracted from an organism or synthesized from oligonucleotides, are fragile and can be damaged by the shear forces that occur during fluid flow. These damages reduce the likelihood of a successful transfer. This problem is particularly acute when nucleic acids are large. For example, a large nucleic acid molecule with more than 100,000 base pairs tends to be more fragile and more susceptible to damage from shear forces, than a smaller nucleic acid.

This microfluidic device allows:

* buffers to move into and out of the chamber to make the recipient cells ready for transplantation and to trigger transplantation;

* gentle transplantation of nucleic acids into cells to occur in microfluidic chambers (e.g., by diffusion) shielded from shear forces that would damage the contents of the chamber during pipetting, or shaking;

* the chemical environment in the chamber to be controlled by fluidic methods;

* hundreds or thousands of side chambers to be arranged perpendicularly to flow channels for high throughput;

* a microscope to be used for high spatiotemporal visualization of large DNA and cells during the transfer process.

For example, for culturing cells (e.g., donor, recipient or transplanted cells), the microfluidic device delivers a steady stream of a nutrient-rich solution. Waste material produced by the cells, as well as cells that overgrow the chambers, flows away. The transplanted genetic material includes a gene for resistance to a particular antibiotic. After the cells recover from the transplantation, a solution containing the antibiotic is introduced to select for the transplanted cells and to eliminate recipient cells that did not receive the donor nucleic acids. The transplanted cells are then cultured until they overflow the chamber and enter a flow channel, from which they can be harvested as they flow out of the microfluidic device. An automated microscope with a camera is used to capture real-time visual data regarding events and objects inside the microfluidic device (such as loading of cells to chambers, lysis, transplantation, and cell culture). In addition, other sensors (e.g., embedded sensors) may be used to take real-time sensor readings of conditions (e.g., pH, temperature, pressure, or capacitance) within the microfluidic device.

Figure 1 below shows a microfluidic device for transplantation of nucleic acids (101).  The device comprises a polydimethylsiloxane (PDMS) slab (103) and a glass (e.g., borosilicate) cover slip (105). Microfluidic channels and chambers are recessed in either the PDMS slab (103) or the glass cover slip (105).  During assembly of the microfluidic device, surfaces of the PDMS slab (103) and glass cover slip (105) are activated with plasma oxygen and then bonded together to seal the microfluidic channels and compartments. A tube (107) interfaces with an inlet (109) in the PDMS slab (103). Another tube (111) interfaces with an outlet (1130 in the PDMS slab (103). An external syringe pump is used to pump fluid through the device.

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 come 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.

Inventors: 
Andreas Mershin, James Pelletier, Neil Gershenfield, John Glass, Elizabeth Strychalski
Patent Number: 
9,834,747
Technology Type(s): 
Analytical Chemistry, Health Care, Biochemical Science, Chemical Sciences, Physical and Chemical Properties, Process Measurement
Internal Laboratory Ref #: 
14-026
Patent Issue Date: 
December 5, 2017
Lab Representatives
Share to Facebook Share to Twitter Share to Google Plus Share to Linkedin