Methods and apparatus for transplantation of nucleic acid molecules

Patent metadata

Field	Value
Publication number	US-9834747-B2
Application number	US-201414449106-A
Country	US
Kind code	B2
Filing date	Jul 31, 2014
Priority date	Jul 31, 2013
Publication date	Dec 5, 2017
Grant date	Dec 5, 2017

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.

First claim

What is claimed is:

1. A method comprising, in combination:
(a) providing a microfluidic chamber;
(b) moving, into the chamber, one or more donor sources that contain nucleic acids;
(c) moving one or more lysis agents from a region external to the chamber into the chamber;
(d) triggering, by the lysis agents, lysis of the one or more donor sources, such that the lysis occurs inside the chamber;
(e) moving the lysis agents out of the chamber;
(f) moving one or more recipient cells into the chamber;
(g) moving one or more transplantation agents into the chamber; and
(h) triggering, by the transplantation agents, transplantation of the nucleic acids into the recipient cells, such that the transplantation occurs inside the chamber;
wherein at all times during steps (a), (b), (c), (d), (e), (f), (g) and (h), no more than one cell portal of the chamber exists.

2. The method of claim 1 , wherein the nucleic acids are large nucleic acids.

3. The method of claim 1 , wherein a cavity is located in the chamber, which cavity has a volume of less than one nanoliter.

4. The method of claim 1 , wherein the moving in steps (c), (e), (f) and (g) is by dead-end loading of the chamber or by diffusion.

5. The method of claim 1 , wherein neither the recipient cells nor the nucleic acids are attached to a wall of the chamber prior to or during the transplantation.

6. The method of claim 1 , wherein:
(i) the cell portal has first dimension, which first dimension is the maximum inner rim-to-inner rim distance of the cell portal;
(ii) the chamber is elongated and has a longitudinal axis along the length of the chamber;
(iii) the chamber has a second dimension, which second dimension is the maximum inner wall-to-inner wall distance of the chamber in any direction that is perpendicular to the longitudinal axis; and
(iv) the first dimension is less than the product of 0.8 and the second dimension.

7. The method of claim 1 , wherein:
(i) the chamber is elongated along a first longitudinal axis;
(ii) the cell portal is an opening into a first channel that is external to the chamber;
(iii) the first channel is elongated along a second longitudinal axis; and
(iv) the first longitudinal axis is at an angle of at least 45 degrees relative to the second longitudinal axis.

8. The method of claim 1 , wherein at least one sphere exists, such that (i) the cell portal subtends less than 3.14 steradians as seen from the center of the sphere, and (ii) the center of the sphere is located in the interior of the chamber.

Assignees

• Mershin Andreas
• Pelletier James
• Gershenfeld Neil
• Glass John
• Strychalski Elizabeth
• Massachusetts Inst Technology

Inventors

• Mershin Andreas
• Pelletier James
• Gershenfeld Neil
• Glass John
• Strychalski Elizabeth

Classifications

• C12N15/87

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title

• C12M35/08 Primary

  Chemical, biochemical or biological means, e.g. plasma jet, co-culture · CPC title