Microfluidic dual-well device for highthroughput single-cell capture and culture

Patent metadata

Field	Value
Publication number	US-10000732-B2
Application number	US-201615159712-A
Country	US
Kind code	B2
Filing date	May 19, 2016
Priority date	Nov 20, 2015
Publication date	Jun 19, 2018
Grant date	Jun 19, 2018

Abstract

A microfluidic dual-well device is disclosed. The device comprises: (a) a first substrate having a first end, a second end, and a culture microwell forming portion; (b) a plurality of culture microwells; (c) a second substrate having a first end, a second end, and a capture microwell forming portion, the two ends of the second substrate being respectively bounded to the two ends of the first substrate; (d) a plurality of capture microwells; (e) a microfluidic channel; (f) a microfluidic inlet port; and (g) a microfluidic outlet port; wherein the microfluidic channel is in fluidic connections with the culture microwells, the capture microwells, and the inlet and outlet ports. Methods of capturing and transferring a single cell or a single cell colony for culture, and method of transferring a target cell from a polydimethylsiloxane (PDMS) structure of culture microwells to a culture plate for culture are also disclosed.

First claim

What is claimed is:

1. A microfluidic dual-well device comprising:
(a) a first substrate, having a first end, a second end opposite to the first end, and a culture microwell forming portion with a thickness of t1 located between the two ends, each end having a first surface and a second surface and a thickness of T1, the culture microwell forming portion having a first surface and a second surface opposite to the first surface wherein the thickness T1 of each end of the first substrate is greater than the thickness t1 of the culture microwell forming portion;
(b) a plurality of culture microwells, each microwell having a diameter of w1 and a depth of d1, extending from the second surface toward the first surface of the culture microwell forming portion;
(c) a second substrate with a thickness of t2, having a first surface and a second surface opposite to the first surface of the second substrate, the second substrate having a first end, a second end opposite to the first end, and a capture microwell forming portion located between the two ends of the second substrate, the two ends of the second substrate being respectively bounded to the two ends of the first substrate with its second surface facing toward the second surface of the first substrate at a distance of h;
(d) a plurality of capture microwells, each microwell having a diameter of w2 and a depth of d2, extending from the second surface toward the first surface of the second substrate, wherein the capture microwells are in alignment with the culture microwells so that each microwell is corresponding to one culture microwell and the projected area of each capture microwell is within the projected area of the corresponding culture well, wherein the diameter w2 of each capture microwell is smaller than the diameter w1 of each culture microwells;
(e) a microfluidic channel with a length of l, a width of w3 and a height of h, formed between the two second surfaces of culture and capture microwell forming portions;
(f) a microfluidic inlet port defining an opening in the first surface of the first substrate; and
(g) a microfluidic outlet port defining an opening in the first surface of the first substrate, opposite to the microfluidic inlet port;
wherein the microfluidic channel is in fluidic connections with the culture microwells, the capture microwells, and the inlet and outlet ports.

2. The microfluidic dual-well device of claim 1 , wherein the surfaces of the microfluidic channel are coated with albumin.

3. The microfluidic dual-well device of claim 1 , wherein the surfaces of the microfluidic channel are coated with bovine serum albumin.

4. The microfluidic dual-well device of claim 1 , further comprising a fitting adapted to seal the inlet and outlet ports.

5. The microfluidic dual-well device of claim 1 , further comprising cells suspended in a medium within the microfluidic channel, wherein the size of each well of the capture microwells is adapted to capture a single cell from the cells within the microfluidic channel.

6. The microfluidic dual-well device of claim 1 , further comprising single cells in the capture microwells.

7. The microfluidic dual-well device of claim 6 , wherein the inlet and outlet ports are sealed liquid-tight.

8. The microfluidic dual-well device of claim 1 , further comprising single cells in the culture microwells.

9. The microfluidic dual-well device of claim 8 , wherein the inlet and outlet ports are sealed liquid-tight.

10. The microfluidic dual-well device of claim 8 , wherein more than one well of the culture microwells comprise a single cell, and each well of the culture microwells has space adapted for the single cell to attach, grow and/or proliferate.

11. The microfluidic dual-well device of claim 8 , wherein more than one well of the culture microwells comprise a single-cell colony.

12. The microfluidic dual-well device of claim 1 , further comprising a tubing adapted to connect to the inlet and/or outlet ports.

13. The microfluidic dual-well device of claim 1 , wherein each well of the capture microwells has a depth of no greater than 30 μm.

14. The microfluidic dual-well device of claim 13 , wherein each well of the capture microwells has a depth of less than 30 μm.

15. The microfluidic dual-well device of claim 1 , wherein the first and second substrates are made of a material selected from the group consisting of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), and polycarbonate (PC).

16. A method of capturing and transferring a single cell or a single cell colony for culture, comprising:
(a) providing the microfluidic dual-well device of claim 2 with the first substrate on the top and the second substrate at the bottom;
(b) loading cells suspended in a culture medium into the microfluidic channel via the inlet port;
(c) allowing the cells in the microfluidic channel to settle into and captured by the capture microwells and/or to settle on the second surface of the second substrate;
(d) washing away uncaptured cells by delivering a washing medium into the microfluidic channel via the inlet port; and
(e) flipping over the microfluidic dual-well device to place the first substrate at the bottom and the second substrate on the top to transfer the captured cells from the capture microwells to the culture microwells for culture.

17. The method of claim 16 , wherein the first and second substrates are made of polydimethylsiloxane (PDMS).

18. The method of claim 16 , wherein step (c) further comprises the step of:
sweeping the cells settled on the second surface of the second substrate by delivering the culture medium into the microfluidic channel to increase the probability of cell-docking in the capture microwells, wherein the flow rate of the medium during the sweeping step is slower than that during the washing step.

Assignees

• National Health Res Inst

Inventors

• Hsu Chia-Hsien
• Lin Ching-Hui
• Chang Hao-Chen
• Chiu Ing-Ming

Classifications

• B01L3/502707

  characterised by the manufacture of the container or its components · CPC title

• B01L2300/0887

  Laminated structure · CPC title

• B01L2300/0829

  Multi-well plates; Microtitration plates · CPC title

• C12M33/00 Primary

  Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus (chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00) · CPC title

• C12M23/50

  Means for positioning or orientating the apparatus (C12M41/08 takes precedence) · CPC title