CELL-TRAPPING DEVICE, APPARATUS COMPRISING IT AND THEIR USE FOR MICROINJECTION INTO CELLS

Abstract

A cell-trapping device includes a microchannel portion for trapping a plurality of cells with an average diameter of at most 25 μm for high-throughput microinjection of an injectant into the cells. The cell-trapping device includes a microchannel portion having formed therein a cell-trapping area including a plurality of cell-trapping microchannels configured to trap one cell per cell-trapping channel. A method for preparing the cell-trapping device and an apparatus for high-throughput microinjection is also provided. Further provided is a method for injecting an injectant into a plurality of cells. The cell-trapping device, apparatus, and method allow for a rapid and highly reproducible microinjection into small cells with high productivity, high accuracy and a good cell survival rate.

Claims

1. A cell-trapping device for trapping a plurality of cells with an average diameter of at most 25 μm for high-throughput microinjection of an injectant into the cells, said cell-trapping device comprising a microchannel portion having formed therein a cell-trapping area comprising a plurality of cell-trapping microchannels configured to trap one cell per cell-trapping microchannel.

2. The cell-trapping device of claim 1, wherein the cell-trapping area comprises more than 200 cell-trapping microchannels.

3. The cell-trapping device of claim 1, wherein the cell-trapping microchannels are arranged substantially in parallel at regular intervals in a row along a linear axis through the cell-trapping device.

4. The cell-trapping device of claim 1, wherein the microchannel portion further comprises an inlet area having an inlet constructed for receiving the plurality of cells in a fluid and an outlet area having outlet microchannels for directing the fluid along with untrapped cells smaller than the cell-trapping microchannels to an outlet for releasing the fluid along with the untrapped cells, wherein the cell-trapping area is arranged between outlet area and inlet area.

5. The cell-trapping device of claim 4, wherein the cell-trapping microchannels of the microchannel portion proceed into the outlet microchannels of the outlet portion and the microchannel portion is formed by a first layer with a height of up to about 20 μm arranged on a second layer with a height of up to about 5 μm and wherein the first layer and the second layer comprise polydimethylsiloxane.

6. The cell-trapping device of claim 5, wherein the cell-trapping microchannels and the outlet microchannels are formed by recesses in the first layer and/or in the second layer, which recesses are formed substantially perpendicular to the horizontal dimensions of the first layer and second layer and proceed substantially parallel to both horizontal dimensions of the first layer and/or the second layer.

7. The cell-trapping device of claim 6, wherein the recesses forming the outlet microchannels have a height between about 0.8 and about 1×the average cell diameter and a width of at least about 1×the average cell diameter, and wherein the cell-trapping microchannels have a cell receiving part formed by recesses with a height and a width of between about 0.8 and about 1×the average cell diameter and a fluid transfer part formed by recesses with a width of between about 0.8 and about 1×the average cell diameter and a high of at most about 0.5×the average cell diameter.

8. The cell-trapping device of claim 7, wherein the fluid transfer part is formed by recesses with a height of at most about 0.25×the average cell diameter and wherein the cell-trapping device further comprises at least one of a cover portion or a base portion with cover portion and base portion comprising glass.

9. The cell-trapping device of claim 1 which is transparent for visible light and which comprises at least 356 cell-trapping microchannels in the cell-trapping area.

10. An apparatus for high-throughput microinjection of an injectant into a plurality of cells with an average diameter of at most 25 μm comprising: a cell-trapping device as claimed in claim 1; and an injection needle with a tip arranged to be stuck into the cells trapped in the cell-trapping area of the cell-trapping device to inject the injectant into the trapped cells.

11. The apparatus of claim 10 for high-throughput microinjection with a throughput of at least about 30 cells/min into more than 100 cells, the cells consisting of human cells having an average diameter of less than about 25 μm and wherein the injectant is selected from at least one of DNA, RNA, polypeptides or proteins.

12. The apparatus of claim 10 further comprising: a device carrier member for carrying the cell-trapping device; a needle holding member for supporting the injection needle; a control unit for guiding the injection needle to the trapped cells; a cell-detection unit to detect the trapped cells and to generate a signal for initiating the microinjection; a pressure-based microinjector; and anti-vibration means.

13. The apparatus of claim 12, wherein the device carrier member has a device carrying surface facing towards the cell-trapping device which is in a horizontal position substantially parallel to an X-Y plane which is parallel to level ground, and wherein the device carrier member is arranged such that it can move the cell-trapping device at least along a X direction and along an Y direction perpendicular to the X direction in the X-Y plane.

14. The apparatus of claim 13, wherein the injection needle is mounted on the needle holding member on a surface of the needle holding member which is arranged substantially perpendicular to the X-Y plane.

15. The apparatus of claim 14 comprising: a control unit comprising a computer and a motion controller for controlling the position of the device carrier member in the X-Y plane and/or of at least a portion of the needle holding member in a Z direction perpendicular to the X-Y plane; and a cell detection unit comprising a vision detector, microscopic means and a light source providing illumination to the microscopic means, which cell-detection unit is arranged on top of the cell-trapping device facing towards the surface of the cell-trapping device which is opposite to the surface of the cell-trapping device facing towards the device carrying surface of the device carrier member; and an anti-vibration member onto which the device carrier member with the surface opposite to the device carrying surface and the needle supporting member are placed.

16. The apparatus of claim 12, wherein the microinjector is connected to the cell-trapping device and the injection needle and provides negative pressure to the cell-trapping device for trapping the cell, and positive pressure to the injection needle.

17. A method for microinjection of an injectant into a plurality of cells having an average diameter of at most 25 μm comprising steps of: (i) providing an apparatus as claimed in claim 10; (ii) introducing a plurality of cells into the cell-trapping device; (iii) trapping the cells in the cell-trapping microchannels in the cell-trapping area in the microchannel portion of the cell-trapping device such that a cell-trapping microchannel traps one cell; (iv) inserting an injection needle with the tip into the cell-trapping area in the microchannel portion of the cell-trapping device and injecting the injectant subsequently into a plurality of trapped cells.

18. The method of claim 17, wherein the microchannel portion of the cell-trapping device further comprises an inlet area having an inlet constructed for receiving the plurality of cells in a fluid and an outlet area having outlet microchannels for directing the fluid along with untrapped cells smaller than the cell-trapping microchannels to an outlet for releasing the fluid along with the untrapped cells, wherein the cell-trapping area is arranged between outlet area and inlet area; and wherein the cell-trapping device comprises at least 200 cell-trapping microchannels in the cell-trapping area; and wherein step (ii) comprises applying the cells in the fluid to the inlet of the cell-trapping device; step (iii) comprises applying a negative pressure of about 124.6 Pa to less than about 400 Pa at the outlet of the cell-trapping device for cell trapping in the cell-trapping microchannels in the cell-trapping area; and wherein inserting the injection needle into the cell-trapping area in step (iv) includes bending the injection needle while inserting the tip into the cell-trapping area of the cell-trapping device for obtaining a needle tilt angle of more than 70°.

19. The method of claim 17, wherein the provided apparatus further comprises a device carrier member for carrying the cell-trapping device; a needle holding member for supporting the injection needle; and wherein step (iv) comprises steps of: (a) inserting the injection needle with the tip into the cell-trapping area in the microchannel portion of the cell-trapping device; (b) aligning a first trapped cell with the tip and moving the cell-trapping device in the direction of the trapped cell by moving the cell-trapping device in a direction to the tip and perpendicular to said direction such that the tip is stuck into the first trapped cell; (c) injecting the injectant into the first trapped cell; (d) moving the cell-trapping device away from the tip and to a second trapped cell by moving the cell-trapping device in a direction opposite to the tip and subsequently perpendicular to said direction such that the tip is in front of the second trapped cell, (d) aligning the second trapped cell with the tip and moving the cell-trapping device in the direction of the second trapped cell by moving the cell-trapping device in a direction to the tip and perpendicular to said direction such that the tip is stuck into the second trapped cell; (e) injecting the injectant into said second trapped cell; and repeating steps (d) to (e) with a third and any further trapped cells until all trapped cells have received the injectant.

20. The method of claim 17, wherein step (iv) further comprises steps of: searching the position of an uninjected trapped cell by calculating the correlation of edge information between a template image and each pattern region on the sample image; determining whether the correlation is larger than a set threshold; and if this condition is met, proceeding with the injection of the injectant into the cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0132] FIGS. 1A, 1B, and 1C illustrate embodiments of the apparatus of the present invention, wherein, FIG. 1A is a schematic diagram of an apparatus according to one embodiment of the present invention; FIG. 1B is a schematic diagram of an arrangement of the cell-trapping device, device carrier member, needle holding member and anti-vibration means in one embodiment of the apparatus of the present invention with a bent form of the injection needle having a needle tilt angle α of more than 10°; and FIG. 1C illustrates an arrangement of the cell-trapping device, device carrier member, needle holding member and anti-vibration means in one embodiment of the apparatus of the present invention wherein the device carrier member can move the cell-trapping device in X direction and Y direction and at least a portion of the needle holding member can be moved in Z direction.

[0133] FIGS. 2A, 2B, 2C, 2D, and 2E illustrate embodiments of the cell-trapping device of the present invention, wherein FIG. 2A is a top view and illustrates one embodiment of the cell-trapping microchannels in the cell-trapping device with a cell receiving part and a fluid transfer part; FIG. 2B is a front view and illustrates one embodiment of the cell-trapping microchannels in the cell-trapping device; FIG. 2C is a top view of the microchannel portion of the cell-trapping device and illustrates an embodiment with inlet area, cell-trapping area and outlet area; FIG. 2D illustrates one embodiment of the cell-trapping device of the present invention with full cell loading; and FIG. 2E is a side view and schematic representation of one embodiment of the cell-trapping device with top portion and cover portion and microchannel portion with first layer and second layer.

[0134] FIGS. 3A, 3B, 3C, and 3D illustrate embodiments of the method of the present invention for microinjection of an injectant into a plurality of cells, wherein FIG. 3A illustrates the introduction of cells suspended in a fluid into the cell-trapping device; FIG. 3B illustrates the cell trapping in the cell-trapping area in the cell-trapping device; FIG. 3C illustrates the step of inserting the injection needle into the cell-trapping area of the cell-trapping device; and FIG. 3D illustrates an automated cell injection.

[0135] FIG. 4 shows a flowchart of embodiments of the method of the present invention for microinjection of an injectant into a plurality of cells including a cell recognition strategy.

[0136] FIG. 5 illustrates the coordinate frames of vision detector and device carrier member in embodiments of the apparatus of the present invention.

[0137] FIG. 6 shows a visual-guided position control scheme for automated cell injection of one embodiment of the present invention.

[0138] FIGS. 7A, 7B, 7C, and 7D illustrate the identification of target cells in an embodiment of the method of the present invention for microinjection of an injectant into a plurality of cells, wherein FIG. 7A shows a template image; FIG. 7B shows an original (sample) image; FIG. 7C shows the reprocessed image; and FIG. 7D shows the recognition result after applying an edge template matching algorithm.

[0139] FIG. 8 illustrates an injection path plan that includes three paths for aligning a cell with the micropipette, moving the cell toward the micropipette, and moving the cell back to its original position.

[0140] FIG. 9 illustrates the method for preparing the cell-trapping device in one embodiment of the present invention.

[0141] FIGS. 10A, 10B, 10C, and 10D show simulation results of the fluid flow velocity for single cell trapping, wherein FIG. 10A shows the simulated region in case of empty cell-trapping microchannels (top view); FIG. 10B shows the simulated region in case of empty cell-trapping microchannels (side view); FIG. 10C shows the simulated region in case of occupied cell-trapping microchannels (top view); and FIG. 10D shows the simulated region in case of occupied cell-trapping microchannels (side view).

[0142] FIGS. 11A, 11B, 11C, and 11D illustrate a microinjection into HFF cells with the method of the present invention, wherein FIG. 11A shows the trapped HFF cell moving into the direction of the micropipette; FIG. 11B illustrates the tip of the micropipette penetrating the cell membrane of the trapped HFF cell; FIG. 11C shows a further step of moving the cell-trapping device in a direction opposite to the tip and subsequently perpendicular to said direction such that the tip reaches the next trapped cell; and FIG. 11D shows the trapped HFF cell moving into the direction of the micropipette.

[0143] FIG. 12 shows the HFF cell trapping by a cell-trapping device of the present invention.

[0144] FIGS. 13A, 13B, 13C, and 13D show photographs of HFF cells before and after the automated injection of TRITC-Dextran, wherein FIG. 13A is a bright field image; FIG. 13B shows a fluorescent image of the trapped HFF cells before microinjection; FIG. 13C is a bright field image; and FIG. 13D shows a fluorescent image of the trapped HFF cells after microinjection of TRITC-Dextran.

[0145] FIGS. 14A, 14B, 14C, and 14D show images of the HFF cells after injection of TRITC-Dextran and incubation for 24 h, wherein FIG. 14A and FIG. 14B are a bright field images; and FIG. 14C and FIG. 14D are fluorescent overlaid images. The arrows indicate dead cells.

[0146] FIG. 15 illustrates the effect of the negative pressure on the cell-trapping efficiency.

DESCRIPTION OF THE INVENTION

[0147] For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. A person skilled in the art will understand that features specifically mentioned for the cell-trapping device in the context of preferred embodiments are also applicable in the apparatus of the present invention and vice versa.

[0148] The usage of words indicating preferences, such as “preferably,” refers to features and aspects that are present in at least one embodiment, but which are optional for some embodiments.

[0149] The technical terms used in the present patent application have the meaning as commonly understood by a respective skilled person unless specifically defined otherwise.

[0150] As used herein and in the claims, “comprising” means including the following elements but not excluding others. “Essentially consisting of” means that it consists of the respective element(s) along with usually and unavoidable impurities. “Consisting of” means that something solely consists of, i.e. is formed by respective element(s).

[0151] Any reference to prior art contained herein is not to be taken as an admission that the information is common general knowledge, unless otherwise indicated.

[0152] And although various specific quantities such as specific values of parameters may be stated herein, such specific quantities are presented as examples only, and further, unless otherwise noted, are approximate values, and should be considered as if the word “about” prefaced each quantity.

[0153] The invention refers in an aspect to an apparatus for high-throughput microinjection of an injectant into a plurality of cells with an average diameter of at most 25 μm comprising:

[0154] a cell-trapping device; and

[0155] an injection needle with a tip arranged to be stuck into the cells trapped in the cell-trapping area of the cell-trapping device to inject the injectant into the trapped cells. The cell-trapping device comprises a microchannel portion having formed therein a cell-trapping area comprising a plurality of cell-trapping microchannels configured to trap one cell per cell-trapping microchannel.

[0156] According to FIG. 1A the apparatus of the present invention 10 comprises in an embodiment a cell-trapping device 24. Automated microinjection is conducted by a device carrier member 26 namely a motorized X-Y device carrier member and a needle holding member 18 namely a motorized needle holding member. A cell-trapping device 24, namely vacuum-based cell-trapping device, is placed on the device carrier member 26. A pressure-based microinjector 12 is connected to the outlet of the cell-trapping device 24, providing sufficient and adjustable negative pressure for cell trapping.

[0157] The microinjector 12 also provides adjustable positive pressure to an injection needle 20, namely a bent micropipette, which is mounted on a needle holding member 18, which is a Z needle holding member. The diameter of the tip of the injection needle 20 is 0.5 μm. The automated cell injection system also includes a cell-detection unit: a vision detector 14, which is a CCD camera, is mounted on microscopic means 22, namely a microscope, which is placed on top of the cell-trapping device 24. A light source 30 provides illumination to the microscopic means 22. A control unit 16, which comprises a personal computer, can control the position of the X-Y device carrier member and Z needle holding member through the motion controller 32. The device carrier member 26 has a device carrying surface 26a facing towards the cell-trapping device and a surface 26b opposite to said surface. The control unit 16 is also connected to the microinjector 12 and can trigger the positive pressure applied to the bent micropipette for cell injection. In addition, the CCD camera as vision detector 14 combined with an image processing method can be coupled to the control unit 32 to locate the target cells, automating the manipulation of the entire apparatus 10. The automated cell injection apparatus 10 is installed on anti-vibration means 28 in form of an anti-vibration table.

[0158] As shown in FIG. 1B, the micropipette is bent to a bent form with a needle tilt angle α compared to the straight form of the micropipette of at least 10°.

[0159] FIG. 1C refers to an embodiment of the apparatus of the present invention comprising the cell-trapping device 24 carried by the device carrier member 26 which is a X-Y device carrier member with a device carrying surface 26a of the device carrier member, wherein surface 26b faces towards the anti-vibration means 28. The apparatus comprises a needle holding member 18 in form of a Z needle holding member holding an injection needle 20 in form of a micropipette. The X-Y plane 34 is the plane parallel to level ground. The X-Y device carrier member can move the cell-trapping device 24 in the X and Y direction. The needle holding member or a portion thereof can be moved in Z direction.

[0160] FIG. 2A to FIG. 2C show an embodiment of the cell-trapping device of the present invention with a microchannel network with cell-trapping microchannels 40. The microchannel portion is formed by a first layer 44 and a second layer 42, namely a thin second layer 42 (3-5 μm) and a thick first layer 44 (10-15 μm) . The microchannel network includes a cell-trapping area 54, which consists of 256 cell-trapping microchannels 40. The two layers display the same binary tree-like branching structure, except at the cell-trapping area 54. The dimensions of the recesses forming the cell-trapping microchannels 40 (including both second and first layer) , namely height 48 and width 50, in the cell receiving part 38 are the same or slightly smaller than the size of the cells 70 to be trapped. In this embodiment, width 50 and height 48 of the recesses forming the cell receiving part are 0.8-1×the average cell diameter. In this way, a relatively large friction force can be produced to hold the cell tightly during injection. A small channel may impose large stress on the cell, which degrades cell vitality. The thin second layer 42 is designed to prevent cells from entering the fluid transfer part 38. The negative pressure provided by the microinjector 12 generates a current that flows from the cell-trapping area 54 to the outlet 52 in the outlet area 58 with outlet microchannels 56. The cells move toward the cell-trapping area 54 along with the fluid but cannot pass through the fluid transfer part of the cell-trapping microchannels 46 because of size exclusion. An adherent cell is so flexible that it can easily squeeze through the fluid transfer part if the height is not small enough. Thus, the width 50 of the recesses forming the microchannels in the fluid transfer part is smaller or equal to the average diameter of the cells 70 to be trapped, wherein the height 48a is up to about 0.25×the average diameter of the cells 70 to be trapped.

[0161] In the embodiment shown in FIG. 2E the cell-trapping device includes a microchannel portion 46 with a second layer 42 and a first layer 44, further it includes a base portion 66 and a cover portion 64.

[0162] In an embodiment of the cell-trapping device of the present invention, the cell-trapping device is transparent for the visible light for clear observation. In one embodiment, the material used for the first and the second layer is poly(dimethylsiloxane) (PDMS) and the fabrication method used is soft lithography. The forming member with a microfluidic channel network is created by transferring the shadow ultraviolet (UV)-mask to the spin-coated negative photoresist film that displays a certain depth. PDMS mixed with the included curing agent at a 10:1 ratio is degassed and poured onto the forming member. An optically transparent replica is prepared to obtain the reverse structure of the forming member after curing. Holes are then punched to provide the outlet area by using a sharpened syringe needle, and the microchannel portion is trimmed to the proper size under the microscope. The bottom glass layer, typically a cover slip, is bonded to the microchannel portion in the oxygen plasma to form an irreversible seal.

[0163] In a further aspect, the present invention provides a method for microinjection of an injectant into a plurality of cells having an average diameter of at most 25 μm comprising steps of:

[0164] (i) providing an apparatus as claimed in claim 10;

[0165] (ii) introducing a plurality of cells into the cell-trapping device;

[0166] (iii) trapping the cells in the cell-trapping microchannels in the cell-trapping area in the microchannel portion of the cell-trapping device such that a cell-trapping microchannel traps one cell;

[0167] (iv) inserting an injection needle with the tip into the cell-trapping area in the microchannel portion of the cell-trapping device and injecting the injectant subsequently into a plurality of trapped cells.

[0168] An embodiment of the method of the present invention for microinjection is shown in FIG. 3A to 3D. Cells 70 suspended in a fluid are introduced near the inlet 62 in the inlet area 60 of the cell-trapping device 24 by using a liquid transfer pipette 72. A negative pressure is applied at the outlet 52 of the cell-trapping device 24, creating a fluid flow toward the cell-trapping microchannels 40 (FIG. 3A). The cells 70 are then transported toward the cell-trapping microchannels 40 (FIG. 3B). After the cell-trapping device is fully loaded, the injection needle 20 in form of a micropipette is bent and inserted with its tip into the cell-trapping area 54 (FIG. 3C). The automated cell injection procedure then starts (FIG. 3D). First, a template of cell image is inputted into the system by selecting the region of interest. The edge information of the selected region is used as a template to locate other target cells. The system will search through the whole image. If the correlation of the sample image and the template image is larger than the set threshold, the sample image will be considered as a target cell.

[0169] FIG. 4 illustrates one embodiment of steps of the method of the present invention in which the microinjection is automated. After the cells are trapped in the cell-trapping microchannels and after loading the micropipette into the cell-trapping area, the position of the microscope is adjusted to bring the inlet of the cell-trapping area into focus. An image containing both trapped cells and micropipette is captured (S1). The operator inputs the region of a target cell on the captured image, and the target cell region will be used as template image. The operator also inputs the position of the micropipette tip, which will initialize the automated injection progress (S2). Following initialization, a sample image is captured (S3). The sample image is preprocessed with a low-pass Gaussian filter (S4) and then converted into a binary image (S5). In addition, the edge information of the binary image is extracted using the Sobel edge detector (S6). The extracted contours are further smoothened through morphological operation (S7). The template image is processed using the same procedure (S4-S7), and the edge information of the template image is also obtained. The central position of the region on sampled image, where the edge information is similar to that of the template image, is located (S8). The correlation of edge information between the template image and each pattern region on the sampled image is calculated. It is determined whether the correlation is larger than a threshold (S9). If the condition in S9 is satisfied, the center of the matched pattern region in the sampled image is used to define the cell position. The position of the cell after injection will be removed from the system, and the target position of the cell with the smallest distance from the tip position will be chosen. If the condition in S9 cannot be satisfied or if all of the target cells are injected, the injection process stops. Once the position of the target cell is obtained, the target cell is aligned with the position of the micropipette tip in the x-axis by controlling the movement of the X-Y device carrier member (S10). The system will check whether alignment is achieved or not (S11). If alignment is achieved, the target cell is moved toward the micropipette tip by controlling the X-Y device carrier member (S12). A signal is sent to the microinjector, which will trigger the injection event (S13). During the injection event, a predefined pressure is applied at the end of the micropipette opposite to the tip for a predefined duration. A certain amount of substance in the micropipette will then be delivered into the cell. Once injection is completed, the target cell is moved back to its previous position that is, aligned with the tip of the injection needle. The system will inject all the cells trapped in the cell-trapping device by looping S3-S14.

[0170] FIG. 7A to FIG. 7D further illustrates the image processing in embodiments of the present invention, wherein FIG. 7A refers to the template image provided, FIG. 7B shows an original (sample) image, FIG. 7C shows the reprocessed image, and FIG. 7D shows the recognition result after applying the edge template matching algorithm. By applying the above edge matching algorithm, the correlation between the actual (sample) image and the template image can be obtained. If the correlation is large enough, the center of the template is accepted as the position of the cell center (FIG. 7D). The performance of the image processing algorithm can be evaluated according to five criteria: true positive (TP), false positive (FP), true negative (TN), false negative (FN), and accuracy (ACC), which are defined as follows:

[0171] TP=no. of occupied cell-trapping microchannels recognized as targets

[0172] FP=no. of empty cell-trapping microchannels recognized as valid targets

[0173] TN=no. of empty cell-trapping microchannels ignored

[0174] FN =no. of occupied cell-trapping microchannels ignored

[00003]$\mathrm{ACC}=\frac{\mathrm{TP}+\mathrm{TN}}{\mathrm{no}..Math.\mathrm{of}.Math..Math.\mathrm{total}.Math..Math.\mathrm{cell}.Math.\text{-}.Math.\mathrm{trapping}.Math..Math.\mathrm{microchannels}}$

[0175] In an embodiment, a visual-guided position control scheme is applied in the method of the present invention. O.sub.c-X.sub.cY.sub.cZ.sub.c is defined as the camera coordinate frame, where the origin O.sub.c is defined at the top left corner of the image captured by the microscope as microscopic means. O-XYZ is defined as the cell coordinate frame, which is the same as that of an X-Y-Z positioning members. FIG. 5 shows the two coordinate frameworks. The relationship between the two coordinate frames is

[00004]$\begin{array}{cc}\left[\begin{array}{c}X\\ Y\\ Z\end{array}\right]=K\left[\begin{array}{c}{X}_c\\ Y_c\\ Z_c\end{array}\right]+\left[\begin{array}{c}0\\ 0\\ d_z\end{array}\right].Math..Math.\mathrm{where}.Math..Math.K=\left[\begin{array}{ccc}{k}_x& 0& 0\\ 0& k_y& 0\\ 0& 0& k_z\end{array}\right]& \left(1\right)\end{array}$

[0176] represents a diagonal transformation matrix and d.sub.z is the vertical distance between the origins of the vision detector like the camera frame and the coordinate frame.

[0177] The dynamics of a 3-DOF micromanipulation framework can be determined using Lagrange's equation of motion [8]

[00005]$\begin{array}{cc}M\left[\begin{array}{c}\stackrel{\mathrm{.Math.}}{X}\\ \stackrel{\mathrm{.Math.}}{Y}\\ \stackrel{\mathrm{.Math.}}{Z}\end{array}\right]+B\left[\begin{array}{c}\stackrel{.}{X}\\ \stackrel{.}{Y}\\ \stackrel{.}{Z}\end{array}\right]+\left[\begin{array}{c}0\\ 0\\ -m_z.Math.g\end{array}\right]=\left[\begin{array}{c}{\tau }_x\\ {\tau }_y\\ {\tau }_z\end{array}\right].Math..Math.\mathrm{where}.Math..Math.M=\left[\begin{array}{ccc}{m}_x+m_y& 0& 0\\ 0& m_y& 0\\ 0& 0& m_z\end{array}\right]& \left(2\right)\end{array}$

[0178] denotes the inertia matrix of the system; m.sub.x, m.sub.y, m.sub.z are the mass of the X-Y, and Z positioning members, respectively; B represents the effect of friction and system damping; −m.sub.zg is the gravitational force;

[τ.sub.xτ.sub.yτ.sub.z].sup.T

[0179] is the input force to the X-Y-Z positioning elements. In this embodiment, dc-brushed motors are used to actuate the X-Y-Z positioning members. Given that the input forces are proportional to the currents to the motors,

[τ.sub.xτ.sub.yτ.sub.z].sup.T=[K.sub.m.sub.xI.sub.xK.sub.m.sub.xI.sub.yK.sub.m.sub.xI.sub.z].sup.T   (3)

[0180] Is obtained where K.sub.mx, K.sub.my, and K.sub.mz are constants which depend on the armature coil and magnetic flux density; I.sub.x, I.sub.y, and I.sub.z are the currents flowing through the motors for the X-Y-Z positioning elements.

[0181] A visual-guided position control scheme, as shown in FIG. 6, is applied in embodiments to achieve automated cell injection. Initially, the injection needle such as the micropipette and the cell-trapping device are aligned with the Z-axis direction to stay in the focused plane of the CCD camera. After alignment, the positions of the micropipette and the cell-trapping device in the Z-axis remain unchanged throughout the injection process, and the automated injection is accomplished in the X-Y plane only. The control scheme includes the identification of cell position using image processing technology and the control of the X-Y device carrier member to drive the cell-trapping device to complete the injection process. Considering that the frame rate of the CCD camera is only 60 Hz, the use of visual feedback in the controller reduces the sampling frequency and degrades injection performance. To solve this problem, a motor encoder mounted on the X-Y device carrier member is used in embodiments to measure positions with high sampling frequency (for example 1000 Hz). Image acquired by the CCD camera is used to locate the cell and determine the destination of the injection motion. The information is then used for guiding the X-Y device carrier member to move toward the micropipette.

[0182] The control algorithm for each motion axis employs a simple feedforward plus PID feedback control in the form of

[00006]$\begin{array}{cc}I=K_p.Math.e_p+K_i.Math.{\int }_0^t.Math.e_p\left(\tau \right).Math.\mathrm{dr}+K_d.Math.\frac{{\mathrm{de}}_p\left(t\right)}{\mathrm{dt}}+K_f.Math.{\stackrel{\mathrm{.Math.}}{x}}_d& \left(4\right)\end{array}$

[0183] where I denotes the current control input, e.sub.p is the position error, K.sub.p, K.sub.i, and K.sub.d are PID control gains, K.sub.f is a feedforward control gain, and

{umlaut over (x)}.sub.d

[0184] is the desired acceleration set by the controller. The current control input I then goes to an inner current control loop, which is designed with a PI control scheme.

[0185] FIG. 8 illustrates an embodiment with an injection path plan that includes three paths for 1) aligning the cell with the micropipette (Path 1), 2) moving the cell toward the micropipette (Path 2), and 3) moving the cell back to its original position (Path 3). In this embodiment, the micropipette is fixed, and the cell located in one cell-trapping microchannel of the cell-trapping device moves straight toward the micropipette (for example, along the Y-axis direction) to complete one single-cell injection. The use of a straight-line path can minimize the damage to the cell during injection. Some sophisticated path planning algorithms (Wu, Y., IEEE/ASME Trans. Mechatronics, 2012, 18, 706-713, Wang, J., IEEE/ASME Trans. Mechatronics, 2013, 19, 549-558, Suzuki, H. and Minami, M., IEEE/ASME Trans. Mechatronics, 2005, 10, 352-357, Conticelli, F. and Allotta, B., IEEE/ASME Trans. Mechatronics, 2001, 6, 356-363) have been proposed, which may be used in embodiments for cell injection; however, given that the regular structure of the proposed cell-trapping device design can greatly simplify the process, the following simplified path plan can be applied in a preferred embodiment:

[0186] Define Δ.sub.xi and Δ.sub.yi as the horizontal and vertical distances between the micropipette tip (P.sub.tip) and the i.sup.th target cell, respectively. In the experiment, Δ.sub.xi and Δ.sub.yi vary among different cells, as shown in FIG. 7A to FIG. 7D. The travel distance of the cell-trapping device to complete one single-cell injection is:

d.sub.f=Δx.sub.i+2Δy.sub.i.

[0187] To complete the injection of n cells, the total traveled distance of the cell-trapping device is

[00007]$D_i=\underset{i=6}{\overset{N}{.Math.}}.Math.\Delta .Math..Math.x_i+2.Math.\Delta .Math..Math.y_i.$

[0188] Before injection, the tip position of the micropipette (P.sub.tip) and the template image of the filled cell-trapping microchannels are determined. All the cell-trapping microchannels occupied by cells are checked to trigger injection. The first cell-trapping microchannel is aligned vertically with the micropipette, and a pulse is sent to the microinjector after the micropipette inserts the cell. The injection pressure and time are adjusted. The cell-trapping device is moved away from the micropipette, and the system starts to search for the second cell-trapping microchannel. The process repeats until all the cells in cell-trapping microchannels are injected.

[0189] Although the invention is described with reference to the specific embodiment described above, the invention is not intended to be limited to the above-mentioned details. Various modifications and improvements can be made according to certain applications without departing from the invention. The following non-limiting examples demonstrate the advantages of the invention.

EXAMPLES

Example 1

Preparation of a Cell-Trapping Device of the Present Invention

[0190] A cell-trapping device of the present invention was prepared by a soft lithography replica molding technology with PDMS (SYLGARD). The fabrication process is illustrated in FIG. 9. Prior to fabrication, two UV masks with features of two layers were printed on a transparency with a high-resolution printer. For the second layer, which had a thickness of 3-5 μm, mold fabrication was initiated by spin coating SU-8 negative photoresist (GM1050, Gersteltec Sarl) on a clean 3-in silicon wafer 74, where the thickness of SU-8 photoresist 76 was dependent on the size of the target cells. The diameter of the target cells was set to be 10-25 μm. Hence, 5-μm SU-8 was spin coated on the silicon wafer to fabricate the cell-trapping device. The SU-8 photoresist was then heated on a hotplate (AccuPlate, Labnet) (step 1). The temperature increased from room temperature to 95° C. in 5 min. It was baked at 95° C. for 3 min. After it was cooled to room temperature, it was covered with a first UV mask 78 and irradiated by UV light (365 nm) (step 2). After UV exposure, it was heated on the hotplate (from room temperature to 80° C. in 5 min) and then baked at 80° C. for 2 min. The exposed area of SU-8 photoresist formed crosslinks 80 during postexposure baking. After it was cooled to room temperature, the unexposed area of photoresist on was removed using the SU-8 developer (step 3).

[0191] The same procedures were used for the first layer (steps 4 to 6), which had a height of 10-15 μm. Before the second UV exposure, a second UV mask 78 was aligned precisely with the second layer using a mask aligner (MA6, Karl Suss).

[0192] The cell-trapping device was fabricated by replica molding with PDMS (SYLGARD 184, DowCorning) and the forming member (step 7). The PDMS was mixed with its curing agent in 10:1 (w/w) and poured on the forming member. The forming member with PDMS was degassed to remove air bubbles inside PDMS. The PDMS mixture 82 was cured by baking in an oven. The cured PDMS was peeled off from the forming member (step 8) and trimmed under a microscope with a 5×objective (Mitutoyo, Japan), which was followed by punching the outlet on the PDMS. The trimmed PDMS sample was cleaned and bonded on a glass surface as cover portion 64 or base portion 66 using the plasma bonding technique (step 9).

Example 2

Simulations with a Cell-Trapping Device of the Present Invention

[0193] Simulations were performed to test preliminary pressure setting parameters and to verify the effectiveness of the cell-trapping device. The finite-element analysis software, Comsol Multiphysics, was used for the simulation. The incompressible Naïve-Stoke equation was used to simulate the velocity and pressure distribution, in which the cell was assumed to be a perfect sphere with a diameter of 19 μm. In the simulation, the height and the width of the channel were set as 20 μm. The fluid flow was assumed to be laminar. The simulation was performed in two steps. In the first step, the three-dimensional structure of the whole cell-trapping device (see FIG. 2C) was modeled, which was used to determine the suitable pressure for trapping cells with minimal deformation while moving the cells into the traps at a reasonable speed. Note that the typical force required to deform a cell is in the order of 10 pN. Assuming that the fluid flow is laminar, the drag force acting on the cell can be estimated by Stokes' law, so the fluid velocity can be determined. The inlet fluid velocity was set to 50 μm/s to minimize the deformation of the trapped cell. The outlet was set as an open boundary. The difference between the outlet and the inlet pressures was estimated as −157.6 Pa in simulation. The actual outlet pressure applied in the experiment may be smaller such that the cell will not squeeze through the cell-trapping microchannels. In the second step, the cell-trapping microchannels of the cell-trapping device were modeled to verify the effectiveness of the cell-trapping device in trapping only one single cell at each cell-trapping microchannel. Two simulations have been performed to obtain the velocity distributions of the occupied and the empty cell-trapping microchannels, respectively, in which the inlet fluid velocity was set as 50 μm/s, and the outlet was set as open boundary.

[0194] FIG. 10 shows the simulation results, where the slice plot represents the flowing velocity inside the cell-trapping channel.

[0195] When the cell-trapping microchannel is empty, flow velocity is high.

[0196] According to Stokes' law, the dragging force is proportional to the flow velocity; hence, the flow drags the cell to the cell receiving part. When the cell-trapping microchannel is occupied, flow velocity is low. The fluid velocity of the empty microchannels is about two times larger than that of the occupied microchannel. As a result, the flow redirects the incoming cells to other empty microchannels, preventing the occupied microchannel from being overloaded. The follow-on experiments have also verified that each cell-trapping microchannel traps only one single cell.

Example 3

[0197] Assembly of an Apparatus of the Present Invention

[0198] An apparatus is provided comprising an X-Y device carrier member in form of a X-Y stage (PIM-L01, Physik Instrumente Co., Ltd.) , a needle holding member in form of a Z-axis linear table (KR30H06A, THK CO., LTD.), a micropipette (BF-100-50-15, Sutter Instrument), a cell-trapping device, and a pressure-based microinjector (IM-300, NARISHIGE). The motorized X-Y device carrier member has a resolution of 0.2 μm. The cell-trapping device is placed inside a petri dish, which is fixed in the X-Y device carrier member with two clamps. A glass micropipette, with an outer diameter of 1.0 mm and an inner diameter of 0.5 mm, is heated and pulled using a laser-based micropipette puller (P-2000, Sutter instrument). The micropipette is mounted on the Z needle holding member and connected to the microinjector via a pressure tube. The diameter of the micropipette tip is approximately 0.5 μm. The microinjector is connected to the outlet of the cell-trapping device and provides a negative pressure. The control unit consists of a computer and a motion controller (DCT0040, Dynacity Tech. Ltd.), with a sampling frequency of 4 kHz. The cell-detection unit consists of a CCD camera (STC-700, SENTECH) and a 20× objective (Mitutoyo, Kawasaki, Japan), which are mounted at the two ends of an observation tube (Infinity Tube, Boulder, Colo., USA). A light source (PL-800, Fiber-Lite) provides illumination. The image is captured using the PC2-Vision frame grabber (OC-PC2MVUM00, Dasal Corp.) and displayed with the image-processing library (Sapera Essential, Dasal Corp.). Both the injection module with the X-Y device carrier member, the cell-trapping device and the Z needle holding member with the micropipette and the cell-detection unit are placed inside a PMMA chamber, which is mounted on an anti-vibration table.

Example 4

Microinjection into HFF Cells

[0199] Human foreskin fibroblasts (HFF) were used in the cell injection experiment. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 U/mL streptomycin in a humidified atmosphere of 37° C. and 5% CO.sub.2. Before experiments, the cells were enzymatically detached from the cell culture plate and isolated to single cells. The cells were then suspended in the stated culture medium.

[0200] The cell-trapping device was sterilized by flowing 70% ethanol through the microchannels for 10 min. The microchannels were rinsed by flowing DMEM for 10 min. The cell-trapping device was then exposed to UV for 30 min and filled with the cell culture medium by connecting its outlet to a 1-mL syringe (BD Falcon). The cell-trapping device was degassed and fixed in a petri dish filled with the cell culture medium. The negative pressure was applied to the cell-trapping device by connecting the outlet to the microinjector through polyethylene tubing.

[0201] During experiments, the cells were trapped into cell-trapping microchannels of the cell-trapping device within 10 min upon their introduction. The cells that were not trapped were removed by gently flushing the cell culture medium near the cell-trapping area using a pipette. To easily verify the injection effect, tetramethylrhodamine isothiocyanate (TRITC) was injected into the cells. The micropipette was bent and backfilled with 1-mg/mL TRITC-Dextran before it was mounted on the Z needle holding table. The micropipette tip was carefully aligned and inserted into the cell-trapping device. About 20 μL of the cell solution (˜1000 cells/μL) was transferred into the petri dish using a pipette. The effect of the negative pressure on the trapping efficiency was determined for optimizing the cell-trapping performance, with data summarized in FIG. 15. The trapping efficiency of the cell-trapping device attained a maximum of 87% when the negative pressure was —124.6 Pa. As observed from the microscope, the trapped cells were largely deformed when the negative pressure was higher than 249 Pa. When all the channels were occupied by cells, the negative pressure was reduced to 24.9 Pa for holding the cell in the channel. The pressure was maintained by the microinjector throughout the whole injection process. Before the cell injection experiment, the ratio of pixel to actual length k was calibrated as 2.13 pixels/μm. The relationship between the encoder count and the micrometer was calibrated as 15 counts/μm. The maximum speed and acceleration of the X-Y device carrier member were 0.22 mm/s and 1.76 mm/s.sup.2, respectively.

[0202] The cell was aligned with the micropipette (FIG. 11A to FIG. 11D). The cell-trapping device was moved toward the micropipette and then stopped for injection (FIG. 11B). After injection was completed, the cell-trapping device was moved back to the original position (FIG. 11C) and moved horizontally so that the next cell was aligned with the micropipette. The process was repeated until all the trapped cells were injected.

[0203] The total injection process for one single cell, including detecting the cell in the cell-trapping microchannel, moving the cell-trapping device on the device carrier member, and performing injection, took approximately 1.7 s. This finding was equivalent to an operation speed of 35.3 cells/min, which was much higher than other existing approaches (for example, 6 cells/min in Becattini, G. et al., IEEE J. Biomed. Health Informat., 2014, 18, 83-93).

[0204] To verify the cell recognition efficiency, a total of 377 cells were processed in the experiment. The cell recognition results are given in Table 1.

TABLE-US-00001 TABLE 1 Image processing results TP TN FP FN ACC (%) 340 9 23 5 92.6

[0205] The accuracy of the cell recognition algorithm was 92.6%, indicating that the system can detect the occupied cell-trapping microchannels as targets and skip the empty cell-trapping microchannels efficiently. The system only incorrectly treated 7.4% of the examined cell-trapping microchannels. TN, FN, and FP are rare because most of the cell-trapping microchannels are occupied. In the above data set, the correlation threshold was set as 70%.

[0206] An image of the loaded cell-trapping device in the experiment is shown in FIG. 12. A summary of the trapping efficiency is given in Table 2.

TABLE-US-00002 TABLE 2 Cell-trapping results No. of Cell-trapping cell-trapping No. of Trapping Cell microchannel microchannels trapped efficiency type width (μm) observed cells (%) HFF 20 735 608 82.7

[0207] The trapping efficiency is defined as the ratio of the number of the filled cell-trapping microchannels to the number of total cell-trapping microchannels. The measured efficiency was 82.7% (HFF) in the experiments. Notably, a high cell-trapping efficiency can help reduce FP in the recognition results. When most of the cell-trapping microchannels are occupied by cells, the chance of miscounting empty cell-trapping microchannels decreases.

[0208] To further examine the injection effect, the injected cells were analyzed using a fluorescent microscope. HFFs with a diameter of 15 μm to 20 μm were applied and the height and width of the cell-receiving part of the cell-trapping microchannels were 15 μm and 20 μm, respectively. The loading cell concentration was ˜1000 cells per μL. The negative pressure applied to the cell-trapping device was 1.5 iH.sub.2O (about 373 Pa), which generates a fluid flow that drags cells toward the cell holder outlet. The typical cell trapping time was 10 min. An injected cell should show red fluorescence if the dye is successfully injected into the cell. FIG. 13A to 13D show the images of HFF before and after injection of TRITC-Dextran.

[0209] FIG. 13A shows the bright field image of HFFs before injection, whereas FIG. 13B shows the fluorescent overlaid image of HFFs before injection. Before injection, no fluorescence signal was detected. FIG. 13C shows the bright field image of HFF after injection, whereas FIG. 13D shows the fluorescent overlaid image of HFF cells after injection. The overall injection efficiency was 88%, and the survival rate was 81.5%. The summary of the cell injection performance is given in Table 3.

TABLE-US-00003 TABLE 3 Cell-injection results No. of No. of Injection injected fluorescent efficiency Cell type cells cells (%) HFF 657 581 88.4

[0210] The injection efficiency is defined as the ratio of the number of the fluorescent cells to the number of the total injected cells, namely

[00008]$\mathrm{Injection}.Math..Math.\mathrm{efficiency}=\frac{\mathrm{no}..Math.\mathrm{of}.Math..Math.\mathrm{fluorescent}.Math..Math.\mathrm{cells}}{n.Math.o..Math.\mathrm{of}.Math..Math.\mathrm{injected}.Math..Math.\mathrm{cells}}$

[0211] The overall injection efficiency was 88% for HFF, which value is better than that of manual injection performed by trained operators, which was about 40% as reported in Wang, W. et al.(Rev. Sci. Instrum., 2008, 79, 104302-1-104302-6). Furthermore, this result was better than two existing methods of flow constriction (Sharei, A. et al., Proc. Nat. Acad. Sci., 2013, 110, 2082-2087) and femtosecond laser delivery (Chakravarty, P. et al., Nature Nanotechnol., 2010, 5, 607-611), which has an efficiency of 70% (delivering 70-kDa dextran) and 35% (delivering FITC-BSA), respectively.

[0212] After cell injection, the injected cells were incubated for 24 h to examine the cell survival rate. The survival rate is defined as

[00009]$\mathrm{Survival}.Math..Math.\mathrm{rate}=\frac{\mathrm{no}..Math.\mathrm{of}.Math..Math.\mathrm{fluorescent}.Math..Math.\mathrm{cells}.Math..Math.\mathrm{after}.Math..Math.24.Math..Math.h.Math..Math.\mathrm{after}.Math..Math.\mathrm{injection}}{\mathrm{no}..Math.\mathrm{of}.Math..Math.\mathrm{fluorescent}.Math..Math.\mathrm{cells}.Math..Math.\mathrm{right}.Math..Math.\mathrm{after}.Math..Math.\mathrm{injection}}$

The survival rate for HFF was 81.5%. FIG. 14A to 14D show images of the cells at 24 h after injection. The survived cells exhibited normal morphology and were attached to the chip bottom, indicating that cell damage induced by injection was small. FIGS. 14B and 14D illustrate the dead cells, which became round and were detached from the device bottom.

LIST OF REFERENCE SIGNS

[0213] 10 Apparatus [0214] 12 Microinjector [0215] 14 Vision detector [0216] 16 Control unit [0217] 18 Needle holding member [0218] 20 Injection needle [0219] 22 Microscopic means [0220] 24 Cell-trapping device [0221] 26 Device carrier member [0222] 26a Device carrying surface [0223] 26b Surface opposite to device carrying surface [0224] 28 Anti-vibration means [0225] 30 Light source [0226] 32 Motion controller [0227] 34 X-Y plane [0228] 36 Cell receiving part [0229] 38 Fluid transfer part [0230] 40 Cell-trapping microchannels [0231] 42 Second layer [0232] 44 First layer [0233] 46 Microchannel portion [0234] 48 Height of microchannel in cell receiving part and the outlet microchannels [0235] 48a Height of microchannel in fluid transfer part [0236] 50 Width of microchannel [0237] 52 Outlet [0238] 54 Cell-trapping area [0239] 56 Outlet microchannels [0240] 58 Outlet area [0241] 60 Inlet area [0242] 62 Inlet [0243] 64 Cover portion [0244] 66 Base portion [0245] 68 Bent micropipette [0246] 70 Cell [0247] 72 Liquid transfer pipette [0248] 74 Silicon wafer [0249] 76 SU-8 photoresist [0250] 78 UV mask [0251] 80 Cross-linked photoresist [0252] 82 Mixture of PDMS with curing agent [0253] 84 Cured PDMS