Method For Mechanical and Hydrodynamic Microfluidic Transfection and Apparatus Therefor

Abstract

Methods for introducing exogenous material into a cell are provided, which include exposing the cell to a transient decrease in pressure in the presence of the exogenous material. Also provided are devices for performing the method of the invention.

Claims

1. A method for introducing an exogenous material into a cell, comprising exposing said cell to a transient decrease in pressure and unsteady flow in the presence of said exogenous material to thereby introduce said exogenous material into said cell.

2. The method of claim 1, wherein the cell is viable after being exposed to the transient decrease in pressure and unsteady flow.

3. The method of claim 1, wherein said cell is selected from the group consisting of a bacterial cell, a mammalian cell, a yeast cell, a plant cell and an insect cell.

4. The method of claim 1, wherein said exogenous material is selected from the group consisting of a small organic molecule, a nucleic acid, a nucleotide, an oligonucleotide, a protein, a peptide, an amino acid, a lipid, a polysaccharide, a quantum dot, a carbon nanotube, a nanoparticle, a gold particle, a monosaccharide, a vitamin and a steroid.

5. The method of claim 1, wherein said exogenous material is introduced into the cytoplasm of said cell.

6. The method of claim 1, wherein said cell is a mammalian cell, a yeast cell or an insect cell and said exogenous material is introduced into the nucleus of said cell.

7. The method of claim 1, wherein said cell is exposed to said transient decrease in pressure in the presence of said exogenous material for at least 10 nanoseconds.

8. The method of claim 1, wherein said exogenous material and said cell are in a liquid when being exposed to said transient decrease in pressure and unsteady flow.

9. The method of claim 8, wherein said cell is exposed to said transient decrease in pressure and unsteady flow within an enclosed channel with dimensions configured to allow the flow of said liquid comprising said exogenous material and said cell therethrough.

10. The method of claim 9, wherein said flow of said liquid in said channel has a fluctuating velocity.

11. The method of claim 9, wherein said channel is configured to influence unsteady flow of said liquid.

12. The method of claim 11, wherein the flow of liquid is influenced by one or more flow diverters within said enclosed channel.

13. The method of claim 12, wherein the object Reynolds number (Re.sub.o) of the unsteady flow of the liquid around a flow diverter in at least one of said regions within the channel is sufficient to induce unsteady flow.

14. The method of claim 13, wherein said one or more regions within the channel where unsteady flow of said liquid is downstream of said flow diverter.

15. The method of claim 14, wherein said cell is exposed to said transient decrease in pressure downstream of said flow diverter.

16. The method of claim 12, wherein said flow diverter is an obstacle placed within said enclosed channel.

17. The method of claim 1, wherein unsteady flow is at least one of a laminar vortex street, a transitional vortex street, a turbulent vortex street, transitional flow or turbulent flow.

18. A device for use in a method for introducing exogenous material into a cell in a liquid, comprising: an at least partially enclosed channel with dimensions configured to allow a flow of said cell and exogenous material suspended in a liquid therethrough; and one or more flow diverters within said channel; wherein the flow diverter results in at least one region of decreased pressure and unsteady flow immediately downstream of said flow diverter.

19. The device of claim 18, wherein the device is a microfluidic device.

20. A kit comprising a device for use in a method for introducing exogenous material into a cell in a liquid, including: an at least partially enclosed channel with dimensions configured to allow a flow of said cell and exogenous material suspended in a liquid therethrough; and one or more flow diverters within said channel; wherein the flow diverter results in at least one region of decreased pressure and unsteady flow immediately downstream of said flow diverter.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0139] FIG. 1 shows an overview of unit geometry of a device according to an embodiment of the invention.

[0140] FIG. 2 shows an overview of the pressure changes that occur during simulations of an embodiment of a method of the invention.

[0141] FIG. 3 is an overview of experimental transfection data taken by fluorescent microscopy (left) and optical microscopy (right) at a magnification of 20×, wherein HEK293 cells were transfected with pcDNA 3.1 in accordance with the parameters shown in Table 1.

[0142] FIG. 4 is a schematic diagram of a microfluidic device containing three columns of posts (n.sub.c=3) and four rows of posts (n.sub.r=4) according to one embodiment of the invention.

[0143] FIG. 5 is a schematic diagram of a microfluidic device containing three columns of posts (n.sub.c=3) and four rows of posts (n.sub.r=4) according to another embodiment of the invention.

[0144] FIG. 6 is a schematic diagram of a microfluidic device containing three columns of posts (n.sub.c=3) and four rows of posts (n.sub.r=4) according to yet another embodiment of the invention.

[0145] FIG. 7 is a schematic diagram of a microfluidic device containing three columns of posts (n.sub.c=3) and four rows of posts (n.sub.r=4) according to yet a further embodiment of the invention.

[0146] FIG. 8 is a sectional view of a device design according to a preferred embodiment of the invention. Panel A is an exploded view of the array design (3× magnification) whilst Panel B is an exploded view of the post design present on the array (9× magnification).

[0147] FIG. 9 is a sectional view of a device design according to another preferred embodiment of the invention. Panel A is an exploded view of the array design (3× magnification) whilst Panel B is an exploded view of the post design present on the array (9× magnification).

[0148] FIG. 10 is a sectional view according to yet another preferred embodiment of the invention. Panel A is an exploded view of the array design (3× magnification) whilst Panel B is an exploded view of the post design present on the array (9× magnification). The diagrammatic representations in FIG. 10 are not drawn to scale.

[0149] Some figures contain color representations or entities. Color illustrations are available from the Applicant upon request or from an appropriate Patent Office. A fee may be imposed if obtained from the Patent Office.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0150] Although the invention has been described with reference to certain embodiments detailed herein, other embodiments can achieve the same or similar results. Variations and modifications of the invention will be obvious to those skilled in the art and the invention is intended to cover all such modifications and equivalents.

[0151] The invention is further described by the following non-limiting examples.

Example 1

[0152] A method and device of the invention was assessed by transfecting a cell model with pcDNA 3.1 (Invitrogen™), which expresses green fluorescent protein (GFP). The device used was a microfluidic device configured with an array of posts, wherein the gap between posts was greater than the cell diameter.

Methods

Simulation & Analysis

[0153] Simulation by computation fluid dynamics (CFD) with the finite-volume method was employed to examine the microenvironment around the gaps between posts for the parameters shown in Table 1 and the device geometry shown in FIG. 1. FIG. 1 shows an overview of unit geometry of a device according to the invention, where a liquid with a velocity (Q) enters the enclosed channel at an inlet, along with cells with a diameter (d.sub.c) suspended in a liquid, wherein dc that is less than the gap width (g). Other variables represent post diameter (d.sub.p), channel width (w) and channel height (h). Three-dimensional geometry was built in SolidWorks with an inlet length of 100 μm and an outlet length of 1000 μm for solving purposes. A structured mesh was constructed in ICEM CFD 14.5 and element quality was checked using the determinant, angle and aspect ratio. Solutions were obtained using ANSYS FLUENT 14.5 on a Windows 7 Enterprise 64-bit computer with an Intel Core i5-3470 CPU at 3.20 GHz and 16.0 GB of RAM. A coupled pressure-velocity solver was used to solve for velocity, pressure and shear stress contours. The channel Reynolds number (Rec) was calculated according to the parameters in Table 1, using the interior dimensions of the constriction and the equation below:

Rec=2ρQ/μ(g+h)

[0154] The boundary conditions for the channel top, bottom and walls defined by posts were set to no slip. Boundary conditions for fluidic sidewalls were set to zero shear. Inlet velocity was defined by an average velocity and the outlet was set to a zero pressure boundary condition.

TABLE-US-00001 TABLE 1 Summary of experimental parameters. Parameter Value Cell type HEK293 Cell density 1 × 10.sup.5 cells ml.sup.−1 Cell diameter (d.sub.c) 13 μm Floure scent molecule pcDNA 3.1 (GFP plasmid) Molecule density 890 ng ml.sup.−1 Channel height (h) 40 μm Channel width (w) 400 μm  Post diameter (d.sub.p) 20 μm Post gap (g) 30 μm Row shift (s)  0 μm Row pitch (p.sub.r) 50 μm Rows (n.sub.r) 9 posts Column pitch (p.sub.c) N/A Columns pitch (n.sub.c) 1 post Media viscosity (μ) 7.987 × 10.sup.−4 Pa s Media density (ρ) 1,006 kg m.sup.−3 Flow rate (Q) 5 ml min.sup.−1 Channel Reynolds number (Re.sub.c) 375 Object Reynolds number (Re.sub.o) 131 Oscillating frequency (ƒ) 44.4 kHz

Transfection

[0155] Master moulds of microfluidic devices were fabricated using standard photolithography techniques, while devices were replicated using soft lithography and bonded to glass using oxygen plasma. An overview of the device design and transfection parameters are shown in FIG. 1 and Table 1 respectively.

[0156] HEK293 (Human embryonic kidney 293) cells were suspended in cell media at a density of 1×10.sup.5 cells ml.sup.−1, and pcDNA 3.1 GFP plasmids were seeded at a density of 890 ng ml.sup.−1. This suspension was loaded into a syringe and pumped into the microfluidic device with a flow rate of 5 ml min.sup.−1, which corresponds to a Re.sub.c of 375 at the gap between posts as the flow cell contained an array of 8 units separated with a 20 μm diameter post with a gap between posts of 30 μm. This also corresponds to a Re.sub.o of 131. Subsequently, cells were incubated for a period of 6 days then imaged via both fluorescent and optical microscopy to examine green fluorescent protein gene expression.

Results: Simulation & Analysis

[0157] Simulations indicated a high-pressure region occurs just upstream of the posts in the device of FIG. 1 and a decreased pressure region occurs just downstream of the posts. This means that as the cells flow past the posts, they are exposed a sudden and transient decrease in pressure. Additionally, these simulations were run as transient to determine if unsteady flow occurs. FIG. 2 shows an overview of the pressure changes that occur during simulations of an embodiment of the method of the invention showing (a) pressure contours, (b) velocity magnitude, (c) x-direction velocity of the liquid, which can be used to approximate cell velocity and (d) the y-direction liquid velocity with alternating jets due to unsteady flow. As shown in FIG. 2, there are significant flow velocities in the x-direction perpendicular to bulk flow in the y-direction in the enclosed channel, meaning unsteady flow is occurring.

[0158] According to the simulations, as a cell passed through a gap between posts positioned in the enclosed channel of the device, it moves from a surrounding zone with a localised pressure of 43.5 kPa, is exposed to a transient decrease in pressure of 94.3 kPa as it enters a zone of relatively lower pressure, which has a minimum pressure of 50.8 kPa. The magnitude of the transient decrease in pressure may vary depending on the phase of the oscillation. Additionally, cell velocity in the liquid is estimated to be 15 m s.sup.−1 during this transient decrease in pressure, which occurs over a distance of approximately 40 μm for a transient decrease in pressure (dP/dt) of −35.4×10.sup.6 kPa s.sup.−1, wherein dP/dt is the change is pressure (dP) over change in time (dr). dP is change in pressure between local maxima and local minima, dt is change in time between local maxima pressure and local minima.

[0159] Subsequently, the unsteady flow conditions subject the cell rapidly changing flow velocities in the direction orthogonal (y-direction) to the direction the cell is moving (x-direction), as shown in FIG. 2d, where peak y-direction velocity ranges from −8.5 m s.sup.−1 to 8.3 m s.sup.−1 and these localised unsteady flows are approximately 20 μm wide. The magnitude of the localised unsteady flow decays as the cell moves away from the posts and decays completely after approximately 500 μm—in this space a cell is pulsed by approximately 5 unsteady flows with a velocity magnitude of between 3.4 m s.sup.−1 and 8.5 m s.sup.−1. During this period cell velocities are estimated to be between 10 m s.sup.−1 and 15 m s-1 and unsteady flow widths are approximately 20 μm, indicating pulse times range between 2.0 μs and 1.3 μs. After the cell is pulsed with a transient decrease in pressure and the unsteady flow, the pressure increases to the same pressure as the outlet as the cell exits the microfluidic device or as the cell moves away from the gap.

[0160] The simulations suggest the exposure to unsteady flow creates a pressure drop across the cell membrane where the local extracellular pressure is greater than the local intracellular pressure, thereby facilitating active (mechanical) delivery. Additionally, the increase in pressure as the cell moves towards the device outlet suitably to facilitates active delivery due to the pressure drop across the permeabilised cell membrane.

Transfection

[0161] As shown in FIG. 3, the use of a transient decrease in pressure and unsteady flow conditions through a post array can be used to transfect HEK293 cells with pcDNA 3.1 GFP plasmids. The images in the top row are taken from the same field of view as the images in the bottom row. Bright spots in images on the left-hand side of the panel represent HEK293 cells successfully transfected with pcDNA 3.1, which were viable and continued to express green fluorescent protein 6 days after transfection.

[0162] The simulations allow for unsteady flow, and preliminary simulations were used to determine which velocity was the most appropriate for calculating Re.sub.o based on the transition from laminar flow conditions to unsteady flow conditions. The velocity of the liquid used for calculating Re.sub.o varies in the literature, however, previous simulations confirm the mean upstream velocity is the appropriate velocity. For example, for liquid flow around a cylindrical post the object Reynolds number (Re.sub.o) may be calculated with the equation below:

Re.sub.o=ρv∞d/μ

where v∞ refers to the velocity of a bulk liquid relative to the cylindrical post, and in this case, the mean upstream velocity of the liquid before the cylindrical post. This would be 8.68 m s.sup.−1 for the parameters shown in Table 1, resulting in an Re.sub.o of 131.2.

[0163] In order to estimate the frequency of oscillation, the correlation shown below is used as it applies to flow of liquid around cylindrical posts, where the Re.sub.o is between 40 and 190. The Strouhal number (Sr) (a dimensionless number used to describe unsteady flow) maybe calculated from Re.sub.o with the following correlations for flow around a cylindrical post:

Sr=0.2665−1.018/√Re.sub.o for (40<Re.sub.o<190)

[0164] This calculation results in a Sr of 0.17, and the frequency of oscillation (f) may be calculated with the equation below with the liquid velocity (v) and characteristic length (L), which is equal to the diameter of the post (d.sub.p):

f=Sr v/d.sub.p

[0165] For the parameters described above, it is estimated the unsteady flow oscillates at a frequency of 44.4 kHz. These unsteady oscillations are also known to induce structural vibrations within the posts themselves. Thus, it is believed cells may be exposed to a transient decrease in pressure, 44.4 kHz unsteady flow along with induced structural vibrations.

[0166] Laminar flow (Re>>1) between one or more flow diverters, such as (but not limited to) posts, may be used to create a region of transiently decreased pressure substantially immediately downstream of the posts. This may be used to suddenly and temporarily decrease ambient pressure surrounding a cell as it flows past the posts of devices such as those shown in FIGS. 1, and 4 to 10. Additionally, if Re.sub.o>40 then these flow characteristic are known to induce unsteady flow, and in the example describe above, this pulses cells with (1) a transient decrease in pressure and (2) unsteady flow. Moreover, this may be achieved using channel dimensions that are greater than cell dimensions (g>d.sub.c) to mitigate clogging issues. This facilitates the transfer of exogenous material across the cell membrane and into the cytoplasm. According to Pawell et al (Pawell R. S., et al. (2013). Limits of parabolic flow theory in microfluidic particle separation: a computational study. ASME 4th International Conference on Micro/Nanoscale Heat and Mass Transfer, Hong Kong, China. December 11-14.) for channel Reynolds numbers above 100 (Re.sub.c>100) between posts this creates a region of negligible shear stress. That is, under these conditions, membrane permeabilisation is not due to shear stress, which indicates that transfection may be a result of the transient decrease in pressure and unsteady flow conditions along with any conditions induced by the unsteady flow, such as structural vibrations in the posts, as observed by Renfer et al. (Renfer A., et al. (2013) Vortex shedding from confined micropost arrays. Microfluidics and Nanofluidics. 15(2):231-242).

Example 2

[0167] Experiments were performed to investigate the extent to which the magnitude and duration of the decrease in pressure affects transfection.

Methods

[0168] Two cultures of HEK293 cells were seeded at a density of 100,000 cells ml.sup.−1, wherein culture 1 contained HEK293 cells and green fluorescent protein pcDNA 3.1 seeded at a density of approximately 900 ng 10.sup.−5 cells, and culture 2 contained HEK293 cells and 25-based pair oligonucleotides seeded at a density of 100 ng 10.sup.−5 cells. Both cultures were placed in a vacuum dessicator and the pressure decreased to −95 kPa over the course of 2 minutes. The vacuum was then released and returned to atmospheric pressure over the course of 10 seconds.

Results & Discussion

[0169] This experiment using a prolonged decrease in pressure resulted in nil transfection. No cells expressed GFP and the co-localisation of oligonucleotides and cells was negligible. When compared to Example 1, the magnitude of the decrease in pressure was substantially greater (a 95 kPa decrease, as opposed to a 20 kPa decrease in Example 1). However, the rate of decrease was substantially slower. In Example 1, it is estimated that the rate in which the transient decrease in pressure occurs (dP/dt) is −35.4×10.sup.6 kPa s.sup.−1. In the present example, the dP/dt is approximately −0.8 kPa s.sup.−1. Accordingly, dP/dt may play a role in permeabilising the cell membrane as the cell membrane is gas permeable, such that if dP/dt is too low gas transfer will occur naturally through the cell membrane without permeabilising the membrane. Once dP/dt is sufficient, it is thought that the physical properties of cell membrane will not be able to accommodate for rapid gas transfer from the intracellular environment to the extracellular environment. Thus, the cell membrane may be stressed to a point where pores form, thereby allowing the introduction of exogenous material into the cell.

Example 3

[0170] FIG. 4 is a schematic diagram of a microfluidic device containing three columns of posts (n.sub.c=3) and four rows of posts (n.sub.r=4). The array is configured such that the diameter of the posts (d.sub.p) is equal to the gap (g) between posts (d.sub.p=g), and the posts for each column is shifted a sufficient distance to bifurcate flow from the previous gap where the shift distance (s) is equal to half row pitch (s=p.sub.r/2) and the column pitch (p.sub.c) is equal to the row pitch (p.sub.c=p.sub.r). The width of the channel, number of columns (n.sub.c) and number of rows (n.sub.r) will vary for each specific device using this or a similar design.

[0171] FIG. 5 is a schematic diagram of a microfluidic device containing three columns of posts (n.sub.c=3) and four rows of posts (n.sub.r=4). The array is configured such that the diameter of the posts (d.sub.p) is greater than the gap between posts (g), and the posts for each column is shifted a sufficient distance to bifurcate flow from the previous gap where the shift distance (s) is equal to half row pitch (s=p.sub.r/2) and the column pitch (p.sub.c) is equal to the row pitch (p.sub.c=p.sub.r). The width of the channel, number of columns (n.sub.c) and number of rows (n.sub.r) will vary for each specific device using this or a similar design.

[0172] FIG. 6 is a schematic diagram of a microfluidic device containing three columns of posts (n.sub.c=3) and four rows of posts (n.sub.r=4). The array is configured such that the diameter of the posts (d.sub.p) is less than the gap (g) between posts (d<g), and the posts for each column is shifted slightly from the previous gap where the shift distance (s) is less than half the row pitch (s<p.sub.r/2) and the column pitch (p.sub.c) is greater to the row pitch (p.sub.c>p.sub.r). The width of the channel, number of columns (n.sub.c) and number of rows (n.sub.r) will vary for each specific device using this or a similar design.

[0173] FIG. 7 is a schematic diagram of a microfluidic device containing three columns of posts (n.sub.c=3) and four rows of posts (nr=4). The array is configured such that the diameter of the posts (d.sub.p) is less than the gap (g) between posts (d.sub.p<g), and the posts for each column is shifted slightly from the previous gap where the shift distance (s) is less than half the row pitch (s<p.sub.r/2) and the shift direction switches with each row. The column pitch (p.sub.c) is greater to the row pitch (pc>p.sub.r). The width of the channel, number of columns (n.sub.c) and number of rows (n.sub.r) will vary for each specific device using this or a similar design.

[0174] Preferred embodiments of a device design of the invention are depicted in FIGS. 8, 9 and 10. Both embodiments contain a single inlet and a single outlet with different internal post figurations that are particularly shown in Panels A and B of each figure. In these embodiments, all substrates are fused silica with a substrate thickness (t.sub.s) of 700 μm. The unit includes a lid with 2 through-holes, each having a diameter (D.sub.h) of 700 μm. The lid and substrate bond strength or burst pressure should be greater than (>>) 10 atm and once bonded, the total device has a thickness (t.sub.d) of 1.40 mm. The device footprint of 4.80 mm×9.80 mm accounts for a dicing width of 200 μm. It is contemplated that 7×6 devices are arrayed across 70 mm×30 mm jig for a total of 42 devices. The bottom piece of the device is deep reactive ion-etched fused silica, bonded to a laser-machined fused silica wafer using a bulk material bond. For the embodiments shown in FIGS. 8 and 9, the substrate etched is to create a channel having a width of 1.5 mm, length of 7.5 mm and a depth of 40.0 μm. For the embodiment shown in FIG. 10, the substrate etched is to create a channel having a width of 0.6 mm, length of 5.5 mm and a depth of 40.0 μm.

[0175] According to the embodiment shown in FIG. 8, the array design (Panel A) includes thirty (30) posts in the x-direction (n.sub.x) and one (1) row of posts in the y-direction (n.sub.y) with an array pitch of 50.0 μm in the x-direction (P.sub.x; otherwise referred to as the column pitch p.sub.c). In this embodiment, the post design as shown in Panel B, is configured such that the diameter of the posts (d.sub.p=20 μm) is less than the 30.0 μm gap of between the posts (gap=P.sub.x−d.sub.p) that is present in this embodiment.

[0176] According to the embodiment shown in FIG. 9, the array design (Panel A) includes thirty (30) posts in the x-direction (n.sub.x) and three (3) rows of posts in the y-direction (n.sub.y) with an array pitch of 50.0 μm in the x-direction (P.sub.x; otherwise referred to as the column pitch p.sub.c) and 750 μm in the y-direction (Py; otherwise referred to as the row pitch p.sub.r). In this embodiment, the post design as shown in Panel B, is configured such that the diameter of the posts (d.sub.p=20 μm) is less than the 30.0 μm gap of between the posts (gap=P.sub.x−d.sub.p) that is present in this embodiment.

[0177] According to the embodiment shown in FIG. 10, the array design (Panel A) includes twelve (12) posts in the x-direction (n.sub.x) and three (3) rows of posts in the y-direction (n.sub.y) with an array pitch of 50.0 μm in the x-direction (P.sub.x; otherwise referred to as the column pitch p.sub.c) and 500 μm in the y-direction (Py; otherwise referred to as the row pitch p.sub.r). In this embodiment, the post design as shown in Panel B, is configured such that the diameter of the posts (d.sub.p=20 μm) is less than the 30.0 μm gap of between the posts (gap=P.sub.x−d.sub.p) that is present in this embodiment.

[0178] Suitable ranges for particularly preferred embodiments of the invention as shown in the figures are provided below:

[0179] Post diameter range (d.sub.p): 10 nm-5 mm;

[0180] Number of columns (n.sub.c): 1-10,000;

[0181] Number of rows (n.sub.r): 3-10,000;

[0182] Gap range (g): 10 nm-5 mm;

[0183] Shift (s): 0-5 mm;

[0184] Column pitch (p.sub.c): 30 nm 50 mm; and

[0185] Row pitch (p.sub.r) 30 nm-50 mm

[0186] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0187] The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

[0188] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the invention. All such modifications and changes are intended to be included within the scope of the appended claims.