DETAILED ACTION
Preliminary Amendment filed on 04/04/2024 is acknowledged. Claim 21 is canceled. Claims 1-20 are pending in the application and are considered on merits.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1-3, 5-13, 15 and 19-20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Huang et al. (Microfluidics and Nanofluidics, 2018) (Huang) in view of Behroodi et al. (Scientific Reports, 2020, IDS) (Behroodi).
Regarding claim 1, Huang discloses microfluidic device (abstract), comprising:
a channel layer configured for flow of a fluid medium (Fig. 1, page 2, par 3); and
a plurality of microwells in fluid communication with the channel layer (Fig. 1, page 2, par 3).
Huang further teaches that “a deeper microwell structure (15 μm in diameter and 25 μm in depth) is preferred to ensure trapped particles would not be flushed out during the washing step,” (page 5, par 0), and that “[t]he vortex streamline indicates a stable particle confinement in the microwell.” (Fig. 2, page 5, par 0). Huang therefore teaches microwells having a well depth and a well diameter selected to substantially isolate the contents of the microwells from the primary channel flow.
To the extent Huang does not expressly characterize this reduced or recirculating flow as “prevent[ing] fluid flow” from the channel into the microwells, Behroodi teaches that microwell depth and geometry directly control fluid velocity and shear within the microwells. Behroodi states that “with increasing depth of the microwell, the flow rate decreases,” that a “secondary flow is generated” in deeper microwells (page 10, par 1), and that this secondary flow “prevents the escape of cells from the microwells.” (page 11, par 1). Behroodi further explains that increasing microwell depth “reduces the shear stress of the fluid flow to the cells and prevents the spheroid inside the microwell from directly exposing to the fluid flow.” (page 11, par 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date to configure Huang’s microwell depth and diameter in view of Behroodi’s teachings concerning the relationship between microwell geometry and internal fluid velocity, thereby reducing or substantially preventing the circulating channel flow from entering and disturbing the microwells. Both references concern microfluidic microwells used to retain biological particles or cells while fluid is circulated through an adjacent channel. A person of ordinary skill would have been motivated to apply Behroodi’s geometry-dependent flow teachings to Huang’s device to improve retention of magnetically captured cells during high-speed washing and to reduce shear-induced cell loss.
Regarding claim 2, Huang teaches that the motion and retention of particles depend on both microwell geometry and the surrounding flow conditions (Fig. 2, page 5, par 0). Huang states that the motion of magnetic particles in the continuous-flow environment is determined in part by “flow velocity” and explains that “[b]y changing the value of each parameter, the final particle seeding position can be adjusted” (page 3, par 0). Huang further evaluates loading rates of 30, 50, and 70 μL/min and a washing rate of 500 μL/min, while selecting a microwell diameter and depth that retain particles during the washing operation (page 6, par 1).
Behroodi more expressly teaches determining microwell geometry based on expected fluid-flow conditions. Behroodi describes microwell depth as “an essential parameter for microwell design since it directly affects the shear stress of the fluid flow,” (page 2, par 6), performs numerical flow simulations at specified loading and washing flow rates, and compares microwell depths from 100 to 500 μm to determine their effect on internal flow and cell retention (page 3, par 2). Behroodi reports that the flow profiles at 50 μL/min and 30 μL/min were evaluated and that the internal secondary flow depended on “the chip geometry.” (page 11, par 0).
It would have been obvious to determine Huang’s well depth and diameter based on the expected loading and washing flow rates, as taught by Behroodi, because microwell dimensions were known result-effective variables that affect internal velocity, shear stress, and particle retention. Optimizing these dimensions for the anticipated channel flow would have been no more than the predictable use of known geometry-flow relationships to ensure that captured particles remain confined during circulation.
Regarding claim 3, Huang teaches that wherein, for a given microwell, the well depth and the well diameter of the given microwell together cause fluid medium present in the given microwell to form one or more vortices during circulation of the fluid medium in the fluid channel, wherein the one or more vortices prevent the fluid medium from flowing into the given microwell from the channel layer (Fig. 2, page 5, par 0).
Regarding claim 5, Huang teaches that the microfluidic device further comprising a magnet configured to pull cells labeled with magnetic nanoparticles into the plurality of microwells during a cell separation process (Fig. 1, page 3, par 0).
Regarding claim 6, Behroodi discloses a microfluidic chip having a chamber containing the microwells and states that “[t]he height of 200 μm was taken into account for the chamber.” (page 3, par 2). Behroodi further describes the chamber as connected to inlet and outlet microchannels and carrying the flowing medium across the microwell region (page 3, par 2).
Thus, Behroodi teaches a channel or chamber layer that is 200 micrometers thick.
Regarding claim 7, Behroodi discloses microwells having depths ranging from 100 to 500 µm, including a group of microwells having a depth of 100 µm (page 3, par 2). Behroodi states that “the depth of each group of seven microwells was changed from 100 to 500 µm in increments of 100 µm to investigate the effect of depth on the cell trapping and the spheroid formation.” (page 3, par 2).
Thus, Behroodi expressly teaches a microwell depth of approximately 100 micrometers.
Regarding claim 8, Huang discloses that wherein the well diameter is between 10-100 micrometers (Fig. 1, page 5, par 0).
Regarding claim 9, Huang discloses that wherein the channel layer and microwells are formed from polydimethylsiloxane (PDMS) (page 2, par 3).
Regarding claim 10, Huang teaches a method of labeling cells with a microfluidic device (abstract), the method comprising:
introducing a fluid medium into a channel and microwells of the microfluidic device, the microwells being in fluid communication with the channel (Fig. 1);
orienting the microwells of the microfluidic device to be above a channel of the microfluidic device (Fig. 1, page 3, par 0);
introducing cells into the fluid medium, wherein a first portion of the cells are labeled with magnetic particles, and wherein a second portion of the cells are not labeled with the magnetic particles (Fig. 1, page 3, par 3);
applying a magnetic force to the microfluidic device to pull the first portion of the cells into the microwells, wherein the second portion of the cells remain in the channel (Fig. 1, page 3, par 0); and
circulating the fluid medium in the channel to remove the second portion of the cells (Fig. 1, page 5, par 1).
Huang further teaches that “a deeper microwell structure (15 μm in diameter and 25 μm in depth) is preferred to ensure trapped particles would not be flushed out during the washing step,” (page 5, par 0), and that “[t]he vortex streamline indicates a stable particle confinement in the microwell.” (Fig. 2, page 5, par 0). Huang therefore teaches microwells having a well depth and a well diameter selected to substantially isolate the contents of the microwells from the primary channel flow.
To the extent Huang does not expressly state that the microwell geometry prevents fluid flow from the channel into the microwells, Behroodi teaches that microwell geometry controls and substantially reduces fluid flow within the wells (page 11, par 0-1). Behroodi states that “with increasing depth of the microwell, the flow rate decreases,” that increasing depth reduces shear stress and prevents retained cells from being directly exposed to the main fluid flow, and that geometry-induced secondary flow prevents cells from escaping during washing (page 11, par 3).
Both Huang and Behroodi concern retaining cells or particles in microfluidic microwells while fluid is circulated through an adjacent channel, and the modification would predictably improve retention of magnetically captured cells during removal of unlabeled cells. It would have been obvious to one of ordinary skill in the art to configure Huang’s microwells according to Behroodi’s geometry-dependent flow teachings so that the channel washing flow is substantially prevented from entering and disturbing the microwells.
Regarding claim 11, Behroodi teaches the additional limitation of claim 11. Behroodi explains that microwell depth is an important design parameter because it “directly affects the shear stress of the fluid flow and the diffusion of nutrients, respiratory gases, and growth factors.” (page 2, par 6). Behroodi further performs flow simulations using specified inlet flow rates and compares microwells having different depths to determine how the geometry affects flow within the microwells (page 10, par 1).
Behroodi specifically evaluates flow at 50 μL/min for cell loading and 30 μL/min for removing unseeded cells and reports that increasing microwell depth decreases the flow rate within the microwell and can generate secondary flow (page 11, par 0). Thus, Behroodi teaches selecting or determining the microwell geometry in view of the expected operating flow conditions so as to reduce flow and retain cells within the microwells.
Regarding claim 12, Huang teaches that wherein the flow rate is between 1 micrometer per second and 10 centimeters per second (page 4, par 0). Huang performs its flow simulation using an inlet velocity of 1.11 × 10⁻³ m/s (page 4, par 0), which is 1.11 mm/s and falls within the claimed range of 1 µm/s to 10 cm/s. Huang further circulates fluid through the device during cell or particle loading and washing.
Regarding claim 13, Huang teaches that the geometry of each microwell includes both a microwell width and a microwell depth. In particular, Huang discloses microwells having a 15-µm diameter and a 25-µm depth, and explains that the deeper microwell geometry is selected to retain particles during the washing step (page 5, par 0).
Behroodi likewise teaches microwell geometry defined by lateral dimensions and depth and explains that microwell depth affects the internal flow rate, shear stress, and cell retention (page 11, par 3).
Thus, the combination teaches that the geometry used to reduce or substantially prevent channel flow from disturbing the microwells comprises a microwell width and microwell depth.
Regarding claim 15, Huang teaches the additional vortex limitation of claim 15. Huang discloses a deeper microwell structure having a 15-µm diameter and a 25-µm depth and states that “[t]he inset in Fig. 2d shows the streamline profile inside the microwell” and that “[t]he vortex streamline indicates a stable particle confinement in the microwell.” (page 5, par 0). Huang further explains that the deeper microwell geometry is preferred so that trapped particles are not flushed out during washing (page 5, par 0).
Behroodi likewise teaches that microwell geometry produces secondary recirculating flow within deeper microwells. Behroodi states that “with increasing depth of the microwell, the flow rate decreases,” that “a secondary flow is generated” in sufficiently deep microwells (page 10, par 1), and that “[s]econdary flow prevents the escape of cells from the microwells.” (page 11, par 1). Figure 7(c) further illustrates recirculating streamlines within the microwells.
Thus, Huang and Behroodi teach that the width and depth of a microwell cause vortex or secondary recirculating flow to form during circulation of fluid through the adjacent channel, thereby confining retained particles or cells and substantially isolating them from the main channel flow.
Regarding claim 19, the limitation that “at least 95% of the cells that are labeled with magnetic particles are pulled into the microwells [and] subsequently extracted from the microwells” merely states an intended result or degree of efficiency resulting from performance of the previously recited cell-capture and cell-extraction steps. The limitation does not recite any additional manipulative step, operating parameter, or structural feature by which the claimed percentage is achieved and therefore does not further distinguish the claimed method from the method taught or suggested by the cited references. A statement of intended result in a method claim is not entitled to patentable weight where it does not impose a manipulative difference in the recited process steps. See Minton v. National Association of Securities Dealers, Inc., 336 F.3d 1373, 1381 (Fed. Cir. 2003); Bristol-Myers Squibb Co. v. Ben Venue Laboratories, Inc., 246 F.3d 1368, 1376 (Fed. Cir. 2001).
Regarding claim 20, Huang teaches a method of forming a microfluidic device from first and second PDMS layers. Huang states that the device “is composed of two polydimethylsiloxane (PDMS) layers: (1) the microchannel layer and (2) the microwell layer.” (page 2, par 3). Huang further teaches fabricating silicon molds using photolithography, casting the respective PDMS layers from those molds, aligning the two PDMS layers, and irreversibly bonding them using oxygen-plasma treatment page 3, par 1). Huang also teaches punching inlet and outlet openings so that fluid can be introduced into and removed from the microchannel connected to the microwell region (page 3, par 1).
Huang therefore teaches obtaining a first PDMS layer having a channel, obtaining a second PDMS layer having a plurality of photolithographically formed microwells, bonding the two PDMS layers, and forming fluid connections to the channel and microwells (page 3, par 1). Huang additionally teaches selecting a deeper microwell geometry—15 µm in diameter and 25 µm in depth—because it is “preferred to ensure trapped particles would not be flushed out during the washing step,” and Figure 2D shows a vortex streamline providing stable confinement within the microwell (page 5, par 0).
To the extent Huang does not expressly characterize the microwell diameter and depth as preventing fluid flow from the channel into the microwells, Behroodi teaches that microwell geometry controls internal flow. Behroodi explains that microwell depth directly affects fluid-flow shear stress, that “with increasing depth of the microwell, the flow rate decreases,” (page 10, par 1), and that sufficiently deep microwells generate secondary flow that prevents retained cells from escaping during washing (page 11, par 1). Behroodi also teaches fabricating a PDMS microfluidic chip from separately formed PDMS portions and bonding the portions by plasma treatment (Fig. 6).
It would have been obvious to one of ordinary skill in the art to select the diameter and depth of Huang’s photolithographically formed microwells according to Behroodi’s geometry-dependent flow teachings so as to reduce or substantially prevent channel flow from entering and disturbing the microwells during circulation. Both references concern PDMS microfluidic microwell devices configured to retain cells or particles while fluid is circulated through an adjoining channel, and the modification would predictably improve retention during washing.
Claim(s) 4 and 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Huang in view of Behroodi and Le et al. (Nucleic Acids Research, 2015) (le).
Regarding claim 4 and 14, Huang in view of Behroodi teaches the microfluidic device of claim 1 for the reasons discussed above. Huang further teaches circulating fluid through a microchannel containing microwells and explains that microwell geometry affects the internal flow pattern and retention of particles during washing (page 5, par 0). Huang discloses a simulated inlet velocity of 1.11 × 10⁻³ m/s, or 1.11 mm/s, which falls within the claimed range of 1 µm/s to 10 cm/s (page 4, par 0).
Behroodi teaches microwells having a depth of 100 µm, stating that the tested microwell depths ranged “from 100 to 500 µm in increments of 100 µm.” (page 3, par 2). Behroodi also teaches that microwell depth affects the flow rate, shear stress, and formation of secondary flow within the microwells (page 11, par 3).
However, Huang and Behroodi do not expressly teach the claimed relationship between microwell diameter and the number of vortices. Le teaches this additional limitation. Le discloses simulations of microwells having a fixed depth of 100 µm and diameters of 20, 40, 50, 60, 80, and 100 µm (Fig. 2). Le states that, “for microwells with diameters of 20–100 µm and a fixed height of 100 µm,” flow vortices develop within the microwells during channel flow (page 2, par 7). Figure 2 of Le illustrates that decreasing the microwell diameter increases the number of vertically arranged recirculating vortices: the 20-µm well includes three or more recirculating regions, the 40- and 50-µm wells include two recirculating regions, and the 60-, 80-, and 100-µm wells include one principal recirculating vortex.
Le further simulates channel flow at approximately 16 cm/s and experimentally applies flow at approximately 10 cm/s. Thus, Le teaches operation at, or immediately adjacent to, the upper boundary of the claimed range. Huang independently teaches an operating velocity well within the claimed range.
It would have been obvious to one of ordinary skill in the art to configure the microwells of Huang, as modified by Behroodi, according to the diameter-dependent vortex teachings of Le. Huang, Behroodi, and Le are analogous references concerning microfluidic devices having microwells exposed to fluid circulation, and each addresses reducing flow disturbance or retaining material within the microwells. One of ordinary skill would have been motivated to use Le’s disclosed 100-µm well depth and diameter-dependent vortex configurations in the device of Huang and Behroodi to reduce flow velocity near the well bottom, protect captured cells from the main channel flow, and improve retention during washing. The resulting device would have a 100-µm well depth and, depending on the selected well diameter, three or more vortices for a diameter within 10–30 µm, two vortices for a diameter within 40–50 µm, or one vortex for a diameter within 60–100 µm, while operating at a flow rate within the claimed range.
Claim(s) 16 is/are rejected under 35 U.S.C. 103 as being unpatentable over Huang in view of Behroodi and Marson et al. (US 2020/0048606) (Marson).
Regarding claim 16, Huang in view of Behroodi teaches the method of claim 10 for the reasons discussed above. However, Huang and Behroodi do not expressly teach performing cell labeling by circulating a second fluid medium containing fluorophore-conjugated antibodies through the channel and allowing the antibodies to diffuse into the microwells.
Marson teaches this additional limitation. Marson discloses a microfluidic device having a microfluidic channel and sequestration pens containing cells. Marson explains that fluid flow in the channel does not enter the isolation regions of the pens and that “diffusion is the only mechanism by which components in a first medium 254 in the microfluidic channel 264 can move from the microfluidic channel 264 into a second medium 258 in an isolation region 270 of a sequestration pen 266.” (par [0142]). The isolation regions of Marson’s sequestration pens correspond to microwells in fluid communication with the channel.
Marson further teaches labeling cells retained in the pens using fluorescently labeled antibodies, stating:
“Fluorescently-labeled anti-CXCR4 antibody was imported into the chip, and media flow was interrupted to allow diffusion of the antibody into the pens.” (par [0295]).
Marson then incubates the cells with the antibody, flushes the chip with fresh medium to remove excess free antibody, and obtains fluorescent images to determine which cell colonies express CXCR4 (par [0295]). Thus, Marson teaches introducing a second fluid medium containing fluorophore-conjugated antibodies into the microfluidic channel and allowing the antibodies to diffuse into the cell-containing recesses to label the retained cells.
It would have been obvious to one of ordinary skill in the art before the effective filing date to perform Marson’s on-chip fluorescent-antibody labeling process on the cells retained in the microwells of Huang, as modified by Behroodi. Huang, Behroodi, and Marson are analogous art because each concerns retaining and processing cells within recessed regions of a microfluidic device while fluids or reagents are supplied through an adjacent channel. One of ordinary skill would have been motivated to introduce fluorescently labeled antibodies through Huang’s channel and permit the antibodies to diffuse into the microwells, as taught by Marson, to identify, characterize, or immunophenotype the magnetically captured cells without first removing the cells from the microfluidic device.
Claim(s) 17 is/are rejected under 35 U.S.C. 103 as being unpatentable over Huang in view of Behroodi and Hakanson et al. (Integrative Biology, 2010) (Hakanson).
Regarding claim 17, Huang in view of Behroodi teaches the method of claim 10 for the reasons discussed above. Huang teaches magnetically trapping labeled cells in microwells of a microfluidic device, while Behroodi teaches retaining and culturing cells within microfluidic microwells during continued fluid handling. However, Huang and Behroodi do not expressly teach that the microwells are coated with fibronectin.
Hakanson teaches this additional limitation. Hakanson discloses a PDMS microwell array used for culturing cells in a three-dimensional environment and explains that, before cell seeding, “subtractive microcontact printing of fibronectin was used to specifically functionalize the wells.” (Fig. 1). Hakanson further discloses that the individual microwells were approximately 34 µm in diameter and 10 µm deep and that MCF-7 breast carcinoma cells were cultured within the fibronectin-coated microwells (page 33, par 1), where the cells contacted the bottoms and sidewalls of the wells and formed three-dimensional cell clusters (Fig. 3, page 33, par 1).
Hakanson also states that “the cells were cultured for 24 h in 34 µm wide fibronectin-coated microwells,” and reports continued cell culture and proliferation within those wells (Fig. 3). Thus, Hakanson expressly teaches culturing cells in microwells coated with fibronectin.
It would have been obvious to one of ordinary skill in the art before the effective filing date to coat the microwells of Huang, as modified by Behroodi, with fibronectin and culture the captured cells therein, as taught by Hakanson. Huang, Behroodi, and Hakanson are analogous art because each concerns retaining and processing biological cells within microfluidic or microwell structures. One of ordinary skill would have been motivated to use Hakanson’s fibronectin coating in Huang’s microwells to promote cell adhesion to the microwell surfaces and facilitate continued culture and analysis of the magnetically captured cells. Hakanson expressly teaches that its fibronectin-functionalized microwells permit cells to adhere to the bottoms and sidewalls of the wells and form three-dimensional clusters, thereby providing a predictable benefit for maintaining captured cells within the microwells.
Claim(s) 18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Huang in view of Behroodi and Beemiller et al. (WO 2020/081875 (Beemiller).
Regarding claim 18, Huang in view of Behroodi teaches the method of claim 10 for the reasons discussed above. Huang further teaches extracting magnetically captured cells from the microwells by reversing the direction of the applied magnetic field (page 7, par 3). Huang explains that the permanent magnet may be moved from above the microwell layer to below the microchannel and that, “[b]y applying the magnetic field from opposite direction, the trapped particles/cells can be pulled out from the microwell and then be flushed into the collection tube.” (page 7, par 3). Thus, Huang teaches applying a magnetic force that pulls the cells from the microwells into the channel and subsequently flushing the cells from the channel.
However, Huang does not expressly teach inverting the microfluidic device during extraction. Beemiller teaches this additional feature. Beemiller discloses a microfluidic device having a flow path and sequestration pens and teaches that a tilting device may rotate the microfluidic device “180° relative to the x-axis or the y-axis in order to fully invert the microfluidic device.” (par [00504]), further teaches changing the orientation so that the flow path is positioned above or below the sequestration pens (par [00505]).
It would have been obvious to one of ordinary skill in the art to invert Huang’s microfluidic device before applying the reversed magnetic force and flushing the released cells, as taught by Beemiller. Huang and Beemiller are analogous art because both concern manipulating cells between recessed cell-retention regions and an adjoining microfluidic flow path by controlling the physical orientation of the device. Inverting Huang’s device would reposition the microwells relative to the channel and gravity, thereby assisting transfer of the released cells from the microwells into the channel before collection.
Conclusion
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/XIAOYUN R XU, Ph.D./ Primary Examiner, Art Unit 1797
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