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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 03/03/2026 has been entered.
1. Claims 1 – 3, 9 – 13, 15, 16, 19, 28, and 30 – 32 remain pending and are under consideration.
Withdrawn Claim Rejections
2. The rejection of claims 1, 3, 9 – 13, 15, 16, and 19 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1 requiring a perfusion-enabled bioreactor and perfusing the cells.
3. The rejection of claim 2 under 35 U.S.C. 103 is withdrawn in view of the amendment to claims 1 and 2.
4. The rejection of claim 29 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancellation of the claim.
Maintained Claim Rejections
5. Claim(s) 28, 30, 21, and 32 remain rejected under 35 U.S.C. 103 as being unpatentable over Angelini-2018 (WO-2018085823-A1; previously cited), hereinafter Angelini-2018 as evidenced by Kim (Kim, Jong-Yun, et al. Colloid and Polymer Science 281.7 (2003): 614-623; previously cited) hereinafter Kim in view of Derda (Derda, Ratmir, et al. PloS one 6.5 (2011): e18940; previously cited), hereinafter Derda. Although maintained, the rejection is revised in view of the amendment to claim 28 and cancellation of claim 29.
Regarding “3D culture media” of claim 28, Angelini-2018 teaches an apparatus (100 in Figure 1) comprising a container (110 in Figure 1) for placing groups of cells (“plurality of cells”) in a 3D cell growth medium (120 in Figure 1) (“3D culture media”) that may include extracellular matrix proteins (page 14, lines 14 – 25; Figure 1). Angelini-2018 teaches an embodiment in Figure 2A where a 3D cell growth medium (220 in Figure 2A) comprising a plurality of spheroids (230 in Figure 2A) is disposed in a container of the bioreactor (210 in Figure 2A) (page 14, lines 32 – 35; Figure 2A) and where spheroids are in a sheet in Figure 4D in which the longest dimension extends from the bottom of the container to the top of the container (“vertically-oriented flat sheets have a longest dimension extending from a bottom of the bioreactor to a top opening of the bioreactor”). Angelini-2018 does not teach the sheet of cells is “flat” but does teach groups of cells may be placed within a 3D cell growth medium according to any suitable shape, geometry, and/or pattern and cells may be arranged in high aspect ratio objects (page 10, lines 30 – 36; page 11, lines 1 – 6).
Regarding “perfusion-enabled bioreactors” of claim 28, Angelini-2018 teaches an apparatus (350 in Figure 3B) with perfusion tubing, 3D cell growth material (304 in Figure 3B), and biological cells (302 in Figure 3B) (Figure 3B; page 16, lines 14 – 20).
Regarding claims 30 and 31, Angelini-2018 teaches the 3D cell growth medium may include a Herschel-Bulkley yield stress material (claim 30) with a yield stress of 20 Pa (claim 31) (page 14, lines 6 – 7; page 14, lines 22 – 24; page 13, line 4 – 5).
Regarding claim 32, Angelini-2018 teaches a 3D cell growth medium comprises Carbopol particles (page 12, line 33 – 34). Carbopol is a cross-linked polyacrylic acid polymer as evidenced by Kim (page 615, left col. paragraph 1 and 3).
Angelinii-2018 does not teach the sheet of cells is “flat” of claim 28. However, Angelini-2018 teaches cells can be deposited in a pattern within the 3D cell growth medium that can be a granular gel (page 2, lines 25 – 28). Angelini-2018 teaches the 3D cell growth medium is tunable for controlling the environment of cells such that the medium may have mechanical properties which are tuned to be similar to those found in vivo so that the cells 3D growth medium may emulate the natural environment of the cells (page 7, lines 30 – 34). Angelini-2018 teaches the cells in the granular gel can be in a well plate, such as a 96 well plate and the cells can be cultured, the cells can be exposed to a stimulus, and samples can be collected and analyzed for potential changes in the cells or extracellular microenvironment including imaging in the bioreactor (page 2, lines 33 – 35; page 3, lines 1 – 36; Figure 4D). Angelini-2018 teaches the cells may contain fluorescent gene reporters that fluoresce or induce fluorescence (page 17, lines 6 – 24; Figure 4C). Angelini-2018 teaches that an advantage of the system is that samples can be taken without disrupting the 3D geometry of the cells (page 3, lines 30 – 32).
Regarding “flat sheet” of claim 28, Derda teaches a 3D culture of cells comprising a plurality of flat sheets of cells in Matrigel in paper where the cells are in a 96 zone array (page 2, right col. para. 3 – 4; Figure 1). Derda teaches the flat sheets of cells can be stacked to create a multi-zone multi-layer culture and thus the composition of the constructs can be controlled (page 4, left col. para. 3 and right col. para. 1 – 2; Figure 2). Derda teaches treating the 3D culture of stacked flat sheets of cells where the cells expressed fluorescent markers with mitomycin C and analyzing cell division at different locations on the sheets and cell migration (page 4, right col. last para.; page 5; Figure 3 – 4). Derda teaches that the paper layers must be peeled apart for analysis (page 4, right col. para. 3; page 12, left col. para. 4). Derda teaches that the approach can be expanded to any cell types that can be cultured inside ECM hydrogels and any analysis of cell responses that can be measured using fluorescent readout (page 11, left col. para. 1). Derda teaches the simplicity of the patterning and stacking technology will make it possible for researchers in the biomedical community to use this approach to design custom platforms for high-throughput 3D cultures for specific applications (page 11, left col. para. 1).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Angelini-2018 regarding a composition comprising a perfusion-enabled bioreactor, 3D culture media, a plurality of cells, and an extracellular matrix component, where the cells are in a sheet having a longest dimension extending from a bottom of the bioreactor to a top opening of the bioreactor with the teachings of Derda regarding flat sheets of cells and extracellular matrix to arrive at the claimed invention where a cell manufacturing kit comprises a bioreactor, 3D culture media, a plurality of cells, ECM components, where the plurality of cells are provided as one or more vertically-oriented flat sheets, wherein the one or more vertically-oriented flat sheets have a longest dimension extending from a bottom of the bioreactor to a top opening of the bioreactor. One would have been motivated to combine to combine the teachings of Angelini-2018 and Derda in a kit for creating custom 3D cell cultures for testing the effect of compounds or analyzing growth patterns as Angelini-2018 teaches the cells in the granular gel can be cultured, exposed to a stimulus, and samples can be collected and analyzed for potential changes in the cells or extracellular microenvironment including imaging in the bioreactor and Angelini-2018 teaches that an advantage of the system is that samples can be taken without disrupting the 3D geometry of the cells. One would have a reasonable expectation of success in combining the teachings as Angelini-2018 teaches groups of cells may be placed within a 3D cell growth medium according to any suitable shape, geometry, and/or pattern and cells may be arranged in high aspect ratio objects, the cells may contain fluorescent reporters, and analysis of the cells can occur in the container and Derda teaches analysis of fluorescence of cells in flat sheets.
Objections/Rejections Necessitated by Amendment
Claim Objections
6. Claim 1 is objected to because of the following informalities: in line 3 “wherein the bioreactor” is repeated twice and one recitation should be deleted. Appropriate correction is required.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
7. Claim(s) 1, 3, 9 – 13, 15, 16, and 19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Sawyer (WO2016182969A1; Filed 05/07/2016; Published 11/17/2016), hereinafter Sawyer which is cited on the IDS filed 12/22/2021 as evidenced by ATCC (ATCC: (08/16/2021). "MCF 10A." https://www.atcc.org/products/crl-10317. Accessed 05/27/2026.), hereinafter ATCC Bhattacharjee, Tapomoy, et al. ACS Biomaterials Science & Engineering 2.10 (2016): 1787-1795), hereinafter Bhattacharjee which is cited on the IDS filed 05/17/2022 in view of Angelini-2018 (WO-2018085823-A1;Filed 11/07/2017, Published 05/11/2018; previously cited), hereinafter Angelini-2018.
Regarding “printing a composition comprising a plurality of cells into a flat sheet” of step a and “re-printing” of step g of claim 1, Sawyer teaches in Figure 6a, 3D printing a flat sheet of cells (“flat sheet”) using a dispenser that moves in x, y, and z directions (“printing device”) in a container (“bioreactor”) (page 19, para. 0075). Sawyer teaches 3D printing with a computer-controlled injector tip may trace out a spatial path within a 3D cell growth medium and inject cells at locations along the path to form a desired 3D pattern or shape (page 9, para. 0037; Figure 3). Sawyer teaches an array of automated cell dispensers may be used to inject cells into a container of 3D growth medium (page 8, para. 0034; Figure 3). Sawyer teaches movement of the tip of the placement device through the 3D growth medium may cause yielding such that the 3D growth medium flows to accommodate the group of cells (page 8, para. 0034; Figure 3). Sawyer teaches in Figure 1 an apparatus (100) for placing groups of cells in a 3D cell growth medium (120) comprising a container (110) holding the 3D cell growth medium and an injector (150) that displaces the 3D cell growth medium with a plurality of cells (160) (page 16, para. 0065). Sawyer teaches in Figure 2 an injector (150) comprising a capillary with a microscale tip (155) sweeping out a pattern in space as a material (160 is injected into a 3D cell growth medium (120) (Figure 2; page 17, para. 0067). Sawyer teaches a 3D cell growth medium with 0.9% Carbopol swollen particles dispersed in cell growth media and homogenized in a high-speed centrifugal mixer in which cells were placed in this medium using a 3D printing technique (page 11, para. 0044). Sawyer teaches the cells may be placed within a 3D cell growth medium according to any suitable shape, geometry, and/or patten (page 8, para. 0036). Sawyer teaches the cells may be deposited into shapes that correspond to geometries of biological structures (page 9, para. 0036; page 17, para. 0069; Figure 3). Sawyer does not teach “a longest dimension extending from a bottom of the bioreactor to a top opening of the bioreactor” of step a or step g of claim 1.
Regarding “the bioreactor comprises a 3D cell culture medium comprising a plurality of packed hydrogel particles and a liquid cell culture medium, wherein the packed hydrogel particles are swelled with the liquid cell culture medium to form a granular gel and wherein the 3D cell culture medium comprises a network of perfusable pore space” of step a of claim 1, Sawyer teaches the sheet of cells in Figure 6a are printed in a 3D culture medium of packed microgel particles (“3D cell culture medium comprising a plurality of packed hydrogel particles and a liquid cell culture medium”) swollen with liquid cell growth medium (“swelled with the liquid cell culture medium”) having a mesh size of approximately 100 nm which allows for the nearly unimpeded diffusion of nutrients, waste and small molecules (“the 3D cell culture medium comprises a network of perfusable pore space”) (Figure 6a and 6c; page 19, para. 0075). Sawyer teaches in Figure 1 an apparatus (100) for placing groups of cells in a 3D cell growth medium (120) comprising a container (110) holding the 3D cell growth medium and an injector (150) that displaces the 3D cell growth medium with a plurality of cells (160) (page 16, para. 0065). Sawyer teaches an example of a 3D cell growth medium comprising Carbopol particles (Lubrizol) that are mixed with and swell with any suitable liquid cell growth medium (page 11, para. 0043). Sawyer teaches a 3D cell growth medium with 0.9% Carbopol swollen particles dispersed in cell growth media and homogenized in a high-speed centrifugal mixer in which cells were placed in this medium using a 3D printing technique (page 11, para. 0044). Sawyer teaches packed granular microgels have recently been adopted as a robust medium for precise 3D fabrication of delicate materials (page 4, para. 0021). Sawyer teaches the hydrogel particles may swell with the liquid growth medium to form a granular gel material (page 5, para. 0025). Sawyer teaches a method for preparing a 3D cell growth medium (Figure 5; page 19, para. 0074). Sawyer does not teach “a perfusion-enabled bioreactor”.
Regarding “printing one or more extra-cellular matrix (ECM) structures with the printing device in the bioreactor before incubating” of step a of claim 1, Sawyer teaches one or more compounds may be deposited in conjunction with and/or adjacent to cells including structural proteins such as collagens and laminins (page 18, para. 0073). Sawyer teaches 3D printed co-cultures of HuH-7 hepatocytes and MS1 endothelial cells mixed with extracellular matrix precursor showed calcein staining for two weeks and exhibited physical integrity when manually transferred between culture dishes (page 20, para. 0076).
Regarding step b and “incubating” of step g of claim 1, Sawyer teaches once the cells are deposited, the medium containing the cells may be incubated which may alter its chemical properties and it turn modify the growth environment of the 3D cultures contained within (page 18, para. 0072). Sawyer teaches examples in which the cells in the 3D medium may be incubated in low oxygen or hypoxic environments (page 18, para. 0072). Sawyer teaches MDCK, MCF10A, MS1, HAEC, HuH-7, CTLL-2, mesenchymal stem cells, and osteosarcoma cells show about a 90% viability after 24 hours in LLS 3D culture medium (page 20, para. 0076).
Regarding step d of claim 1, Sawyer teaches serially passaging cultures of MCF10A cells into containers of fresh LLS growth media (page 20, para. 0076). Sawyer teaches manually transferring 3D printed co-cultures of cells between culture dishes (page 20, para. 0076). Sawyer teaches a 3D cell growth medium made from a yield stress material may allow for facile retrieval of cells from within the cell growth medium via a reversal of the steps used to deposit the cells (page 9, para. 0038). Sawyer teaches cells may be removed by moving a tip of a removal device such as a syringe or pipette to a location where a group of cells is disposed and applying suction to draw the cells from the cell growth medium (page 9, para. 0038). Sawyer teaches such an approach may be used as part of a test process in which multiple cell samples are deposited in 3D growth medium (page 9, para. 0038). Sawyer teaches those deposited cells may be cultured under the same conditions, but different ones of the samples may be exposed to different drugs or other treatments and one or more samples may be harvested at different times to test impact of the treatment conditions on the cells (page 9 – 10, para. 0038).
Regarding step e and step f of claim 1, Sawyer teaches serially passaging MCF10A cells into containers of fresh LLS growth media (page 20, para. 0076). Passaging MCF10A cells is accomplished by digesting cells with trypsin and diluting the cells and the recommended subcultivation ratio of 1:3 to 1:4 as evidenced by ATCC (page 3, last para.).
Regarding claim 3, Sawyer teaches in Figure 6a, placing cells in a flat sheet using a dispenser that moves in x, y, and z directions (page 19, para. 0075).
Regarding claim 9, Sawyer teaches a 3D cell growth medium comprising Carbopol particles such that when a stress is applied which exceeds the yield stress, the cell growth medium transforms to a liquid-like phase (page 11, para. 0043). Sawyer teaches the ability of LLS materials to gracefully and repeatedly transition between fluidized and solid states facilitates the harvesting of selected cell groups from specific locations in space (page 4, para. 0022).
Regarding claim 10, Sawyer teaches a yield stress material may have an elastic modulus on the order of 10 Pa (page 7, para. 0031). Sawyer teaches in Figure 7a a 3D cell culture medium with 0.9% Carbopol particles having a yield stress of 9 Pa (Figure 7a; page 11, para. 0044). Sawyer teaches soft a low polymer concentration and correspondingly large mesh-size gives LLS made from packed microgel particles swollen with liquid cell growth medium a yield stress of about 10 Pa (page 19, para. 0075). Sawyer teaches such a low yield stress limits the build-up of pressure during cell migration and division which may be a key contributor to long duration cell viability in 3D (page 19, para. 0075). Sawyer teaches the yield stress of the 3D culture medium may control the quality of 3D printed cell assemblies and the support for 3D cell culture (page 11, para. 0045).
Regarding claim 11, Sawyer teaches a particular liquid cell growth medium may be chosen depending on the types of cells which are to be placed within the 3D cell growth medium and a 3D cell growth medium may comprise 0.5% to 1% hydrogel particles by weight (page 5, para. 0023). Sawyer teaches the 3D cell growth medium comprising approximately 0.2% to about 0.7% Carbopol particles (page 11, para. 0043). Sawyer teaches another 3D cell growth medium with 0.9% Carbopol swollen particles dispersed in cell growth media and homogenized in a high-speed centrifugal mixer (page 11, para. 0044). Sawyer teaches a 1% (w/w) granular gel in liquid cell growth medium (page 19, para. 0075).
Regarding claims 12 and 13, Sawyer teaches the swollen particles have a characteristic size of about 1 µm to about 10 µm or about 5 µm in diameter (page 11, para. 0043; page 15, para. 0061page 17, para. 0067). Sawyer teaches another 3D cell growth medium with 0.9% Carbopol swollen particles dispersed in cell growth media and homogenized in a high-speed centrifugal mixer (page 11, para. 0044).
Regarding claim 15, Sawyer teaches MDCK, MCF10A, MS1, HAEC, HuH-7, CTLL-2, mesenchymal stem cells, and osteosarcoma cells show about a 90% viability after 24 hours in LLS 3D culture medium (page 20, para. 0076).
Regarding claim 16, Sawyer teaches incubating MCF10A cells for 24 hours and serially passaging the cells (page 20, para. 0076). MCF10A cells are incubated at 37 °C as evidenced by ATCC (page 3, para. 5).
Regarding claim 19, Sawyer teaches cells may be removed by moving a tip of a removal device such as a syringe or pipette to a location where a group of cells is disposed and applying suction to draw the cells from the cell growth medium (page 9, para. 0038).
Sawyer does not teach “a longest dimension extending from a bottom of the bioreactor to a top opening of the bioreactor” of step a or step g or step c and “perfusing” of step g of claim 1 or “perfusion-enabled bioreactor” of claim 1. However, Sawyer teaches the 2D environment of conventional cell cultures is often a poor substitute for the 3D environment experienced by cells in vivo as the behavior of a cell is often highly dependent on the microenvironment around the cell (page 1, para. 0004). Sawyer teaches current 3D culturing techniques are typically expensive and/or time consuming, and may be limited in the specific structures or geometries of tissues which may be grown and/or tested (page 2, para. 0005). Sawyer teaches the 3D growth medium may allow for cell growth environment which more closely mimics the complex in vivo growth environment and culturing cells in a 3D culture may facilitate cell-cell interactions and allow for easy placement and/or retrieval of groups of cells, which may enable rapid and/or high throughput testing which may reduce or eliminate the need for pre-clinical animal testing as part of new drug development (page 3, para. 0019). Sawyer teaches the gentle yielding and rapid solidification behavior of the LLS culture medium allows the unrestricted placement and retrieval of cells (page 4, para. 0022). Sawyer teaches since the cells are fixed in place in the 3D growth medium, they may be retrieved from the same location at a later time for assaying or testing by causing a phase change in the yield stress material and removing the cells (page 6, para. 0030).
Regarding “a longest dimension extending from a bottom of the bioreactor to a top opening of the bioreactor” of step a or step g of claim 1, Bhattacharjee teaches a method of 3D printing cells into a flat sheet (Figure 1a) with a printing device comprising a syringe pump stage with three linear translation stages for injecting cells into a 3D culture medium of 0.9% Carbopol with a yield stress of about 10 Pa where MCF10A cells are printed in a flat sheet with the longest dimension extending vertically where the vertical height is greater than the width as shown in Figure 5c (page 1788, right col. para. 1 – 2; page 1789, right col. last para.; page 1790, left col. last para.; Figure 1a and 5; Figure S3; page 1792, left col. para. 2). Bhattacharjee teaches when 3D printing MCF10A cells, hyaluronidase must be added to prevent cell aggregation within the injection syringe (page 1792, right col. para. 2). Bhattacharjee teaches printing cell structures with a thickness as small as a few cell diameters (page 1791, left col. para. 2; Figure 4). Bhattacharjee teaches it will be essential to investigate the integration of cells into continuously interconnected, tissue-like structures by studying the potential formation of cell-cell adhesions or indirect connections through shared extracellular matrix (page 1791, left col. last para. and right col. para. 1). Bhattacharjee teaches for cells requiring attachment through integrins or a stiffer microenvironment, natural or synthetic ECM can be mixed into the LLS medium, improving viability (page 1790, right col. para. 2; Figure S1). Bhattacharjee teaches the need is clear for screening and analysis methods using 3D cell culture (page 1793, right col. para. 1). Bhattacharjee teaches the use of LLS as a 3D culture medium allows the tools of 2D molecular and cell biology to be repurposed for 3D without technical barriers (page 1793, right col. para. 1). Bhattacharjee teaches the reversible fluid−solid transition of LLS facilitates the tasks of 2D culture such as seeding, feeding, and passaging to be performed by hand or in well-plates with high-throughput robotic pipetting and incubation systems, as well as fluorescence or luminescence assaying with plate-readers (page 1793, right col. para. 2). Bhattacharjee teaches cells can be 3D printed into arbitrarily complex structures or randomly dispersed directly into well plates or any culture vessel (page 1793, right col. para. 2). Bhattacharjee teaches transport and material properties can be tuned by varying the polymer concentration in the LLS. Imaging can be performed directly in the LLS or cells and cell assemblies at targeted locations can be harvested for analysis without disturbing surrounding material (page 1793, right col. para. 2). Bhattacharjee teaches an unbounded multitude of applications of LLS 3D culture can be envisioned, from screening for personalized cancer treatments to tissue engineering, to fundamental cell biology (page 1793, right col. para. 2). Bhattacharjee teaches uptake of dye molecules in the LLS (Figure 6). Bhattacharjee does not teach “perfusion-enabled bioreactor” or step c or “perfusing” of step g of claim 1. One would have been motivated to combine the teachings of Sawyer and Bhattacharjee where MCF10A cells are printed in a flat sheet with a longest dimension extending from a bottom of the bioreactor to a top opening of the bioreactor for easy removal of the cells without disturbing 3D printed neighboring cell sheets because both teach passaging of cells in the 3D culture medium and Sawyer teaches the method allows for easy placement and/or retrieval of groups of cells, which may enable rapid and/or high throughput testing and Bhattacharjee teaches the method facilitates the tasks of 2D culture such as seeding, feeding, and passaging and both teach the same method using the same 3D culture media.
Regarding “perfusion-enabled bioreactor” and step c and “perfusing” of step g of claim 1, Angelini-2018 teaches a perfusion bioreactor (“perfusion-enabled bioreactor”) where cells are 3D printed in a 3D cell culture medium within the bioreactor (page 16, lines 13 – 20; Figure 3A, 3B). Angelini-2018 teaches perfusion tubing (360) permits dispensing of one or more materials into the 3D cell growth material (304) and the materials that may be dispensed include a cell growth material (“perfusing” of step c and step g), pharmaceuticals, or other compounds (page 16, lines 15 – 20). Angelini-2018 teaches the materials may be dispensed at particular locations within the 3D cell growth medium, or form a concentration gradient (page 16, lines 21 – 25). Angelini-2018 teaches by forming a gradient, different cells may be exposed to different concentration of a material and following exposure, the cells may be inspected (within or outside of the 3D cell growth medium) to determine the impact of different concentrations of the materials on the cells (page 16, lines 25 – 28). Angelini-2018 teaches the pump (312) may be used to remove materials (byproduct of cellular activity, waste, protein) for the 3D cell growth medium for any suitable purpose (page 16, lines 32 – 33). Angelini-2018 teaches the pump may be used to draw materials through the 3D cell growth medium (page 17, lines 1 – 2). Angelini-2018 teaches a method of depositing cells into a granular gel, culturing the cells, exposing the cells to an agent, collecting a sample from the granular gel, and analyzing the sample to evaluate potential changes in the cells (page 2, lines 25 – 35; page 3). Angelini-2018 teaches an advantage of the method is that samples can be taken without disrupting the 3D geometry of the cells (page 3, lines 30 – 32).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Sawyer regarding a method of 3D printing a flat sheet of cells in a granular gel 3D culture medium contained in a bioreactor where the cells are printed with ECM and passaging the printed cells with the teachings of Bhattacharjee regarding a method of 3D printing a flat sheet of cells in a granular gel 3D culture medium with a longest dimension extending from a bottom of the bioreactor to a top opening of the bioreactor and passaging the cells with the teachings of Angelini-2018 regarding a method of 3D printing cells in a granular gel 3D culture medium in a perfusion bioreactor to arrive at the claimed method comprising: a) printing a composition comprising a plurality of cells into a flat sheet into a bioreactor with a printing device, wherein the bioreactor, wherein the bioreactor is a perfusion-enabled bioreactor and wherein the bioreactor comprises a 3D cell culture medium comprising a plurality of packed hydrogel particles and a liquid cell culture medium, wherein the packed hydrogel particles are swelled with the liquid cell culture medium to form a granular gel and wherein the 3D cell culture medium comprises a network of perfusable pore
space; wherein printing the flat sheet further comprises printing one or more extra-cellular matrix (ECM) structures with the printing device in the bioreactor before incubating; wherein the flat sheet in which the one or more ECM structures are printed has a longest dimension extending from a bottom of the bioreactor to a top opening of the bioreactor; b). incubating the flat sheet comprising the plurality of printed cells in the bioreactor; c) perfusing the plurality of printed cells with the liquid cell culture medium to feed the plurality of printed cells; d) collecting the incubated plurality of printed cells; e) digesting the collected plurality of printed cells to produce a digested plurality of cells; f) splitting the digested plurality of cells to produce a diluted plurality of cells; and g) re-printing, incubating, and perfusing the diluted plurality of cells in a second flat sheet according to steps a)-c) to expand cell populations, wherein expanding cell populations results in at least a doubling of a number of cells compared to a starting flat sheet, wherein the second flat sheet has a longest dimension extending from a bottom of the bioreactor to a top opening of the bioreactor. One would have been motivated to combine the teachings of Sawyer, Bhattacharjee, and Angelini-2018 in a method of 3D culture of cells in a 3D culture medium with a granular gel to test the effect of agents on cell growth as Bhattacharjee teaches the need is clear for screening and analysis methods using 3D cell culture and Sawyer teaches the 3D growth medium may allow for cell growth environment which more closely mimics the complex in vivo growth environment and culturing cells in a 3D culture may facilitate cell-cell interactions and allow for easy placement and/or retrieval of groups of cells, which may enable rapid and/or high throughput testing which may reduce or eliminate the need for pre-clinical animal testing as part of new drug development and Bhattacharjee teaches an unbounded multitude of applications of LLS 3D culture can be envisioned, from screening for personalized cancer treatments to tissue engineering, to fundamental cell biology. One would have a reasonable expectation of success in combining the teachings as Sawyer teaches since the cells are fixed in place in the 3D growth medium, they may be retrieved from the same location at a later time for assaying or testing and Bhattacharjee teaches uptake of dye molecules by the cells in the 3D culture medium and Angelini-2018 teaches perfusion tubing permits dispensing of one or more materials into the 3D cell growth material and the materials that may be dispensed include a cell growth material, pharmaceuticals, or other compounds and Angelini-2018 teaches an advantage of the method is that samples can be taken without disrupting the 3D geometry of the cells.
8. Claim(s) 2 is/are rejected under 35 U.S.C. 103 as being unpatentable over Sawyer (WO2016182969A1; Filed 05/07/2016; Published 11/17/2016), hereinafter Sawyer which is cited on the IDS filed 12/22/2021 as evidenced by ATCC (ATCC: (08/16/2021). "MCF 10A." https://www.atcc.org/products/crl-10317. Accessed 05/27/2026.), hereinafter ATCC in view of Bhattacharjee (Bhattacharjee, Tapomoy, et al. ACS Biomaterials Science & Engineering 2.10 (2016): 1787-1795.), hereinafter Bhattacharjee which is cited on the IDS filed 05/17/2022 in view of Angelini-2018 (WO-2018085823-A1;Filed 11/07/2017, Published 05/11/2018; previously cited), hereinafter Angelini-2018 as applied to claims 1, 3, 9 – 13, 15, 16, and 19 above, and further in view of Brennan (Brennan, Martin D., et. al. PLoS One 10.9 (2015): e0137631; previously cited), hereinafter Brennan.
Sawyer as evidenced by ATCC and in view of Bhattacharjee and Angelini-2018 make obvious the limitations of claim 1 as set forth above. Sawyer, ATCC, Bhattacharjee, and Angelini-2018 do not teach the perfusion bioreactor comprises “a well-plate perfusion chamber insert” of claim 2. However, Sawyer teaches examples in which the cells in the 3D medium may be incubated in low oxygen or hypoxic environments (page 18, para. 0072). Angelini-2018 teaches cells in the medium may be incubated in low oxygen or hypoxic environments (page 15, lines 14 – 18). Angelini-2018 teaches atmospheric gas exchange can be evaluated to determine positive and negative effects on microtissues (page 3, lines 15 – 20; page 5, lines 32 – 36).
Regarding “well-plate perfusion chamber insert” of claim 2, Brennan teaches a device that nests into a 24-well culture plate to control gas in each row of the plate (page 1, paragraph 1; Figure 1). Brennan teaches perfusion of gas through the insert and culture of cells with the insert at various percentages of oxygen (page 4, paragraph 2- 5; page 5, last paragraph). Brennan teaches the device effectively controls oxygen as determined by changes in expression of VEGFA relative to different oxygen concentrations (page 6, last paragraph; Figure 3). Brennan teaches oxygen control in cell studies is often overlooked by researchers but is important for mimicking conditions experienced by cells in vivo (page 2, paragraph 3). Brennan teaches studying cancer cells under controlled hypoxic conditions is important in understanding the pathophysiology because research has shown hypoxia may enhance aggressive phenotypes, tumor progression, metastasis, and resistance to therapy and to better study the role of oxygen levels in cancer gene expression, a gas controlled culture system is required (page 2, paragraph 3).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Sawyer regarding a method of 3D printing a flat sheet of cells in a granular gel 3D culture medium contained in a bioreactor where the cells are printed with ECM and passaging the printed cells with the teachings of Bhattacharjee regarding a method of 3D printing a flat sheet of cells in a granular gel 3D culture medium with a longest dimension extending from a bottom of the bioreactor to a top opening of the bioreactor and passaging the cells with the teachings of Angelini-2018 regarding a method of 3D printing cells in a granular gel 3D culture medium in a perfusion bioreactor with the teachings of Brennan regarding perfusion of gas through a culture plate insert and culture of cells with the insert at various percentages of oxygen to arrive at the claimed method wherein the perfusion-enabled bioreactor comprises a well-plate perfusion chamber insert. One would have been motivated to combine the teachings of Sawyer, Bhattacharjee, Angelini-2018, and Brennan in a method of 3D culture of cells in a 3D culture medium with a granular gel to test the effect of different oxygen levels on cell growth as Bhattacharjee teaches the need is clear for screening and analysis methods using 3D cell culture and Bhattacharjee teaches an unbounded multitude of applications of LLS 3D culture can be envisioned, from screening for personalized cancer treatments to tissue engineering, to fundamental cell biology and Brennan teaches oxygen control in cell studies is often overlooked by researchers but is important for mimicking conditions experienced by cells in vivo and Brennan teaches studying cancer cells under controlled hypoxic conditions is important in understanding the pathophysiology because research has shown hypoxia may enhance aggressive phenotypes, tumor progression, metastasis, and resistance to therapy and to better study the role of oxygen levels in cancer gene expression, a gas controlled culture system is required. One would have a reasonable expectation of success in combining the teachings as Brennan teaches the device effectively controls oxygen as determined by changes in expression of VEGFA relative to different oxygen concentrations and Sawyer and Angelini-2018 the cells in the 3D medium may be incubated in low oxygen or hypoxic environments and Angelini-2018 teaches atmospheric gas exchange can be evaluated to determine positive and negative effects on microtissues.
Applicant’s Arguments/ Response to Arguments
9. Applicant Argues: Applicant asserts that a flat sheet is by definition a two dimensional structure.
Response to Arguments: It is unclear how a flat sheet of cells is a two-dimensional structure because cells are three-dimensional. Applicant’s definition of “sheet” is a composition comprising a plurality of printed cells into a three-dimensional structure having a width less than the length or height at page 11 – 12).
Applicant Argues: Applicant asserts that a desired outcome of claim 1 is expansion of cell populations from flat vertical sheets and there would be no cell differentiation in the present claims.
Response to Arguments: The previous rejection of claims 1, 3, 9 – 13, 15, 16, and 19 using the teachings of Angelini, Hakimi, Kolesky, and Takahashi have been withdrawn and therefore arguments addressing the cited teachings are moot. The amendment to claim 1 reciting “wherein expanding cell populations results in at least a doubling of a number of cells compared to a starting flat sheet” is interpreted as an intended result and not given patentable weight. Further, claim 1 does not recite the number of cells printed, collected, or an active step of culturing the second flat sheet or any limitations regarding the culturing conditions that yield the desired outcome (number of cells that are harvested, the dilution factor, or the number of cells re-printed). The claims do not limit the type of cells printed and do not limit the culturing conditions.
Applicant Argues: Applicant asserts that Derda is not directed to expansion of cell populations and thus cultures the tissues in a different way and are not an equivalent material to the vertically oriented flat sheets and using them in the apparatus of Angelini-2018 will not result in the presently claimed kits.
Response to Arguments: Claim 28 has been amended to recite an intended use of the kit and this is not given patentable weight because it is an intended use. Claims 28, 31, and 32 are drawn to a kit and not a method and therefore the kit may be used to culture cells in any way.
Conclusion
No claims allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZANNA M BEHARRY whose telephone number is (571)270-0411. The examiner can normally be reached Monday - Friday 8:45 am - 5:45 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Peter Paras can be reached at (571)272-4517. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/ZANNA MARIA BEHARRY/Examiner, Art Unit 1632
Prosecution Insights