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 .
DETAILED ACTION
The claims filed on February 12, 2026, have been acknowledged. Claims 3, 5, and 21 were cancelled. Claims 1, 15, and 22-23 were amended. Claims 1-2, 4, 6-20, and 22-24 are pending and examined on the merits.
Rejections and/or objections not reiterated from the previous office action mailed October 21, 2025, are hereby withdrawn. The following rejections and/or objections are either newly applied or are reiterated and are the only rejections and/or objections presently applied to the instant application.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Priority
The applicant claims domestic priority from U.S. provisional application No. 62/183273, filed on June 23, 2015. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Claims 1-2, 4, 6-20, and 22-24 receive domestic benefit from U.S. provisional application No. 62/183273, filed on June 23, 2015.
Information Disclosure Statement
The information disclosure statement (IDS) filed on November 30, 2025, has been considered.
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.
Claims 1-2, 4, 6-10, and 12-19 are rejected under 35 U.S.C. 103 as being unpatentable over Ballotta et al. (Biomaterials 35: 9100-9113. 2014), Zhao et al. (Biotechnology and Bioengineering 96: 584-595. 2007), Ahsan et al. (Tissue Engineering Part A 16: 3547–3553, 2010), United States Patent Application No. 20140120608 (Carter), and United States Patent Application No. 20080032380 (Kleis),as evidenced by Wolfe et al. (Biotechnology and Bioengineering 110: 1231-1242. 2013) and SensorsOne (Dyne per Square Centimetre Pressure Unit). This is a new rejection made in response to Applicant’s amendments to claim 1 that is substantially similar to a previous rejection of record. Any aspect of Applicant’s traversal that is relevant to the rejection as newly written is addressed below.
Regarding claims 1 and 17-18, Ballotta teaches a method of culturing stem cells on a substrate while applying a shear stress force.
Scaffolds were cut to size from the electrospun sheets and sterilized using a 70% ethanol solution. Cells were seeded using fibrin gel as a carrier. BMSC [bone marrow-derived mesenchymal stromal cells (i.e. mesenchymal stem cells)] and ADSC [adipose-derived mesenchymal stromal cells ] were used at passage 5 at a concentration of 8x103 cells/ml. PBMC [peripheral blood mononuclear cells] were seeded at a concentration of 1x105 cells/ml. The cells were suspended in fibrinogen (10 mg/ml), after which the suspension was mixed with thrombin (10 IU/ml) and immediately seeded into the PCL-U4U scaffolds. Rectangular strips (10 x 15 mm2) of the preseeded scaffolds were mounted into a parallel-plate flow chamber, which is a type of bioreactor. Specifically, Applicant’s specification provides no special definition of a “bioreactor”, and Wolfe evidences that parallel-plate flow chambers are a type of a bioreactor (page 1232, column 2, paragraph 4). Ballotta teaches PBMC were suspended in complete RPMI medium (5x106 cells/ml) and added to the syringes of a Fluidic Unit with a total volume of 10 ml per sample. The cell suspension was driven along the scaffold in a pulsatile flow using a modified pressure pump. Flow conditions were set at a frequency of 1 Hz, with peak shear stress and peak pressure of approximately 1.6 Pa and 100 mm Hg, respectively, resembling typical hemodynamic conditions of human small-diameter arteries. Samples of the remaining cell suspension were taken during flow via in-line injection ports on the outlet port of the flow chambers (at 25 min, 1 h and 4 h) (page 9102, column 1, paragraph 3 and column 2, paragraph 1). Figure 9 depicts a heatmap of protein secretion by PBMC (PB), BMSC (BM), ADSC (AD)-seeded or unseeded (fibrin) PCL-U4U scaffolds exposed to circulating PBMC in shear flow, after 1 and 4 h of incubation. Release of inflammatory and immunomodulatory species in MSC-seeded groups was enhanced in flow conditions with respect to the static control and the total amount of proteins was comparable to the unseeded group. For example, IL-10 expression increased from 0 pg/mL in the BMSC and ADSC groups in static conditions to ~500 pg/mL in shar stress conditions (Figures 9-10).
Ballota does not teach the structure of the parallel plate flow chamber.
However, Zhao teaches a closed loop perfusion bioreactor system for applying shear stress to stem cells (abstract). Figure 1 of Zhao teaches that their perfusion system had 4 different perfusion chambers and each perfusion chamber comprised two fluid inlets, two fluid outlets, two sides, two ends, a first conduit, and a second conduit, a media reservoir, and three peristaltic pumps. The reservoir is connected to the first fluid inlet by a conduit and the second fluid outlet connects to the reservoir via a separate conduit. The whole system, except for the pumps, was placed in a 37°C incubator (a base) to maintain a constant operating temperature. Two chambers were exposed to the media flow at 0.1 mL/min, and another two at 1.5 mL/min (page 585, column 2, paragraph 2-page 586, column 1, paragraph 1). Zhao teaches that their study utilized the advantages afforded by the modular design of the perfusion bioreactor system in which multiple cellular samples over an extended period were obtained from the same initial cell population and under the same seeding and culturing conditions (page 591, column 2, paragraph 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have chosen the closed loop parallel plate bioreactor of Zhao to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to choose the parallel plate bioreactor of Zhao with a reasonable expectation of success because Ballotta specifically contemplates performing their method in a parallel plate bioreactor and Zhao uses their closed loop parallel plate bioreactor to stimulate stem cells with shear stress. Therefore, Ballota and Zhao both use the parallel plate bioreactor to stimulate stem cells (MSCs in Ballota and Zhao) attached to a substrate (PCL-U4U scaffolds in Ballota and PET matrices in Zhao). Furthermore, Zhao teaches that their study utilized the advantages afforded by the modular design of the perfusion bioreactor system in which multiple cellular samples over an extended period were obtained from the same initial cell population and under the same seeding and culturing conditions. As such, it would have been obvious that one could use the modular system of Zhao with multiple chambers as this would allow for multiple experiments to be run simultaneously while reducing the amount of confounding variables. As such, it would have been obvious that the parallel plate bioreactor of Zhao could be used by Ballotta. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Zhao does not teach wherein their modular perfusion system comprises a base plate nor wherein there are intermediate plates with a distribution channel and gathering channel.
However, Ahsan teaches a closed system recirculating parallel plate bioreactor that comprises an aluminum frame (a base plate), a cell preparation apparatus that comprises a flow block (an inlet fluid feed plate), a rubber gasket, a tubing that connects the medium reservoir with the flow block (a conduit), a spacer and a glass slide placed between the flow block and the rubber gasket, a reservoir, and a peristaltic pump. The flow block comprises a first fluid inlet and a first fluid outlet. Ahsan teaches that they used their parallel plate reactor to apply shear stress to early differentiating embryonic stem cells attached to a glass slide (abstract and page 3548, column 1, paragraph 3-column 2, paragraph 2 and Figure 1).
Carter teaches a multilayered cell culture vessel comprising a stacked formation of multiple cell culture insert layers for culturing cells in each of the individual chambers wherein the vessel comprises a base (a base plate), multiple insert layers, and a lid that are assembled to form a leak proof, closed vessel for the culturing of cells (whole document).
Kleis teaches a bioreactor for culturing cells in a laminar flow configuration wherein there are at least two laminar flow zones. The bioreactor is ideally suited to studying short, moderate and long term studies of cell cultures and the response of cell cultures to stressors such as shear stress (abstract, Figures 1-2 and paragraphs 0073, 0087, and 0134). Kleis teaches that for mechanotaxis studies, the method can include the step of adjusting a shear level or shear gradient using different flow rates for different inlets (paragraph 0134). Kleis teaches that the volume 284 includes four laminar flow zones 286. Each zone 286 includes an inlet 288 having a diffuser 290, where the diffuser 290 is adapted to spread a flow of fluid as it enters its corresponding Zone 286. Each zone 286 also includes an outlet 292 having a collector 294, where the collector 294 is adapted to reduce flow constrictions of fluid as it exits its corresponding zone 286 (Figure 2F and paragraph 0125).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of the closed loop parallel plate device of Ballota and Zhao by stacking the four perfusion chambers of Zhao, as identified by Ahsan, Carter, and Kleis, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because the bioreactors of Zhao, Ahsan, and Kleis involve applying shear stress to cells. Furthermore, Ahsan successfully reduces to practice that a parallel plate bioreactor can be coupled to a base with other components between the inlet fluid feed plate and the base plate, Carter successfully reduces to practice that multiple cell culture inserts can be stacked as part of a larger cell culture vessel connected to a base for culturing cells in each of the individual chambers, and Kleis successfully reduces to practice a bioreactor for examining cell response to shear stress wherein there are multiple laminar flow zones with their own inlets and outlets for controlling shear gradient using different flow rates for different inlets. Therefore, it would have been obvious that the perfusion chambers of Zhao could be assembled as a stacked cell culture vessel wherein each of the perfusion chambers maintain their own inlets and outlets that can maintain the different shear stress levels of Zhao, as shown by Kleis. This would result in a cell culture vessel with an inlet fluid feed plate (the top stacked perfusion chamber), a base plate (the bottom stacked perfusion chamber) and a plurality of intermediate plates (the other two stacked perfusion chambers). Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Furthermore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the four perfusion chambers of Zhao to include a diffuser and a collector for each inlet and outlet, respectively, as identified by Kleis, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because the bioreactors of Zhao and Kleis involve applying shear stress to cells. Furthermore, Kleis teaches the diffuser is adapted to spread a flow of fluid as it enters its corresponding zone, providing a more even distribution of fluid flow rates and the collector is adapted to reduce flow constrictions of fluid as it exits its corresponding zone. Therefore, it would have been obvious to include a diffuser (a distribution channel) and collector (a gathering channel) to more evenly distribute the fluid flow rate for the samples and limit constriction when the fluid exits. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding the distribution channel being proximal to the first end and the gathering channel proximal to the second end, applicant does not provide a definition for proximal. Therefore, under the broadest reasonable interpretation, proximal is considered to mean situated close to and as can be seen in Figure 2E and 2F of Kleis, the diffuser and collector are proximal to the first end and second end, respectively. Furthermore, as can be seen in Figures 2E and 2F, the diffuser and collector extend between the first side and second side of the laminar flow zone.
Regarding the allowing cell adhesion in a monolayer limitation, Ahsan teaches that they seeded their embryonic stem cells on glass slides coated with collagen type IV and allowed the cells to attach for 48 hours before placing the glass slide seeded with ESCs in the parallel plate apparatus (page 3548, column 1, paragraph 3-column 2, paragraph 2). Ahsan teaches that the cells were seeded in a monolayer (Figure 1). Ahsan teaches that the seeded cells had altered protein expression under shear stress conditions (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the PCL-U4U scaffolds of Ballota with the collagen IV coated glass slides of Ahsan to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to substitute with a reasonable expectation of success because Ballota, Zhao, and Ahsan are interested in stimulating stem cells (MSCs in Ballota and Zhao and ESCs in Ahsan) under shear stress in a parallel plate bioreactor and both resulted in changes in protein expression, it would have been obvious that one could have used glass coated with collagen IV as the substrate for attachment rather than the PCL-U4U scaffolds of Ballota as it is a known alternative for cell attachment for use in a parallel plate bioreactor. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 2, the specification does not define the term automated. The fact that Ballotta teaches that the flow conditions were set at a frequency of 1 Hz, with peak shear stress and peak pressure of approximately 1.6 Pa and 100 mm Hg shows that there is automation within their mesofluidics setup and anticipates the instant claim (page 9102, column 2, paragraph 1).
Regarding claim 4, Figure 1 of Ballotta clearly shows that the growth medium passes over the stem cells (Figure 1).
Regarding claims 6-9, Ballotta teaches that the flow conditions were set at a frequency of 1 Hz, with peak shear stress and peak pressure of approximately 1.6 Pa and 100 mm Hg (page 9102, column 2, paragraph 1). SensorsOne teaches that 1 Dyne per SquareCentimetre equals 0.1 pascals. As such, Ballotta teaches that their shear stress was 16 Dynes per square centimeter.
Regarding claim 10, Ballotta teaches that the BDSC and ADSC cells were from humans (abstract and page 9101, column 2, paragraph 2).
Regarding claim 12, Ballotta teaches that cells were seeded using fibrin gel as a carrier. BMSC [bone marrow-derived mesenchymal stromal cells (i.e. mesenchymal stem cells)] and ADSC [adipose-derived mesenchymal stromal cells] were used at passage 5 at a concentration of 8x103 cells/ml (page 9102, column 1, paragraph 3).
Regarding claims 13-14 and 16, Ballotta teaches that they used BMSC [bone marrow-derived mesenchymal stromal cells (i.e. mesenchymal stem cells)] and ADSC [adipose-derived mesenchymal stromal cells]. As such, these cells were isolated from bone marrow or adipose tissue. Specifically, in regard to the cells being autologous, it must be noted that this limitation is directed to the intended use of the cells and does not necessary change the structure of the cells themselves. Nevertheless, Ballotta teaches the BMCS were derived from human donor in a hospital, and thus could have been used in an autologous transplant.
Regarding claim 15, Figure 9 of Ballotta depicts a heatmap of protein secretion by PBMC (PB), BMSC (BM), ADSC (AD)-seeded or unseeded (fibrin) PCL-U4U scaffolds exposed to circulating PBMC in shear flow, after 1 and 4 h of incubation. Release of inflammatory and immunomodulatory species in MSC-seeded groups was enhanced in flow conditions with respect to the static control and the total amount of proteins was comparable to the unseeded group. For example, IL-10 (an anti-inflammatory gene) expression increased from 0 pg/mL in the BMSC and ADSC groups in static conditions to ~500 pg/mL in shear stress conditions (Figures 9-10).
In regard to claim 19, as stated supra, cells in shear stress conditions secrete factors such as TNF alpha (Figures 9-10). Thus, the growth media would comprise TNF alpha.
Response to Arguments
Applicant's arguments filed February 12, 2026, are acknowledged.
Applicant argues that Ballotta and Zhao are related to the application of shear stress to MSCs cultured in three dimensions rather than in a two-dimensional setting (e.g., as a cellular monolayer), as is presently claimed. Accordingly, Applicants submit that neither Ballotta nor Zhao discloses or suggests (1) a method of producing a conditioned composition (as defined in the instant specification, at paragraph 0011, a population of conditioned pluripotent cells or a media, e.g., a cell-free media comprising secreted factors from pluripotent cells that have been subjected to a controlled sheer stress; (2) the culture of the cells as a monolayer in a bioreactor, (3) nor the bioreactor as claimed in claim 1. That is, Applicants submit that one of skill in the art would not rely on Ballotta nor Zhao when attempting to develop a method for producing a condition composition from cell cultures as a monolayer. Furthermore, Applicant argues that none of Ahsan, Carter, Kleis and Cytomat teach items (1)-(3) as described above. That is at least because one of skill in the art would find no motivation, guidance or otherwise suggestion in the cited references to construct a bioreactor comprising an inlet fluid feed plate comprising a first fluid inlet, a first fluid outlet, a second fluid inlet and a second fluid outlet; where the reservoir is coupled to the first fluid inlet via the first conduit; the reservoir is coupled to the second fluid outlet via the second conduit, as is presently claimed. (page 8, paragraph 3-page 11, paragraph 1 and page 12, paragraphs 2-3)
Applicant’s arguments have been fully considered but they are not persuasive.
As an initial matter, in response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Regarding whether the combined teachings of Ballotta, Zhao, Ahsan, Carter, and Kleis teach a conditioned composition, Applicant’s identified “definition” for a conditioned population in the arguments is not a controlling definition and is not considered limiting as the recited section states “In further aspects, the conditioned composition is a media (e.g. a cell-free media) comprising secreted factors from pluripotent cells that have been subjected to a controlled sheer stress”. As the statement is prefaced with in further aspects, this is not considered a controlling and is just considered a potential embodiment. Paragraph 0011 of the specification does state that “a conditioned composition refers to a composition that has been subjected to the conditioning effects of mechanical forces”. This is considered the controlling definition for a conditioned composition. As Ballotta, Zhao, Ahsan, and Kleis all apply shear stress (a mechanical force) to cells, this would lead to a conditioned composition.
Regarding whether the combined teachings of Ballotta, Zhao, Ahsan, Carter, and Kleis teach the culture of the cells as a monolayer in a bioreactor, as stated in the rejection above, Ahsan teaches that they seeded their embryonic stem cells on glass slides coated with collagen type IV and allowed the cells to attach for 48 hours before placing the glass slide seeded with ESCs in the parallel plate apparatus (page 3548, column 1, paragraph 3-column 2, paragraph 2). Ahsan teaches that the cells were seeded in a monolayer (Figure 1). Ahsan teaches that the seeded cells had altered protein expression under shear stress conditions (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the PCL-U4U scaffolds of Ballota with the collagen IV coated glass slides of Ahsan to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to substitute with a reasonable expectation of success because Ballota, Zhao, and Ahsan are interested in stimulating stem cells (MSCs in Ballota and Zhao and ESCs in Ahsan) under shear stress in a parallel plate bioreactor and both resulted in changes in protein expression, it would have been obvious that one could have used glass coated with collagen IV as the substrate for attachment rather than the PCL-U4U scaffolds of Ballota as it is a known alternative for cell attachment for use in a parallel plate bioreactor. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Therefore, there is motivation for substituting the scaffolds of Ballota with the glass substrate of Ahsan for adherence of the cells in a monolayer before applying shear stress in the parallel plate bioreactor.
Regarding whether the combined teachings of Ballotta, Zhao, Ahsan, Carter, and Kleis teach the bioreactor as claimed in claim 1, this has been discussed in the rejection above and will not be repeated here. When considering the rejection above, the combined teachings of Ballotta, Zhao, Ahsan, Carter, and Kleis do teach all of the limitations of the claimed bioreactor. Therefore, Applicant’s arguments are considered moot.
Claims 1-2, 4, 10, and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Obi (Am J Physiol Cell Physiol 303: C595–C606, 2012) and further in view of Zhao et al. (Biotechnology and Bioengineering 96: 584-595. 2007), Wolfe et al. (Biotechnology and Bioengineering 110: 1231-1242. 2013), Ahsan et al. (Tissue Engineering Part A 16: 3547–3553, 2010), United States Patent Application No. 20140120608 (Carter), and United States Patent Application No. 20080032380 (Kleis). This is a new rejection made in response to Applicant’s amendments to claim 1 that is substantially similar to a previous rejection of record. Applicant’s traversal has been addressed above.
Obi teaches a method of culturing CD133+ (a known stem cell marker) endothelial progenitor cells in shear stress. Human CD133-positive cells were prepared from freshly obtained human umbilical cord blood samples. CD133-positive cells (3x105) were cultured at 37°C under a 5% CO2 atmosphere in Stem Span media with 50 ng/ml VEGF, 20 ng/ml interleukin-6, 100 ng/ml stem cell factor, 20 ng/ml thrombopoietin, 100 ng/ml Fms-related tyrosine kinase 3 ligand, and 1% penicillin-streptomycin in a suspended manner for 7 days. Ex vivo expanded EPCs keep approximately half of the characteristics of freshly isolated EPCs. To investigate the effect of shear stress ex vivo, expanded EPCs were applied on culture dishes coated with 100 μg/ml solution of human fibronectin (i.e. a substrate that would allow cell adhesion in a monolayer) and cultured in M199 medium with 5% fetal bovine serum (FBS), EGM2 (VEGF, fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor-1, and ascorbic acid), and 10% dextran for 1 or 2 days. Ex vivo expanded EPCs were exposed to laminar shear stress with a rotating-disk-type flow-loading device for 1 or 2 days. A dish containing cultured EPCs was placed on the stage of the device, and a stainless steel disk was placed in the dish. The rotation of the disk caused the medium to flow in concentric circles, thereby exerting laminar shear stress on the cells (page C596, column 1, paragraph 3-column 2, paragraph 1). The EPC colony-forming assay revealed that shear stress induced differentiation of circulating colony-forming EPCs. Moreover, shear stress increased adhesion and expression levels of endothelial markers through the PI3K/Akt/mTOR pathway. Furthermore, shear stress activates the VEGF-R2 phosphorylation in a ligand-independent manner. Applicant teaches that a conditioned composition refers to a composition that has been subjected to the conditioning effects of mechanical forces (paragraph 0011). As such, the method of Obi produces a conditioned composition.
Obi teaches that their rotating-disk device generates a gradient of shear stress that is dependent on the distance from the center of the dish, and the shear stress applied varied across regions of the dish at various radii, thus the results of the assays represent averaged cell responses to different levels of shear stress within the above range (C596, column 2, paragraph 1).
Obi does not teach wherein the shear stress is applied in a bioreactor.
However, Zhao teaches a closed loop perfusion bioreactor system for applying shear stress to stem cells (abstract). Figure 1 of Zhao teaches that their perfusion system had 4 different perfusion chambers and each perfusion chamber comprised two fluid inlets, two fluid outlets, two sides, two ends, a first conduit, and a second conduit, a media reservoir, and three peristaltic pumps. The reservoir is connected to the first fluid inlet by a conduit and the second fluid outlet connects to the reservoir via a separate conduit. The whole system, except for the pumps, was placed in a 37°C incubator (a base) to maintain a constant operating temperature. Two chambers were exposed to the media flow at 0.1 mL/min, and another two at 1.5 mL/min (page 585, column 2, paragraph 2-page 586, column 1, paragraph 1). Zhao teaches that their study utilized the advantages afforded by the modular design of the perfusion bioreactor system in which multiple cellular samples over an extended period were obtained from the same initial cell population and under the same seeding and culturing conditions (page 591, column 2, paragraph 1).
Wolfe teaches that shear stress effects are often difficult to interpret in scalable systems due to complex patterns of fluid flow. Instead, use of simpler configurations, which allow application of a well-defined shear stress, can help decipher the effect of shear stress parameters. For example, the parallel plate system, which applies a uniform shear stress to cells attached to a surface, is a suitable surrogate to study the effect of shear stress magnitude and protein substrate for adherent cell culture (page 1232, column 1, paragraph 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the rotating-disk-type flow-loading shear stress device of Obi with the parallel plate bioreactor of Zhao to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to substitute with a reasonable expectation of success because both devices are used to generate shear stress on cells and Wolfe teaches that simpler configurations that apply uniform shear stress, such as the parallel plate bioreactor, can help decipher the effect of shear stress on cells. As such, it would have been obvious to substitute the devices as the parallel plate provides a uniform shear stress to all cells and can provide more accurate data for analysis. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Zhao does not teach wherein their modular perfusion system comprises a base plate nor wherein there are intermediate plates with a distribution channel and gathering channel.
However, Ahsan teaches a closed system recirculating parallel plate bioreactor that comprises an aluminum frame (a base plate), a cell preparation apparatus that comprises a flow block (an inlet fluid feed plate), a rubber gasket, a tubing that connects the medium reservoir with the flow block (a conduit), a spacer and a glass slide placed between the flow block and the rubber gasket, a reservoir, and a peristaltic pump. The flow block comprises a first fluid inlet and a first fluid outlet. Ahsan teaches that they used their parallel plate reactor to apply shear stress to early differentiating embryonic stem cells attached to a glass slide (abstract and page 3548, column 1, paragraph 3-column 2, paragraph 2 and Figure 1).
Carter teaches a multilayered cell culture vessel comprising a stacked formation of multiple cell culture insert layers for culturing cells in each of the individual chambers wherein the vessel comprises a base (a base plate), multiple insert layers, and a lid that are assembled to form a leak proof, closed vessel for the culturing of cells (whole document).
Kleis teaches a bioreactor for culturing cells in a laminar flow configuration wherein there are at least two laminar flow zones. The bioreactor is ideally suited to studying short, moderate and long term studies of cell cultures and the response of cell cultures to stressors such as shear stress (abstract, Figures 1-2 and paragraphs 0073, 0087, and 0134). Kleis teaches that for mechanotaxis studies, the method can include the step of adjusting a shear level or shear gradient using different flow rates for different inlets (paragraph 0134). Kleis teaches that the volume 284 includes four laminar flow zones 286. Each zone 286 includes an inlet 288 having a diffuser 290, where the diffuser 290 is adapted to spread a flow of fluid as it enters its corresponding Zone 286. Each zone 286 also includes an outlet 292 having a collector 294, where the collector 294 is adapted to reduce flow constrictions of fluid as it exits its corresponding zone 286 (Figure 2F and paragraph 0125).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of the closed loop parallel plate device of Obi and Zhao by stacking the four perfusion chambers of Zhao, as identified by Ahsan, Carter, and Kleis, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because the bioreactors of Zhao, Ahsan, and Kleis involve applying shear stress to cells. Furthermore, Ahsan successfully reduces to practice that a parallel plate bioreactor can be coupled to a base with other components between the inlet fluid feed plate and the base plate, Carter successfully reduces to practice that multiple cell culture inserts can be stacked as part of a larger cell culture vessel connected to a base for culturing cells in each of the individual chambers, and Kleis successfully reduces to practice a bioreactor for examining cell response to shear stress wherein there are multiple laminar flow zones with their own inlets and outlets for controlling shear gradient using different flow rates for different inlets. Therefore, it would have been obvious that the perfusion chambers of Zhao could be assembled as a stacked cell culture vessel wherein each of the perfusion chambers maintain their own inlets and outlets that can maintain the different shear stress levels of Zhao, as shown by Kleis. This would result in a cell culture vessel with an inlet fluid feed plate (the top stacked perfusion chamber), a base plate (the bottom stacked perfusion chamber) and a plurality of intermediate plates (the other two stacked perfusion chambers). Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Furthermore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the four perfusion chambers of Zhao to include a diffuser and a collector for each inlet and outlet, respectively, as identified by Kleis, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because the bioreactors of Zhao and Kleis involve applying shear stress to cells. Furthermore, Kleis teaches the diffuser is adapted to spread a flow of fluid as it enters its corresponding zone, providing a more even distribution of fluid flow rates and the collector is adapted to reduce flow constrictions of fluid as it exits its corresponding zone. Therefore, it would have been obvious to include a diffuser (a distribution channel) and collector (a gathering channel) to more evenly distribute the fluid flow rate for the samples and limit constriction when the fluid exits. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding the distribution channel being proximal to the first end and the gathering channel proximal to the second end, applicant does not provide a definition for proximal. Therefore, under the broadest reasonable interpretation, proximal is considered to mean situated close to and as can be seen in Figure 2E and 2F of Kleis, the diffuser and collector are proximal to the first end and second end, respectively. Furthermore, as can be seen in Figures 2E and 2F, the diffuser and collector extend between the first side and second side of the laminar flow zone.
Regarding claim 2, Zhao, as stated supra, teaches that the parallel plate provides a steady fluid flow rate of 0.1 ml/min or 1.5 ml/min (Figure 1). As such, the uniform flow rate would require automation to achieve for an extended period of time.
Regarding claim 4, Figure 1 of Zhao shows that the fluid flow occurs above and below the cells attached to the substrate. As such, the growth medium would pass over the stem cells.
Regarding claim 10, Obi teaches that Human CD133-positive cells were prepared from freshly obtained human umbilical cord blood samples (page C596, column 1, paragraph 3).
Regarding claim 16, as per the EPCs being autologous, it must be noted that this limitation is directed to the intended use of the cells and does not necessary change the structure of the cells themselves. Nevertheless, Obi teaches the EPCs were derived from human donor, and thus could have been used in an autologous transplant.
Claims 1, 4, and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Obi (Am J Physiol Cell Physiol 303: C595–C606, 2012) and further in view of Zhao et al. (Biotechnology and Bioengineering 96: 584-595. 2007), Wolfe et al. (Biotechnology and Bioengineering 110: 1231-1242. 2013), Ahsan et al. (Tissue Engineering Part A 16: 3547–3553, 2010), United States Patent Application No. 20140120608 (Carter), and United States Patent Application No. 20080032380 (Kleis) as applied to claims 1 and 4 above, and further in view of Zhu et al. (PLoS One 6: 1-10. 2011). This is a new rejection made in response to Applicant’s amendments to claim 1 that is substantially similar to a previous rejection of record. Applicant’s traversal has been addressed above.
The teachings of Obi, Zhao, Wolfe, Ahsan, Carter, and Kleis are as discussed above.
The combined teachings of Obi, Zhao, Wolfe, Ahsan, Carter, and Kleis do not teach wherein the growth medium comprises prostaglandins.
However, Zhu teaches that endothelial progenitor cells are known as a subset of circulating bone marrow mononuclear cells that have the capacity to differentiate into endothelial cells. Treatment with PGE2 (BMCs were pretreated with PGE2 (1 μM) for 24 hr.) significantly increased the differentiation and migration of BMCs (abstract and Figure 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the shear stress-based differentiation media of EPC cells of the combined teachings of Obi, Zhao, Wolfe, Ahsan, Carter, and Kleis with the PGE2 of Zhu to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to combine with a reasonable expectation of success because both media involve differentiating endothelial progenitor cells. As such, it would have been obvious to combine them into a single media to improve the rate of differentiation of the endothelial progenitor cells. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success. Furthermore MPEP 2144.06 states "It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition to be used for the very same purpose.... [T]he idea of combining them flows logically from their having been individually taught in the prior art." In re Kerkhoven, 626 F.2d 846, 850, 205 USPQ 1069, 1072 (CCPA 1980) (citations omitted).
Claims 1, 4, and 19-20 are rejected under 35 U.S.C. 103 as being unpatentable over Obi (Am J Physiol Cell Physiol 303: C595–C606, 2012) and further in view of Zhao et al. (Biotechnology and Bioengineering 96: 584-595. 2007), Wolfe et al. (Biotechnology and Bioengineering 110: 1231-1242. 2013), Ahsan et al. (Tissue Engineering Part A 16: 3547–3553, 2010), United States Patent Application No. 20140120608 (Carter), United States Patent Application No. 20080032380 (Kleis), and Zhu et al. (PLoS One 6: 1-10. 2011) as applied to claims 1, 4, and 19 above, and further in view of National Cancer Institute (16,16-dimethyl prostaglandin E2) and NCBI (16,16-Dimethylprostaglandin E2). This is a new rejection made in response to Applicant’s amendments to claim 1 that is substantially similar to a previous rejection of record. Applicant’s traversal has been addressed above.
The teachings of Obi, Zhao, Wolfe, Ahsan, Carter, Kleis, and Zhu are as discussed above.
The combined teachings of Obi, Zhao, Wolfe, Ahsan, Carter, Kleis, and Zhu does not teach wherein the growth medium comprises 16,16-dimethyl prostaglandin E2.
National Cancer Institute teaches that 16,16-dimethyl prostaglandin E2 is a stable derivative of prostaglandin E2 (page 1, paragraph 1) and NCBI teaches that 16,16-Dimethylprostaglandin E2 has been available since at least 1991 (page 1, paragraph 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted PGE2 of the combined teaching of Obi, Zhao, Wolfe, Ahsan, Carter, Kleis, and Zhu with 16,16-dimethyl prostaglandin E2 as identified by National Cancer Institute to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to substitute with a reasonable expectation of success because 16,16-dimethyl prostaglandin E2 is a known stable derivative of PGE2. As such, it would have been obvious to substitute 16,16-dimethyl prostaglandin E2 for PGE2 because using a stable derivative would increase the rate of differentiation of the epithelial progenitor cells compared to a non-stable PGE2. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 1-2, 4, 10-11 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Circulation Research: 1125–1136, 2012) and further in view of Zhao et al. (Biotechnology and Bioengineering 96: 584-595. 2007), Wolfe et al. (Biotechnology and Bioengineering 110: 1231-1242. 2013), Ahsan et al. (Tissue Engineering Part A 16: 3547–3553, 2010), United States Patent Application No. 20140120608 (Carter), and United States Patent Application No. 20080032380 (Kleis), as evidenced by Wolfe et al. (Biotechnology and Bioengineering 110: 1231-1242. 2013). This is a new rejection made in response to Applicant’s amendments to claim 1 that is substantially similar to a previous rejection of record. Applicant’s traversal has been addressed above.
Zhang teaches a method of culturing Pluripotent stem cells (PSCs; human induced pluripotent stem cells and human embryonic stem cells) on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. Zhang teaches that the pluripotent stem cells were seeded on Matrigel-coated plate at the density of 100 000 cells/cm2 in mTeSR1 medium supplemented with 10 μmol/L Rho kinase inhibitor (Y-27632). The medium was changed daily, and after 3 to 4 days when the monolayer of cells reached 80% to 90% confluence, a thin layer of Matrigel was overlaid by freshly mixing Matrigel (growth factor reduced), 0.5 mg (faster growing lines, ie, DF19-9-11T, DF19-9-7T, IMR90 C4, or H9) or 1 mg (slower growing lines, ie, DF6-9-9T and H1), in 15 mL ice-cold mTeSR1 medium and replacing the medium in each well of a 6-well plate with 2.5 mL of Matrigel containing mTeSR1. Therefore, the Matrigel of Zhang allows the pluripotent stem cells to adhere in a monolayer. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells (abstract and page 1126, column 1, paragraph 4-column 2, paragraph 1). Zhang teaches that the matrix sandwich protocol also is amenable to scale-up using large parallel plate bioreactors technology, because the protocol starts with single-cell seeding (as a monolayer) and does not require further cell processing until harvest of the CMs (page 1135, column 1, paragraph 1).
Although Zhang identifies parallel plate bioreactors as useful for scale-up they do not use it in their initial method.
However, Zhao teaches a closed loop perfusion bioreactor system for applying shear stress to stem cells (abstract). Figure 1 of Zhao teaches that their perfusion system had 4 different perfusion chambers and each perfusion chamber comprised two fluid inlets, two fluid outlets, two sides, two ends, a first conduit, and a second conduit, a media reservoir, and three peristaltic pumps. The reservoir is connected to the first fluid inlet by a conduit and the second fluid outlet connects to the reservoir via a separate conduit. The whole system, except for the pumps, was placed in a 37°C incubator (a base) to maintain a constant operating temperature. Two chambers were exposed to the media flow at 0.1 mL/min, and another two at 1.5 mL/min (page 585, column 2, paragraph 2-page 586, column 1, paragraph 1). Zhao teaches that their study utilized the advantages afforded by the modular design of the perfusion bioreactor system in which multiple cellular samples over an extended period were obtained from the same initial cell population and under the same seeding and culturing conditions (page 591, column 2, paragraph 1).
Ahsan teaches a method of applying fluid shear stress using a parallel plate apparatus (Wolfe evidences that the parallel plate system of Ahsan is a bioreactor system, specifically citing the Ahsan paper when referring to it as a parallel plate bioreactor system (page 1232, column 2, paragraph 4)). Ahsan teaches a closed system recirculating parallel plate bioreactor that comprises an aluminum frame (a base plate), a cell preparation apparatus that comprises a flow block (an inlet fluid feed plate), a rubber gasket, a tubing that connects the medium reservoir with the flow block (a conduit), a spacer and a glass slide placed between the flow block and the rubber gasket, a reservoir, and a peristaltic pump. The flow block comprises a first fluid inlet and a first fluid outlet. Ahsan teaches that they used their parallel plate reactor to apply shear stress to early differentiating embryonic stem cells attached to a glass slide (abstract and page 3548, column 1, paragraph 3-column 2, paragraph 2 and Figure 1). Using this system allows for a steady laminar shear stress of 15 dyn/cm2 (page 3548, column 2, paragraph 1). Ahsan teaches that fluid flow can differentially influence cell fate decisions of pluripotent stem cells and their derivatives. Shear stress in bioreactors can increase the number of cardiomyocytes (page 3552, column 1, paragraph 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the method of differentiating iPSCs into cardiomyocytes of Zhang with the parallel plate bioreactor of Zhao to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to combine with a reasonable expectation of success because Zhang specifically contemplates performing their sandwich method in a parallel plate bioreactor and Ahsan teaches that it was already known that pluripotent stem cells can have increased differentiation into cardiomyocytes when applied a shear stress. As such, it would have been obvious to combine the two to increase the rate of cardiomyocyte differentiation even more than when the two methods are done separately. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Zhao does not teach wherein their modular perfusion system comprises a base plate nor wherein there are intermediate plates with a distribution channel and gathering channel.
However, Carter teaches a multilayered cell culture vessel comprising a stacked formation of multiple cell culture insert layers for culturing cells in each of the individual chambers wherein the vessel comprises a base (a base plate), multiple insert layers, and a lid that are assembled to form a leak proof, closed vessel for the culturing of cells (whole document).
Kleis teaches a bioreactor for culturing cells in a laminar flow configuration wherein there are at least two laminar flow zones. The bioreactor is ideally suited to studying short, moderate and long term studies of cell cultures and the response of cell cultures to stressors such as shear stress (abstract, Figures 1-2 and paragraphs 0073, 0087, and 0134). Kleis teaches that for mechanotaxis studies, the method can include the step of adjusting a shear level or shear gradient using different flow rates for different inlets (paragraph 0134). Kleis teaches that the volume 284 includes four laminar flow zones 286. Each zone 286 includes an inlet 288 having a diffuser 290, where the diffuser 290 is adapted to spread a flow of fluid as it enters its corresponding Zone 286. Each zone 286 also includes an outlet 292 having a collector 294, where the collector 294 is adapted to reduce flow constrictions of fluid as it exits its corresponding zone 286 (Figure 2F and paragraph 0125).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of the closed loop parallel plate device of Zhang and Zhao by stacking the four perfusion chambers of Zhao, as identified by Ahsan, Carter, and Kleis, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because the bioreactors of Zhao, Ahsan, and Kleis involve applying shear stress to cells. Furthermore, Ahsan successfully reduces to practice that a parallel plate bioreactor can be coupled to a base with other components between the inlet fluid feed plate and the base plate, Carter successfully reduces to practice that multiple cell culture inserts can be stacked as part of a larger cell culture vessel connected to a base for culturing cells in each of the individual chambers, and Kleis successfully reduces to practice a bioreactor for examining cell response to shear stress wherein there are multiple laminar flow zones with their own inlets and outlets for controlling shear gradient using different flow rates for different inlets. Therefore, it would have been obvious that the perfusion chambers of Zhao could be assembled as a stacked cell culture vessel wherein each of the perfusion chambers maintain their own inlets and outlets that can maintain the different shear stress levels of Zhao, as shown by Kleis. This would result in a cell culture vessel with an inlet fluid feed plate (the top stacked perfusion chamber), a base plate (the bottom stacked perfusion chamber) and a plurality of intermediate plates (the other two stacked perfusion chambers). Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Furthermore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the four perfusion chambers of Zhao to include a diffuser and a collector for each inlet and outlet, respectively, as identified by Kleis, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because the bioreactors of Zhao and Kleis involve applying shear stress to cells. Furthermore, Kleis teaches the diffuser is adapted to spread a flow of fluid as it enters its corresponding zone, providing a more even distribution of fluid flow rates and the collector is adapted to reduce flow constrictions of fluid as it exits its corresponding zone. Therefore, it would have been obvious to include a diffuser (a distribution channel) and collector (a gathering channel) to more evenly distribute the fluid flow rate for the samples and limit constriction when the fluid exits. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding the distribution channel being proximal to the first end and the gathering channel proximal to the second end, applicant does not provide a definition for proximal. Therefore, under the broadest reasonable interpretation, proximal is considered to mean situated close to and as can be seen in Figure 2E and 2F of Kleis, the diffuser and collector are proximal to the first end and second end, respectively. Furthermore, as can be seen in Figures 2E and 2F, the diffuser and collector extend between the first side and second side of the laminar flow zone.
Regarding claim 2, Zhao, as stated supra, teaches that the parallel plate provides a steady fluid flow rate of 0.1 ml/min or 1.5 ml/min (Figure 1). As such, the uniform flow rate would require automation to achieve for an extended period of time.
Regarding claim 4, Figure 1 of Zhao shows that the fluid flow occurs above and below the cells attached to the substrate. As such, the growth medium would pass over the stem cells.
Regarding claim 10-11, Zhang, as stated supra, teaches that they used human iPSCs and ESCs for their differentiation experiments (page 1126, column 1, paragraph 4-column 2, paragraph 1).
Regarding claim 16, as per the iPSCs being autologous, it must be noted that this limitation is directed to the intended use of the cells and does not necessary change the structure of the cells themselves. Nevertheless, Zhang teaches the iPSCs were derived from human donor, and thus could have been used in an autologous transplant.
Claims 1, and 22-23 are rejected under 35 U.S.C. 103 as being unpatentable over Ballotta et al. (Biomaterials 35: 9100-9113. 2014), Zhao et al. (Biotechnology and Bioengineering 96: 584-595. 2007), Ahsan et al. (Tissue Engineering Part A 16: 3547–3553, 2010), United States Patent Application No. 20140120608 (Carter), and United States Patent Application No. 20080032380 (Kleis), as applied to claim 1 above and further in view of Sin et al. (Biotechnol. Prog. 20: 338-345. 2004). This is a new rejection made in response to Applicant’s amendments to claim 1 that is substantially similar to a previous rejection of record. Applicant’s traversal has been addressed above.
The teachings of Ballotta, Zhao, Ahsan, Carter, and Kleis are as discussed above.
The combined teachings of Ballotta, Zhao, Ahsan, Carter, and Kleis do not teach wherein the pump of each modular cell preparation is coupled to the first fluid outlet and the second fluid inlet.
However, Sin teaches a shear stress device (μCCA device) wherein fluid from multiple tissue chambers are taken out of the device through an outlet and a flexible conduit connected to a peristaltic pump that recirculates the fluid back into the chamber through an inlet through a flexible conduit connected to the pump (page 341, column 2, paragraph 8-page 342, column 1, paragraph 3 and Figures 2-3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the parallel plate bioreactor used in the method of conditioning stem cells in Ballotta, Zhao, Ahsan, Carter, and Kleis by connecting the first fluid outlet and second fluid inlet to a peristaltic pump by a flexible conduit to recirculate the conditioned media, as identified by Sin to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Ballotta, Zhao, Ahsan, Kleis, and Sin are all focused on generating sheer stress on cells and Sin successfully reduces to practice that a fluid inlet and fluid outlet can be connected to a peristaltic pump by a flexible conduit to recirculate the media in the device. It would have been obvious to make this modification because recirculation allows for the conditioned media generated by the shear stress to again flow over the stem cells to generate a greater effect on the cells compared to only using fresh media for generating the shear stress. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
As part of the recirculation, the pump would draw a fluid from the reservoir through the first fluid inlet and the first fluid outlet and direct the fluid through the second fluid inlet, across a plurality of intermediated plates, out the second inlet, and back to the reservoir as this is necessary for recirculation to occur.
Claims 1, 15 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Ballotta et al. (Biomaterials 35: 9100-9113. 2014), Zhao et al. (Biotechnology and Bioengineering 96: 584-595. 2007), Ahsan et al. (Tissue Engineering Part A 16: 3547–3553, 2010), United States Patent Application No. 20140120608 (Carter), and United States Patent Application No. 20080032380 (Kleis), as applied to claims 1 and 15 above and further in view of Yu et al. (BioResearch Open Access 1: 124-136. 2012). This is a new rejection made in response to Applicant’s amendments to claim 1 that is substantially similar to a previous rejection of record. Applicant’s traversal has been addressed above.
The teachings of Ballotta, Zhao, Ahsan, Carter, and Kleis are as discussed above. Ballota teaches that they used complete RPMI media for their shear stress experiments (page 9102, column 2, paragraph 1). Zhao teaches that after MSCs that experienced shear stress exposure at 1.5 mL/min had improved osteoinductive capabilities, such as increased alkaline phosphatase and calcium levels (Figure 9).
The combined teachings of Ballotta, Zhao, Ahsan, Carter, and Kleis do not teach wherein there is 6-fold higher expression of Cox-2 in the conditioned composition.
However, Yu teaches that they applied MSCs in a collagen solution to a PCL scaffold, then cultured the cell and scaffold under a flow perfusion system with osteogenic media at a constant rate of 0.6 mL/min (Figure 1 and page 126, column 1, paragraph 1). Yu teaches that their culture method increased COX2 expression levels by greater than a 6-fold increase after three days of culture (Figure 11).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the complete RPMI media and 1 Hz flow rate of Ballota with the osteogenic media and 0.6 mL/min flow rate of Yu to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to substitute with a reasonable expectation of success because Ballota, Zhao, Ahsan, and Yu are interested in stimulating stem cells (MSCs in Ballota, Zhao, and Yu and ESCs in Ahsan) under shear stress and resulted in changes in protein/mRNA expression, it would have been obvious that different medias, such as complete RPMI media and osteogenic media, and flow rates can be used for generating the flow conditions, as Ballota, Zhao, and Yu each used different flow rates depending on the experiments being run. A shown by Yu, this set up would lead to a greater than 6-fold expression in COX-2 expression (a gene involved in the initial intracellular signal transduction after mechanical stimulation during culture). It would naturally flow that the method of the combined teachings of Ballotta, Zhao, Ahsan, Carter, Kleis, and Yu would also exhibit a greater than 6 fold increase in COX-2 expression as a result of mechanical stimulation. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/KEENAN A BATES/Examiner, Art Unit 1631
/JAMES D SCHULTZ/Supervisory Patent Examiner, Art Unit 1631
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