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
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 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.
Election/Restrictions
Applicant’s election without traverse of Invention I, claims 1-8 and 19-21 in the reply filed on Oct. 30, 2025 is acknowledged.
Claims 1-9 and 11-21 remain pending in the current application, claims 9 and 11-18 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention.
The requirement for the restriction of Inventions BLANK and BLANK is still deemed proper and is therefore made FINAL.
Claims 1-8 and 19-21 have been considered on the merits.
Status of the Claims
Claims 1-9 and 11-21 are currently pending.
Claims 9 and 11-18 have been withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Invention, there being no allowable generic or linking claim.
Claim 10 is cancelled.
Claims 1-9 and 11-21 have been considered on the merits.
Specification
The disclosure is objected to because of the following informalities: the use of trademarks.
The use of the terms: Ecoflex® on pg. 7 line 1, pg. 21 lines 11-13, 21, 24, pg. 23 line 9, pg. 24 line 2, 8, 13, 15, 21-23, pg. 25 line 21, pg. 26 line 8, pg. 27 line 1, 7, 14, 26, pg. 28 line 29, pg. 29 line 1, pg. 30 line 14, pg. 32 line 6, pg. 33 lines 13, 14, 23, 24, pg. 34 lines 24-25, 26, 28-29, 31, pg. 35 lines 2, 6, 9, pg. 35 lines 17, 19-20; Pluronic® on pg. 16 line 31, pg. 30 line 12, pg. 37 lines 31 and 33, pg. 38 lines 2 and 4; Glutamax™ on pg. 17 line 10, pg. 30 line 23; Matrigel® on pg. 25 line 16, pg. 30 line 1, 4, 10, pg. 37 line 22 and 27; TrypLE™ reagent on pg. 29 line 30; Triton®-X on pg. 30 lines 31 and 33; and Tween® 20 on pg. 31 line 1 and 3; Alexa Fluor® 488 on pg. 31 line 2, which are a trade names or a marks used in commerce, have been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the terms.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Appropriate correction is required.
Claim Objections
The disclosure is objected to because of the following informalities:
Claim 19 is objected to in the recitation of “wherein the manufactured microstructure is used to generate muscle tissue by:”, and in the interest of improving claim form, it is suggested that the recited phrase be deleted and replaced with “further comprising using the microstructure to generate muscle tissue, the method further comprising:”. In other words it is suggested to clarify that claim 19 is adding additional steps to method disclosed in claim 1. Presently, the claim almost reads as an intended use.
In claim 20, line 4, it is suggested to add the word “and” following the phrase “extracellular matrix protein,” to clarify that the hydrogel includes fibrinogen, extracellular matrix protein and myogenic progenitor proliferation medium.
In claim 20, in lines 10 and 11, it is suggested to only use the acronym for FBS and FGF2, since both terms are spelled out previously in the claim in lines 5-6.
Claim 20 is objected to because the first time an acronym is utilized in a claim-set, said acronym should be spelled out in its entirety followed by said acronym in parenthesis (e.g. Insulin-Transferrin-Selenium-Ethanolamine (ITS-X)).
Appropriate correction is appreciated.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 20 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
In claim 20, lines 5-6, the phrase: “antibiotics, fetal bovine serum (FBS) and fibroblast growth factor 2 (FGF2), in a concentration of 80-120 ng/ml”, renders the claim and its dependents indefinite, since it is unclear whether the concentration between the two commas is referring to the FGF2, the FBS and/or the antibiotics. For the purposes of compact prosecution, the phrase in the claim will be interpreted to mean “and fibroblast growth factor 2 (FGF2)[[
In claim 20, lines 10-13, the phrase:
“a proliferation medium comprising antibiotics, fetal bovine serum (FBS) and fibroblast growth factor 2 (FGF2) in the concentration of 80-120 ng/ml, comprising 6-aminocaproic acid in a concentration of 0.5-5 mg/ml, or aprotinin in a concentration of 60-100 µg/ml, or
a differentiation medium, such as DMEM or DMEM high glucose, comprising antibiotics, ITS-X in a concentration of 0.5-2.5% v/v, knock-out serum replacement in a concentration of 0.5-2.5% v/v, L-glutamine and 6-aminocaproic acid in a concentration of 0.5-5 mg/ml, or aprotinin in a concentration of 60-100 µg/ml.”
It is unclear which components are require for both the proliferation and the differentiation medium due to the layout of the list of components and the use of words “and” and “or”. In addition, in line 11, there is a second use of the word “comprising” for describing the proliferation medium. For the sake of compact prosecution the claim will be interpreted as: “a proliferation medium containing antibiotics, FBS, FGF2 in the concentration of 80-120 ng/ml, 6-aminocaproic acid in a concentration of 0.5-5 mg/ml or aprotinin in a concentration of 60-100 µg/ml; or a differentiation medium containing antibiotics, ITS-X in a concentration of 0.5-2.5% v/v, knock-out serum replacement in a concentration of 0.5-2.5% v/v, L-glutamine, 6-aminocaproic acid in a concentration of 0.5-5 mg/ml or aprotinin in a concentration of 60-100 µg/ml.”
Claim 20, line 14, recites “a differentiation medium, such as DMEM or DMEM high glucose”. The metes and bounds of the phrase “such as” is unclear because it raises confusion as to whether the optional language was intended to describe the type of medium that is required. Appropriate clarification is required. Moreover, the MPEP 2111.04 states that “a claim scope is not limited by claim language that suggests or makes optional but does not require steps to be performed, or by claim language that does not limit a claim to a particular structure”. Therefore, in this case for the purpose of compact prosecution, the media, “DMEM or DMEM high glucose” will be interpreted as the optional components and therefore is/are non-limiting or not required.
In claim 20, lines 18-19, the phrase: “whereby the culture medium is initially the proliferation medium and after 1.5-3 day is replaced by the differentiation medium”, renders the claim and its dependents indefinite, since it is unclear whether this is a step that is intended to be performed or intended uses of the proliferation and differentiation media. It is further confusing since the claim only requires one of the proliferation or the differentiation medium and does not require both media.
Appropriate correction is required.
Claim Rejections - 35 USC § 103
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.
Claims 1, 4-8 and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Osaki et al. (Nature Protocols, Feb. 2020) (ref. of record) in view of Balmert et al. (Journal of Controlled Release, Jan. 10, 2020) (ref. of record) as evidenced by Rubino et al. (Journal of Polymer Science Part B: Polymer Physics, 2016), Long et al. (Advanced Laser Processing and Manufacturing VIII, 2024), and Hong et al. (Polymer, 2018).
With respect to claim 1, Osaki teaches a method of manufacturing micropillars for generating and analyzing muscle tissue (pg. 422 para. 2-4).
With respect to the first recited step of claim 1, Osaki teaches using a photolithography process to generate a first SU-8 master mold fabrication, a first negative mold (pg. 438 “SU-8 master mold fabrication (photolithography)” and Fig. 2 b). Osaki teaches filling this SU-8 master mold with PDMS to make a first positive template to which caps are place on the pillars (pg. 439 “NMJ chip fabrication (soft lithography)”). Osaki teaches the microstructure contains a set of two pillars in a microchamber for holding liquid (Fig. 2 and Fig. 4).
With respect to the second recited step of claim 1, Osaki teaches filling the first positive PDMS template with a second material, an epoxy resin (Smooth-Cast 310), to generate a second negative mold (pg. 439 “NMJ chip fabrication (soft lithography)”).
With respect to the third recited step of claim 1, Osaki teaches filling the negative mold structure with a third material, PDMS, to form the microstructure (pg. 439 “NMJ chip fabrication (soft lithography)”).
With respect to the fourth recited step of claim 1, Osaki teaches releasing the microstructure from the negative mold structure (pg. 439 “NMJ chip fabrication (soft lithography)”).
Osaki does not teach the method where the second material used to make the negative mold has a Young’s modulus less than the first material used to make the first positive mold structure as recited in the second recited step of claim 1. Similarly, Osaki does not teach the method where the negative mold structure is stretched during the releasing to facilitate the release of the microstructures from the negative mold as recited in claim 1. Additionally, Osaki does not teach the method where the positive mold is formed using a 3D printing process comprising stereo-lithography and first material is a liquid polymeric resin that is solidified by a laser spot or other light pattern as recited in claim 8.
However, Balmert teaches a method of manufacturing a microneedle array (MNA) (microstructures) (abstract). With respect to the first recited step of claim 1, Balmert teaches using a 3D printing process to form a fabricated master MNA (microneedle array) (positive mold structure) using photoresist IP-S (a printable first material) wherein the positive mold structure is a positive of the microstructure to be manufactured (Fig. 2 and pg. 338 Col. 1 last para. to Col. 2 para. 3).
With respect to claim 8, Balmert teaches the 3D printing process comprises stereo-lithography and the first material contains a liquid polymeric resin that is solidified by a laser spot or other light pattern (pg. 338 Col. 2 para. 3)
With respect to the second recited step of claim 1, Balmert teaches filling the master MNA (positive mold structure) with a second material that is an elastomer, polydimethylsiloxane (PMDS), to form a negative PDMS mold (elastically deformable negative mold) (Fig. 2, pg. 338 Col. 1-2 bridging para. and Col. 2 para. 4). The second material, PDMS, has a lower Young’s modulus than the first material than IP-S photoresist as evidenced by Rubino and Long. Rubino reports that PDMS has Young’s modulus of 1-2.6 and 0.6-1.1 MPa depending on mixing ratios (abstract). Long reports that IP-S photoresist has an Young’s modulus of 2.1 GPa (pg. 2 para. 1).
With respect to the third recited step of claim 1, Balmert teaches filling the negative model structure with a third material, UV-curable resin, to form the MNA replicas (microstructures) (Fig. 2 and pg. 338-339 bridging para.).
With respect to the fourth recited step of claim 1, Balmert teaches releasing the MNA replicas from the negative PDMS mold (Fig. 2 and pg. 338-339 bridging para.).
Balmert teaches the MNA can be directly removed from the flexible production molds (pg. 341 Col. 1 para. 2 and pg. 345 Col. 1 last para.), but does not explicitly teach the method where the negative mold structure is stretched during the releasing to facilitate release of the microstructure from the negative mold structure as recited in claim 1. However, this would be inherent to the negative mold structure made from PDMS taught by Balmert, PDMS is a silicone elastomer as required by claim 7 for the second material (pg. 338 Col. 2 para. 3).
Balmert teaches “elastomer molding with PDMS is a well-established technique for rapid, accurate and reproducible replication of high-fidelity micron-scale structures” (pg. 338 Col. 2 para. 3).
Accordingly, at the effect time of filing of the claimed invention, one of ordinary skill in the art would have been motivated to modify the method of Osaki so that the second material used to make the negative mold has a Young’s modulus less than the first material used to make the first positive mold structure and where the negative mold structure is stretched during the releasing to facilitate the release of the microstructures from the negative mold for the benefit of generating with high-fidelity the desired microstructures, the micropillars with the caps, as taught by Balmert. It would have been obvious to one of ordinary skill in the art to modify the method of Osaki to make the negative mold flexible as taught by Balmert, since Balmert teaches that this a well-known technique for generating reproducible micron scaled structures. Furthermore, one of ordinary skill in the art would have had a reasonable expectation of success in making such a modification to the method of manufacturing microstructures of Osaki, since Osaki teaches a method of using a negative mold to make the microstructures and Balmert teaches as similar method of manufacturing microstructures and teaches the benefits of using flexible negative molds to make microstructures.
Osaki is silent with respect to the size of the microchambers and does not teach explicitly teach the microchamber has a capacity for holding between 5 to 100 µl of the liquid as recited in claim 4. Although Osaki is silent with respect to the size of the microchambers, one of ordinary skill in the art would recognize that the size of the microchambers is a result effective variable and that the size of the microchambers would be matter of routine optimization depending on the size of the desired tissue being culture or number of cells to be cultured and the desired purpose of the microchamber with the microstructures.
Osaki does not teach the method where the second material is more flexible than the first material forming the 3D printed positive mold structure and the third material forming the microstructure and where the third material is more flexible than the first material forming the 3D positive mold structure as recited in claim 5. Similarly, Osaki does not teach the method where the second material forming the elastically deformable negative mold structure is reversibly stretchable by at least a factor of three without breaking as recited in claim 6. Although, Osaki teaches the method the final structure with the pillars is formed from PDMS, a biocompatible elastomer that is a polymeric organosilicon compound (pg. 439 “NMJ chip fabrication (soft lithography)”) as recited in claim 7, Osaki does not teach the method where the second material forming the negative mold is a silicone elastomer as recited in claim 7.
However, Balmert teaches the method where second material is PDMS which is more flexible than the first material, IP-S photoresist, forming the 3D printed positive mold structure and where the third material, UV-curable resin, VeroWhiteplus-RGD835, forms the microstructure which is more flexible than the first material as evidenced by Hong (Fig. 2 and pg. 338 Col. 1 last para. to Col. 2 para. 6). Hong reports that the Young’s modulus of VeroWhitePlus-RGD835 is 2000-3000 MPa (Table 1). As stated previously, Rubino reports that PDMS has Young’s modulus of 1-2.6 and 0.6-1.1 MPa depending on mixing ratios (abstract) and Long reports that IP-S photoresist has an Young’s modulus of 2.1 GPa (pg. 2 para. 1). Young’s modulus indicates flexibility with higher values indicating more stiffer the material and lower values indicating more flexible the material. In addition, Balmert teaches that the second material can be made more flexible by adjusting the crosslinker ratio and/or curing temperature when it is PDMS or another hyper-elastic material such as Ecoflex can serve as more flexible alternative to PDMS (pg. 342 Col. 2 para. 1). Ecoflex is reversibly stretchable by at least a factor of three without breaking and a silicone elastomer as evidenced by the instant specification (0009 and 0076 of published application.)
Accordingly, at the effect time of filing of the claimed invention, one of ordinary skill in the art would have been motivated to modify the method of Osaki so that the second material used to make the negative mold more flexible than the first material used to make the first positive mold structure and the third material used to make the microstructures and where the third material is more flexible than the first material and where the second material is Ecoflex for the benefit of generating with high-fidelity the desired microstructures, the micropillars with the caps using the method as taught by Balmert. It would have been obvious to one of ordinary skill in the art to modify the method of Osaki so that the materials used to form the molds and the microstructures have the claimed characteristics recited in claims 5-7, since Balmert teaches using materials with the claimed characteristics to generate reproducible micron scaled structures. Furthermore, one of ordinary skill in the art would have had a reasonable expectation of success in making such a modification to the method of manufacturing microstructures of Osaki, since Osaki teaches a method of using a negative mold to make the microstructures and Balmert teaches as similar method of manufacturing microstructures, teaches the benefits of using flexible negative molds to make microstructures and using materials with the claimed characteristics.
With respect to claim 21, Osaki teaches the method the final structure with the pillars is formed from PDMS, a biocompatible elastomer that is a polymeric organosilicon compound (pg. 439 “NMJ chip fabrication (soft lithography)”).
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective time of filing of the invention, especially in the absence of evidence to the contrary.
Claims 2 and 3 are rejected under 35 U.S.C. 103(a) as being unpatentable over Osaki in view of Balmert as evidenced by Rubino, Long and Hong (as applied to claims 1, 4-8 and 21 above), and further in view of Chen et al. (US 2014/0220555 A1) (ref. of record).
The teachings of Osaki and Balmert can be found in the previous rejection above.
With respect to claim 2, Osaki teaches the micropillars have a first diameter of less than one millimeter and the top of the micropillars has a cap which is larger than the first diameter of the micropillar (Fig. 2). Osaki is silent with respect to the size of cap compared to pillar and does not teach it has a diameter that is larger by at least 50% of the pillar diameter (Fig. 2).
However, Chen teaches a similar microstructure with microchambers containing micro-cantilevers (micropillars) for culturing muscle tissue (0010, 0012 Fig. 1a and Fig. 2). Chen teaches the cantilevers with a cap can have dimensions in the X and Y dimensions that are greater than the X and Y dimension of the post (0048). Chen teaches the X-Y dimensions for the post can range from 5 to 1000 µm (0048). Chen teaches the caps aid in anchoring the tissue (0048). Chen teaches the micropillar rectangular with the X-Y dimensions of 45 x 100 µm (4500 µm2) and the cap with the X-Y dimensions of 85 x 140 µm (11,900 µm2).
In further support Osaki teaches that the pillar caps prevent the tissue from slipping off the posts (pg. 422 last para. and Fig. 4).
Accordingly, at the effect time of filing of the claimed invention, one of ordinary skill in the art would have been motivated to modify the method of Osaki so that the cap has a diameter that is larger by at least 50% of the pillar diameter for the benefit of keeping the muscle tissue attached to the microstructure as taught by Chen. It would have been obvious, to one of ordinary skill in the art to modify the micropillars of Osaki so that the cap is has a diameter that is larger by at least 50% of the pillar diameter, since similar method of culturing muscle tissues on micropillars with caps were known to have such dimensions as taught by Chen. Additionally, one of ordinary skill in the art would have had a reasonable expectation of success in making such a modification to the method of manufacturing microstructures of Osaki, since Osaki teaches the caps prevent the tissue from slipping off the posts or pillars and Chen likewise teaches the caps aid in anchoring the tissue.
Osaki does not teach the method where the micropillars have a widening section below the cap where a diameter of the widening section gradually decreases in a downward direction from the second diameter of the cap to the first diameter of the micropillar below as recited in claim 3.
However, Chen teaches the posts can be cylindrical and can taper from the base to the tip (0046). Chen teaches the geometry of the microcantilever cap can affect the tissue stability and maturation, and the width of the micropillar and cap can have an effect. Chen teaches wider micropillars form tissues that are wide around the pillars and narrow in the middle and narrow micropillars tend to form tissues with a more uniform cross-section (0089).
Accordingly, at the effect time of filing of the claimed invention, one of ordinary skill in the art would have been motivated to modify the method of Osaki so that the micropillars have a widening section below the cap where a diameter of the widening section gradually decreases in a downward direction from the second diameter of the cap to the first diameter of the micropillar below for the benefit of adjusting the shape of the tissue as taught by Chen. Although Chen does not teach the tapering of the diameter of the micropillars from the cap to the base, Chen teaches adjusting and modifying the diameter of the pillars to control the shape of the tissue formed. Accordingly, it would have been obvious to one of ordinary skill based on these teachings in Chen to modify the micropillars so that the diameter of the micropillars tapirs from the cap to the base so to form a particular shape of muscle. It would have been obvious, to one of ordinary skill in the art to modify the micropillars of Osaki so that the diameters of the micropillars decreases in size from the cap to the base, since similar method of culturing muscle tissues on micropillars with caps were known to have micropillars with diameters that change across the length as taught by Chen. Additionally, one of ordinary skill in the art would have had a reasonable expectation of success in making such a modification to the method of manufacturing microstructures of Osaki, since Chen micropillars with diameters that change the length of the pillar for controlling the shape of the tissue cultured on the microstructures.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective time of filing of the invention, especially in the absence of evidence to the contrary.
Claim 19 is rejected under 35 U.S.C. 103(a) as being unpatentable over Osaki in view of Balmert as evidenced by Rubino, Long and Hong (as applied to claims 1, 4-8 and 21 above), and further in view of Zimmermann et al. (US 2024/0076620 A1, priority to Oct. 14, 2019).
The teachings of Osaki and Balmert can be found in the previous rejection above.
With respect to claim 19, Osaki teaches the method where muscle tissue is generated by providing the microchamber with skeletal myoblasts, a hydrogel and a culture medium and culturing the myoblasts to generate the muscle tissue (pg. 441 “skeletal muscle tissue formation in the NMJ chip”). Osaki does not teach that the cells are myogenic progenitor cells as recited in claim 19.
However, Zimmerman teaches a similar in vitro method of producing artificial skeletal muscles tissue by culturing pluripotent stem cells or myogenic progenitor cells to generate the muscle tissue for the benefit to produce engineered models (abstract, 0005, and 0012).
Accordingly, at the effect time of filing of the claimed invention, one of ordinary skill in the art would have been motivated to modify the method of Osaki so that the myoprogenitor cells are provided and cultured to generate the muscle tissue for the benefit of generating muscle tissue as taught by Zimmerman. It would have been obvious to one of ordinary skill to make such a modification to Osaki based on these teachings in Zimmerman that myoprogenitor cells can be used to generate muscle tissue in vitro. Additionally, one of ordinary skill in the art would have had a reasonable expectation of success in making such a modification to the method of manufacturing microstructures and generating muscle tissue of Osaki, since myoprogenitor cells were known to be successful used to generate muscle tissue in culture as taught by Zimmerman.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective time of filing of the invention, especially in the absence of evidence to the contrary.
Claim 20 is rejected under 35 U.S.C. 103(a) as being unpatentable over Osaki in view of Balmert as evidenced by Rubino, Long and Hong and in further view of Zimmermann (as applied to claim 19 above), and further in view of Chen et al. (US 2014/0220555 A1) (ref. of record), Perlingeiro et al. (WO 2019/118768 A1) and Zimmermann et al. (US 2024/0076620 A1, priority to Oct. 14, 2019).
The teachings of Osaki, Balmert and Zimmermann can be found in the previous rejection above.
With respect to claim 20, Osaki teaches the method where the hydrogel comprises collagen cell matrix type 1-A and Matrigel (extracellular matrix proteins) and Ham/F12 10X medium (a culture medium) (pg. 441 “skeletal muscle tissue formation in the NMJ chip”).
Osaki does not teach the hydrogel containing 0.2-4 mg/ml fibrinogen or a myogenic progenitor proliferation medium containing antibiotics, fetal bovine serum (FBS) and 80-120 ng/ml of fibroblast growth factor 2 (FGF2), as claim 20 is being interpreted as explained in the rejections under 35 USC §112(b).
However, Chen teaches a similar microstructure with microchambers containing micro-cantilevers (micropillars) for culturing muscle tissue (0010, 0012 Fig. 1a and Fig. 2). Chen teaches providing cells, a hydrogel, and a medium, and where the hydrogel can include any type of natural extracellular matrix protein including fibronectin and fibrin (0011, and 0069-0071). Chen does not teach the concentration of the fibrinogen is 0.2-4 mg/ml. However, one of ordinary skill in the art would recognize that the concentration of hydrogel components such as fibrinogen are result effective variables and that the concentration of the various components of the hydrogel, such as fibrinogen, would be matter of routine optimization depending on the desired properties of the gel as evidence by Chen. Chen teaches changing the density of the prepolymer matrix or hydrogel to control the time it takes the cells to contract (0088). Chen further teaches providing a medium containing 10% horse serum, penicillin, and streptomycin (antibiotics) (0081).
Chen does not teach the medium containing fetal bovine serum (FBS) or fibroblast growth factor 2 (FGF2).
Perlingeiro teaches culturing myogenic progenitor cells in an expansion medium and supplementing the medium with FBS (pg. 16 lines 1-7, and pg. 26 line 18 to pg. 27 line 11). Perlingeiro teaches sera that can be included include horse serum and FBS, which are functional equivalents (pg. 16 lines 1-7). Accordingly, it would have been obvious to one of ordinary skill in the art to substitute known serum equivalents in the art and to substitute the horse serum for FBS in the medium taught by Chen.
Zimmermann teaches media for culturing myogenic progenitor cells in a medium containing FGF2 (0014-0016, and 0091). Zimmermann teaches media containing FGF2 to induce myogenic specification (0224). Zimmermann does not teach the FGF2 is in a concentration of 80-120 ng/ml as claimed. However, one of ordinary skill in the art would recognize that the concentration of cell culture media components is a result effective variable and that the concentration of the various components, such as FGF2, would be matter of routine optimization as evidence by Zimmermann. Zimmermann teaches optimizing the concentration of the substances of the media including FGF2 (0089).
Accordingly, at the effective time of filing of the claimed invention one of ordinary skill in the art would have been to modify the method of Osaki to include the claimed components in the hydrogel for their known purpose in culturing myogenic progenitor cells and muscle tissues in vitro and based on the combined teachings of Chen, Perlingeiro, and Zimmermann. It would have been obvious to combine the instant ingredients for their known benefit, as disclosed by the cited references above, since each is well known in the art for their claimed purpose and for the following reasons. This rejection is based on the well-established proposition of patent law that no invention resides in combining old ingredients of known properties where the results obtained thereby are no more than the additive effect of the ingredients. The idea for combining them flows logically from their having been used individually in the prior art.
Osaki is silent with respect to the concentration of cells provided and does not teach 106 -109 cells/ml. However, Chen teaches the method where 1,000 to 100,000,000 cells/ml are added to the micro-well or microchamber (0077). Furthermore one of ordinary skill in the art would recognize that the concentration or number of cells added to the microchamber is a result effective variable and that the concentration or number of cells added, would be matter of routine optimization as evidence by Chen. Chen teaches an appropriate cell density can be varied depending on the desired application and the particular size and dimension of the micro-wells (0077).
Osaki does not teach the method where the culture medium provided to generate the muscle tissue is either a proliferation medium containing antibiotics, FBS, FGF2 in the concentration of 80-120 ng/ml, 6-aminocaproic acid in a concentration of 0.5-5 mg/ml or aprotinin in a concentration of 60-100 µg/ml or a differentiation medium containing antibiotics, ITS-X in a concentration of 0.5-2.5% v/v, knock-out serum replacement in a concentration of 0.5-2.5% v/v, L-glutamine, 6-aminocaproic acid in a concentration of 0.5-5 mg/ml or aprotinin in a concentration of 60-100 µg/ml as claim 20 is presently being interpreted as explained in the rejections under 35 U.S.C. §112 (b).
However, Perlingeiro teaches culturing myogenic progenitor cells in an expansion medium containing 2 mg/ml ɛ-aminocaproic acid (6-aminocaproic acid) to prevent fibrin degradation (pg. 26 line 18 to pg. 27 line 11). Accordingly, it would have been obvious to one of ordinary skill in the art to include 6-aminocaproic acid in the culture medium when fibrinogen or fibrin are used for culturing with a hydrogel and generating the muscle tissue for the benefit of preventing the degradation of the hydrogel as taught by Perlingeiro. It would have been obvious to make this modification to the method of Osaki, since Chen teaches fibrinogen and fibrin based hydrogels for generating muscle tissue spanning and connected to micropillars and Perlingeiro teaches 6-aminocaproic acid prevents degradation of fibrin. For these reasons and since 6-aminocaproic acid was known to be included in cell cultures for generating muscle tissue, one of ordinary skill in the art would have add a reasonable expectation of success in making such a modification to the method of Osaki.
Osaki does not teach the method whereby the culture medium is initially the proliferation medium and after 1.5-3 day is replaced by the differentiation medium as claim 20 is presently being interpreted as explained in the rejections under 35 U.S.C. §112 (b).
However, Perlingeiro teaches an in vitro method of producing skeletal muscle cells by culturing pluripotent stem cells or myogenic progenitor cells to generate muscle cells for the benefit to produce models to study diseases (abstract, pg. 1 lines 12-32). Perlingeiro further teaches replacing the expansion medium with the differentiation medium after 3 days of culturing (pg. 26 line 18 to pg. 27 line 11).
In further support, Zimmerman teaches a similar in vitro method of producing artificial skeletal muscles tissue by culturing pluripotent stem cells or myogenic progenitor cells to generate the muscle tissue for the benefit to produce engineered models (abstract, 0005, and 0012).
Accordingly, at the effect time of filing of the claimed invention, one of ordinary skill in the art would have been motivated to modify the method of Osaki so that the myoprogenitor cells are provided and cultured to generate the muscle tissue and where the expansion medium is replaced with a differentiation medium after 3 days of culturing for the benefit of generating muscle cells as taught by Perlingeiro. It would have been obvious to one of ordinary skill to make such a modification to Osaki based on these teachings in Zimmerman that myoprogenitor cells can be used to generate muscle tissue in vitro and Perlingeiro teaches a similar method of generating muscle cells from progenitors where the expansion medium is replaced with a differentiation medium after 3 days of culturing. Additionally, one of ordinary skill in the art would have had a reasonable expectation of success in making such a modification to the method of manufacturing microstructures and generating muscle tissue of Osaki, since myoprogenitor cells were known to be successful used to generate muscle tissue in culture as taught by Zimmerman and where the expansion medium is replaced with a differentiation medium after 3 days of culturing as taught Perlingeiro.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective time of filing of the invention, especially in the absence of evidence to the contrary.
Conclusion
No claims are allowed.
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/EMILY A CORDAS/Primary Examiner, Art Unit 1632