DETAILED ACTION
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 .
Remarks
The amendments and remarks filed on 10/31/2025 have been entered and considered. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior office action. The rejections and/or objections presented herein are the only rejections and/or objections currently outstanding. Any previously presented objections or rejections that are not presented in this Office Action are withdrawn. Claims 1-5, 7-13, 20-25, and 28 are pending; Claims 6, 14-19, 26-27, and 29-36 are cancelled; Claims 1-5, 7-10, 12-13, and 20-25 are amended; and Claims 1-5, 7-13, 20-25, and 28 are under examination.
Withdrawal of Objections
The objection to Claims 10, 13-14, 17 and 19 is withdrawn due to amendment to or cancellation of the claims.
Withdrawal of Rejections
The rejection of claims 1-29 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph in the previous office action is withdrawn due to the amendment to or cancellation of the claims.
The rejection of claim 29 under 35 U.S.C. 112(d) is withdrawn due to the cancellation of the claim.
The rejection of claim 9 under 35 U.S.C. 103 as being unpatentable over Lehmann et al. in view of Martinez et al., Mizuno et al., Hassler et al., and Kosuke et al. is withdrawn due to the amendment to the claim.
Claim Rejections - 35 USC § 112(b), or 112, Second Paragraph
Claims 13 and 25 are 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 pre-AIA the applicant regards as the invention. This rejection is maintained.
Claim 13 is indefinite due to the recitation of “the chondrogenic cells in step a) and/or … spheroids in step b) are submerged in another cell culture medium”. The term “another” implies a cell culture medium is previously defined in the step a) and/or step b) of Claim 1. However, no cell culture medium is defined in the step (a) or (b) of Claim 1. It is unclear whether the step a) or b) comprises using two different culture media, and which specific culture medium can be considered as “another cell culture medium”. For the purpose of examination with respect to the prior art, the term “another cell culture medium” will be interpreted as “a cell culture medium”.
Claim 25 is indefinite due to the recitation of “the cells in step a) are propagated in another hypoxic environment”. The term “another” implies a hypoxic environment is previously defined in the step a) of Claim 1. However, no hypoxic environment is defined in the step (a) of Claim 1. It is unclear whether the step (a) in the claim comprises the use of two different hypoxic environments. For the purpose of examination with respect to the prior art, the term “another hypoxic environment” will be interpreted as “a hypoxic environment”.
Claim Rejections - 35 USC § 103
Claims 1-5, 7-8, 11-13, 20-25, and 28 are rejected under 35 U.S.C. 103 as being unpatentable over Lehmann et al. (US 2015/0166960, 2015, cited in IDS) in view of Martinez et al. (Cell Transplantation, 2008, Vol. 17, pages 987–996, cited in IDS) and Mizuno et al. (WO 2006022671, 2006, cited in IDS).
Lehmann et al. teach a method for in vitro production of a functional fusion tissue, specifically a functional cartilage tissue (reading on the “cohesive cartilage construct” or “fused cartilage spheroids” in claim 1) (abstract), comprising: isolating chondrocytes (reading on “chondrogenic cells” in claims 1 and 11) from a cartilage tissue of a human; propagating the chondrocytes in a culture medium in an agarose-coated plate to form spheroids having a diameter of 800 – 1400 uM (reading on the “cartilage spheroids” in claim 1); and putting a plurality of the spheroids in a culture medium in 3D environment and fusing the spheroids with each other to produce a functional fusion cartilage tissue in the presence of a differentiation inducer (Claims 1-6, 8 and 10; Examples 1-3 and 9.3; paras 0018-0027, 0031-32, 0046, 0055, 0059-60, 0105;), wherein the differentiation inducer is a mechanical differentiation inducer, and the fusing/putting step is carried out in motion with mechanical stimuli/stimulation, such as cultivation in rotating wall vessel or spinner flasks, and pressure application (with compression and decompression), wherein contact/closeness of spheroids to each another facilitates fusion of the spheroids as well as cell redifferentiation corresponding to native functionality of native tissue (native cartilage tissue) (Claim 8, paras 0045/lines 4-15, 0049, 0055/lines 1-8, 0105/lines 9-10 from bottom); wherein the chondrocyte cells and spheroids are cultivated and submerged in a liquid culture medium (para 0124/lines 1-4; para 0125/lines 6-10 and last 4 lines); and wherein the functional fusion cartilage tissue corresponds largely or is identical to a native tissue (paras 0032/lines 1-2, 0035, 0037-0040, 0042).
The method of Lehmann et al. differs from the method of claims 1 and 11-12 in that Lehmann et al. do not teach subjecting the chondrocytes, spheroids, or fused cartilage tissue to a mechanical stimulation in a hypoxic environment.
Martinez et al. teach a process for in vitro production/formation of three-dimensional cartilage tissue (chondrospheroids) from isolated human chondrocytes, comprising culturing the chondrocytes (and also the chondro-spheroids formed during this process) in the presence of low (2.5%) oxygen (i.e. in a hypoxic environment), wherein a technique of hanging-drop cultivation is used for the culturing, and the chondrocytes and chondro-spheroids are submerged in a culture medium in the hypoxic environment (abstract, page 988/right col/para 2, page 990/left col/last para, Fig. 1). Martinez et al. further teach that the cartilage tissue generated in the low oxygen environment has a bigger size, enhanced matrix deposition (abstract/lines 11-12, page 995/last para/lines 1-6, Fig. 1), with a high rate of cell viability (page 990: left col/last 3 lines, right col/para 1/last 3 lines). Martinez et al. further teach developing a better quality of cartilage tissue by combining an environmental factor such as mechanical stress/stimulation with the low oxygen condition used in their method (the sentence spanning pages 995 and 996).
Mizuno et al. teach cartilage constructs for treating damaged cartilage and a method of preparation thereof (abstract, claim 30). Mizuno et al. also teach that in order to promote growth and/or formation of chondrocytes and cartilage constructs (produced from the chondrocytes) it is advantageous and necessary to change environmental conditions, which include cyclic hydrostatic pressure, changing flow rate of a medium, and changing oxygen concentration (specifically a low-oxygen concentration with less than 20% saturation of oxygen, i.e. a hypoxic environment) (page 34/last para, page 35/lines 6-9, page 39/lines 6-17, pages 49-50). Mizuno et al. further investigate these environmental conditions to determine their beneficial effect on the chondrocytes and cartilage constructs while they are submerged in a culture medium (pages 36-50). Mizuno et al. teach that in a low-oxygen environment (2% O2), sGAG accumulation and production of extracellular matrix in cell constructs are significantly increased (page 49/para 3 – page 50/para 3, table 4); that a flow of liquid culture medium at low rate results in a higher extracellular matrix accumulation (page 37/lines 16-17); and that cyclic hydrostatic pressure stimulates chondrocyte proliferation and metabolism, leading to increased S-GAG and extracellular matrix accumulation of cell constructs (page 38/lines 10-14, page 43/lines 25-30). Mizuno et al. further teach using a combination of low-oxygen tension, cyclic hydrostatic pressure and/or a flow of culture medium for culturing and producing chondrocytes and cartilage constructs (page 50/lines 13-27, page 51/lines 17-20); and demonstrated that the combination of low-oxygen tension with cyclic hydrostatic pressure stimulates accumulation of S-GAG extracellular matrix (page 50/lines 18-21). It is noted that cyclic hydrostatic pressure and a culture medium flow taught by Mizuno et al. are mechanical inducers or stimulations in the method taught by Lehmann et al.
It would have been obvious to one of ordinary skill in the art to modify the method of Lehmann et al. by subjecting fused cartilage tissue and spheroids to a mechanical stimulation/motion in a hypoxic environment for in vitro production of the functional fusion cartilage tissue, because Lehmann et al. teach that extracellular matrix produced in fusion tissue is key for the biological functionality of the cartilage tissue (para 0012, lines 7-8), and a hypoxic environment enhances extracellular matrix production in cell constructs and generates a larger size of the cartilage tissue, as supported by Martinez et al. and Mizuno et al. In addition, it is well known in the art to use a combination of a mechanical stimulation with a low-oxygen/hypoxic environment for in vitro production of a better-quality cartilage tissue, as supported or taught by Martinez et al. and Mizuno et al.
Regarding the limitation about increasing and facilitating contacts of spheroids in the step b) of the claim 1, this limitation is directed to the outcome of the active step of putting spheroids in motion in a hypoxic environment. The active step suggested by the cited prior art is the same as the step b). It is presumed that substantially the same steps are capable of performing substantially the same outcomes.
Regarding the step c) recited in the claim 1, Lehmann et al. teach that the pressure application comprises compression and decompression. It is noted that the compression and decompression would respectively increase and decrease the pressure, thus leading to a fluctuating pressure. Lehmann et al. also teach applying mechanical stimulation via such a pressure application and stimulating synthesis of extracellular matrix components for obtaining a functional fusion tissue with natural property (para 0105/lines 10-14 and last 3 lines). Furthermore, the cyclic hydrostatic pressure taught by Mizuno et al. is a since wave form of pressure, i.e. a fluctuating pressure (page 14, lines 13-17). Given Mizuno et al. teach that a combination of hypoxic environment and fluctuating pressure stimulates accumulation and production of extracellular matrix, it would have been obvious to subject the cell culture medium submerged with the fused cartilage spheroids to a fluctuating pressure in a hypoxic environment in the method suggested by the cited prior art for increasing extracellular matrix content in the fused spheroids for obtaining a functional fusion tissue with native property. Regarding the limitation about the culture medium having dissolved oxygen less than 100% air saturation in Claim 1, Martinez et al. and Mizuno et al. respectively teach partial oxygen pressures 2.5% and 2%. As evidenced by the disclosure of the specification of the instant application (page 20, lines 16-20), the partial pressure of oxygen of air in the atmosphere is 20.9%. Thus, the low-oxygen environment (2% or 2.5%) taught by the cited prior art generates an amount of dissolved oxygen in the culture medium less than 100% air saturation (compared to those generated by 20.9% O2 in air), meeting the limitation of the claim. Thus, the teachings of the cited prior art render the claims 1 and 11-12 to be obvious.
Regarding the claim 2, it would have been obvious to propagate chondrocyte cells in a hypoxic environment in the method suggested by the cited prior art for improving quality of the formed spheroids, because Martinez et al. and Mizuno et al. teach that hypoxic environment enhances extracellular matrix accumulation and promotes large cartilage construct production.
Regarding the claims 3 and 4, Martinez et al. and Mizuno et al. respectively teach partial oxygen pressures are 2.5% and 2%, which read on the claimed partial oxygen pressure ranges of less than 20% and less than 10%.
Regarding the claims 5 and 25, it would have been obvious to propagate chondrocyte cells via hanging drop cultivation in the method suggested by the cited prior art for producing chondro-spheroids, because the hanging drop cultivation is a technique well established in the art for forming spheroids, as supported by Martinez et al. Regarding the further limitation about hypoxic environment in the claim 25, Martinez et al. and Mizuno et al. respectively teach partial oxygen pressures 2.5% and 2%, which read on the claimed range of less than 10%.
Regarding claims 7 and 8, Lehmann et al. teach that the mechanical stimulation is the cultivation in a rotating vessel or spinner flask. Rotating or spinning is a process in circular motion, which can tilt about one direction/axis. Thus, the teachings of Lehmann et al. render the claims obvious.
Regarding Claim 13, Lehmann et al., Martinez et al. and Mizuno et al. teach that the chondrocyte cells are cultivated and submerged in a liquid base culture medium; and Martinez et al. and Mizuno et al. teach a low-oxygen environment that generates an amount of dissolved oxygen in the culture medium less than 100% air saturation, as indicated above.
Regarding claims 20-24, it is well known in the art that application of a fluctuating pressure not more than 5 MPa is effective for increasing chondrocyte metabolism and promoting S-GAG and extracellular matrix accumulation, as supported by Mizuno et al., who teach that an effective cyclic hydrostatic pressure is in a range of 0.5 – 3.0 MPa or specifically 0.5 or 3.0 MPa (page 40/lines 9-10, page 42/lines 1-2, tables 1 and 2). Thus, it would have been obvious to apply a fluctuating hydrostatic pressure at a level below 5 MPa in the method suggested by the cited prior for carrying out the fluctuating pressure application.
Regarding the claim 28, Martinez et al. and Mizuno et al. teach that hypoxic environment, mechanical stimulation/motion, and fluctuating pressure provide benefits of promoting extracellular matrix accumulation, enhancing cell viability, increasing sizes and improving quality of fused tissue. Thus, it would have been obvious to combine treatment of hypoxic environment, mechanical stimulation/motion, and fluctuating pressure in the same step in the method suggested by the cited prior art for generating a functional fusion tissue.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Lehmann et al. (US 2015/0166960, 2015, cited in IDS) in view of Martinez et al. (Cell Transplantation, 2008, Vol. 17, pages 987–996, cited in IDS) and Mizuno et al. (WO 2006022671, 2006, cited in IDS), as applied to Claims 1-5, 7-8, 11-13, 20-25, and 28, further in view of Hassler et al. (EP 3498310, published on 6/19/2019, cited in IDS) and Kosuke et al. (Drug Delivery System, 2013, 28(1): 45-53, cited in IDS).
The teachings of Lehmann et al. as modified by Martinez et al., and Mizuno et al. are described above.
Regarding Claim 10, the modified Lehmann et al. do not expressively teach subjecting spheroids to tilting about two or more axes. However, Lehmann et al. teach that contact of spheroids to each another facilitates fusion of the spheroids as well as their redifferentiation corresponding to native functionality of native cartilage tissue. Lehmann et al. also teach using rotation or spinning as a mechanical stimulation and differentiation inducer to achieve the contact of spheroids to each another (which renders the limitation of tilting about one axis to be obvious as indicated above). Whether subjecting spheroids to tilting about one axis or tilting about two or more axes is deemed merely a matter of an obvious design choice and routine optimization for facilitating their contact with each other, their fusion and redifferentiation, and production of fused cartilage tissue with better functionality in the method suggested by the cited prior art. Examiner notes that the techniques for tilting cells or cell constructs about one, two or more axes to contact them with each other are well established, as supported by Hassler et al., who teach a method for generating a cohesive cartilage construct by forming chondrocyte aggregates and then fusing the aggregates to a cohesive cartilage tissue, in a process involved with application of mechanical stimulation and motion (paras 0029, 0032-0034, 0039-0042, and 0055-0058), specifically with oscillation and/or rotation (para 0032/last 2 lines) (Note: oscillation and/or rotation comprise tilting spheroids about two or more axes); and as further supported by Kosuke et al., who teach a method/technique for generating 3D spheroids for therapy application, wherein the method comprises using agarose-coated plates for culturing cells and generating spheroids (title; translated page 47: left col./last para, right col/para 1), and specifically teach using shaking (with a shaker) for increasing cell components to contact with each other for achieving a high efficiency of spheroid production (page 47, left col, lines 1-3) (Note: this teaching renders the limitation of using a shaker for tilting about one axis to be obvious because shaking in tilting about one axis allows cell components or spheroids to contact with each other). Overall, it is considered that the rotation or spinning of Lehmann et al. can be readily modified or replaced by other well established techniques in the art (such as shaking with a shaker or a process comprising oscillation) through routine optimization for tilting spheroids about one, two or more axes for maximizing biological functionality of fused cartilage tissue products in corresponding to native cartilage tissue. It is well settled that routine optimization is not patentable, even though it results in significant improvement over the prior art (see MPEP 2144.05).
Examiner notes that Kosuke et al. further teach and render the “hanging drop cultivation” in Claims 5 and 25 to be obvious. Specifically, Kosuke et al. teach hanging drop cultivation is a technique well established in the art (see the para spanning translated pages 47 and 48), and hanging drop cultivation has an advantage that it’s easy to create uniform cell spheroids with a constant number of cells per spheroids when compared to the technique involved with agarose-coated plates in the method of Lehmann et al. (see Kosuke et al.: translated page 48/left col/lines 7-8, translated page 47/right col/para 1/last 4 lines).
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Lehmann et al. (US 2015/0166960, 2015, cited in IDS) in view of Martinez et al. (Cell Transplantation, 2008, Vol. 17, pages 987–996, cited in IDS) and Mizuno et al. (WO 2006022671, 2006, cited in IDS), as applied to Claims 1-5, 7-8, 11-13, 20-25, and 28, further in view of Dankbar et al. (US 2013/0059288, 2013) and Kosuke et al. (Drug Delivery System, 2013, 28(1): 45-53, cited in IDS).
The teachings of Lehmann et al. as modified by Martinez et al., and Mizuno et al. are described above.
Regarding Claim 9, the modified Lehmann et al. do not expressively teach using a rocker shaker to subject the spheroids to tilting about one axis. However, Lehmann et al. teach that contact of spheroids to each another facilitates fusion of the spheroids as well as their redifferentiation corresponding to native functionality of native cartilage tissue. Lehmann et al. also teach using the motion of rotation or spinning as mechanical stimulation and differentiation inducer to increase the contact between spheroids (which renders the limitation of tilting about one axis to be obvious as indicated above).
Kosuke et al. teach a method/technique for generating 3D spheroids for therapy application, wherein the method comprises using agarose-coated plates for culturing cells and generating spheroids (title; translated page 47: left col./last para, right col/para 1). Kosuke et al. specifically teach using shaking (with a shaker) for increasing cell components to contact with each other for achieving a high efficiency of spheroid production (page 47, left col, lines 1-3) (Note: this teaching renders the limitation of tilting about one axis to be obvious because shaking in tilting about one axis allows cell components to contact with each other).
Dankbar et al. teach using a rocking platform shaker (i.e. rocker shaker) to agitate a cell-containing suspension, thus to put cells in motion for improving the binding of target cells to a separation surface, wherein the rocking platform shaker is tilted at a tilt angel 5o (i.e. subjecting cells to tilting about one axis) (paras 0104, and 0051/last 8 lines). Dankbar et al. further teach the rocking motion of cells may advantageously be accomplished by means of a rocking shaker (i.e. rocker shaker).
It would have been obvious to replace the rotation or spinning in the method of Lehmann et al. with other well established techniques in the art, such as shaking with a shaker, specifically a rocker shaker, for subjecting the spheroids to tilting about one axis, thus increasing the contact and fusion between the spheroids for producing a functional fusion cartilage tissue. This is because it is well known in the art to use a shaker to subject cell components to tilting about one axis to increase the probability for them to contact with each other for forming a cell construct, as supported by Kosuke et al.; and a rocker shaker is a conventional device commonly used in the art for performing tilting about one axis, and this shaker is advantageously effective at delivering a rocking motion of cells via tilting about one axis, as supported by Dankbar et al.
It is noted that Kosuke et al. further teach the “hanging drop cultivation” in Claims 5 and 25, as described above.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Response to Arguments
Applicant's arguments about the claim objection as well as the claim rejections under 35 U.S.C. 112(b) and 112(d) in the response filed on 10/31/2025 (pages 8-11) have been fully considered but they are moot because these objection and rejections have been withdrawn as indicated above.
Applicant's arguments about the rejection of Claims 1-8 and 11-29 under 35 U.S.C. 103 over Lehmann et al. in view of Martinez et al. and Mizuno et al. in the 10/31/2025 response (pages 11-18) have been fully considered but they are not persuasive for the following reasons.
First, Applicant’s arguments about unexpected results/effects based on Exhibit A throughout pages 11-17 of the response are not persuasive. Although the results of Experiments A and B in the Exhibit A show treatments of hypoxic condition, motion and/or non-fluctuating pressure improve fusion of spheroids, Applicant failed to provide any factual evidence to support this improvement is indeed unexpected in the prior art. Furthermore, the objective evidence in Exhibit A, which is based on treatments involved with tilting about one axis and a non-fluctuating pressure (i.e. a constant pressure of 1.4 MPa for 1 hour per day), is not commensurate in scope with the claim 1. Specifically, the claim 1 does not recite any limitation to limit the claimed “motion” to be the “tilting about one axis” that is used in the Exhibit A; and the pressure required in the claim 1 is a “fluctuating pressure”, not the non-fluctuating pressure used in the Exhibit A.
Moreover, it appears that there are some conflictions about effects of the motion of tilting about one axis between Experiments A and B, because the motion in Experiment A improved fusion of spheroids when comparing the group D with motion/tilting to the group C without motion/tilting, whereas the motion in Experiment B did not improve fusion of spheroids when comparing the group C with motion/tilting to the group B without motion/tilting (see the statement, in last page, last para of Exhibit A, that there was no significant histologic difference between group C with titling and group B without tilting).
Second, Applicant’s arguments about teachings of Lehmann throughout pages 12-17 of the response are not persuasive and misleading. Applicant picked a single embodiment in para [0047] and used it to represent the overall entire disclosure of Lehmann; and asserts that Lehmann’s method requires incubating spheroids on a concave surface in the presence of a chemical differentiation inducer and requires static spheroid-to-spheroid contact (i.e. not putting spheroids in motion) for production of a cohesive cartilage construct. Examiner reminds Applicant that the para [0047] is only one of multiple different embodiments taught by Lehmann et al., and no teachings of Lehmann et al. indicate that their method is limited to use a concave surface and a chemical differentiation inducer under static incubation for contact of spheroids. In fact, Lehmann et al. expressively teach preferably using a mechanical differentiation inducer, which includes the motion and fluctuating pressure recited in the instant claims, for promoting fusion of spheroids and formation of fusion tissue. See the para [0055]: “In the sense of this method, "differentiation inducers" are preferably mechanical … differentiation inducers. By way of example, the cultivation in 3D environment with at least 5 spheroids, the cultivation in the presence of mechanical stimuli, such as pressure application, cultivation in bioreactors such as a rotating wall vessel or spinner flasks are suitable as mechanical differentiation inducers”; and see the para [0105]: “In particular for cartilage tissue, this includes the mechanical stimulation for example via pressure application (compression and decompression) or the cultivation in special bioreactors and spinner flasks in order to thus simulate the natural processes and effective forces in the body” (emphasis added). These teachings clearly indicate in the method of Lehmann et al. the spheroids are subjected to the motion, such as in a rotating wall vessel or a spinner flask, for promoting contact and fusion between spheroids. As such, Applicant’s arguments based on static spheroid-to-spheroid contact used by Lehmann’s method are misleading and unpersuasive. Finally, in response to Applicant’s arguments about mechanical stimuli the paragraph spanning pages 15 and 16 of the response, Examiner notes that the invention of Lehmann is not limited to the disclosed examples; and Lehmann expressively teaches that the mechanical stimuli is a preferred differentiation inducer, which comprises pressure application (comprising compression and decompression, delivering fluctuating pressure) and motion/rotating (by using a device such as a rotating wall vessel or spinning flask); and these pressure and motion applications simulate natural processes of functional fusion tissue and are all suitable as mechanical differentiation inducer, in particular for cartilage tissue. In view of the teachings of Lehmann, it would have been obvious to one of ordinary skill in the art to apply mechanical stimuli via the rotating/spinning motion and compression/decompression pressure application as mechanical differentiation inducer in the method of Lehmann for improving spheroid fusion as well as function of the resulted functional fusion tissue. It is further noted that devices used for mechanical stimuli such as rotating wall vessels and spinning flasks are all conventional and commercially available devices and procedures for handling them are well known and established in the art. One of ordinary skill in the art certainly has a reasonable expectation of success at carrying out the mechanical stimuli in the method of Lehmann.
Furthermore, Examiner disagrees with Applicant’s assertation in pages 13-14 that the treatment group B in the Experiment A of the Exhibit is similar to what was done in Lehmann. Even assuming that the method of Lehmann is limited to the single embodiment of [0047] (continued to [0048]), there is still nothing similar between Group B and Lehmann about the setting of the surface and spheroids. Specifically, in the embodiment of paras 0047-48 of Lehmann, the 5 spheroids having a diameter of at least 800 mm are applied to a concave cultivation surface in a well of a 96-well plate, wherein the concave surface is constructed by pouring 100 ul of agarose-dissolved cell culture medium to the well of the 96-well plate. In contrary to Lehmann, the treatment of group B in the Experiment A comprises applying 250 spheroids having undefined diameters or sizes onto an undefined conventional dish without any agarose-coated surface, and the dish is left at a 40 degrees slope angle, which is a setting distinct from that of the concave surface of Lehmann. As such, the group B in the Experiment A cannot be considered as being similar or equivalent to the embodiment of [0047] of Lehmann. Given the structure of concave surface, sizes and numbers of spheroids, as well as dimensions of a cultivation dish greatly affect the contact and fusion between spheroids, the differences between the group B and the group D revealed in the Experiment A are not sufficient to support the claimed subject matter represents improvement over the embodiment of [0047] of Lehmann.
In response to Applicant’s arguments based on the teachings of Martinez et al. in page 17 of the response, Martinez et al. teach application of hypoxic conditions has the benefits of enhancing extracellular matrix and maintaining a high rate of cell viability as well as leading to formation of cartilage tissue having a larger size through redifferentiation of chondrocytes. Given Lehmann teaches the fusion between spheroids in their method involves redifferentiation of cells/chondrocytes, it would have been obvious to apply a hypoxic condition to the method of Lehmann for obtaining the benefits taught by Martinez et al.
In response to Applicant’s arguments based on the teachings of Mizuno in pages 17-18 of the response, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). As indicated above, Mizuno et al. teach that changes in environmental conditions such as a low-oxygen concentration (i.e. a hypoxic environment) and a cyclic hydrostatic pressure (i.e. a fluctuating pressure) have the advantage of promoting cell growth and formation of cartilage constructs from chondrocytes; and Mizuno et al. demonstrate that a combination of hypoxic environment and cyclic hydrostatic pressure/fluctuating pressure stimulates accumulation and production of sGAG/extracellular matrix in chondrocytes. Lehmann teach that extracellular matrix produced in fusion tissue is key for the biological functionality of a cartilage tissue, and specifically teaches stimulating synthesis of extracellular matrix components for obtaining a functional fusion tissue with natural property (para 0105/last 3 lines). It would have been obvious to apply a combination of hypoxic environment and fluctuating pressure taught by Mizuno et al. to the process of fusing spheroids in the method of Lehmann for increasing accumulation and production of extracellular matrix in chondrocytes, thus obtaining a functional fusion cartilage tissue with improved extracellular matrix content and natural property.
Overall, the method of Lehmann et al. differs from the method of claim 1 in that Lehmann does not teach using a hypoxic environment condition, as indicated above.
However, it is well known in the art that hypoxic condition, mechanical stimulation/motion, and fluctuating pressure provide benefits such as promoting extracellular matrix accumulation, enhancing cell viability, increasing sizes of fused tissues formed from spheroids, and improving fused tissue formation, as supported by the cited prior art Martinez and Mizuno. The conclusion of the obviousness of the instant claims has been established for all the reasons indicated above. Finally, in response to Applicant’s arguments in page 18/para 3, Examiner notes that this 103 rejection is solely based on the combined teachings of the cited prior art, not on Applicant’s own disclosure.
Applicant's arguments about the rejection of Claims 9 and 10 under 35 U.S.C. 103 over Lehmann et al. in view of Martinez et al., Mizuno et al., Hassler et al., and Kosuke et al. in the 10/31/2025 response (pages 19-21) have been fully considered but they are not persuasive for the following reasons.
In response to Applicant’s arguments about the teachings of Hassler et al. in pages 19-20 of the response, Examiner disagrees with Applicant’s assertation that the shear force or movement parallel to the Applicator’s surface does not put the cells in motion. It is noted that Hassler et al. teach that the cells and cell aggregates (i.e. spheroids) are in a liquid culture medium, specifically a liquid DMEM culture medium is applied in both cell culture step and the mechanical stimulation step (see page 6: lines 3-6, Table 1/last 2 rows, and also “cell aggregate” in lines 1 and 3). Hassler et al. also teach during the mechanical stimulation step applying a force of shaking and mechanical solicitation (page 6/lines 4-6) or a force of oscillation and rotation (page 4/line 26) to the cells or cell aggregates in disk. As such, these forces would put the cells or spheroids/cell aggregates to move around in the liquid culture medium, thus increasing a number of times for the cells or spheroids to contact with each other. With regarding to Applicant’s remaining arguments based on the claimed limitations about dissolved oxygen less than 100% and a fluctuating pressure in page 20 of response, they are not persuasive because these limitations have been taught by the combined teachings of Lehmann et al., Martinez et al., and Mizuno et al. render, as indicated above.
In response to Applicant’s arguments about the teachings of Kosuke et al. in page 20 of the response, it is noted that the primary reference Lehmann teaches fusion of spheroids into cohesive cartilage construct and putting the spheroids into closeness and motion during the fusion process, as indicated above. In view of the combined teachings of Lehmann, Kosuke et al. and other cited prior art, it would have been obvious that the shaking device taught by Kosuke et al. is readily appliable to the method of Lehmann for increasing the probability for spheroids to contact each other, for the reasons indicated above.
In response to Applicant’s arguments in the paragraph spanning pages 20 and 21 in the response, Examiner notes that the rejection of the claims 9 and 10 under 35 U.S.C. 103 is solely based on the combined teachings of the cited prior art, not on Applicant’s own disclosure.
Overall, the conclusion of the obviousness of the claims 9 and 10 has been established for all the reasons indicated above.
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 extension fee 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 date of this final action.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to Qing Xu, Ph.D., whose telephone number is (571) 272-3076. The examiner can normally be reached on Monday-Friday from 9:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath N. Rao, can be reached at (571) 272-0939. Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to the receptionist whose telephone number is (571) 272-1600.
/Qing Xu/
Patent Examiner
Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656