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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 11/10/2025 has been entered.
Claims 5, 12 and 19 have been canceled, claim 21 is newly added, and claims 1-4, 6-11, 13-18 and 20-21 have been considered on the merits. All arguments have been considered.
Claim Rejections - 35 USC § 102
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.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1 and 6-8 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Brown et al. (2016, Tissue Engineering; IDS ref.).
Regarding claim 1 and 6, Brown et al. teach a method of treating fetal ovine articular chondrocytes (foACs) or juvenile bovine articular chondrocytes (jbAC) contaminated with red blood cell (RBC) using the hypotonic ACK buffer (ammonium-chloride-potassium buffer) to selectively eliminate RBCs and additionally reduced the number of apoptotic chondrocytes in the cell isolates (Abstract; p.896, Materials and Methods). It is submitted that the population of foACs or jbAC of Brown et al. would inherently contain non-pre-apoptotic cartilage cells, pre-apoptotic cartilage cells and apoptotic cartilage cells and contaminated with RBC. This is because the population of Brown et al. would be enriched with healthy chondrocytes removing contaminated RBC and apoptotic cells. Brown et al. teach both cytoskeletal breakdown and loss of cell membrane through blebbing make chondrocytes on the apoptotic pathway more susceptible to ACK buffer-induced cell rupture (p.901, 1st col.). While Brown et al. do not teach that the ACK buffer treatment induces cell swelling, however, Brown et al. teach the ACK buffer induces cell rupture (p.901, 1st col.), and thus, it is expected that the hypotonic solution inherently swell the cells. It is the Examiner’s understanding that all the cells in the population would be swelled upon the treatment but only those having weaker cell membrane would be ruptured and removed. Thus, regardless of how the cells being classified (e.g. pre-apoptotic or apoptotic), those cells killed by the ACK buffer would inherently include pre-apoptotic and apoptotic as well as RBCs, and thus enriching healthy cartilage cells.
The resulting viable cells after the ACK buffer treatment of Brown et al. are considered to be enriched and enhanced because the cell purity and resulting neotissue functional properties are increased (Abstract). The resulting cell population of Brown et al. is devoid of RBC and apoptotic cells and thus, the resulted population would meet the method of enhancing a cartilage cell population.
Regarding claim 7, Brown et al. teach that primary foACs and jbACs treated with ACK buffer were self-assembled into engineered neocartilage constructs (p.896, 2nd col., last para.). Thus, this teaching meets “directly using the fraction of cartilage cells produced in (c) of claim 1.
Regarding claim 8, Brown et al. teach the ACK buffer treated cells are self-assembled into engineered neocartilage constructs in nonadherent agarose wells, and the agarose wells were filled with chemically defined chondrogenic medium containing 1% PSF (penicillin/streptomycin/fungizone), 1% ITS (insulin-transferrin-selenium), dexamethasone, etc. (p.896-897: Neocartilage construct seeding and culture). This teaching is considered to meet the limitation of claim 8 directed to the step of subjecting the fraction of cartilage cells produced in (c) in to treatment comprising hormones (insulin) or toxic compounds (PSF as they can be toxic at higher concentration, etc.).
Thus, the reference anticipates the claimed invention.
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, 6-11, 13-18 and 20-21 are rejected under 35 U.S.C. 103 as being unpatentable over Brown et al. (supra) in view of Eslaminejad et al. (2009, Iranian Journal of Biotechnology; of record) and Naughton et al. (US 5,842,477; IDS ref.).
Brown et al. teach the subject matter of claims 1 and 6-8 and thus render them obvious (see above).
Regarding directed to the cartilage cells are non-articular cartilage cells (claims 2, 10, 16-18 and 20), the source of the cartilage cells being a portion of a rib (claims 4, 11, and 18) or the cartilage cells being human (claims 3, 9-11, 13-15 and 17), Brown et al. do not teach the limitation.
However, it would have been obvious to a person skilled in the art to use non-articular chondrocytes such as costal chondrocytes or any other chondrocytes isolated from human sources in producing neocartilage using the method of Brown et al. with a reasonable expectation of success. A person of ordinary skilled in the art would have been motivated to do so because one skilled in the art would recognize that the effect of hypotonic buffer to selectively burst apoptotic cells is not limited to articular chondrocytes, as any apoptotic cells would have breakdown of cytoskeletal structure and membrane blebbing, etc. as these features are considered universal to any apoptotic cells not limiting to articular chondrocytes. Brown et al. teach that the loss of excess cell membrane through blebbing may not allow for the surface area expansion that takes place in normal chondrocytes under hypotonic conditions (p.901, 1st col.). As these structural features during apoptosis are not limited to chondrocyte, let alone articular chondrocyte, one skilled in the art would recognize that the effect of ACK buffer or hypotonic buffer is applicable to any apoptotic cells including different type of cartilage cells as well as those from different species, and thus, the same effect in producing neocartilage having improved properties.
Naughton et al. teach a method of repairing cartilage in humans (col. 6, lines 35-42) by using chondrocytes from various sources including any type of cartilage including hyaline cartilage, costal cartilage, fibrous cartilage, etc. (col. 10, lines 49-53). Furthermore, it is known in the art that costal chondrocytes can replace articular chondrocytes for their property in producing engineered cartilage (see Eslaminejad et al.). Eslaminejad et al. teach costal chondrocytes isolated from rib was compared to articular chondrocyte isolated from knee of an animal, and costal chondrocytes are viable alternative to articular chondrocytes for manufacturing 3D cartilage scaffold (Abstract; Discussion).
Considering the effect of removing RBCs and apoptotic cells by ACK buffer taught by Brown et al. is applicable to any cartilage cell preparation in producing neocartilage intended for cartilage repair, and the use of any cartilage cells, chondrocytes, from any source in repairing cartilage tissue in human as taught by Naughton et al. and the teaching of costal chondrocytes being a suitable alternative to articular chondrocytes as taught by Eslaminejad et al., a person skilled in the art would have sufficient reason to try to human costal chondrocytes in producing neocartilage for the method of Brown et al. with a reasonable expectation of success.
Regarding the limitations of claims 14-15 directed to the additional method steps, Brown et al. teach that primary foACs and jbACs treated with ACK buffer were self-assembled into engineered neocartilage constructs (p.896, 2nd col., last para.). Thus, this teaching meets “directly using the fraction of cartilage cells produced in (c) of claim 9. Brown et al. teach the ACK buffer treated cells are self-assembled into engineered neocartilage constructs in nonadherent agarose wells, and the agarose wells were filled with chemically defined chondrogenic medium containing 1% PSF (penicillin/streptomycin/fungizone), 1% ITS (insulin-transferrin-selenium), dexamethasone, etc. (p.896-897: Neocartilage construct seeding and culture). This teaching is considered to meet the limitation of claim 15 directed to the step of subjecting the fraction of cartilage cells produced in (c) in to treatment comprising hormones (insulin) or toxic compounds (PSF as they can be toxic at higher concentration, etc.).
Regarding claim 21, the claimed limitation is substantially similar to claim 10, and the limitation of “removes pre-apoptotic and apoptotic cells” in step (b) is considered the same as “enriches for non-pre-apoptotic cells” of claim 10. As Brown et al. teach the selective lysis of apoptotic cells by ACK buffer, this limitation is met by the teaching of Brown et al. As discussed above, the combined teachings of Brown et al. in view of Naughton et al. and Eslaminejad et al. would meet the limitations of claim 21.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention.
Response to Amendment/Argument
The declaration under 37 CFR 1.132 filed 11/10/2025 by Dr. Guilak is insufficient to overcome the 103 rejection of claims 1-4, 6-11, 13-18 and 20 based upon Brown in view of Eslaminejad and Naughton as set forth in the last Office action.
The declaration discussed the 103 rejection based on Brown, Eslaminejad and Naughton, and stated that the treatment of human non-articular cartilage cells with a hypotonic solution to produce cells that are capable of neocartilage production is not obvious. The rationales of this opinion is based on the alleged variability depending on source, tissue depth and culturing conditions.
The declaration stated that the response of chondrocytes to a hypotonic solution is not uniform across all cell types, and the increased susceptibility of apoptotic cells to hypotonic lysis reported in Brown cannot be assumed to occur in other chondrocyte populations. The Examiner respectfully disagrees with this allegation. The swelling of cells under hypotonic solution is universal regardless of cell types due to the osmosis, and eventually leading to cell lysis. This is extremely well established fact. Thus, one skilled in the art would understand that any mammalian cell would behave the same way (i.e. swelling and eventual cell lysis in the hypotonic solution) regardless the cell type. However, there are parameters that change the effect of hypotonic solution including, but not limited to: 1) the different rigidity of cell membrane based on cell types or the health of the cells; 2) concentration of solutes in the hypotonic solution (osmotic pressure); and 3) the duration of hypotonic treatment.
The rigidity of cell membrane is known to be changed in the apoptotic cells of the same cell types, and this is evident from the same chondrocytes shown by Brown et al. Brown teaches that apoptotic cells in the population of chondrocytes which have lost excess cell membrane through blebbing (p.901, 1st col.).
The claim rejection to claims 2, 10 and 16, and now including claim 21, directed to the non-articular cartilage cell is based on the physiological fact that a hypotonic solution would render any mammalian cell swelling and lysis (osmotic rupture) as evident by the teaching of Brown is applicable to any other types of cell including non-articular chondrocytes. In other words, the hypotonic solution would selectively lyze apoptotic cells as they would lyze apoptotic cells faster than the healthy and normal cells. The hypotonic buffer taught by Brown is ACK buffer which is designed to lyse red blood cells and Brown showed that the ACK buffer would be able to selectively lyse apoptotic chondrocytes. In the absence of any evidence that non-articular chondrocytes would be lysed by the ACK buffer or the apoptotic non-articular chondrocytes would not be lysed by the ACK buffer, it is reasonable to expect that the apoptotic cells of non-articular chondrocytes would be lysed by the ACK buffer because they would have less rigid cell membrane than healthy counterparts as shown in articular chondrocytes by Brown et al.
It is noted that the effect of hypotonic solution is compared within the population and the effect of the hypotonic solution would be different based on each individual cell’s cell membrane rigidity. As it is well established that apoptotic cells would have less rigid cell membrane than normal healthy cells, the selective effect of a hypotonic solution is expected in apoptotic cells vs. healthy cells in the population regardless of the cell types, tissue depth or culture condition as they are in the same cell population.
Thus, it is the Examiner’s position that one skilled in the art would have a reasonable expectation of success in selectively lysing apoptotic cells over healthy cells in the population of non-articular chondrocytes.
The declaration further alleged that it is an overstatement to say that Eslaminejad shows that “costal chondrocytes can replace articular chondrocytes for their property in producing engineered cartilage because Eslaminejad does not produce cartilage or evaluate any functional aspects. The Examiner respectfully disagrees with this allegation. The claim rejection did not state that Eslaminejad produces cartilage or evaluate any functional aspects. Rather the claim rejection stated that Eslaminejad teaches that costal chondrocytes are viable alternative to articular chondrocytes for manufacturing 3D cartilage scaffold, and thus, it would have been obvious to a person skilled in the art to try costal chondrocytes for the method of Brown et al. with a reasonable expectation of success. It is noted that the 103 rejection requires a reasonable expectation of success and some degree of predictability, but not a conclusive proof of evidence (See MPEP2143.02). The combined teaching of Brown and Eslaminejad as presented in the 103 rejection is based on a reasonable expectation of success and the predictability of using non-articular costal chondrocytes for tissue engineering of Brown replacing articular chondrocytes. There is no evidence presented in the declaration that the costal chondrocytes of Eslaminejad cannot replace articular chondrocytes of Brown. Rather the arguments are based on the differences between the costal chondrocytes and articular chondrocytes. This is not persuasive as Eslaminejad acknowledged the differences in chondrocytes from different sources. However, Eslaminejad also teach that costal and articular chondrocytes are similar in their propagation patterns and aggrecan and type II collagen gene expression, and they suggested that costal chondrocytes could be alternative to articular chondrocytes in tissue engineering of 3D cartilage constructs. Based on the teachings of Eslaminejad, one skilled in the art would have a reasonable expectation of success in replacing articular chondrocytes of Brown et al. with costal chondrocytes for the method of Brown et al.
Based on the above discussion, it is the Examiner’s position that the declaration is not effective to overcome the 103 rejection.
Applicant's arguments filed 11/10/2025 have been fully considered but they are not persuasive.
Regarding the 102 rejection, applicant’s argument directed to the presence of pre-apoptotic cells not always being present in cell populations, this argument has been addressed in the previous OA mailed on 8/11/2025.
Claim 1 discloses in step (b) such that the sample of cartilage cells from (a) is subjected to a treatment that enriches for non-pre-apoptotic cells, and the claim discloses that adding a hypotonic solution to the sample of cells to induce cell swelling is the treatment. Thus, this limitation is understood that when the sample of cartilage cells is treated with a hypotonic solution, then the sample of cartilage cells would inherently enrich for non-pre-apoptotic cells. As Brown teaches the step of treating fetal ovine articular chondrocytes (foACs) or juvenile bovine articular chondrocytes (jbAC) contaminated with red blood cell (RBC) using the hypotonic ACK buffer (ammonium-chloride-potassium buffer) to selectively eliminate RBCs and additionally reduced the number of apoptotic chondrocytes in the cell isolates, the treating with the hypotonic ACK buffer would meet the step (b) of claim 1.
Applicant argued that it is impossible to surmise that the population of foACs or jbACs of Brown contained pre-apoptotic cells or not. Applicant asserted that pre-apoptosis is a distinct, but semi-related condition and it cannot be assumed that pre-apoptotic cells are also inherently present in a cell population.
It is submitted that claim 1, step (a) requires obtaining a sample of cartilage cells, and there is no active step of determining if the cartilage cell comprises a mixed population of non-pre-apoptotic cartilage cells and pre-apoptotic cartilage cells. The wherein clause of Claim 1, step (b), is interpreted as the inherent feature of the cartilage cell sample. Furthermore, Brown et al. teach that the ACK treatment would reduce the number of apoptotic chondrocytes in the cell isolates (Abstract). This teaching of Brown et al. would indicate that the isolated cartilage cells of Brown et al. inherently comprise the cells in the apoptotic process. Still further, Heraud et al. (2000, Ann. Rheum. Dis.) teach that normal as well as osteoarthritic human articular cartilage contain apoptotic cells (Abstract). As the presence of pre-apoptotic cells is a prerequisite of apoptosis (i.e. an earlier stage of apoptosis), the sample of cartilage cells taught by Brown et al. must contain pre-apoptotic cells. Thus, when one obtains a sample of cartilage cells, it is expected that the sample of cartilage cells would inherently comprise mixture of normal non-apoptotic cells as well as those in the apoptotic process.
Applicant cited several prior art including Gibson et al., Pettenuzzo et al., Bhola et al., and Sabbagh et al., however, there were no copies of these cited references attached to the instant amendment. Thus, the Examiner has not fully reviewed the contents of the cited references but relied on what applicant discussed in the remarks. Based on the applicant’s arguments, it is understood that these references were cited in support of showing conditions and/or other factors that determine the apoptosis in different cells, and concluded that apoptosis characteristics are not being universal to all cell types and cells within a single population. The Examiner respectfully disagrees with this allegation. Applicant implies that each different cell type would have different characteristics of apoptosis. However, it is submitted that the process and the characteristics of apoptosis is extremely well established in the art including membrane blebbing, breakdown/degradation of the cytoskeleton, etc. and apoptosis is highly conserved mechanism by which eukaryotic cells commit suicide, and apoptosis occurs normally during development and aging and as a homeostatic mechanism to maintain cell populations in tissues according to Pucci et al. (2000, Neoplasia) and Elmore (2007, Toxicol. Pathol.).
The effect of ACK buffer taught by Brown et al. is mainly affecting the cells based on osmotic pressure as a hypotonic solution. Brown et al. teach that ACK buffer induced cell rupture is more susceptible in the chondrocytes having cytoskeletal breakdown and loss of cell membrane through blebbing (p.901, 1st col.). While the effect of ACK buffer would be obviously different from cell to cell based on cell type and condition, however, when cells undergo the apoptotic process, they would have a weaker cellular structure due to breakdown of cytoskeletal and membrane blebbing that render them more susceptible to the osmotic pressure caused by ACK buffer compared to the normal healthy counterpart. Thus, one skilled in the art would expect that the effect of a hypotonic solution causing osmotic pressure like the ACK buffer would be universal to the cells undergoing apoptotic process. Thus, whether or not the articular chondrocytes are different from the costal chondrocytes, the effect of the hypotonic ACK buffer to the sample of costal chondrocytes would be the same, i.e. pre-apoptotic or apoptotic cells are easier to be ruptured than non-apoptotic healthy cells within the cell population of costal chondrocytes as for the population of articular chondrocytes.
Applicant stated that the term “pre-apoptotic” of “pre-apoptosis” is rarely used in the research, and thus, it is not obvious that the hypotonic buffer would burst pre-apoptotic cells or not. As discussed in the previous OA, the Examiner has acknowledged that Brown et al. do not particularly use the term “pre-apoptotic cells”, and the “pre-apoptotic cells” are understood as the cells in the state between healthy and apoptotic cells, and they can be apoptotic or healthy (p.7 of the remarks of the OA mailed on 4/1/2025). However, the instant claims do not require a population of “pre-apoptotic cells” are needed to be ruptured by the ACK buffer. Claim 1 only requires that the sample of cartilage cells are enriched for non-pre-apoptotic cells by adding a hypotonic solution to the sample of cells to induce cell swelling.
Regarding the argument directed to the teachings of Eslaminejad or Naughton, as discussed in the previous OA, they are cited to address chondrocytes from different sources and one skilled in the art would utilize the ACK buffer treatment to generate purer and improved properties of cartilage tissue prepared by the costal chondrocytes and human chondrocytes. As discussed above, one skilled in the art would recognize that the treatment of ACK buffer is considered to be effective in any cells by selective killing those in the apoptotic pathway, and thus, utilize other type of chondrocytes and/or human sources with a reasonable expectation of success.
There is no evidence that the increased susceptibility of the apoptotic cells or cells in the apoptotic pathway than healthy counterparts to the ACK buffer shown in Brown et al. is different in human costal chondrocytes. In the absence of such evidence and the discussion presented above, it is the Examiner’s position that the teachings of Brown et al. anticipate the subject matter of claims 1 and 6-8, and the combined teachings of the cited reference render the claimed invention of claims 1-4, 6-11, 13-18 and 20-21 obvious.
Conclusion
No claims are allowed.
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/TAEYOON KIM/Primary Examiner, Art Unit 1631