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 .
Request for Continued Examination
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicants’ submission filed on 07/23/2026 has been entered.
DETAILED ACTION
Claims 1-3, 5-7, 10, 12 and 13 are pending in the Claim Set filed 7/23/2026.
Claims 1-3, 7 and 12 have been amended.
Claims 4, 8, 9 and 11 are cancelled.
Herein, claims 1-3, 5-7, 10, 12 and 13 are for examination.
Withdrawn Rejections
The rejection of claim 12 under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends is withdrawn in view of the claim amendments. Applicant’s arguments are moot in view thereof.
The rejection of claims 1-3, 5-7, 10, 12 and 13 under 35 U.S.C. 103 as being unpatentable over Matthews (US20150315540, cited in IDS filed 3/01/2023) in view of Sawada et al (Cell removal with supercritical carbon dioxide for acellular artificial tissue, J. Chem. Technol. Biotechnol. p.943, 2008, of record), Allaire (Cell-free arterial grafts: Morphologic characteristics of aortic isografts, allografts, and xenografts in rats, Journal of Vasculature Surgery, March, 445, 1994), Woods et al (Matrix Alteration and Not Residual Sodium Dodecyl Sulfate Cytotoxicity Affects the Cellular Repopulation of a Decellularized Matrix, Tissue engineering, p.2975; 2006, of record), Booth et al (Tissue engineering of cardiac valve protheses I: Development and histological characterization of an acellular porcine scaffold, The Journal of Heart Valve Disease, p.457, 2002, of record), Pang et al (A rabbit anterior cornea replacement derived from acellular porcine cornea matrix, epithelial cells and keratocytes, Biomaterials, July, p.7257, 2010) and Fu et al (Decellularization of porcine skeletal muscle extracellular matrix for the formulation of a matrix hydrogel: a preliminary study, Jan., p.740, 2016) is withdrawn in favor of the new grounds of rejection.
The 35 USC § 112(a) rejection set forth below has been reformulated.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(a):
(a) IN GENERAL - The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), first paragraph:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-3, 5-7, 10, 12 and 13 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention. Rejection is reformulated that is necessitated by claim amendments.
There is lack of written description for claim 1.
Claim 1 (Currently Amended) recites:
A method for decellularizing an aorta, the method comprising:
placing the aorta in a pretreatment chamber that is separate from the environmental chamber;
pretreating the aorta, with a surfactant comprising sodium dodecyl sulfate under agitation in the pretreatment chamber; wherein the aorta is pretreated with the surfactant in the pretreatment chamber for a time of less than 48 hours;
forming a decellularization solution in a presaturation chamber in an environmental chamber that is separate from the pretreatment chamber, wherein the decellularization solution comprises carbon dioxide, water, and one or more polar solvents at a temperature greater than 31.1 °C, wherein the one or more polar solvents comprises ethanol, methanol, isopropanol, acetic acid, or a combination thereof, wherein the carbon dioxide is maintained at a pressure greater than 7.38 megapascals to form supercritical carbon dioxide;
placing the aorta in a treatment chamber located in the environmental chamber; and treating the aorta with the decellularization solution from the presaturation chamber, wherein one hour after treating an aorta with the supercritical carbon dioxide of the decellularization solution the treated an aorta contains less than about 0.004 volume % of surfactant,
wherein water retention of the aorta is greater than 97.3%,
wherein the method facilitates removal of cells from the aorta, so that the aorta treated with the supercritical carbon dioxide of the decellularization solution contains less than 0.05 micrograms of DNA per milligram of dry tissue after the aorta is exposed to the decellularization solution.
The MPEP states that the purpose of the written description requirement is to ensure that the inventor had possession, at the time the invention was made, of the specific subject matter claimed. The courts have stated:
"To fulfill the written description requirement, a patent specification must describe an invention and do so in sufficient detail that one skilled in the art can clearly conclude that "the inventor invented the claimed invention." Lockwood v. American Airlines, Inc., 107 F.3d 1565, 1572, 41 USPQ2d 1961, 1966 (Fed. Cir. 1997); In re Gostelli, 872 F.2d 1008, 1012, 10 USPQ2d 1614, 1618 (Fed. Cir. 1989) ("[T]he description must clearly allow persons of ordinary skill in the art to recognize that [the inventor] invented what is claimed."). Thus, an applicant complies with the written description requirement "by describing the invention, with all its claimed limitations, not that which makes it obvious," and by using "such descriptive means as words, structures, figures, diagrams, formulas, etc., that set forth the claimed invention." Lockwood, 107 F.3d at 1572, 41 USPQ2d at 1966." Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
Further, for a broad generic claim, the specification must provide adequate written description to identify the genus of the claim. In Regents of the University of California v. Eli Lilly & Co. the court stated:
"A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) ("In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus ...") Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
Firstly, the term ‘aorta’, encompasses a broad genus of aorta that includes an aorta from various species, e.g. Sun et al (USP 8,637,067) teaches decellularized aorta [col.4, lns.4-1; col.4, lns.53-61] comprising aorta from human, non-human primates (e.g. monkeys, baboons, or chimpanzees), pig, cow, horse, goat, sheep, dog, cat, rabbit, guinea pig, gerbil, hamster, rat, or mouse ([col.3, lns.60-64; col.8, lns.1-6]; See entire document). Also, Niklason et al (US2009/0028817) teaches decellularized aortas of different species comprising human, cow, pig, monkey, rat and dog ([0151-0164]; See entire document)
MPEP §2163 does state that for a generic claim the genus can be adequately described if the disclosure presents a sufficient number of representative species that encompass the genus: aorta. If the genus has a substantial variance, the disclosure must describe a sufficient variety of species to reflect the variation within that genus. A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, or chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 197 3).
In the reply filed 7/23/2026, Applicants argue that claim 1 has been amended as shown (Claim Set filed 7/23/2026) to recite a method for decellularizing a natural tissue. Support for said amendments may be found at least in paragraphs [0044]; [0049]-[0050] and [0062]-[0068], and in FIGs. 3 and 5-11 of the originally filed application.
Specification and Figures disclose the following:
[0044] The present invention is directed to a system and method for decellularizing tissue (e.g., porcine aorta) for use in tissue engineering applications using supercritical CO2 and one or more polar solvents (e.g., ethanol, methanol, isopropanol, water, acetic acid, or a combination thereof). The present invention is also directed to a decellularization solution for removing cellular matter from the tissue. The system, method, and decellularization solution of the present invention can allow for decellularization of a native scaffold while maintaining ECM fiber integrity, which is a problem seen during conventional detergent and saline washing methods. Further, the system and method can include a pretreatment solution or wash that includes a surfactant such as sodium dodecyl sulfate. In some embodiments, the pretreatment solution can be referred to as a first part of the decellularization solution, and the supercritical CO2 and the one or more polar solvents (e.g., ethanol, methanol, isopropanol, water, acetic acid, or a combination thereof) can be referred to collectively as a second part of the decellularization solution, where the tissue can be exposed to the first part of the decellularization solution ( e.g., the surfactant) in a separate chamber from the second part of the decellularization solution. The system and method facilitate removal of cells from the tissue so that tissue treated with decellularization solution contains less than 0.05 micrograms of DNA per milligram of dry tissue after the tissue is exposed to the decellularization solution.
Examiner Remarks:
[0044] discloses decellularizing porcine aorta (i.e. pig (porcine)) but fails to disclose a sufficient number of representative species that encompass the genus: aorta. The disclosure must describe a sufficient variety of species to reflect the variation within that genus (MPEP §2163).
[0049] Specifically, and referring to FIG. 1, in one particular embodiment, the decellularization system 100 of the present invention can include a supply of liquid carbon dioxide 1, a high-pressure valve 2, a pump 3 (e.g., a syringe pump), an environmental chamber 4, a presaturation chamber 5 containing a stir bar 12 to mix the CO2 and polar solvent (e.g., ethanol and water) to form a decellularization solution 13, a treatment chamber 6 containing the tissue to be decellularized (e.g., a porcine aorta) 7, a CO2 hand pump 8, a pressure gauge 9, a back pressure regulator 10, a treatment chamber valve 14, and an emergency vent 11. The system can also include a pretreatment chamber 15 that can include a pretreatment solution 16. In one embodiment, the pretreatment solution 16 can be a surfactant such as sodium dodecyl sulfate (SDS).
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[0050] Generally, to decellularize the tissue 7, such as a porcine aorta, the tissue 7 is loaded into the treatment chamber 6 of the decellularization system 100. The treatment chamber 6 is located in an environmental chamber 4. Then, liquid carbon dioxide 1 can be compressed in a chilled syringe pump 3 or any other suitable pump and slowly bubbled into a first high-pressure vessel, which can be referred to as the presaturation chamber 5, which is also located in the environmental chamber 4. In the presaturation chamber 5, additives (e.g., one or more polar solvents including water and ethanol) can be mixed with the carbon dioxide 1 using a stir bar 12 until the one or more polar solvents is fully dissolved in the carbon dioxide 1 to form the decellularization solution 13. The carbon dioxide 1 and additive(s) (e.g., the one or more polar solvents) can be mixed for a time period ranging from about 1 minute to about 30 minutes, such as from about 5 minutes to about 25 minutes, such as from about 10 minutes to about 20 minutes. In one particular embodiment, the carbon dioxide and additive(s) (e.g., the one or more polar solvents) can be mixed for a time period of about 10 minutes to about 15 minutes. Next, the valve 14 to the treatment chamber 6, which contains the tissue 7 to be decellularized, can be opened, and the CO2 flow through the treatment chamber 6 at a rate ranging from about 0.1 milliliters per minute to about 5 milliliters per minute, such as from about 0.2 milliliters per minute to about 3 milliliters per minute, such as from about 0.5 milliliters per minute to about 2.5 milliliters per minute. In one particular embodiment, the CO2 flow rate through the treatment chamber 6 can be about 1 milliliter per minute. In addition, the tissue 7 can be treated for a time frame ranging from about 1 minute to about 2 hours, such as from about 2 minutes to about 90 minutes, such as from about 4 minutes to about 1 hour. In one particular embodiment, the tissue 7 can be treated for about 1 hour.
[0062] Porcine aorta was obtained from a local slaughterhouse and the surrounding fatty tissue was removed. The aortic tissue was cut into thin rectangles (approximately 3 centimeters by 2 centimeters) and stored in phosphate-buffered saline (PBS) at 4°C for up to 48 hours prior to use. Each tissue specimen was dried for 15 minutes under a light vacuum using filter paper and a Buchner funnel to remove free saline prior to weighing and treatment. Drying in a vacuum oven (37°C, 38.1 centimeters of mercury (cm Hg) vacuum) was used as a negative control; changes in mass were recorded after 1, 2, 3, 6, and 24 hours. The treatment ratio and other conditions used (including temperature, pressure, and depressurization rate) were chosen to be analogous to the conditions used by K. Sawada, et al. in "Cell removal with supercritical carbon dioxide for acellular artificial tissue," Journal of Chemical Technology and Biotechnology, 83 (2008) 943-949, to allow for comparison.
. Further, Specification at paragraph [0075] states: [0075] Results for the control (dry CO2) and presaturated supercritical CO2 treatments of porcine aorta are also shown in FIG. 3. The average mass retentions are 78.6% ± 4.6% with dry CO2 and 97.3% ± I .4% with presaturated CO2; this difference is highly significant. It is evident from these results that using presaturated CO2 considerably reduces the amount of mass lost during treatment, as expected based on theory and the analogous hydrogel results.
Figure 3 taken from Drawings filed 7/8/2021 is shown below:
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Thus, paragraphs [0049]-[0050], FIG. 1 and FIG. 3 are directed to a single type of tissue: porcine aorta (as the natural tissue), without presenting further disclosure of more species of natural tissue.
For a broad generic claim, the specification must provide adequate written description to identify the genus of the claim. In Regents of the University of California v. Eli Lilly & Co. the court stated:
"A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) ("In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus ...") Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
Although the MPEP does not define what constitute a sufficient number of representative species, the courts have indicated what do not constitute a representative number of species to adequately describe a broad generic. In Gostelli, the courts determined that the disclosure of two chemical compounds within a subgenus did not describe that subgenus. In re Gostelli, 872, F.2d at 1012, 10 USPQ2d at 1618.
Furthermore, Specification at page 3 states: [0009] As such, what is needed is an improved supercritical CO2 decellularization method that also maintains the hydration state of the treated tissue. The objectives are as follows: (1) to develop a system and method that can presaturate supercritical CO2 with water; (2) to treat two model scaffolds (a model hydrogel and porcine aorta) with dry and presaturated CO2 and compare the level of dehydration observed. Also, ‘porcine aorta’ is stated in the Specification at 29 times: [0009]; [0033-0040]; [0044]; [0045]; [0049]; [0050]; [0057]; [0061]; [0062]; [0064]; [0067]; [0073]; [0075]; [0076]; [0082]; [0100], without mentioning any other type(s) of aorta.
Furthermore, the term ‘aorta’’ is broad and encompasses a wide array of different aortas, e.g., Sun et al (USP 8,637,067) teaches decellularized aorta [col.4, lns.4-1; col.4, lns.53-61] comprising aorta from human, non-human primates (e.g. monkeys, baboons, or chimpanzees), pig, cow, horse, goat, sheep, dog, cat, rabbit, guinea pig, gerbil, hamster, rat, or mouse ([col.3, lns.60-64; col.8, lns.1-6]; See entire document). Also, Niklason et al (US2009/0028817) teaches decellularized aortas of different species comprising human, cow, pig, monkey, rat and dog ([0151-0164]; See entire document)
Therefore, paragraphs [0044]; [0049]-[0050]; [0062]; and FIG. 1 and FIG. 3, fail to provide support for the broad genus term ‘aorta’ (claimed in Claim Set filed 7/23/2026).
Examiner Remarks:
[0049]; [0050]; [0062]; and FIG. 1 and FIG. 3 discloses decellularizing porcine aorta (i.e. pig (porcine)) but fails to disclose a sufficient number of representative species that encompasses the genus: aorta. The disclosure must describe a sufficient variety of species to reflect the variation within that genus (MPEP §2163).
Moreover, as stated in the Specification on page 18 at para. [0080]: Currently, there is no universally accepted standard for evaluating the extent of decellularization. This is not surprising because tissues vary greatly in stiffness, cell density, ECM composition, and numerous other characteristics, so decellularization processes must be tailored to the specific tissue of interest.
In addition, Specification (in part) at paragraph [0073] states: [0073] Hydrogels were treated with dry (control) and presaturated supercritical CO2 at 37°C and 50°C and at 13.8 megapascals (2000 psi); porcine aorta was treated at 37°C only. Thus, as shown above, Fig. 3 shows the porcine aorta was treated at 37oC.
Thus, the Disclosure of Instant Application provides support for a single species of an aorta: porcine aorta, that is treated at 37°C.
Furthermore, regarding the Hydrogel presented in Fig. 3, Specification on page 13 states the following:
Biomaterial Selection and Preparation
[0061] To further validate the overall presaturation concept, a synthetic biomaterial (a hydrogel) and a natural tissue, porcine aorta, were utilized. The hydrogel was poly(acrylic acidcoacrylamide) potassium salt (Sigma-Aldrich, St. Louis, MO), a hydrogel used previously to establish the ability of CO2 to achieve sterilization. Hydrogel powder was hydrated in excess water at 4°C for 24 hours. Excess water was removed from each hydrogel specimen by drying for 30 minutes under a light vacuum, using filter paper and a Buchner funnel. Each hydrogel was blotted onto a nylon filter and sealed inside the treatment chamber prior to the start of each trial. The weight of each gel was approximately 0.2 grams.
Accordingly, para. [0061] describes the hydrogel as the poly(acrylic acid-co-acrylamide) potassium salt. Therefore, the hydrogel is not an ‘aorta’, because the hydrogel is a synthetic copolymer. Accordingly, Fig. 3 is singularly directed to a single kind of tissue: porcine aorta.
[0063] All treatments were performed using the apparatus shown in FIG. 1. In these tests, a hydrated (or native) biomaterial was weighed, then contacted with either dry CO2 or presaturated CO2.
[0064] For decellularization treatments, porcine aorta was obtained from a local abattoir, rinsed in phosphate buffered saline (PBS) and cut into ring-shaped sections measuring about 1 centimeter in width. At this point, tissues were stored at -20°C until treatment.
Examiner Remarks:
As described above Fig. 1 is directed to a treatment chamber 6 containing the tissue to be decellularized (e.g., a porcine aorta).
Thus, [0063] discloses decellularizing porcine aorta (i.e. pig (porcine)) and [0064 is directed to porcine aorta but [0063-0064] fail to disclose a sufficient number of representative species that encompass the genus: aorta. The disclosure must describe a sufficient variety of species to reflect the variation within that genus (MPEP §2163).
[0065] is directed to ‘The standard SDS treatment is described as follows.
Examiner Remarks:
[0065] fails to disclose a sufficient number of representative species that encompass the genus: aorta.
[0066] is directed to Tissue was loaded into the treatment chamber of the supercritical CO2 apparatus as described in FIG. 1.
As described above Fig. 1 is directed to a treatment chamber 6 containing the tissue to be decellularized (e.g., a porcine aorta).
[0067] states ‘Once equilibrium was reached, the valve to the treatment chamber, which contained the porcine aorta, was opened and CO2 flow was programmed to 1 milliliter per minute at the pump inlet.’
[0068] describes: Hematoxylin and Eosin (H&E) Staining. After CO2 treatment (i.e., referring to para. [0067, tissues were fixed in 10% neutral buffered formalin for at least 24 hours and embedded in paraffin. Tissues were then cut into 5 μm sections using a microtome and deparaffinized by immersion in xylene (3 times), 100% ethanol, 95% ethanol, 80% ethanol, and finally water. The tissues were stained with hematoxylin for 7 minutes, washed with water and ammonia, and then stained with eosin for 2 minutes before being dehydrated by immersion in 80% ethanol, 95% ethanol, 100% ethanol, and finally xylene (3 times). A coverslip was mounted on slides, which were then viewed using a light microscope (Nikon E600, Tokyo, Japan) after waiting at least 24 hours for the slides to dry.
Examiner Remarks:
Thus, [0067] and [0068] discloses decellularizing porcine aorta (i.e. pig (porcine)) but fails to disclose a sufficient number of representative species that encompass the genus: aorta.
Fig. 3, as described above is directed to porcine aorta. FIG. 3 is directed to a single type of tissue: porcine aorta), without presenting further disclosure of more species of natural tissue. The disclosure must describe a sufficient variety of species to reflect the variation within that genus (MPEP §2163).
Regarding Figs. 5-11:
Specification [0035]: FIG. 5 shows hemotoxylin and eosin (H&E) stained sections of untreated (a, d), SDS treated (b, e), and dry CO2-treated (c, f) porcine aorta at low (top row) and high (bottom row) magnification, where the scale bars represent 50 micrometers.
Specification [0036]: FIG. 6 shows hemotoxylin and eosin (H&E) stained sections of untreated (a,d), water/CO2-treated (b, e), and water/Ls-54/CO2-treated (c, f) porcine aorta at low (top row) and high (bottom row) magnification, where the scale bars represent 50 micrometers.
Specification [0037]: FIG. 7 shows hemotoxylin and eosin (H&E) stained sections of untreated (a, d), ethanol/CO2-treated (b, e), and ethanol/water/CO2-treated (c, f) porcine aorta at low (top row) and high (bottom row) magnification, where the scale bars represent 50 micrometers.
Specification [0038]: FIG. 8 is a graph showing the DNA concentration in micrograms/milligram of porcine aorta decellularized via various methods, where values below the horizontal line indicate complete decellularization.
Specification [0039]: FIG. 9 shows hemotoxylin and eosin (H&E) stained sections of untreated (a, d), SDS treated (b, e), and SDS/CO2-treated (c, f) porcine aorta at low (top row) and high (bottom row) magnification, where the scale bars represent 50 micrometers.
Specification [0040]: FIG. 10 is a graph showing the DNA concentration in micrograms/milligram of porcine aorta decellularized via various methods, where values below the horizontal line indicate complete decellularization; and
Examiner Remarks:
Thus, Figs. 5-10 are directed to a single type of aorta: porcine aorta, without presenting further disclosure of more species of natural tissue. The disclosure must describe a sufficient variety of species to reflect the variation within that genus (MPEP §2163). Further, Fig. 11 is directed to washing SDS.
Specification [0041]: FIG. 11 is a graph showing the results of a residual sodium dodecyl sulfate (SDS) quantitation assay, where SDS was quantified before and after washing with either PBS or supercritical CO2, where 1 hour of supercritical CO2 treatment compares similarly to 24 hours of PBS washing.
Specification [0098]: DNA quantification of the hybrid method, along with the results of the other treatments, can be seen in FIG. 10. The Figure shows a level of DNA removal similar to the standard SDS treatment, below the threshold for decellularization with a concentration of 0.036 μg DNA/mg dry tissue. This is a promising result, as the hybrid method is able to achieve the original objective of decellularizing effectively while avoiding dehydration of the tissue.
Specification [0099]: Residual SDS Quantitation: Next, residual SDS from the standard and hybrid treatments was quantified using an SDS Detection and Estimation Kit (G Biosciences, St. Louis, MO) as described above. Removal of SDS is a consideration for scaffold viability, as cytotoxicity is observed for many cell types at concentrations greater than about 0.002% SDS. Residual SDS was quantified for the standard SDS treatment and the SDS/supercritical CO2 hybrid treatment, as shown in FIG. 11. FIG. 11 shows that one hour of supercritical CO2 treatment removes about as much SDS as 24 hours of washing with PBS, which results in a significant time savings. PBS washes also have diminishing returns, making the wash step last several days in many protocols to reduce SDS below cytotoxic level. Thus, the supercritical CO2 treatment of the present invention could compare even more favorably over longer time periods. This finding also indicates solubility of SDS in the supercritical CO2 treatment solution, and this is not surprising since SDS is an organic molecule with similar molecular weight to other molecules extracted by supercritical CO2, such as caffeine.
Examiner Remarks:
Thus, Figs. 11 is directed to residual SDS Quantitation (See Figs. 10-11). FIG. 10 is a graph showing the DNA concentration in micrograms/milligram of porcine aorta decellularized via various methods, where values below the horizontal line indicate complete decellularization.
Thus, Fig. 11 fails to disclose a sufficient number of representative species that encompass the genus: aorta. The disclosure must describe a sufficient variety of species to reflect the variation within that genus: aorta (MPEP §2163).
If the genus has a substantial variance: aorta, the disclosure must describe a sufficient variety of species to reflect the variation within that genus. A description of what a material does, rather than of what it is, usually does not suffice. Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. One cannot describe what one has not conceived.
Sun et al (USP 8,637,067) teaches decellularized aorta [col.4, lns.4-1; col.4, lns.53-61] comprising aorta from human, non-human primates (e.g. monkeys, baboons, or chimpanzees), pig, cow, horse, goat, sheep, dog, cat, rabbit, guinea pig, gerbil, hamster, rat, or mouse ([col.3, lns.60-64; col.8, lns.1-6]; See entire document). Also, Niklason et al (US2009/0028817) teaches decellularized aortas of different species comprising human, cow, pig, monkey, rat and dog ([0151-0164]; See entire document)
Accordingly, the genus term ‘aorta’ covers many different species, so that disclosing only porcine aorta, as disclosed in the Specification and Drawings, fails to sufficiently represent the full scope of the broad genus: aorta.
Therefore, it is deemed that the Specification and Drawings fail to provide adequate written description for the genus: aorta and does not reasonably convey to one skilled in the relevant art that the inventors, at the time the application was filed, had possession of the entire scope of the claimed invention.
The remaining rejected claims do not resolve the issue regarding the term ‘aorta’ and are rejected because they are dependent on a rejected claim.
Response to Arguments
Applicants argue in the reply filed 7/23/2026 that claim 1 has been amended as shown (Claim Set filed 7/23/2026) to recite a method for decellularizing a natural tissue. Support for said amendments may be found at least in paragraphs [0044]; [0049]-[0050] and [0062]-[0068], and in FIGs. 3 and 5-11 of the originally filed application.
Applicants’ arguments have been fully considered but they are not persuasive, because the Specification at paragraphs [0044]; [0049]-[0050] and [0062]-[0068], and Drawings FIGs. 3 and 5-11, as discussed above, or anywhere else in the Specification and/or Drawings fail to provide adequate written description for the broad genus term ‘aorta’, because Instant Application only discloses a single tissue: porcine aorta. Further, for a broad generic claim, the specification must provide adequate written description to identify the genus of the claim. In Regents of the University of California v. Eli Lilly & Co.
Furthermore, ‘porcine aorta’ is stated in the Specification at 29 times: [0009]; [0033-0040]; [0044]; [0045]; [0049]; [0050]; [0057]; [0061]; [0062]; [0064]; [0067]; [0073]; [0075]; [0076]; [0082]; [0100], without mentioning any other types of aorta. Additionally, Drawings Figs. 1-11 fail to provide adequate written description for the broad genus term ‘aorta’.
Here, the Instant Specification and Drawings do not set forth any description of an aorta other than porcine aorta. Therefore, it is deemed that the Instant Specification and Drawings fail to provide adequate written description for the genus: aorta and does not reasonably convey to one skilled in the relevant art that the inventors, at the time the application was filed, had possession of the genus: aorta.
New Grounds of Rejection
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 of this title, 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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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 under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 1-3, 5-7, 10, 12 and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Matthews (US20150315540, cited in IDS filed 3/01/2023) [Matthew] in view of Allaire (Cell-free arterial grafts: Morphologic characteristics of aortic isografts, allografts, and xenografts in rats, Journal of Vasculature Surgery, March, 445, 1994, of record) [Allaire], Woods et al (Matrix Alteration and Not Residual Sodium Dodecyl Sulfate Cytotoxicity Affects the Cellular Repopulation of a Decellularized Matrix, Tissue engineering, p.2975; 2006, of record) [Woods], Booth et al (Tissue engineering of cardiac valve protheses I: Development and histological characterization of an acellular porcine scaffold, The Journal of Heart Valve Disease, p.457, 2002, of record) [Booth], Sabirzyanov et al (Solubility of Water in Supercritical Carbon Dioxide, High Temperature, p.203, Nov. 2002) [Sabirzyanov] and Sawada et al (Cell removal with supercritical carbon dioxide for acellular artificial tissue, J. Chem. Technol. Biotechnol. p.943, 2008, of record) [Sawada].
Claim Interpretation
The claims are in open format. The transitional term ‘comprising’ is open-ended and does not exclude additional, unrecited elements or method steps. See, e.g. Mars Inc. V. H. J. Heinz Co., 377 F.3d 1369, 1376, 71 USPQ2d 1837, 1843 (Fed. Cir. 2004) (See MPEP 2111.03).
Regarding claims 1, 2, 3, 5, 6, 7, 10, 12 and 13,
Matthews teaches a decellularization method for tissue, wherein the method comprises exposing a natural tissue: porcine aorta, to water-saturated supercritical CO2, wherein the method comprises prior to exposing the tissue to the water-saturated supercritical CO2, saturating a stream of supercritical CO2, wherein the tissue is exposed to the water-saturated supercritical CO2 at a treatment temperature of about 35°C to about 40°C, wherein the natural tissue is porcine aorta, that is exposed to the water-saturated supercritical CO2 at a constant flow rate at about 0.5 mL/min to about 2.5 mL/min, wherein the method comprises exposing porcine aorta to the water-saturated liquid CO2 at a pressure of about 7.38 MPa (megapascals), (Title: PRESATURATION OF SUPERCRITICAL CO2 WITH WATER FOR DECELLULARIZATION OF MATRICES; Abstract; [0031-0032]; [0046-0047]; [0061-0062]; See entire document). Further, the method of decellularization of porcine aorta, as taught by Matthews encompasses using a system as shown below (See Drawings: Figs 1,2,4). Matthew teaches that decellularization environmental chamber comprises a pressure cell, called the treatment chamber, wherein the porcine aorta was loaded (i.e., placed) into the treatment chamber ([0058]; [0061-0062]; FIG. 4). Matthews teaches treatment time was determined by using a treatment ratio of 60 min per 0.25-gram tissue [0062]. e.g., Table 4 shows Native mass of about 0.28 g, which would amount to treating the porcine aorta for about 60 min (1 hour). Furthermore, Matthews teaches that method comprises a pump (syringe pump) that compresses the carbon dioxide (Fig. 4; [0053]). Matthews teaches completely (effectively) saturating super critical CO2, wherein the liquid carbon dioxide was thoroughly mixed with water for about 15 minutes to ensure that the super critical CO2 was really humidified and saturated prior to exposing to tissue ([0031]; [0052-0054]). Matthews teaches that the cold trap experiments show that the presaturation method used can effectively saturate scCO2 with water (0073]; Claims 1-16).
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Thus, the teachings of Matthews renders obvious a method for decellularizing porcine aorta, [0039]; [0040-0045]; [0061-0064]; Table 3; [0072]) comprising forming a decellularization solution in a Presaturation Chamber comprising CO2 and water inside an Environmental Chamber and treating the porcine aorta, with water-saturated supercritical CO2 in the treatment chamber (See Fig. 4: Environmental Chamber includes a Presaturation Chamber and a Treatment Chamber), wherein the decellularization solution comprises carbon dioxide (CO2) and water, wherein the water-saturated supercritical CO2 is provided at a treatment temperature of about 35°C to about 40° C and maintained at a pressure greater than 7.38 (i.e., supercritical CO2 has critical conditions of 31.1°C and 7.38 MPa, as taught by Matthews [0047]) and placing porcine aorta in the Treatment Chamber in an Environmental Chamber and treating the porcine aorta in the Treatment Chamber with the decellularization solution obtained from the Presaturation Chamber in the Environmental Chamber.
Matthew teaches presently disclosed are methods that provide a major step in developing an effective scCO2-based decellularization method by preventing dehydration of porcine aorta. Because of the high-water content in mammalian tissues (e.g., greater than 80% in a porcine aorta) maintenance of the natural hydration state of the tissue is important to fabricating a suitable TE scaffold [0045]. Matthews teaches in the presently disclosed methods; the extraction of water is inhibited from occurring at all. A simple presaturation method is provided by using water saturated scCO2. The presaturated scCO2 is then contacted with the porcine aorta, but water is not substantially extracted from the tissue. That is, the presaturated scCO2 cannot dissolve any additional water due to the amount of water already within the presaturated scCO2 flow stream, since the amount of water presaturated can be close to or at the saturation limit of the scCO2. In particular embodiments, the flow rate can be relatively low, such as less than about 3 mL/min (e.g., about 0.5 mL/min to about 2.5 mL/min) [0046]. This flow rate lies within the decellularization solution is delivered to the treatment chamber at a flow rate ranging from about 0.1 millimeters per minute to about 5 milliliters per minute. In the case where the claimed ranges overlap or lie inside ranges disclosed by the prior art a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257,191 USPQ 90 (CCPA 1976); In re Woodruff, 91 9 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). MPEP 2144.05. Matthews teaches often decellularization researchers will combine multiple agents together to create a unique protocol [0007].
Additionally, Matthews teaches the objective of any decellularization method is twofold: (1) the removal of all cellular material, and (2) the preservation of the physical and chemical properties of the extracellular matrix (ECM) [0005]. The most common form of chemical treatments involves chemical detergents. These can be ionic, nonionic, or zwitterionic. Further, Matthews teaches physical treatment can be used to unattach cells from the ECM. Agitation and sonication can be used to burst cells or shake them off of the ECM [0010]. Matthews teaches a fully decellularized material is characterized by less than 50 nanograms of double-stranded DNA per mg ECM (dry weight) [0016]. Matthews teaches that it is well known that incomplete decellularization of an ECM scaffold prior to implantation will trigger an adverse immune response in the host [0019]. Further, Matthews teaches that a tissue or organ to be used in a graft or implant must be fully acellular and sterile and must also be mechanically and chemically similar to the native tissue or organ [0028].
Matthews differs from claim 1 in that the document does teach placing the porcine aorta in a pretreatment chamber that is separate from the environmental chamber, pretreating the porcine aorta with a surfactant comprising sodium dodecyl sulfate (SDS) under agitation in the pretreatment chamber, wherein the porcine aorta is pretreated with the surfactant in the pretreatment chamber for a time of less than 48 hours (recited in instant claim 1); wherein the tissue is pretreated with the surfactant in the pretreatment chamber for a time period of about 8 hours to less than 48 hours (recited in instant claim 12), wherein one hour after treating the porcine aorta with the supercritical carbon dioxide of the decellularization solution the treated natural tissue contains less than about 0.004 volume % of surfactant (recited in claim 1). Matthews further differs from instant claims in that the document does teach that decellularization solution comprising water-saturated supercritical CO2 further contains ethanol.
However, Allaire, Woods, Booth, Sabirzyanov and Sawada, as a whole, cure the deficiencies.
Allaire teaches we developed a method to remove arterial cells from rat and guinea pig abdominal aortas with sodium dodecylsulfate (SDS). Treatment with SDS results in the formation of an extracellular matrix tube with morphologically intact elastin and collagen networks that is easy to suture and immediately blood tight after in vivo grafting (p.447, first para., left col.). Particularly, Allaire teaches the aortas to be decellularized were incubated for 15 hours (reads on instant claims 1, 12) at 370 C in 0.1 % SDS in distilled water with gentle agitation (p.447, second para., right col.). Allaire teaches treatment with SDS led to a complete loss of cellular structures from the three layers of the arterial wall, but the main components of the extracellular matrix were conserved, including medial elastin and collagen networks (Fig. 1). Smooth muscle cell-specific actin staining was absent. A few compacted nuclear residues were present (Fig. 2). The interface between the lumen and the decellularized matrix consisted of a fine network of proteoglycans and associated glycoproteins, laying on an intact internal elastic lamina as shown on transmission electron microscopic view. Our preliminary studies indicated that less powerful detergents, such as Triton X-IOO (trimethyl ammonium hydroxide), Chaps (3-[(3-Cholamidopropyl) dimethylammonia]-1-propane sulfonate), or Tween, did not produce similar decellularization (p.448-449, right col, Results; See entire document). Moreover, Allaire teaches that sodium dodecylsulfate-induced decellularization appears to reduce immune injury in arterial allografts with preservation of medial elastin and the presence of very few adventitial inflammatory cells. Sodium dodecylsulfate-treated grafts were associated with regular, noninflammatory, elastin-rich intimal thickening (p.455, left col. third para.).
Thus, one of ordinary skill in the art would have been motivated to treat an aorta, e.g., porcine aorta, using SDS for about 15 hours because SDS is more effective at decellularizing an aorta than other detergents such as Triton X-IOO (trimethyl ammonium hydroxide), Chaps (3-[(3-Cholamidopropyl) dimethylammonia]-1-propane sulfonate), or Tween and treatment with SDS led to a complete loss of cellular structures from the three layers of the arterial wall, wherein the main components of the extracellular matrix were conserved, including medial elastin and collagen networks as taught by Allaire.
Woods teaches that it has been suggested that residual cytotoxic sodium dodecyl sulfate (SDS) is responsible for the low levels of cell in-growth observed in SDS decellularized tissues. To determine whether this is the case, we used 2 washing methods to remove residual SDS and extensive biochemical, mechanical, and structural analyses to determine the effects of SDS-based decellularization on porcine anterior cruciate ligament (ACL) tissue and its propensity for cellular repopulation. The level of residual SDS in decellularized tissue was reduced using 2 different washing techniques (pH = 9 buffer, 75% ethanol). After washing in pH = 9 or 75% ethanol, residual SDS concentrations in decellularized tissues were found to be approximately 8 and 23 times less than reported SDS cytotoxic levels, respectively. Further, Woods teaches for tissue from the mid-substance of the ACL, treatment with SDS achieved 100% removal of visible cell nuclei and the cytoskeletal protein vimentin from the mid-substance of B-ACL-B grafts. These observations matched the effectiveness of SDS in removing cellular materials from vascular tissues (encompasses porcine aorta, i.e., an arterial vessel in the circulatory system), heart valves, rat tail tendons, and rabbit bone–patellar bone grafts and in other studies (p.2979, Table 1; Fig. 1; (p.2981, left col. Bottom paragraph; Figs. 1 and 2). Further, Woods teaches that there was significantly more residual SDS in the pH9-washed tissue than in the EtOH (ethanol)-washed tissue (p<0.05). The pH9-washed tissue contained about 1.38 µg SDS/mg dry tissue, whereas the EtOH-washed tissue contained about 0.44 µg SDS/mg dry tissue (p.2979, left col. SEE, Residual SDS and cytotoxicity; p.2980, right col.). The lower cytotoxic threshold for residual SDS in tissue is approximately 10 µg /mg dry tissue. Levels of SDS found in pH9- and EtOH- washed tissue were less than the reported cytotoxic level by approximately 8 and 23 times, respectively. Also, no significant differences were found in the growth of primary porcine ACL fibroblasts after 1 week in the presence of 1.38 mg/mL or 0.44 mg/mL of SDS from that of controls containing no SDS in the cell culture medium (p.2979). Woods teaches cellular repopulation of SDS-treated decellularized ACL matrix was equivalent in samples containing residual SDS with concentrations approximately 8 and 23 times less than the toxicity limit (p/2981, right col.).
Thus, one of ordinary skilled in the art would have been motivated to provide ethanol as a polar solvent to wash SDS from decellularized porcine aorta having a reasonable expectation of success that ethanol would substantially remove SDS from a decellularized aorta.
Booth teaches that sodium dodecyl sulfate (SDS) is effective at decellularization of aortic value leaflets, wherein the major structural components of the valve matrix were maintained (Abstract). Booth teaches that SDS was found to have decellularizing capabilities at 0.03% w/v (p.459, right col.; See Fig. 1b, c, d). Booth teaches that for the complete decellularization of valve leaflets in situ, within the valve root, the concentration of SDS had to be increased to 0.1% (p.460, lns.1-4). Furthermore, Booth teaches that there was no apparent disruption of the overall tissue histoarchitecture and the familiar trilaminar structure consisting of fibrosa, spongiosa and ventricularis had been maintained. The major structural components of the heart valve - collagen I, elastin, and GAG - also appeared to have been preserved within the valve matrix of leaflets decellularized with SDS (0.03-1% w/v) (p.460, right col.; p.461, right col.). Further, Booth teaches that SDS for use in decellularizing vascular prosthesis and tendon (p.461, left col., top). Booth further teaches SDS was found to be capable of totally decellularizing the aortic valve matrix. Moreover, Booth teaches that electron microscopy of fresh and 0.03% (w/v) SDS treated leaflets revealed no loss of collagen fiber integrity, and fibers could be observed to have a similar banding pattern to that of collagen fibers observed in fresh untreated valve leaflet tissue (p.460, right col.; See Fig. 2g, h; See entire document).
Thus, one of ordinary skilled in the art would have been motivated to provide SDS to decellularize an aorta because SDS provided no apparent disruption of the overall tissue histoarchitecture is maintained and SDS was found to be capable of totally decellularizing the aortic valve matrix a taught by Booth.
Woods teaches that it has been suggested that residual cytotoxic sodium dodecyl sulfate (SDS) is responsible for the low levels of cell in-growth observed in SDS decellularized tissues. To determine whether this is the case, we used 2 washing methods to remove residual SDS and extensive biochemical, mechanical, and structural analyses to determine the effects of SDS-based decellularization on porcine anterior cruciate ligament (ACL) tissue and its propensity for cellular repopulation. The level of residual SDS in decellularized tissue was reduced using 2 different washing techniques (pH = 9 buffer, 75% ethanol). After washing in pH = 9 or 75% ethanol, residual SDS concentrations in decellularized tissues were found to be approximately 8 and 23 times less than reported SDS cytotoxic levels, respectively. Further, Woods teaches for tissue from the mid-substance of the ACL, treatment with SDS achieved 100% removal of visible cell nuclei and the cytoskeletal protein vimentin from the mid-substance of B-ACL-B grafts. These observations matched the effectiveness of SDS in removing cellular materials from vascular tissues (encompasses porcine aorta, i.e., an arterial vessel in the circulatory system), heart valves, rat tail tendons, and rabbit bone–patellar bone grafts and in other studies (p.2979, Table 1; Fig. 1; (p.2981, left col. Bottom paragraph; Figs. 1 and 2). Further, Woods teaches that there was significantly more residual SDS in the pH9-washed tissue than in the EtOH (ethanol)-washed tissue (p<0.05). The pH9-washed tissue contained about 1.38 µg SDS/mg dry tissue, whereas the EtOH-washed tissue contained about 0.44 µg SDS/mg dry tissue (p.2979, left col. SEE, Residual SDS and cytotoxicity; p.2980, right col.). The lower cytotoxic threshold for residual SDS in tissue is approximately 10 µg /mg dry tissue. Levels of SDS found in pH9- and EtOH- washed tissue were less than the reported cytotoxic level by approximately 8 and 23 times, respectively. Also, no significant differences were found in the growth of primary porcine ACL fibroblasts after 1 week in the presence of 1.38 mg/mL or 0.44 mg/mL of SDS from that of controls containing no SDS in the cell culture medium (p.2979). Woods teaches cellular repopulation of SDS-treated decellularized ACL matrix was equivalent in samples containing residual SDS with concentrations approximately 8 and 23 times less than the toxicity limit (p/2981, right col.).
Thus, one of ordinary skilled in the art would have been motivated to provide ethanol as a polar solvent to wash SDS from decellularized porcine aorta having a reasonable expectation of success that ethanol would substantially remove SDS from a decellularized aorta
Thus, the teachings of Allaire and Booth, as a whole, render obvious using SDS under agitation as a surfactant for about 15 hours to decellularize an aorta and Woods render obvious using the polar solvent ethanol to substantially wash out SDS following the decellularized aorta using SDS having a reasonable expectation of success.
Therefore, Allaire, Booth and Woods, as a whole render obvious treating an aorta with a surfactant comprising sodium dodecyl sulfate (SDS) under agitation in the treatment chamber, wherein the aorta, e.g., porcine aorta , is treated with the SDS in a treatment chamber for a time of less than 48 hours (recited in instant claim 1); wherein the tissue is pretreated with the surfactant in the pretreatment chamber for a time period of about 8 hours to less than 48 hours (recited in instant claim 12).
However, Allaire, Booth and Woods differ from instant claims in that the documents do not teach placing an aorta, e.g., porcine aorta, in a pretreatment chamber that is separate from the environmental chamber, pretreating the aorta with SDS in a pretreatment chamber, and, wherein a decellularization solution comprising water-saturated supercritical CO2 further contains ethanol.
However, Sabirzyanov and Sawada, as a whole, cure the deficiencies.
Sabirzyanov teaches that water has low solubility in supercritical carbon dioxide (I.e., less than 0.1 mole fraction at about 40OC. See Fig. 3. P.205). Thus, water would not be expected to increase the polarity of supercritical carbon dioxide (nonpolar solvent). Hence, water-saturated supercritical CO2 as taught by Mathews would still be significantly nonpolar and unlikely to extract polar components, e.g., a few compacted nuclear residues remained after treatment with SDS as taught by Allaire, as described above. Nonetheless, Matthews renders obvious a water-saturated supercritical CO2-based decellularization method for preventing tissue dehydration.
Sawada teaches a method for the decellularization of tissue, e.g., porcine aorta, that did not require a long period for completion, wherein decellularization of porcine aorta was performed using a high-pressure reaction apparatus (See, Figure 1 at page 944). The main components of the apparatus include a pressurizing pump, stainless steel vessel, and backpressure regulator. The pressurizing pump supported the flow of carbon dioxide at the desired rate. The vessel was placed in a water bath to control the treatment temperature, wherein the contents of the vessel could be stirred with a Teflon coated bar driven by an outside magnet. The backpressure regulator was a computer-controlled machine and was able to release carbon dioxide at the desired flow rate for the desired period. Liquid carbon dioxide from a cylinder was compressed with a pressurizing pump and made to flow into the reaction vessel until the pressure reached the desired value. Furthermore, Sawada teaches ethanol was preloaded in the bottom of the vessel. Under the supercritical condition the ethanol at the bottom of the vessel dissolves in the upper phase until the carbon dioxide is saturated with ethanol (polar solvent). The aorta was fixed to the upper side of the vessel to prevent direct contact with ethanol. In the supercritical condition, the aorta would be surrounded with supercritical carbon dioxide alone or with a mixture of supercritical carbon dioxide and ethanol fluid, wherein the operating temperature was fixed at 37 °C (p.944, right col.: Procedure).
Sawada teaches that supercritical carbon dioxide alone did not seem to dissolve cell nucleic, but supercritical carbon dioxide that contained ethanol was able to extract cell nuclei. As shown in Fig. 2(c) and (d), cell nuclei in the tissue were completely removed from the extracellular matrix within 1 hour (i.e., for 15 min; for 1 hour: p.945, right col., top), wherein cell nuclei, which did not dissolve in either carbon dioxide or ethanol, appeared to be effectively solubilized in mixed fluid under the supercritical condition. More remarkable was the decellularization that was attained after a short period of treatment. Figure 2(d) demonstrates that cell nuclei were removed within 15 min when the supercritical carbon dioxide/ethanol system was used (p.945, right col., top, Fig. 2(c)). Sawada teaches mixed supercritical fluid (carbon dioxide/ethanol), which rapidly reached the inside of the tissue, seems to have the potential to solubilize and extract both cell nuclei and phospholipid within a short time. Sawada teaches that 100% removal of DNA and 80-90% removal of phospholipids at relatively mild pressures and temperatures See Fig. 1-6).
Thus, in view of Sawada, one of ordinary skill in the art would have understood that supercritical carbon dioxide and ethanol is very effective at removing cell nuclei components.
However, one skilled in the art would have recognized that supercritical carbon dioxide and ethanol cannot be used alone (emphasis added), since ethanol cause dehydration of tissue, as disclosed by Matthews (see below).
Matthew (para. [0023)] teaches that Sawada reported cell removal with supercritical carbon dioxide for acellular artificial tissue, which shows the use of supercritical carbon dioxide containing an ethanol entrainer to decellularize porcine aortas. Sawada teaches 100% removal of DNA and 80-90 %removal of phospholipids at relatively mild pressures and temperatures. Supercritical carbon dioxide is unique in that it has strong penetrating power into tissues and reverts to the gas phase so that it does not remain in the tissue after treatment. Still, problems with tissue dehydration and residual phospholipids were reported. The dehydration seen by Sawada caused the decellularized matrix to become brittle and lose mechanical strength, making it unsuitable for tissue engineering applications. Sawada et al. concluded that the cause was extraction of water by supercritical CO2.
Nonetheless, Matthews teaches presently disclosed are methods to develop an effective water-saturated supercritical CO2 -based decellularization method by preventing the tissue dehydration
Thus, one skilled in the art would have been motivated to modify Matthews to further contain ethanol to provide a co-solvent mixture comprising supercritical carbon dioxide, water and ethanol, i.e., solvent system comprising ethanol, a polar entrainer, wherein ethanol would help dissolve supercritical CO2 and facilitate the removal of polar components, e.g., phospholipids, as taught by Sawada, wherein ethanol would also remove residue SDS as taught by Woods, wherein treatment with SDS would be expected to provide loss of cellular structures from the three layers of the arterial wall (i.e., cell lysis) as render obvious by Allaire and Booth, and wherein the water saturated ssCO2 as taught by Matthes would prevent dehydration of the porcine aorta.
Particularly, Woods teaches residual SDS and cytotoxicity, as described above. The lower cytotoxic threshold for residual SDS in tissue is approximately 10 µg /mg dry tissue. Levels of SDS found in ethanol washed tissue were less than the reported cytotoxic level by approximately 23 times.
Moreover, one skilled in the art would not have combined the steps (i.e., single step) of decellularizing porcine aorta comprising treating porcine aorta with SDS and supercritical carbon dioxide, water and ethanol, because sequential treatment, i.e., treating porcine aorta in a pretreatment chamber with SDS) would optimize cell lysis by SDS while supercritical carbon dioxide, water and ethanol of the decellularizing solution would be expected to remove residual SDS, which a single-step mixture may be too aggressive and uneven, thus, decreasing the ability of SDS to provide cell lysis. Moreover, supercritical carbon dioxide, water combined with ethanol in the second step would be expected to benefit from prior cellular permeabilization (cell lysis) using SDS to penetrate the extracellular matrix and remove away the cellular debris. Further, applying supercritical carbon dioxide, water and ethanol after the SDS treatment would function as an efficient extraction process to strip away residual SDS from the porcine aorta that a simultaneous mixture, e.g., supercritical carbon dioxide, water, ethanol and SDS would likely fail to adequately clear the residual SDS. Moreover, the presence of water in the water saturated ssCO2 would prevent dehydration of the porcine aorta.
Thus, one of ordinary skill in the art would have been motivated to provide a method of decellularizing an aorta, e.g., porcine aorta, by placing the porcine aorta in a pretreatment chamber that is separate from the environmental chamber, pretreating the porcine aorta with sodium dodecyl sulfate (SDS) under agitation in the pretreatment chamber, wherein the porcine aorta is pretreated with SDS in the pretreatment chamber for 15 hours, i.e., renders obvious a time of less than 48 hours (recited in claim 1) and wherein the aorta is pretreated with SDS in the pretreatment chamber for a time period of about 8 hours to less than 48 hours (recited in instant claim 12) in view of Matthews, Allaire, Woods, Booth, Sabirzyanov and Sawada, as a whole.
However, Matthews, Allaire, Woods, Booth, Sabirzyanov and Sawada differ from the claims in that they do not explicitly teach wherein one hour after treating the porcine aorta with the supercritical carbon dioxide of the decellularization solution the treated natural tissue contains less than about 0.004 volume % of surfactant (recited in claim 1).
The method of decellularization of a tissue as taught by Matthews, Allaire, Woods, Booth, Sabirzyanov and Sawada, as a whole, is substantially identical to the claimed method, wherein a tissue is pretreated with SDS in a separate pretreatment chamber for a time period of about 48 hours and subsequently treated in an environment chamber with a decellularization solution comprising carbon dioxide, water and ethanol (process of treating the aorta using SSD followed by treating with decellularization solution are performed sequentially, so it would necessarily follow that the treated tissue contains less than about 0.004 volume% of surfactant, wherein water retention of the treated tissue is greater than 97.3%, so that the method would necessarily facilitate removal of cells from the tissue so that tissue treated with the supercritical carbon dioxide of the decellularization solution contains less than 0.05 micrograms of DNA per milligram of dry tissue after the tissue is exposed to the decellularization solution. Accordingly, the limitations at issue are the 'natural result' of the combination of prior art elements." PAR Pharm., Inc. v. TWI Pharms., Inc., 773 F.3d 1186, 1195 (Fed. Cir. 2014).
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). Mere recognition of latent properties in the prior art does not render nonobvious an otherwise known invention. In re Wiseman, 596 F.2d 1019, 201 USPQ 658 (CCPA 1979). The fact that Applicants have recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious." Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985).
Moreover, one of ordinary skilled in the art would treat the aorta, e.g., porcine aorta, with SDS before supercritical, water and ethanol treatment primarily to enhance decellularization efficiency, facilitate the removal of cellular components, and provide a more efficient method of decellularizing an aorta, wherein pretreatment with SDS would act as a primer to break down cellular structure having a reasonable expectation of success.
Where the claimed and prior art products are identical or substantially identical in structure or composition, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). "When the PTO shows a sound basis for believing that the products of the applicant and the prior art are the same, the applicant has the burden of showing that they are not." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). Therefore, the prima facie case can be rebutted by evidence showing that the prior art products do not necessarily possess the characteristics of the claimed product. In re Best, 562 F.2d at 1255, 195 USPQ at 433.
Therefore, the teachings of Matthews, Allaire, Woods, Booth, Sabirzyanov and Sawada, as a whole, render obvious a method for decellularizing an aorta, as instantly claimed.
Response to Arguments
Applicants’ arguments directed to Pang and Fu are moot because these references are not included in the above rejections.
Applicants argue that Matthews discourages the use of ethanol. Matthews describes Sawada's supercritical carbon dioxide/ethanol approach and reports that it caused "tissue dehydration and residual phospholipids," that the resulting matrix became "brittle and lose[s] mechanical strength," and that it is "unsuitable for tissue engineering applications" (See Matthews, [0023]). Matthews' thesis is that stronger, chemical-laden methods bring "an inability to maintain the chemical and material properties of pre-treatment ECM" ([0026]), and that its benign water-presaturation method is the alternative. A person of ordinary skill starting from Matthews would thus be led away from reintroducing Sawada's ethanol into the supercritical carbon dioxide stream, not toward it.
Applicants’ arguments have been fully considered but they are not persuasive, because in view of Sawada, one skilled in the art would have recognized that supercritical carbon dioxide and ethanol is very effective at removing cell nuclei. Furthermore, Matthews teaches presently disclosed are methods to develop an effective water-saturated supercritical CO2 -based decellularization method by preventing the tissue dehydration. However, one skilled in the art would have clearly recognized that supercritical carbon dioxide and ethanol cannot be used alone (emphasis added), since ethanol cause dehydration of tissue. Thus, one skilled in the art would have been motivated to modify Matthews comprising water saturated supercritical carbon dioxide to further contain ethanol to provide a co-solvent mixture comprising supercritical carbon dioxide, water and ethanol, i.e., solvent system comprising a polar entrainer: ethanol, wherein ethanol would help dissolve supercritical CO2 and facilitate the removal of polar components, e.g., phospholipids, as taught by Sawada.
Applicants argue that the Office Action's contrary reasoning that "[the] carbon dioxide and water in Matthews would inhibit tissue dehydration" supplies the very insight underlying the present invention and is impermissible hindsight. See KSR Int 'l Co. v. Teleflex Inc., 550 U.S. 398 (2007) (an obviousness rejection requires an articulated reasoning with rational underpinning).
In response to Applicants’ argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). Moreover, the teachings of Matthews, Allaire, Woods, Booth, Sabirzyanov and Sawada, as a whole, were known and available to one of ordinary skill before the effective filing date of the claimed invention.
As discussed above, Sawada teaches a method for the decellularization of tissue, e.g., porcine aorta, that did not require a long period for completion, wherein ethanol was preloaded in the bottom of the vessel. Under the supercritical condition, the ethanol at the bottom of the vessel dissolves in the upper phase until the carbon dioxide is saturated with ethanol. The aorta was fixed to the upper side of the vessel to prevent direct contact with ethanol. In the supercritical condition, the aorta would be surrounded with supercritical carbon dioxide alone or with a mixture of supercritical carbon dioxide and ethanol fluid, wherein the operating temperature was fixed at 37 °C. Moreover, Sawada teaches that supercritical carbon dioxide alone did not seem to dissolve cell nucleic, but supercritical carbon dioxide that contained ethanol was able to extract cell nuclei, wherein cell nuclei, which did not dissolve in either carbon dioxide or ethanol, appeared to be effectively solubilized in mixed fluid under the supercritical condition. Moreover, Sawada teaches mixed supercritical fluid (carbon dioxide/ethanol), which rapidly reached the inside of the tissue, seems to have the potential to solubilize and extract both cell nuclei and phospholipid within a short time. However, complete extraction of phospholipid was not attained, wherein some phospholipid remained in the tissue even when the treatment was prolonged (p.946, left col.). Sawada teaches that 100% removal of DNA and 80-90% removal of phospholipids at relatively mild pressures and temperatures See Fig. 1-6). However, Sawada teaches problems with tissue dehydration and residual phospholipids were reported. Accordingly, one skilled in the art would have recognized that supercritical carbon dioxide and ethanol cannot be used alone (emphasis added), since ethanol cause dehydration of tissue. Therefore, Sawada actually provides motivation to provide a co-solvent, e.g., water and ethanol, where the mixture of water along with ssCo2 would be expected to remove any residual SDS having a reasonable expectation of SDS. Furthermore, as taught by Sabirzyanov, water has an extremely low solubility in ssCo2, so that the addition of ethanol would be expected to enhance the solubility of sCO2 and provide much improved extraction of SDS.
Applicants argue Woods teaches a separate aqueous wash (pH 9 buffer or 75% ethanol) performed after decellularization to remove residual SDS. As such, a skilled artisan would not have been motivated to modify Matthews to include ethanol as a washing solvent based on Woods because Matthews cautions that the use of supercritical carbon dioxide containing ethanol may result in problems with tissue dehydration and residual phospholipid. Applicants argue that nowhere does Woods describe a method for decellularizing an aorta that includes a decellularization solution comprising carbon dioxide, water, and one or more polar solvents, wherein the one or more polar solvents comprise ethanol, methanol, isopropanol, acetic acid, or a combination thereof, as set forth in amended independent claim 1.
Applicant’s arguments have been fully considered but they are not persuasive, because one skilled in the art would have looked at the teachings of Wood because Woods teaches the level of residual SDS in decellularized tissue after washing with ethanol, residual SDS concentrations in decellularized tissues were found to be as much as 23 times less than reported SDS cytotoxic levels. Thus, one skilled in art when reading Woods would have recognized the superior ability of ethanol to remove residual SDS from an aorta, e.g., porcine aorta, after being treated with SDS. Accordingly, Woods would provide one skilled in the art sufficient motivation to include ethanol in a decellularization solution comprising carbon dioxide and water to provide a decellularization solution comprising carbon dioxide, water and ethanol having a reasonable expectation of success of effectively removing residual SDS from aorta that had been pretreated with SDS in a pretreatment chamber having a reasonable expectation of success. Moreover, the impressive removal of SDS surfactant using ethanol would clearly outweigh any potential dehydration of the tissue, of which the Method of decellularization as taught by Matthew comprising carbon dioxide and water would be expected to inhibit tissue dehydration. Notably, a reference is analogous art to the claimed invention if the reference is from the same field of endeavor as the claimed invention (even if it addresses a different problem); and/or the reference is reasonably pertinent to the problem faced by the inventor.
Furthermore, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of the teachings of Matthews, Allaire, Woods, Booth, Sabirzyanov and Sawada, as a whole. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Accordingly, the instant 103 rejection is based on the combined teachings of Matthews, Allaire, Woods, Booth, Sabirzyanov and Sawada, as a whole. Thus, in the absence of clear and convincing evidence to the contrary the claimed method of decellularizing an aorta is obvious over the prior art and one of ordinary skill in the art would have had a reasonable expectation of success of providing instantly claimed invention. Applicants are reminded that obviousness does not require absolute predictability, however, at least some degree of predictability is required. Evidence showing there was no reasonable expectation of success may support a conclusion of nonobviousness. In re Rinehart, 531 F.2d 1048, 189 USPQ 143 (CCPA 1976).
Applicants argue that the secondary references fail to cure the deficiencies of Matthew, Sawada, and/or Woods. For instance, the secondary references fail to disclose or suggest pretreating the aorta with a surfactant comprising sodium dodecyl sulfate under agitation in the pretreatment chamber, wherein the aorta is pretreated with the surfactant in the pretreatment chamber for a time period of from about 8 hours to less than 48 hours, as set forth in amended independent claim 1.
Applicants’ arguments have been fully considered but they are not persuasive, because one of ordinary skilled in the art would treat the aorta, e.g., porcine aorta, with SDS before supercritical, water and ethanol treatment primarily to enhance decellularization efficiency, facilitate the removal of cellular components, and provide a more efficient method of decellularizing an aorta, wherein pretreatment with SDS would act as a primer to break down cellular structure having a reasonable expectation of success. Furthermore, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of the teachings of Matthews, Allaire, Woods, Booth, Sabirzyanov and Sawada, as a whole. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Accordingly, the instant 103 rejection is based on the combined teachings of Matthews, Allaire, Woods, Booth, Sabirzyanov and Sawada, as a whole. Moreover, Allaire teaches the aortas to be decellularized were incubated for 15 hours (reads on instant claims 1, 12)
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). Mere recognition of latent properties in the prior art does not render nonobvious an otherwise known invention. In re Wiseman, 596 F.2d 1019, 201 USPQ 658 (CCPA 1979). The fact that Applicants have recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious." Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). Moreover, it would have been obvious to perform the processes sequentially since SDS is known to promote cell lysis comprising solubilizing and disrupting the cellular structures and the decellularization solution can extract the residual SDS and further remove any remaining cellular components from the aorta having a reasonable expectation of success.
Applicants argue that the present application teaches that the method facilitates the removal of cells from the tissue so that tissue treated with the supercritical carbon dioxide of the decellularization solution contains less than 0.05 micrograms of DNA per milligram of dry tissue after the tissue is exposed to the decellularization solution, as set forth in amended independent claim 1.
Applicants’ arguments have been fully considered but they are not persuasive, because there is no unobvious distinction between the structural and functional characteristics of the claimed composition and the composition of the prior art, as evidenced by Matthews, Allaire, Woods, Booth, Sabirzyanov and Sawada, as a whole. Accordingly, the method of decellularization of a tissue as taught by Matthews, Allaire, Woods, Booth, Sabirzyanov and Sawada, as a whole, is substantially identical to the claimed method, so that the method would necessarily facilitate removal of cells from the tissue so that tissue treated with the supercritical carbon dioxide of the decellularization solution contains less than 0.05 micrograms of DNA per milligram of dry tissue after the tissue is exposed to the decellularization solution. Thus, the limitations at issue are the 'natural result' of the combination of prior art elements." PAR Pharm., Inc. v. TWI Pharms., Inc., 773 F.3d 1186, 1195 (Fed. Cir. 2014). Matthew teaches the purpose of a decellularization method is removal of all cellular material. Moreover, Sawada teaches supercritical carbon dioxide that contained ethanol was able to extract cell nuclei, wherein cell nuclei in the tissue were completely removed from the extracellular matrix. Obviousness does not require absolute predictability, but at least some degree of predictability is required. In re Rinehart, 531 F.2d 1048, 189 USPQ 143 (CCPA 1976). In the instant case, Applicants have failed to provide evidence showing there was no reasonable expectation of success.
Moreover, Instant claims (Claim 1) fail to recite concentrations for ssCO2, water and ethanol; thus, it cannot be said that the claimed scope of instantly claimed invention is commensurate with the disclosure. Furthermore, the dependent claims do resolve tis issue with the scope of the claimed invention.
MPEP 716.02(d) Unexpected Results Commensurate in Scope with Claimed Invention
Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980). Thus, in the instant case, the unexpected results are not commensurate in scope with what is instantly claimed, because the scope of Instant Claims encompasses natural tissue. Accordingly, the genus term ‘aorta’ is a broad genus encompassing many different aorta species, so that porcine aorta, by itself, as disclosed, fails to be commensurate in scope with the claims which the evidence is offered to support. In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. That is, the method of decellularizing a natural tissue that generated said alleged unexpected results are not commensurate in scope with the method of decellularizing aorta which is presently claimed (i.e., results generated by the single example, porcine aorta, by itself, is not commensurate in scope with claimed natural tissue. surfactant(s)).
MPEP 716.02(d) I. NONOBVIOUSNESS OF A GENUS OR CLAIMED RANGE MAY BE SUPPORTED BY DATA SHOWING UNEXPECTED RESULTS OF A SPECIES OR NARROWER RANGE UNDER CERTAIN CIRCUMSTANCES
The nonobviousness of a broader claimed range can be supported by evidence based on unexpected results from testing a narrower range if one of ordinary skill in the art would be able to determine a trend in the exemplified data which would allow the artisan to reasonably extend the probative value thereof. In re Kollman, 595 F.2d 48, 201 USPQ 193 (CCPA 1979). Instant Application discloses porcine aorta. Accordingly, a trend is not established by the showing of the results of porcine aorta, by itself. Evidence as to any unexpected benefits must be "clear and convincing" In re Lohr, 137 USPQ 548 (CCPA 1963), and be of a scope reasonably commensurate with the scope of the subject matter claimed, In re Linder, 173 USPQ 356 (CCPA 1972). Here, there is no trend in a single example: aorta.
Conclusions
No claim is allowed.
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/T.W./ Examiner, Art Unit 1619
/SARAH ALAWADI/Primary Examiner, Art Unit 1619
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