Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
Claims 1-3, 5-7, 10, 12 and 13 are pending in the Claim Set filed 1/02/2026.
Claims 1 and 3 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Withdrawn Rejections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 4 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of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends is withdrawn because claim 4 has been cancelled in the Claim Set filed 1/02/2026.
The rejection of claims 1-7, 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 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), Jung et al (US 10,537,663), 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2006, of record), Gratzer (US20130028981, cited in IDS filed 3/01/2023) and Ansari et al (WO2017068336, of record) is withdrawn in view of the claim amendments in favor of the New Grounds of Rejection as set forth below.
The 35 USC § 112(a) rejection set forth below has been reformulated that is necessitated by Applicants’ claim amendments. Rejections are maintained and made again necessitated by Applicants’ claim amendments.
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.
The rejection of 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 is maintained and made again. 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 a natural tissue, the method comprising:
placing the natural tissue in a pretreatment chamber that is separate from the environmental chamber;
pretreating the natural tissue with a surfactant comprising sodium dodecyl sulfate under agitation in the pretreatment chamber; wherein the natural tissue 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 natural tissue in a treatment chamber located in the environmental chamber; and treating the natural tissue with the decellularization solution from the presaturation chamber, wherein one hour after treating the natural tissue with the supercritical carbon dioxide of the decellularization solution the treated natural tissue contains less than about 0.004 volume % of surfactant,
wherein water retention of the natural tissue is greater than 97.3%,
wherein the method facilitates removal of cells from the natural tissue so that the natural 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 natural tissue 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 ‘natural tissue’ encompasses a broad genus of natural tissue that includes natural tissues from various species, e.g. human, bovine, equine, ovine, porcine, seal or kangaroo (typically mammalian), wherein the tissue is decellularized that include xenografts thereof (Holmberg, US 20030139805: [0065], [0066]; [0067]; [0076]; [0079]; 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. 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 1/2/2026, Applicants argue that claim 1 has been amended as shown (Claim Set filed 1/2/2026) to recite a method for decellularizing a natural tissue. Support for said amendments may be found at least in paragraphs [0049]-[0050] and [0062], and in FIG. 3 of the originally filed application.
Specification and Figures discloses the following:
[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 (i.e., 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 tissue.
Furthermore, the term ‘natural tissue’ is broad and encompasses a wide array of varied tissue(s). Natural, i.e. biological, tissue material for use in the invention includes relatively intact tissue as well as decellularized tissue. These natural tissues may be obtained from, for example, native heart valves, portions of native heart valves such as roots, walls and leaflets, pericardial tissues such as pericardial patches, amniotic sacs, connective tissues, bypass grafts, tendons, ligaments, skin patches, blood vessels, cartilage, dura mater, skin, bone, fascia, submucosa, umbilical tissues, and the like. Natural tissues are derived from a particular animal species, typically mammalian, such as human, bovine, equine, ovine, porcine, seal or kangaroo. These tissues may include a whole organ, a portion of an organ or structural tissue components (See Holmberg, US 20030139805, [0066]).
Therefore, paragraphs [0049]-[0050] and FIG. 1 and FIG. 3, fail to provide support for the broad genus term ‘natural tissue’ (claimed in Claim Set filed 1/02/2026).
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 natural tissue: 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 a natural tissue, because the hydrogel is a synthetic copolymer. Accordingly, Fig. 3 is singularly directed to a single kind of tissue: porcine aorta.
Notably, ‘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].
If the genus has a substantial variance: natural tissue, 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.
Accordingly, the genus term ‘natural tissue’ is a broad genus encompassing a vast array of different types of tissues, so that porcine aorta, by itself, as disclosed fails to provide a sufficient representation of species to reflect the variation within the broad genus: ‘natural tissue’.
Therefore, it is deemed that the specification fails to provide adequate written description for the genus: ‘tissue’ of the instant claims and does not reasonably convey to one skilled in the relevant art that the inventor(s), 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 ‘natural tissue’ and are rejected because they are dependent on a rejected claim. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181,26 USPQ2d 1057 (Fed. Cir. 1993).
Response to Arguments
Applicants argue in the reply filed 1/2/2026 that claim 1 has been amended as shown (Claim Set filed 1/2/2026) to recite a method for decellularizing a natural tissue. Support for said amendments may be found at least in paragraphs [0049]-[0050] and [0062], and in FIG. 3 of the originally filed application.
Applicant’s arguments have been fully considered but they are not persuasive, because the Disclosure fails to provide adequate written description for the broad genus term ‘natural tissue’, as thoroughly discussed above) since 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.
The 35 USC § 112(d) rejection set forth below is necessitated by Applicants’ claim amendments.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS - Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), fourth paragraph:
Subject to the [fifth paragraph of 35 U.S.C. 112 (pre-AIA )], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 12 is rejected 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.
Claim 12 is dependent on Instant Claim 1; however, claim 12 does not include a limitation that would further limit the subject matter of claim 1.
Claim 12 recites: The method of claim 1, wherein the tissue is pretreated with the surfactant in the pretreatment chamber for a time period of up to 48 hours.
Claim 1 recites (in part):
A method for decellularizing a natural tissue, the method comprising:
placing the natural tissue in a pretreatment chamber that is separate from the environmental chamber;
pretreating the natural tissue with a surfactant comprising sodium dodecyl sulfate under agitation in the pretreatment chamber; wherein the natural tissue is pretreated with the surfactant in the pretreatment chamber for a time of less than 48 hours;
However, claim 12 recites: The method of claim 1, wherein the tissue is pretreated with the surfactant in the pretreatment chamber for a time period of up to 48 hours. Where, the phrase ‘’up to 48 hours’ includes the full 48 hour duration, whereas claim 1 recites ‘a time of less than 48 hours that excludes 48 hours.
Therefore, claim 12 does not further limit the subject matter of claim 1 upon which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
New Grounds of Rejection necessitated by claim amendments
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) 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).
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).
Matthews teaches: exposing a natural tissue, e.g., porcine aorta, to a water-saturated, supercritical CO2.
Abstract
Decellularization methods for tissue are provided. The method can include: exposing a tissue to a water-saturated,
supercritical CO2. The method can further comprise, prior to exposing the tissue to the water-saturated, supercritical CO2,
saturating a stream of supercritical CO2. The tissue can be exposed to the water-saturated, supercritical CO2 at a treatment
temperature of about 35° C. to about 40° C. (e.g., about 37° C.). In one embodiment, the water-saturated, supercritical
CO2 is completely saturated with water at the treatment temperature. The tissue can be exposed to the water-saturated,
supercritical CO2 at a constant flow rate, such as less than 3 mL/min (e.g., about 0.5 mL/min to about 2.5 mL/min).
Regarding claims 1, 2, 3, 5, 6, 10 and 13,
Matthews teaches a decellularization method for tissue, wherein the method comprises exposing a natural tissue, e.g., 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, e.g., porcine aorta, 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 a natural tissue, e.g., 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). And further, the method of decellularization of natural tissue, e.g., porcine aorta, as taught by Matthews encompasses using a system as shown below (Drawings: Figs 1-4). Matthew teaches that decellularization environmental chamber comprises a pressure cell, called the treatment chamber, wherein the natural tissue, e.g., 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 tissue for about 60 min (I hour). Reads on Claim 7. 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 make prima facie obvious a method for decellularizing tissue (e.g., 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 natural tissue, e.g., 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 a tissue in the Treatment Chamber in an Environmental Chamber and treating the natural tissue, e.g., porcine aorta, in the Treatment Chamber with the decellularization solution obtained from the Presaturation Chamber in the Environmental Chamber.
Further, Matthews teaches presently disclosed are methods provide a major step in developing an effective scCO2-based decellularization method by preventing the tissue dehydration. 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 presently disclosed methods; the extraction of water is inhibited from occurring at all. In particular, a simple presaturation method is provided by using waters saturated scCO2. The presaturated scCO2 is then contacted with the tissue, 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, as instantly claimed in claim 6. 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].
In addition, 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]. Moreover, 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].
Firstly, Matthews differs from claim 1 in that the document does teach that decellularization solution comprising water-saturated supercritical CO2 further comprise one or more polar solvents selected from ethanol, methanol, isopropanol, acetic acid, or a combination thereof.
However, Sawada cures the deficiency.
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, ethanol, when needed, 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 (p.944, right col.: Procedure).
In particular, 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. However, complete extraction of phospholipid was hot 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).
Thus, one of ordinary skill in the art would have been motivated to modify Matthews’ method of decellularized tissue by providing a mixture of supercritical carbon dioxide/ethanol to effectively solubilize the natural tissue that is attained after a short period of treatment to optimize the removal of cellular components, e.g., in about 1 hour; in about 15 min) in order to provide an improved method of decellularization of natural tissue having a reasonable expectation of success. Moreover, Sawada teaches that as shown in Fig. 2, the aorta that had been treated with supercritical carbon dioxide only (Fig. 2(b)) was microscopically similar to the native tissue (Fig. 2(a)).
Additionally, it would have been prima facie obvious to follow the guidance for decellularization of natural tissue as taught by Matthew (primary reference) so that the flow rate of the carbon dioxide in the Presaturation Chamber containing a decellularization solution comprising water-saturated supercritical CO2 and ethanol, i.e., a decellularization solution comprising carbon dioxide, water and ethanol (polar solvent) that is provided at less than about 3 mL/min (e.g., about 0.5 mL/min to about 2.5 mL/min) [0046] having a reasonable expectation of success. This flow rate lies within the decellularization solution that is delivered to the treatment chamber at a flow rate ranging from about 0.1 millimeters per minute to about 5 milliliters per minute, as instantly claimed in claim 6. 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. And, that the method of decellularization comprises a pump (syringe pump) that compresses the carbon dioxide in accordance with the teachings of Matthew (Fig. 4; [0053]). Reads on claim 2. Still more, 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 ([0031-0032]; [0052-0054]), Reads on claim 3; along these lines, it would have been well within the purview of one skilled in the art to continue to stirring the carbon dioxide, water and ethanol decellularization solution for about 1 minute to about 30 minutes by design choice to provide that that the mixture was thoroughly mixed to obtain a it is completely saturated comprising continually mixing the decellularization solution having a reasonable expectation of success (reads on claim 5). Moreover, it would have been prima facie obvious to those skilled in the art to continue mixing the decellularization solution containing the natural tissue to ensure that the tissue was efficiently exposed to decellularizing solution in order to optimized the removal of the cellular structures having a reasonable expectation of success. Reads on claim 13. Optimization of parameters is a routine practice that would be obvious to a person of ordinary skill in the art to employ and reasonably expect success; See In re Aller, 220 F.2d 454, 456, 105 USPQ 233,235 (CCPA 1955). Matthews teaches treatment time was determined by using a treatment ratio of 60 min per 0.25-gram tissue [0062]. Particularly, Mathew teaches 0.2777 gram of natural tissue was treated with decellularization solution in the treatment chamber ([0065]; Table 3), thus, providing the tissue is exposed to the decellularization solution for a time period ranging from about 1 minute to about 2 hours, e.g., 60 minutes. Reads on claim 7. Additionally, Matthews teaches the treatment chamber was used to maintain a slow, controlled depressurization of 50 psi/min (i.e., 0.34 Megapascals/min) after treatment, wherein finally, the cold trap is removed, and the CO2 is simply vented [0058]., of which lies within the claimed. Reads on claim 10. A prima facie case of obviousness typically exists when the ranges of a claimed composition overlap the ranges disclosed in the prior art. In re Peterson, 315 F.3d 1325, 1329 (Fed. Cir. 2003).
Secondly, Matthews differs from claim 1 in that the document does teach placing the natural tissue in a pretreatment chamber that is separate from the environmental chamber, pretreating the natural tissue with a surfactant comprising sodium dodecyl sulfate (SDS) under agitation in the pretreatment chamber, wherein the natural tissue 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 up to 48 hours (recited in instant claim 12), wherein one hour after treating the natural tissue 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).
However, Allaire, Woods and Booth, Pang and Fu, as a whole, cure the deficiencies.
Allaire teaches we developed a method remove arterial cells from rat and guinea pig (i.e., natural tissues) 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 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 a-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.).
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, heart valves, rat tail tendons, and rabbit bone–patellar bone grafts noted in other studies (p.2979, Table 1; Fig. 1; (p.2981, left col. Bottom paragraph; Figs. 1 and 2). Furthermore, Woods teaches that there was significantly more residual SDS in the pH9-washed tissue than in the EtOH-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.).
Matthews (US20150315540) teaches that tissue decellularization has been investigated that involves chemical detergents. A common ionic detergent is sodium dodecyl sulfate (SDS), which was used to decellularize human adipose tissue and to treat porcine brain tissue. However, detergents can eventually cause degradation of the ECM (extracellular matrix) under prolonged treatments [0007-0008]. References are evaluated by what they suggest to one versed in the art, rather than by their specific disclosures. In re Bozek, 163 USPQ 545 (CCPA 1969). Thus, one skilled in the art reading Matthew would not be discouraged from using SDS detergent in method of tissue decellularization, since Matthews merely expresses a general preference to avoid prolonged treatments using SOS, otherwise, Matthew does not 'criticize, discredit, or otherwise discourage' using SDS in method of tissue decellularization. Above all, Woods teaches 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. Thus, one of ordinary skill in the art would recognize the advantage of using SDS to decellularized tissue wherein the surfactant in a pretreatment chamber treatment is less than 48 hours (recited claim 1) and/or for a time period of up to 48 hours, in view of Allaire and Woods, as a whole, having a reasonable expectation of success, since Allaire teaches the aortas (natural tissue) to be decellularized were incubated for 15 hours 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) (See Allaire supra).
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.). Furthermore, Booth teaches that SDS for use in decellularizing vascular prosthesis and tendon (p.461, left col., top). Also, Booth further teaches SDS was found to be capable of totally decellularizing the aortic valve matrix. A higher concentration of SDS was required for decellularizing the valve leaflets in situ (0.1% w/v), compared to excised leaflets which have a second free edge (0.03% w/v) (p.461, left col., bottom). 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).
Pang teaches the aim of this study was to construct a rabbit anterior cornea replacement with an acellular porcine cornea matrix (APCM) as a scaffold. The scaffold was prepared from fresh porcine corneas which were treated with 0.5% (wt./vol.) sodium dodecyl sulfate (SDS) solution and stirred for 24 h in a 4 0C refrigeration chamber. The complete removal of corneal cells was confirmed by H&E and DAPI staining. The stroma structure and mechanical properties were well preserved. The extracts had no cytotoxicity to rabbit corneal keratocytes, epithelial and endothelial cells as determined by MTT assay (Abstract). Pang teaches an acellular porcine cornea matrix (APCM) was developed by using SDS which is an anionic surfactant usually used for cell lysis (p.7258, left col.). Furthermore, an acellular porcine cornea matrix (APCM) was developed by using SDS which was an anionic surfactant usually used for cell lysis, however, quantitative detection of the residual SDS was not taken out after efficient washing (p.7263, See Discussion left col.; right col. second par.; see entire document).
Fu teaches extracellular matrix (ECM) hydrogels are used as scaffolds to facilitate the repair and reconstruction of tissues. This study aimed to optimize the decellularization process of porcine skeletal muscle ECM and to formulate a matrix hydrogel scaffold (Abstract). Fu teaches decellularization of ECM can be achieved by a variety of techniques. These processes generally involve physical methods comprising shaking and chemical reagents (detergents, organic solvents) to dissolve or digest cellular components (p.740, right col.). Sequential combination of these techniques is usually required to achieve the complete removal of cells (p.740, right col. last sentence to p.741, first sentence).
Thus, it would have been obvious to one of ordinary skill in the art that SDS is extremely effective for decellularization of tissue, for example, aortic value leaflets, wherein SDS was found to have decellularizing capabilities at 0.03% w/v when using SDS where there was no apparent disruption of the overall tissue histoarchitecture and the familiar trilaminar structure consisting of fibrosa, spongiosa and ventricularis had been maintained. Accordingly, one skilled in the art would have been motivated to select SDS as a the surfactant for pretreating tissue before applying the method of decellularization of tissue as taught by Matthews (US20150315540), because it is important that a technique developed left no remaining cells, or cell fragments behind in the matrix scaffold, having a reasonable expectation of success, as evidence exists that these components are associated with inflammation and calcification, which may ultimately lead to calcific tissue deterioration and limited valve longevity (See Booth at p.458, left col., second paragraph). Moreover, one skilled in the art would have recognized from the teachings of Booth that low levels of SDS are capable of decellularizing natural tissue. Thus, making SDS the most efficient option for cell membrane disruption in a pretreatment chamber. Moreover, Pang teaches SDS which is used for cell lysis. Additional, Fu teaches decellularization of ECM can be achieved by a variety of techniques that involve physical methods comprising shaking and chemical reagents (detergents, organic solvents) to dissolve or digest cellular components (p.740, right col.). Sequential combination of these techniques is usually required to achieve the complete removal of cells (p.740, right col. last sentence to p.741, first sentence).
Accordingly, one of ordinary skill in the art would have recognized that subsequent washing of tissue using ethanol that had been pre-treated with SDS used for decellularization of tissue was extremely effective at removing residual SDS from the decellularized tissue. Thus, one skilled in the art would have been motivated to use ethanol to remove SDS from treated tissue in the decellularization solution. Moreover, one skilled in the art would have been motivated to use SDS for decellularization tissue because Woods teaches that SDS can be used for decellularization of a wide range of varied tissue, e.g., ACL, vascular tissues, heart valves, rat tail tendons, and rabbit bone–patellar bone grafts. Moreover, Woods teaches that the residual SDS in decellularized tissue can be washed with ethanol (EtOH) to the provide about 23 times residual SDS less than the toxicity limit. Thus, one of ordinary skill in the art would have recognized the benefit of using SDS to decellularize a wide range of different types of tissue wherein the residual SDS remaining in the decellularize tissue can be substantially removed using an ethyl alcohol (EtOH) wash to the extent that the SDS concentration in the decellularized tissue is about 23 times less than the toxicity limit for SDS while having a reasonable expectation of success.
Thus, it would have been obvious to one of ordinary skill in the art that SDS is extremely effective for decellularization of tissue, for example, aortic value leaflets, wherein SDS was found to have decellularizing capabilities at 0.03% w/v when using SDS where there was no apparent disruption of the overall tissue histoarchitecture and the familiar trilaminar structure had been maintained. Accordingly, one skilled in the art would have been motivated to select SDS as a the surfactant for pretreating tissue before applying the method of decellularization of tissue as taught by Matthews (US20150315540), because it is important that a technique developed left no remaining cells, or cell fragments behind in the matrix scaffold, having a reasonable expectation of success, as evidence exists that these components are associated with inflammation and calcification, which may ultimately lead to calcific tissue deterioration and limited valve longevity (See Booth at p.458, left col., second paragraph). Moreover, one skilled in the art would have recognized from the teachings of Booth that low levels of SDS are capable of decellularizing natural tissue. Thus, making SDS the most efficient option for cell membrane disruption in a pretreatment chamber. Moreover, Fu teaches decellularization of ECM can be achieved by a variety of techniques comprising comprise shaking and chemical reagents (detergents, organic solvents), wherein sequential combination of these techniques is usually required to achieve the complete removal of cells.
The method of decellularization of a tissue as taught by Matthew, Sawada, Allaire, Woods and Booth, Pang and Fu, 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 natural tissue 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. 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). Lack of recognition of these features by those skilled in the art is thus not dispositive. 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 natural tissue having a reasonable expectation of success.
All the claimed elements herein are known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded predictable results to one of ordinary skill in the art at the time of the invention.
Therefore, it would have been obvious for one of ordinary skill in the art to provide instantly claimed invention and one of ordinary skill would have had a reasonable expectation of success in producing the claimed invention. Therefore, in the absence of evidence to the contrary, the invention as a whole would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, as evidenced by Matthew, Sawada, Allaire, Woods and Booth, Pang and Fu, as a whole.
Response to Arguments
Applicants argue that Matthews only contemplates the use of water in conjunction with carbon dioxide. Further, Matthews cautions that the use of supercritical carbon dioxide containing an ethanol may result in problems with tissue dehydration and residual phospholipids. Applicants argue that Matthews does not teach placing the natural tissue in a pretreatment chamber that is separate from the environmental chamber; pretreating the natural tissue with a surfactant comprising sodium dodecyl sulfate under agitation in the pretreatment chamber; 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, as set forth in amended independent claim 1.
Applicant’s arguments have been fully considered but they are not persuasive, because Matthews teaches a decellularization method for tissue (e.g., porcine aorta [0039]; [0040-0045]; [0061-0064]; Table 3; [0072]) , wherein the method comprises exposing a tissue 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. Moreover, Matthews claims a decellularization method for tissue, the method comprising exposing a tissue to a water-saturated, supercritical CO2 (See Matthews: claims 1, 1-6, 9-14). Thus, the teachings of Matthews make clear that the carbon dioxide is presaturated with water, i.e., carbon dioxide and water, which is more than just contemplating using water, but necessarily includes water in a method of decellularizing tissue comprising using water-saturated supercritical CO2.
Moreover, Matthews teaches presently disclosed are methods provide a major step in developing an effective scCO2-based decellularization method by preventing the tissue dehydration.
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 in the tissue were completely removed from the extracellular matrix within 1 hour (i.e., for 15 min; for 1 hour), 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. Further, Sawada teaches the more remarkable was the decellularization that was attained after a short period of treatment, wherein cell nuclei were removed within 15 min when the supercritical carbon dioxide/ethanol system was used. 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 hot 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. Thus, the small amount of ethanol and short duration of time required as taught by Sawada would provide even motivation to provide a carbon dioxide/water/ethanol combination in the presaturation chamber in order to minimize and/or greatly lessen the chances of dehydration during decellularization. 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. Furthermore, 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. Therefore, one of ordinary skill in the art would have recognized that the method of decellularizing tissue as taught by Matthew was provided to completely remove all cellular material, so that it would necessarily include removal of phospholipids having a reasonable expectation of success. 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. Furthermore, Allaire teaches a method remove arterial cells from rat and guinea pig (i.e., natural tissues) 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, wherein the aortas to be decellularized were incubated for 15 hours with agitation. Thus, it would have been prima facie obvious to perform the processes sequentially (two-step process) since SDS is known to promote cell lysis comprising solubilizing and disrupting the cellular structures and where the decellularization solution can extract the residual SDS and further remove any remaining cellular components from the natural tissue having a reasonable expectation of success, i.e., makes obvious ‘A method for decellularizing a natural tissue comprising placing the natural tissue in a pretreatment chamber that is separate from the environmental chamber, pretreating the natural tissue with a surfactant comprising sodium dodecyl sulfate (SDS) under agitation in the pretreatment chamber, wherein the natural tissue is pretreated with SDS in the pretreatment chamber for a time period of less than 48 hours, as recited in claim 1.
Applicants argue that from the totality of Booth, a skilled artisan would reasonably understand Booth as disclosing SDS as an adequate decellularizing reagent, however, Booth does not disclose nor suggest pretreating the natural tissue with a surfactant comprising sodium dodecyl sulfate under agitation in the pretreatment chamber, wherein the natural tissue is pretreated with the surfactant in the pretreatment chamber for a time period of less than 48 hours, as set forth in amended independent claim 1.
Applicant’s arguments have been fully considered but they are not persuasive, because one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of the teachings of Matthew, Sawada, Allaire, Woods and Booth, Pang and Fu, 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 Matthew, Sawada, Allaire, Woods and Booth, Pang and Fu, as a whole. Furthermore, one skilled in the art would have looked at the teachings of Booth since 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. 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. Moreover, Booth teaches 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. 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.
Applicants argue that Gratzer does not disclose nor suggest pretreating the natural tissue with a surfactant comprising sodium dodecyl sulfate under agitation in the pretreatment chamber, wherein the natural tissue is pretreated with the surfactant in the pretreatment chamber for a time period of less than 48 hours, as set forth in amended independent claim 1.
Applicant’s arguments have been fully considered but they are not persuasive, because Grtazer is not included in the above rejections; therefore, Applicants’ arguments directed to Gratzer are moot.
Furthermore, Applicants’ arguments directed to Jung and Ansari are moot, because the teachings of Jung and Ansari are not included in the above rejections.
Applicants argue that Woods, for instance, is generally directed to comparing two washing methods to remove residual SDS. 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. Nevertheless, 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. Applicant argue that nowhere does Woods describe a method for decellularizing a natural tissue 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 cannot show nonobviousness by attacking references individually where the rejections are based on combinations of the teachings of Matthew, Sawada, Allaire, Woods and Booth, Pang and Fu, 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 Matthew, Sawada, Allaire, Woods and Booth, Pang and Fu, as a whole. Furthermore, 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 after being treated with SDS. Thus, Woods further provides 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 effective removing SDS from natural tissue that had been treated 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.
Applicants argue that the supercritical CO2 treatment of the present application could compare even more favorably over longer time periods. Unexpectedly, 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.
Applicant’s 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 Matthew, Sawada, Allaire, Woods and Booth, Pang and Fu, as a whole. Accordingly, the method of decellularization of a tissue as taught by Matthew, Sawada, Allaire, Woods and Booth, Pang and Fu, 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.
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 ‘natural tissue’ is a broad genus encompassing a vast array of different types of tissues, 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 tissue 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: porcine aorta.
Conclusions
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/T.W./ Examiner, Art Unit 1619
/SARAH ALAWADI/ Primary Examiner, Art Unit 1619
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