DETAILED CORRESPONDENCE
Status of the Application
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant’s submission filed on 03/16/2026 has been entered.
Claims 1, 4-20 and 22-24 are pending in this application.
Applicant’s amendment to the claims filed 03/16/2026 is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Applicant’s amendment to the specification filed 03/16/2026 is acknowledged.
Applicant’s remarks filed on 03/16/2026 in response to the final rejection mailed on 12/17/2025 are acknowledged and have been fully considered.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Election
The elected subject matter is:
Species A1) the external stimulus comprises a pharmacological compound,
elected without traverse in the reply filed 11/20/2024.
Claims 6-8 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected species, there being no allowable generic or linking claim.
Claims 1, 4-5, 9-20 and 22-24 are being examined on the merits only to the extent they read on the elected subject matter.
Objections to Specification
The objections to the specification are withdrawn in view of the amendments to address the use of trademarks.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph 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.
Claim 23 is newly rejected under 35 U.S.C. 112(a) 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, at the time the application was filed, had possession of the claimed invention.
This is a new matter rejection and is necessitated by amendment.
MPEP § 2163.II.A.3.(b) states, “when filing an amendment an applicant should show support in the original disclosure for new or amended claims.” See also MPEP 714.02. MPEP § 2163.II.A.3.(b) further states, “[i]f the originally filed disclosure does not provide support for each claim limitation, or if an element which applicant describes as essential or critical is not claimed, a new or amended claim must be rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112, para. 1, as lacking adequate written description.” According to MPEP § 2163.I.B, “While there is no in haec verba requirement, newly added claim limitations must be supported in the specification through express, implicit, or inherent disclosure” and “The fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, applicant was in possession of the invention as now claimed. See, e.g., Vas-Cath, Inc., 935 F.2d at 1563-64, 19 USPQ2d at 1117.”
New Claim 23 recites “the method of claim 1, wherein subjecting the vascularized biological system to the external stimulus comprises supplying a perfusion media comprising the external stimulus through vascular conduits of the vascularized biological system at a time-varying change in perfusion media flow-rate”.
According to applicant’s instant remarks in the final paragraph of page 8, “Support for claim 23 may be found … at paragraphs [0135] (‘Depending on the tissue being modeled, the constituents of the in vitro system may include one or more of: mammalian cells, a plurality of mammalian cell types, extracellular tissue, vessels, bacterial cells, cell culture media, perfusion media, and the like’) and [065] (‘Any of the methods and systems provided herein are particularly useful and compatible with dynamic control of the cell culture. For example, the vasculature of the plant tissue readily accommodates changes in perfusion rate, localized changes (e.g., spatially varying) in perfusion, and time-varying changes, such as by changing the flow-rate through the plant conduits or period changes in the perfusate introduced to the plant conduit’)”.
Applicant appears to take the position that the disclosure of example properties of the vasculature of plant tissue recited in [para 065] of the instant specification renders obvious the method of claim 23 comprising “subjecting the vascularized biological system to the external stimulus comprises supplying a perfusion media comprising the external stimulus through vascular conduits of the vascularized biological system at a time-varying change in perfusion media flow-rate”, however MPEP 2163.I states the Federal Circuit has pointed out that, under United States law, a description that merely renders a claimed invention obvious may not sufficiently describe the invention for the purposes of the written description requirement of 35 U.S.C. 112. See Eli Lilly, 119 F.3d at 1567, 43 USPQ2d at 1405. Therefore, there is no apparent descriptive support for the limitation “subjecting the vascularized biological system to the external stimulus comprises supplying a perfusion media comprising the external stimulus through vascular conduits of the vascularized biological system at a time-varying change in perfusion media flow-rate” in the original application as filed. Absent descriptive support, the noted limitation is considered to introduce new matter into the claims. Applicant is invited to show support for the limitation at issue.
Claim Rejections - 35 USC § 103
The rejection of claims 1 and 11-13 under 35 U.S.C. 103 as being unpatentable over Adamski et al. (J Vis Exp, 2018, 135:e57586; cited on the IDS submitted 10/18/2022; herein referred to as Adamski) in view of Casali et al. (J Supercritical Fluids, 2018, 131:72; cited on the IDS submitted 10/18/2022; herein referred to as Casali) and evidentiary reference Neri et al. (Clin Diag Lab Immunol, 2001, 8:1131; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Neri),
the rejection of claims 5 and 14 under 35 U.S.C. 103 as being unpatentable over Adamski in view of Casali, and further in view of Ren et al. (Chem Soc Rev, 2015, 44:5680; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Ren),
the rejection of claims 9-10 under 35 U.S.C. 103 as being unpatentable over Adamski in view of Casali, and further in view of Liu et al. (Curr Opin Chem Eng, 2016, 11:94; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Liu),
the rejection of claims 15-17 under 35 U.S.C. 103 as being unpatentable over Adamski in view of Casali, Bittner et al. (Mat Today, 2018, 21:861; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Bittner) and evidentiary reference Neri,
the rejection of claims 18 and 20 under 35 U.S.C. 103 as being unpatentable over Adamski in view of Casali and Bittner, and further in view of Carlson et al. (Curr Prot Cell Biol, 2008, 19.9.1; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Carlson), and
the rejection of claim 19 under 35 U.S.C. 103 as being unpatentable over Adamski in view of Casali, Bittner and Carlson, and further in view of Gershlak et al. (Biomat, 2017, 125:13; cited on the IDS submitted 10/18/2022; herein referred to as Gershlak) and Hareven et al. (Cell, 1996, 84:735; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Hareven)
are withdrawn in view of the amendment to claims 1 and 15 to recite “a single fluid comprising supercritical CO2, peracetic acid, and ethanol”, as Adamski and Casali do not teach or suggest such a fluid corresponding to claim 1, and Adamski, Casali and Bittner do not teach or suggest such a fluid corresponding to claim 15.
Claims 1, 11-13 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Adamski in view of Casali, Hennessy et al. (JACC: Basic Translat Sci, 2017, 2:71; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as Hennessy) and evidentiary reference Neri.
The instant rejection is maintained from the previous Office Action and any newly recited portions are necessitated by claim amendment.
Claim 1 is drawn to a method of simulating a biological response of a cellular system, the method comprising:
(i) removing at least some DNA-containing material from a vascular plant tissue to produce a vascularized cellulose scaffold;
wherein the removing step comprises submerging the vascular plant tissue in a single fluid comprising supercritical CO2, peracetic acid (PAA) and ethanol, thereby removing at least some DNA-containing material from the vascular plant tissue;
(ii) seeding the vascularized cellulose scaffold with cultured biological cells;
(iii) growing the cultured biological cells on the vascularized cellulose scaffold to produce a vascularized biological system;
(iv) subjecting the vascularized biological system to an external stimulus; and
(v) detecting a chemical response of the vascularized biological system via an instrument.
Adamski discusses methods for decellularization of plant tissues for tissue engineering [title].
Regarding claim 1, Adamski discloses a method of generating a scaffold for tissue replacement using plant tissues comprising the removal of cellular matter to isolate leaf vasculature [abstract], wherein Mesenchymal stem cells (MSCs) were seeded on decellularized scaffold and allowed to incubate for 24 h [p 3, para 5] resulting in the growth of MSCs on the scaffold [Figure 4 title] corresponding to steps (ii)-(iii), and the cells were subsequently treated with calcein to visualize the cells [Figure 4 and Figure 4 legend]. The use of decellularized plant tissue is interpreted to correspond to a vascularized cellulose scaffold, as Adamski discloses plant vascular vessels minimize hydraulic resistance by branching into smaller vessels similar to mammalian vasculature, and decellularized plant tissue maintains the complex network of vessels and interconnected pores which overcomes limitations affecting scaffolds in tissue engineering [p 1, final para]. As disclosed by Adamski, the removal of plant cellular matter encompasses removing DNA-containing material as recited in step (i), and the treatment of cells with calcein is considered to encompass subjecting the system to an external stimulus as recited in step (iv), as the calcein is known in the art to produce fluorescence upon interaction with intracellular enzymes as evidenced by Neri [p 1131, col 2, para 1]. The disclosure Adamski to use of a microscope to visualize the chemical response of MSCs to calcein in [Figure 4] is considered to encompass the limitations in step (v).
Adamski does not teach submerging the vascular plant tissue in a single fluid comprising supercritical CO2, paracetic acid (PAA) and ethanol, thereby removing at least some DNA-containing material from the vascular plant tissue.
Casali discusses scCO2-based decellularization methods for maintaining scaffold hydration and mechanical properties [title] and discloses scCO2 is an alternative to common decellularization methods that damage the tissue microstructure or deposit cytotoxic residue [abstract].
Regarding claim 1, Casali teaches a method of decellularizing tissue using scCO2 using aqueous additives that include mixtures comprising ethanol [p 74, col 1, para 3] wherein scCO2 mixtures were contacted with the sample in the treatment chamber [Figure 1], which is considered to encompass the submerging of tissue in a fluid comprising scCO2 to remove at least some DNA-containing material from tissue.
Hennessy discusses scCO2-based sterilization of decellularized heart valves [title] and discloses that sterilizing tissue using scCO2 is a superior method of sterilizing tissue for grafts that has a promising use with decellularized tissues [abstract].
Regarding claim 1, Hennessy teaches xenografts used for tissue engineering requires decellularization and the removal of immunogenic components, which requires sterilization from bacteria, fungi and other organisms [p 71, col 1, para 1], which is echoed by Casali that tissue engineering scaffolds must be sterile [p 72, col 1, para 2]. To achieve such sterilization, Hennessy teaches the treatment of tissue with scCO2 for decellularization, and subsequent treatment with PAA and ethanol for sterilization [p 72, col 1, para 2, and Table 1]. Hennessy additionally teaches that sterilization may be carried out using scCO2 and a reagent comprising PAA [p 73, col 2, para 4].
As Hennessy and Casali teach decellularization techniques comprising using scCO2 fluids, Casali teaches the scCO2 decellularization fluid may contain additives such as ethanol, and Hennessy teaches sterilization both with fluids comprising PAA and ethanol and with fluids comprising scCO2 and PAA, one of ordinary skill in the art would have reasonably expected to combine the decellularization steps with the sterilization steps as taught by Hennessy through routine optimization (see MPEP 2144.05.II), as it is common in the art to optimize methods to increase their efficiency. One of ordinary skill in the art would have had a reasonable expectation of success because both Casali and Hennessy relate to decellularization protocols, and the steps comprise considerable overlap regarding the respective active components.
In view of Adamski and Casali, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Adamski by using scCO2 for decellularization, as taught by Casali, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the method of Adamski by using scCO2 for decellularization because Casali discloses using scCO2 is an alternative to common decellularization methods that damage the tissue microstructure or deposit cytotoxic residue. One of ordinary skill in the art would have had a reasonable expectation of success because both Adamski and Casali relate to decellularization for the preparation of tissue engineering scaffolds.
In view of Casali and Hennessy, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Adamski and Casali by adding PAA and ethanol to the scCO2 fluid as taught by Casali and Hennessy to arrive at the claimed invention through routine optimization. One of ordinary skill in the art would have been motivated to modify the combined method of Adamski and Casali because Hennessy and Casali teach decellularization techniques comprising using scCO2 fluid, Casali teaches the scCO2 decellularization fluid may contain additives such as ethanol, Hennessy teaches sterilization both with fluids comprising PAA and ethanol and with fluids comprising scCO2 and PAA, and because it is common in the art to optimize methods to increase the efficiency, which would include combining steps using fluids with considerable overlap in respective components. One of ordinary skill in the art would have had a reasonable expectation of success because Adamski, Casali and Hennessy relate to tissue decellularization methods, and Casali and Hennessy relate to such methods comprising the use of scCO2.
Regarding claims 11-12, Casali teaches the decellularization treatment with scCO2 was maintained at 37 °C [p 74, col 1, para 4].
Regarding claim 13, Adamski teaches the seeding of cells on tissue scaffolds derived from plants and culturing the cells [p 3, para 5] which is understood to encompass the supplying of growth media, and further teaches that plant tissues are desirable for such scaffolds because of their efficiency in fluid transport due to their vascular networks being efficiently perfused, which can guide cellular migration and angiogenesis for the creation of replacement tissue [p 6, final para]. Therefore one of skill in the art would reasonably conclude that seeding and growing cells on a vascular plant tissue scaffold would result in the media required for cell growth to be supplied via said vasculature of the tissue scaffold.
Regarding claim 24, Adamski teaches the treatment of cells with calcein [Figure 4], wherein calcein is known in the art to produce fluorescence upon interaction with intracellular enzymes as evidenced by Neri [p 1131, col 2, para 1]. Therefore, as the method of Adamski is adding calcein in order to visualize its reaction with the an enzyme, it is considered to be encompassed by the limitation “detecting expression of a protein biomarker” recited in the claim.
Therefore, the invention of claims 1, 11-13 and 24 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 4 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Adamski in view of Casali and Hennessy as applied to claims 1, 11-13 and 24 above, and further in view of White et al. (J Biotechnol, 2006, 123:504; cited on the Form PTO-892 mailed 03/07/2025; herein referred to as White).
The instant rejection is maintained from the previous Office Action and any newly recited portions are necessitated by claim amendment.
Claim 4 is drawn to the method of claim 1, wherein the fluid comprises at least 80 wt% scCO2; and wherein the ratio of PAA to ethanol is from 1:100 to 1:10.
The teachings of Adamski, Casali and Hennessy as applied to claims 1, 11-13 and 24 are discussed above. These references do not teach the fluid comprises at least 80 wt% scCO2.
White discusses effective terminal sterilization using scCO2 [title], and discloses that scCO2 is appealing for sterilization due to the ease at which the supercritical state is attained, and that scCO2-based sterilization techniques are capable of achieving rapid inactivation of bacterial endospores [abstract].
Regarding claim 4, White teaches a method of sterilizing samples using scCO2 and additives that include ethanol and 5% PAA [Table 1], which corresponds to a fluid comprising at least 80 wt% scCO2. Hennessy additionally teaches a mixture of 96% ethanol and 2% PAA can be used to sterilize tissue [Table 1], which corresponds to a ratio of PAA to ethanol from 1:100 to 1:10.
In view of White, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Adamski, Casali and Hennessy by replacing the 5% PAA component of the PAA/scCO2 mixture of White with a component of 96% ethanol and 2% PAA, as taught by Hennessy, to arrive at the claimed invention, since the simple substitution of one known element for another results in a predictable result. One of ordinary skill in the art would have recognized that both the PAA/scCO2 mixture of White and the mixture of ethanol/PAA mixture of Hennessy can be used for sterilization, and as such both are capable of being incorporated into such methods as described by Adamski, Casali and Hennessy. Thus it would have been obvious to one of ordinary skill in the art to replace the 5% PAA component of the PAA/scCO2 mixture of White with a mixture of 96% ethanol and 2% PAA, as one of ordinary skill in the art would have been able to carry out such a substitution with reasonable expectation of success because both White and Hennessy discuss sterilization techniques comprising scCO2, ethanol and PAA.
Regarding claim 22, White teaches scCO2 with 5% PAA as stated above, which is considered to correspond to 5 wt% PAA, and the solution of White is considered to correspond to a solution comprising 95 wt% scCO2. As Hennessy teaches a solution with a 2% PAA and 96% ethanol, one of skill in the art in substituting the PAA:ethanol solution of Hennessy for the 5 wt% PAA solution of White would be expected to produce a 95 wt% scCO2 solution with 0.1 wt% PAA and 4.8 wt% ethanol. While White and Hennessy do not teach the mixture recited in the claim comprising at least 95 wt% scCO2, 0.1-3 wt% ethanol and 0.01-0.1 wt% PAA, MPEP 2144.05.II.A states where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Therefore, the invention of claims 4 and 22 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 5 and 14 are newly rejected under 35 U.S.C. 103 as being unpatentable over Adamski in view of Casali and Hennessy as applied to claims 1, 11-13 and 24 above, and further in view of Ren.
The instant rejection is newly stated and is necessitated by the amendments to claim 1.
Claim 5 is drawn to the method of claim 1, wherein the external stimulus comprises a pharmacological compound. As the instant specification does not define a pharmacological compound, the term’s plain meaning of a medication or drug is being used in the examination.
The teachings of Adamski, Casali and Hennessy as applied to claims 1, 11-13 and 24 are discussed above. These references do not teach the use of a pharmacological compound.
Ren discusses surface modification and endothelialization of biomaterials as potential scaffolds for vascular tissue engineering [title], and discloses that surface modification and endothelialization of vascular biomaterials are common approaches to resist nonspecific adhesion of proteins, to improve hemocompatibility and hence enhance long-term patency of artificial vascular grafts [p 5680, col 2, para 2]. Regarding the enhancement of long-term patency, Ren teaches that artificial vascular grafts seeded with endothelial cells should help in improving patency rates of these grafts, given that cell spreading and growth can be controlled [p 5683, col 2, para 3], as typically adhesion of platelet cells on endothelial layers results in the detachment of endothelial cells [p 5683, col 2, para 4]. Ren additionally reviews various components involved in tissue modeling including polymers for attachment and protein additives, such as heparin, which is effective for preventing proliferation of vascular smooth muscle cells (VSMCs), as proliferation can lead to graft occlusion and failure [p 5688, col 2, para 2]. Therefore, Ren outlines some of the strategies to overcome known problems in tissue modeling with various cell types.
Regarding claim 5, Ren teaches that rapamycin can be used to suppress local SMC proliferation when supplemented in cultures growing on tissue scaffolds [p 5693, col 2, para 3], wherein rapamycin is considered to be a pharmacological compound.
In view of Ren, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Adamski, Casali and Hennessy by using rapamycin, as taught by Ren, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the combined method of Adamski, Casali and Hennessy by using rapamycin, because Ren discloses using rapamycin can prevent cell types from proliferating in tissue scaffolds that would lead to graft occlusion and failure. One of ordinary skill in the art would have had a reasonable expectation of success because both Adamski and Ren relate to seeding cell cultures on tissue scaffolds.
Regarding claim 14, Ren teaches that artificial vascular grafts seeded with endothelial cells should help in improving patency rates of these grafts, given that cell spreading and growth can be controlled [p 5683, col 2, para 3].
Therefore, the invention of claims 5 and 14 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 9-10 are newly rejected under 35 U.S.C. 103 as being unpatentable over Adamski in view of Casali and Hennessy as applied to claims 1, 11-13 and 24 above, and further in view of Liu.
The instant rejection is newly stated and is necessitated by the amendments to claim 1.
Claim 9 is drawn to the method of claim 1, wherein the cultured biological cells comprise human cancer cells.
The teachings of Adamski, Casali and Hennessy as applied to claims 1, 11-13 and 24 are discussed above. These references do not teach human cancer cells.
Liu discusses modeling tumor microenvironments using custom-designed biomaterial scaffolds [title], and discloses that the tumor microenvironment is critical in promoting resistance and recurrence of tumors, therefore inspiring the application of tissue engineering strategies to identify therapeutic targets and regimens [p 11, col 2, para 1].
Regarding claims 9-10, Liu teaches a workflow for engineering a tumor microenvironment on a tissue scaffold comprising the seeding of human cancer cells and stromal cells on a scaffold to produce a 3D tumor model that represents the tumor microenvironment [Figure 1].
In view of Liu, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Adamski, Casali and Hennessy by using human cancer cells, as taught by Liu, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the combined method of Adamski, Casali and Hennessy by using human cancer cells, because Liu discloses using human cancer cells in tissue engineering strategies can identify potential therapeutic targets and regimens. One of ordinary skill in the art would have had a reasonable expectation of success because both Adamski and Liu relate to seeding cell cultures on tissue scaffolds.
Therefore, the invention of claims 9-10 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claim 23 is newly rejected under 35 U.S.C. 103 as being unpatentable over Adamski in view of Casali, Hennessy and Liu as applied to claims 1, 9-13 and 24 above, and further in view of Gershlak.
The instant rejection is newly stated and necessitated by claim amendment.
Claim 23 is drawn to the method of claim 1, wherein subjecting the vascularized biological system to the external stimulus comprises supplying a perfusion media comprising the external stimulus through vascular conduits of the vascularized biological system at a time-varying change in perfusion media flow-rate.
As claim 23 is dependent from claim 1, it is being discussed here with the other claim 1 dependents.
The teachings of Adamski, Casali, Hennessy and Liu as applied to claims 1, 9-13 and 24 are discussed above. These references do not teach supplying a perfusion media comprising the external stimulus through vascular conduits of the vascularized biological system at a time-varying change in perfusion media flow-rate.
As discussed above, Liu teaches a workflow for engineering a tumor microenvironment on a tissue scaffold comprising the seeding of human cancer cells and stromal cells on a scaffold to produce a 3D tumor model that represents the tumor microenvironment, and shows a step involving the perfusion of cytokines and growth factors to a scaffolded tissue system to generate a 3D tumor model [Figure 1]. As such the teachings of Liu are considered to correspond to the supplying of perfusion media comprising an external stimulus (e.g., growth factors and cytokines).
Gershlak discusses using decellularized plants as perfusable tissue engineered scaffolds [title], and describes perfusion studies using said scaffolds to visualize the effects of previously carried out method steps, wherein perfusion was carried out at 200 µl/min, and wherein multiple solutions were perfused into the scaffold through the vascular conduits at different times such as serial perfusions of various sized microspheres and blank fluid [p 15, col 1, para 1-2]. As Gershlak is supplying multiple solutions over time, the flow-rate of solution is not constant, and therefore is considered to be variable. The supplying of multiple solutions as taught by Gershlak is therefore encompassed by the limitation of “time-varying change in perfusion media flow-rate”. As Liu teaches the perfusion of media containing cytokines and growth factors into a scaffolded tissue model, and Gershlak teaches sequential perfusion of solutions comprising various substances at flow rates that vary over time, the combined teachings of Liu and Gershlak are considered to encompass the limitations of claim 23.
In view of Liu and Gershlak, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Adamski, Casali, Hennessy and Liu by supplying perfusion media to the scaffold comprising external stimuli, as taught by Liu, and at different flow rates, as taught by Gershlak, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the combined method of Adamski, Casali, Hennessy and Liu, because Liu teaches a method of engineering 3D cellular environments wherein media comprising growth factors and cytokines are perfused into the model, and Gershlak teaches perfusion into the vasculature of plant tissue models wherein the perfusion of different solutions corresponds to time-varying flow rates. One of ordinary skill in the art would have had a reasonable expectation of success because Adamski and Liu relate to seeding cell cultures on tissue scaffolds, and Adamski and Gershlak relate to tissue scaffolds generated from decellularized plant leaves.
Therefore, the invention of claim 23 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 15-17 are rejected under 35 U.S.C. 103 as being unpatentable over Adamski in view of Bittner, Casali and Hennessy, and evidentiary reference Neri.
The instant rejection is maintained from the previous Office action, and any newly stated and is necessitated by claim amendment.
Claim 15 is drawn to a method of simulating a biological response of a multilayer cellular system, the method comprising:
(i) removing at least some DNA-containing material from a first vascular plant tissue to produce a first vascularized cellulose scaffold;
(ii) removing at least some DNA-containing material from a second vascular plant tissue to produce a second vascularized cellulose scaffold;
wherein the removing step comprises submerging the vascular plant tissue in a single fluid comprising supercritical CO2, PAA and ethanol, thereby removing at least some DNA-containing material from the vascular plant tissue;
(iii) combining the first and second vascularized cellulose scaffolds to create a multilayered scaffold;
(iv) seeding the multilayered scaffold with cultured biological cells;
(v) growing the cultured biological cells on the multilayer scaffold to produce a multilayered vascularized biological system;
(vi) subjecting the multilayered vascularized biological system to an external stimulus; and
(vii) detecting a chemical response of the vascularized biological system via an instrument.
Regarding claim 15, Adamski discloses a method of generating a scaffold for tissue replacement using plant tissues comprising the removal of cellular matter to isolate leaf vasculature [abstract], wherein Mesenchymal stem cells (MSCs) were seeded on decellularized scaffold and allowed to incubate for 24 h [p 3, para 5] resulting in the growth of MSCs on the scaffold [Figure 4 title] corresponding to steps (iv) and (v), and the cells were subsequently treated with calcein to visualize the cells [Figure 4 and Figure 4 legend]. The use of decellularized plant tissue is interpreted to correspond to a vascularized cellulose scaffold, as Adamski discloses plant vascular vessels minimize hydraulic resistance by branching into smaller vessels similar to mammalian vasculature, and decellularized plant tissue maintains the complex network of vessels and interconnected pores which overcomes limitations affecting scaffolds in tissue engineering [p 1, final para]. As disclosed by Adamski, the removal of plant cellular matter encompasses removing DNA-containing material corresponding to steps (i)-(ii), and the treatment of cells with calcein is considered to encompass the subjection of the system to an external stimuli corresponding to step (vi), as the calcein is known in the art to produce fluorescence upon interaction with intracellular enzymes as evidenced by Neri [p 1131, col 2, para 1]. The disclosure Adamski to use of a microscope to visualize the chemical response of MSCs to calcein in [Figure 4] is considered to encompass the limitations in step (vii).
Adamski does not teach a multilayered scaffold comprising two vascularized cellulose scaffolds each seeded with cultured biological cells; and a single fluid comprising supercritical CO2, PAA and ethanol.
Bittner discusses 3D printing of multilayered tissue engineering scaffolds [title], and discloses that highly complex scaffolds offer a more accurate replication of native tissue properties and architecture, with strong interest in the construction of multilayered scaffolds [abstract].
Regarding claim 15, Bittner teaches techniques of preparing multilayered tissue scaffolds for creating spatially heterogeneous constructs [p 864, col 2, para 2]. Therefore, one of skill in the art would be motivated to carry out the method of Adamski to generate two vascular scaffolds each seeded with cultured biological cells and combine them to form a multilayered scaffold as taught by Bittner, corresponding to step (iii).
Casali discusses scCO2-based decellularization methods for maintaining scaffold hydration and mechanical properties [title] and discloses scCO2 is an alternative to common decellularization methods that damage the tissue microstructure or deposit cytotoxic residue [abstract].
Regarding claim 15, Casali teaches a method of decellularizing tissue using scCO2 using aqueous additives that include mixtures comprising ethanol [p 74, col 1, para 3] wherein scCO2 mixtures were contacted with the sample in the treatment chamber [Figure 1], which is considered to encompass the submerging of tissue in a fluid comprising scCO2.
Hennessy discusses scCO2-based sterilization of decellularized heart valves [title] and discloses that sterilizing tissue using scCO2 is a superior method of sterilizing tissue for grafts that has a promising use with decellularized tissues [abstract].
Regarding claim 15, Hennessy teaches xenografts used for tissue engineering requires decellularization and the removal of immunogenic components, which requires sterilization from bacteria, fungi and other organisms [p 71, col 1, para 1], which is echoed by Casali that tissue engineering scaffolds must be sterile [p 72, col 1, para 2]. To achieve such sterilization, Hennessy teaches the treatment of tissue with scCO2 for decellularization and subsequent treatment with PAA and ethanol for sterilization [p 72, col 1, para 2, and Table 1]. Hennessy additionally teaches decellularization may be carried out by scCO2 [p 73, col 1, penultimate paragraph], and that sterilization may be carried out using scCO2 with a reagent comprising PAA [p 73, col 2, para 4].
As Hennessy and Casali teach decellularization techniques comprising using scCO2 fluid, Casali teaches the scCO2 decellularization fluid may contain additives such as ethanol, and Hennessy teaches sterilization both with fluids comprising PAA and ethanol and with fluids comprising scCO2 and PAA, one of ordinary skill in the art would be reasonably expected to combine the decellularization steps with the sterilization steps as taught by Hennessy through routine optimization (see MPEP 2144.05.II), as it is common in the art to optimize methods to increase the efficiency. One of ordinary skill in the art would have had a reasonable expectation of success because both Casali and Hennessy relate to decellularization protocols, and the steps comprise considerable overlap regarding the respective active components.
In view of Adamski, Bittner and Casali, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Adamski by using scCO2 for decellularization, as taught by Casali, and forming a multilayered tissue scaffold, as taught by Bittner, to arrive at the claimed invention.
One of ordinary skill in the art would have been motivated to modify the method of Adamski by using scCO2 for decellularization because Casali discloses using scCO2 is an alternative to common decellularization methods that damage the tissue microstructure or deposit cytotoxic residue.
One of ordinary skill in the art would have been motivated to modify the method of Adamski by forming a multilayered scaffold because Bittner discloses that highly complex scaffolds such as multilayered scaffolds offer a more accurate replication of native tissue properties and architecture.
One of ordinary skill in the art would have had a reasonable expectation of success to modify the method of Adamski by using scCO2 for decellularization because both Adamski and Casali relate to decellularization for the preparation of tissue engineering scaffolds.
One of ordinary skill in the art would have had a reasonable expectation of success to modify the method of Adamski by forming a multilayered scaffold because both Adamski and Bittner relate to generating tissue scaffolds for tissue engineering.
In view of Casali and Hennessy, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Adamski, Bittner and Casali by adding PAA and ethanol to the scCO2 fluid as taught by Casali and Hennessy to arrive at the claimed invention through routine optimization. One of ordinary skill in the art would have been motivated to modify the combined method of Adamski and Casali because Hennessy and Casali teach decellularization techniques comprising using scCO2 fluid, Casali teaches the scCO2 decellularization fluid may contain additives such as ethanol, Hennessy teaches sterilization both with fluids comprising PAA and ethanol and with fluids comprising scCO2 and PAA, and because it is common in the art to optimize methods to increase the efficiency, which would include combining steps using fluids with considerable overlap in respective components. One of ordinary skill in the art would have had a reasonable expectation of success because Adamski, Casali and Hennessy relate to tissue decellularization methods, and Casali and Hennessy relate to such methods comprising the use of scCO2.
Regarding claim 16, considering the interpretation that the first and the second cell types recited in the claim can be the same cell type, the combined teachings of Bittner and Adamski as discussed above satisfy the limitations of claim 16.
Regarding claim 17, the limitation “the multilayered scaffold comprises a third vascularized cellulose scaffold” is interpreted such that the third scaffold is formed the same way as the other two scaffolds in the multilayered scaffold according to the method of claim 15, and includes cultured cells. Regarding claim 17, as Bittner teaches techniques of preparing multilayered tissue scaffolds for creating spatially heterogeneous constructs [p 864, col 2, para 2], one of skill in the art would be motivated to carry out the method of Adamski to generate a third vascular scaffold seeded with cultured biological cells to combine with the other vascular scaffolds to form a multilayered scaffold as taught by Bittner.
Therefore, the invention of claims 15-17 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 18 and 20 are newly rejected under 35 U.S.C. 103 as being unpatentable over Adamski in view of Bittner, Casali and Hennessy as applied to claims 15-17 above, and further in view of Carlson.
The instant rejection is newly stated and is necessitated by the amendments to claim 15.
Claim 18 is drawn to the method of claim 16, wherein the first cell type is keratinocytes and the second cell type is dermal fibroblasts.
The teachings of Adamski, Bittner, Casali and Hennessy as applied to claims 15-17 are discussed above. These references do not teach keratinocytes or dermal fibroblasts.
Carlson discusses 3D tissue models of normal and diseased skin [title] and discloses that the production of 3D tissue models known as human skin equivalents (HSEs) allows the generation of human tissues that mimic morphology, differentiation and growth of human skin, as well as disease processes of cancer and would re-epithelialization to provide new tools for the study of diseases in humans [abstract].
Regarding claims 18 and 20, Carlson teaches the construction of a 3D HSE [Figure 19.19.1] comprising skin keratinocytes that are grown on a substrate populated with dermal fibroblasts [p 1, final para]. As the tissue model of Carlson is described as a 3D tissue model that is a human skin equivalent to generate human tissues that mimic morphology, differentiation and growth of human skin, it is considered to correspond to a biomimetic model of human skin.
In view of Carlson, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Adamski, Bittner, Casali and Hennessy by using skin keratinocytes and dermal fibroblasts as the two cell types, as taught by Carlson, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the combined method of Adamski, Bittner, Casali and Hennessy by using skin keratinocytes and dermal fibroblasts, because Carlson discloses that the production of human skin equivalents allows the generation of human tissues that mimic morphology, differentiation and growth of human skin, as well as disease processes of cancer and would re-epithelialization to provide new tools for the study of diseases in humans. One of ordinary skill in the art would have had a reasonable expectation of success because Adamski, Bittner and Carlson relate to tissue engineering.
Therefore, the invention of claims 18 and 20 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claim 19 is newly rejected under 35 U.S.C. 103 as being unpatentable over Adamski in view of Bittner, Casali, Hennessy and Carlson as applied to claims 15-18 and 20 above, and further in view of Gershlak and Hareven.
The instant rejection is necessitated by the amendments to claim 15.
Claim 19 is drawn to the method of claim 18, wherein the first layer of the multilayered scaffold is derived from a tomato leaf and the second layer is derived from spinach leaves.
The teachings of Adamski, Bittner, Casali, Hennessy and Carlson as applied to claims 15-18 and 20 are discussed above. These references do not teach the use of tomato leaf and spinach leaf for the construction of the scaffolds.
Regarding claim 19, Adamski discloses a method of using ficus leaves (F. hispada) as a tissue scaffold [p 2, Protocol 1], Gershlak discusses using decellularized plants as perfusable tissue engineered scaffolds [title] and discloses a method of decellularizing spinach leaves [p 14, col 2, para 1], and Hareven discusses genetic manipulation of tomato leaf architecture [title] and discloses that tomatoes have leaves [Figure 1].
In view of Gershlak and Hareven, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Adamski, Bittner, Casali, Hennessy and Carlson by replacing the ficus leaves of Adamski with the spinach leaves of Gershlak and the tomato leaves of Hareven to arrive at the claimed invention, since the simple substitution of one known element for another results in a predictable result. One of ordinary skill in the art would have recognized that the ficus leaves of Adamski, the spinach leaves of Gershlak, and the tomato leaves of Hareven are all leaves, and as such all are capable of being incorporated into such methods as described by Adamski. Thus it would have been obvious to one of ordinary skill in the art to replace the ficus leaves of Adamski with the spinach leaves of Gershlak and the tomato leaves of Hareven, as one of ordinary skill in the art would have been able to carry out such a substitution with reasonable expectation of success because Adamski and Gershlak relate to tissue scaffolds using decellularized plant leaves, and Adamski, Gershlak and Hareven relate to methods involving plant leaves.
Therefore, the invention of claim 19 would have been obvious to one of ordinary skill in the art before the effective filing date.
Response to Remarks: beginning on page 10 of Applicant’s response to rejections under 35 USC 103; Applicant in summary contends the prior art does not teach the use of a single fluid comprising scCO2, ethanol and PAA for the removal of DNA-containing material in a single step; Applicant further contends the prior art does not teach the discovery of the use of scCO-2, PAA and ethanol for decellularizing plant material quickly and efficiently while also preserving the cellulosic materials structure; Applicant further contends the rejections are based on improper hindsight reasoning.
Applicant’s remarks are considered and found not convincing.
Regarding the teachings of the prior art, as stated in the rejection above, Casali teaches a method of decellularizing tissue using scCO2 using aqueous additives that include mixtures comprising ethanol [p 74, col 1, para 3] wherein scCO2 mixtures were contacted with the sample in the treatment chamber. Hennessy discusses scCO2-based sterilization of decellularized heart valves and teaches the treatment of tissue with scCO2 for decellularization, and the subsequent treatment with PAA and ethanol for sterilization [p 72, col 1, para 2, and Table 1] as noted by Applicant in two separate steps. Hennessy additionally teaches that sterilization may be carried out using scCO2 with a reagent comprising PAA [p 73, col 2, para 4]. As Hennessy and Casali teach decellularization techniques comprising using scCO2 fluid, Casali teaches the scCO2 decellularization fluid may contain additives such as ethanol, and Hennessy teaches sterilization both with fluids comprising PAA and ethanol and with fluids comprising scCO2 and PAA, one of ordinary skill in the art would be reasonably expected to combine the decellularization steps with the sterilization steps as taught by Hennessy through routine optimization (see MPEP 2144.05.II), as it is common in the art to optimize methods to increase the efficiency. One of ordinary skill in the art would have had a reasonable expectation of success because both Casali and Hennessy relate to decellularization protocols, and the steps comprise considerable overlap regarding the respective active components.
Regarding the assertion that the prior art does not teach the discovery of the use of scCO-2, PAA and ethanol for decellularizing plant material quickly and efficiently while also preserving the cellulosic materials structure: the use of said fluid for quick and efficient plant material decellularization while preserving the cellulosic materials structure is not required by the claimed method, as this limitation is not recited in the claims. As the prior art teaches the components of the fluid mixture and in view of the obviousness to combine steps as set forth in the rejection above, one of ordinary skill in the art in carrying out the combined method proposed in the rejection would be quickly and efficiently decellularizing plant material while also preserving the cellulosic materials structure.
In response to applicant’s 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). In this case, the rejections take into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and do not include knowledge gleaned only from the applicant’s disclosure and as such, the rejections are proper.
Conclusion
Status of the Application:
Claims 1, 4-20 and 22-24 are pending.
Claims 6-8 are withdrawn.
Claims 1, 4-5, 9-20 and 22-24 are rejected.
No claim is in condition for allowance.
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/JOSEPH R SPANGLER/
Examiner
Art Unit 1656
/David Steadman/Primary Examiner, Art Unit 1656
Prosecution Insights