Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 01/21/2026 has been entered.
Claim Status
1. Claims 1, 3 – 6, 8, 9, 15, 34, 41, 45, 47, 59, 62, 67, 69, 73, and 76 remain pending. Claim 7 has been canceled.
Election/Restrictions
2. Applicant’s election without traverse of Group I (claims 1 – 9, 15, 34, 41, 45, 47, 59, 62, 67, 69, and 73) in the reply filed on 01/30/2025 is acknowledged.
3. Claim 76 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 01/30/2025.
4. Claims 1, 3 – 6, 8, 9, 15, 34, 41, 45, 47, 59, 62, 67, 69, and 73 are under consideration.
Priority
5. This application is a 371 of PCT/AU2020/050882, which claims priority to a foreign application No. 2019903094, filed in Australia on 23 August 2019.
Withdrawn Claim Rejections
6. The rejection of claim 7 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancelation of the claim.
7. The rejection of claims 1, 3, 8, 9, and 15 under 35 U.S.C. 103 is withdrawn in view of Applicant’s arguments at page 6 regarding the surface area of the spheroid at page 7, para. 2 regarding “anomalies in thickness”.
8. The rejection of claims 4 – 6 under 35 U.S.C. 103 is withdrawn in view of Applicant’s arguments cited above.
9. The rejection of claim 34 under 35 U.S.C. 103 is withdrawn in view of Applicant’s arguments cited above.
10. The rejection of claims 41, 45, 47, 62, 67, 69, and 73 under 35 U.S.C. 103 is withdrawn in view of Applicant’s arguments at page 10.
11. The rejection of claim 59 under 35 U.S.C. 103 is withdrawn in view of Applicant’s arguments at page 10.
New Claim Objections and Rejections
Claim Objections
12. Claim 1 is objected to because of the following informalities: in line 3, “throughout the tissue” should read “throughout the bio-printed kidney tissue” to clarify that the nephrons are distributed throughout the bio-printed kidney tissue and not the nephron tissue. Appropriate correction is required.
13. Claim 9 is objected to because of the following informalities: in line 1, “the bio-printed kidney tissue of claim 7” should read “the bio-printed kidney tissue of claim 1” because claim 7 is canceled. Appropriate correction is required. Claim 7 is being examined as depending from claim 1.
14. Claim 41 is objected to because of the following informalities: in line 2, “bio- ink” should read “bio-ink”. Appropriate correction is required.
15. Claim 47 is objected to because of the following informalities: “renal progenitor cells” is first recited on line 2 and repeated on line 5. The first recitation should be deleted because it is redundant. Appropriate correction is required.
16. Claim 62 is objected to because of the following informalities: in line 1, “bio- printing” should read “bio-printing”. Appropriate correction is required.
Claim Interpretation
17. For the purpose of applying prior art, claim 73 is interpreted as bio-printed kidney tissue and recitation of “produced according to claim 41” is interpreted as a product-by-process limitation where the claim is not limited to the manipulation of the recited steps, only the structure implied by the steps (see MPEP 2113).
Claim Rejections - 35 USC § 112
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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.
18. Claims 41, 45, 47, 59, 62, 67, and 69 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method for producing bio-printed kidney tissue comprising (1) bio-printing a pre-determined amount of a bio-ink comprising kidney organoids onto a surface, and (2) inducing the bio-printed kidney organoids to form bio-printed kidney tissue by culturing the bio-printed kidney organoids in culture media containing CHIR99021, FGF9, and heparin, wherein the bio-ink is bio-printed in a layer that is about 50 µm high or less and wherein the bio-printed kidney tissue comprises a surface area of nephron tissue of greater than 0.2 mm2 per 10,000 cell printed does not reasonably provide enablement for a method for producing bio-printed kidney tissue comprising bio-printing any bio-ink comprising a plurality of any cells and any method of induing the bio-printed bio-ink to form bio-kidney tissue. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(a)). The court in Wands states: "Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue experimentation. The key word is 'undue,' not 'experimentation.' " (Wands, 8 USPQ2d 1404). Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. "Whether undue experimentation is needed is not a single, simple factual determination, but rather is a conclusion reached by weighing many factual considerations." (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required include: (a) the breadth of the claims, (b) the nature of the invention, (c) the state of the prior art, (d) the level or ordinary skill in the art, (e) the level of predictability in the art, (f) the amount of direction provided by the inventor, (g) the existence of working examples, and (h) the quantity of experimentation needed to make use of the invention based on the content of the disclosure. While all of these factors are considered, a sufficient amount for a prima facie case are discussed below.
(a) The breadth of the claims: The claims broadly recite “the bio-ink comprises a plurality of cells” and “inducing the bio-printed, pre-determined amount of the bio-ink to form bio-printed kidney tissue” and therefore the claims read on any bio-ink comprising any cells and any method of inducing the formation of kidney tissue. Consequently, the breadth of the claims is expansive.
It is noted that the instant rejection is based on the following issue:
(i) absence of an enabling disclosure of bio-printing any bio-ink comprising a plurality of any cells to form bio-printed kidney tissue.
(b) The nature of the invention: The nature of the invention relates to bio-printed kidney tissue and methods of manufacturing the same where the bio-printed tissue and methods may be used in a variety of applications such as disease modeling, drug screening, drug testing, renal replacement, tissue engineering, and regenerative medicine (page 1, para. 0001).
(c) The state of the prior art:
Homan (Homan, Kimberly A., et al. "Bioprinting of 3D convoluted renal proximal tubules on perfusable chips." Scientific reports 6.1 (2016): 34845.) teaches a printed renal proximal tubule where an ECM scaffold in the shape of a tubule was printed and then seeded with proximal tubule epithelial cells (page 2; Figure 1; page 3, para. 2 – 6; page 9, para. 2 – 5). Homan teaches the inks for printing do not comprise cells (page 8, last para.). Homan teaches after seeding the scaffold with cells, the construct is perfused with PTEC media and the cells reach confluency at around 3 weeks post seeding (page 10, para. 2).
King (King, Shelby M., et al. in physiology 8 (2017): 123) teaches a method of bioprinting proximal tubule tissues by combining cultured renal fibroblasts and HUVECs at a 50:50 ratio in NovoGel Bio-Ink followed by bioprinting the cells followed by culturing the cells in EGM-2 media with EBM-2 supplements followed by adding primary RPTEC cells in RPTEC media (page 3, left col. 3). King teaches tissues were then maintained for up to 30 days in 3D PT Tissue media and FBS (page 3, left col. 3).
Nguyen (US94818868; Filed 10/06/2015; Published11/01/2016; previously cited) teaches multiple examples of bioprinting renal tubules similar to King in which various ratios renal fibroblasts to HUVECs are combined in a bio-ink and bioprinted and the resulting tissue is cultured in renal cell media (col. 27 – 31).
Thus, the state of the prior art teaches bioprinting kidney tissue with a bio-ink that comprises renal cells and HUVECs and culturing the printed cells in renal cell culture media. The state of the prior art does not teach bio-inks comprising any other types of cells (e.g., stem cells) for producing a kidney tissue and does not teach any other methods (e.g., differentiation) for inducing the formation of bio-printed kidney tissue apart from standard culture media for renal cells.
(d) The level skill in the art:
The level of skill in the art of bioprinting tissue is high, as an artisan in this art needs specialized knowledge such as a postgraduate degree (Ph.D. and/or M.D.).
(e) The level of predictability in the art:
Regarding absence of an enabling disclosure of bio-printing any bio-ink comprising a plurality of any cells to form bio-printed kidney tissue, while bio-inks comprising renal cells and HUVECs is disclosed in the prior art, the use of bio-inks with any cell type within the scope of the claim is not predictable. Murphy (Murphy, Sean V., and Anthony Atala. "3D bioprinting of tissues and organs." Nature biotechnology 32.8 (2014): 773-785) teaches that one of the main challenges in the 3D bioprinting field has been to find materials that are not only compatible with biological materials and the printing process but can also provide the desired mechanical and functional properties for tissue constructs (page 778, right col. para. 2). Murphy teaches the choice of cells for tissue or organ printing is crucial for correct functioning of the fabricated construct (page 780, left col. last para.). Murphy teaches current options for printing cells involve either the deposition of multiple primary cell types into patterns that faithfully represent the native tissue or printing stem cells that can proliferate and differentiate into required cell types (page 780, right col. para. 1). Murphy teaches cells used for bioprinting applications must be robust enough to survive the bioprinting process and many published bioprinting studies use cell lines that are known to be capable of substantial proliferation and are very robust (page 781, left col. last para.).
Ouyang (Ouyang, Liliang, et al. "Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells." Biofabrication 8.3 (2016): 035020.) teaches bioprinting parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensitivity (Abstract). Ouyang teaches bioink printability, namely the ability to form 3D structure with good fidelity and integrity, and cell viability, namely cell survival rate post printing, are viewed as primary representative criteria of the physical and physiological properties, respectively (page 2, left col. para. 2). Ouyang teaches to achieve a successful 3D cell printing procedure, all parameters need to be carefully tuned to ensure both good printability and high cell viability (page 2, right col. para. 1). Ouyang teaches assessing the rheological properties and printability of 3 different bioinks for bioprinting embryonic stem cells (ESCs) (page 4, right col. last para.; Figure 2A; page 6; Figure 5). Ouyang teaches assessing cell viability after bioprinting and that viability decreased with holding time and viability depended on the composition of the bioink (page 7, right col. para. 2; Figure 7). Ouyang teaches assessing cell viability as a function of shear stress during bioprinting where higher shear stress resulted in lower cell viabilities (page 7, right col. para. 3; Figure 8). Ouyang teaches printability and viability are influenced by printing temperature, bioink concentration and holding time (page 7, right col. last para.; page 8, left col. Figure 9). Therefore, the predictability of any bio-ink comprising any plurality of cells encompassed by the claim would be low given that identification of bioprinting conditions with a specific bioink and specific cell might not be suitable for other types of cells.
(f) The amount of direction provided by the inventor:
The specification shows that kidney organoids containing kidney progenitor cells and fully differentiated kidney cells derived from stem cells comprising fully segmented nephrons with a collecting duct network surrounded by renal interstitium and endothelial cells can be bioprinted into kidney tissue with functional proximal tubules. Therefore, there is a nexus between kidney organoids and bioprinted kidney tissue, but not to the full scope of the method as claimed.
(g) The existence of working examples:
The specification provides multiple examples of bioprinting kidney tissue with each example using a bio-ink of differentiated cells that are kidney organoids derived from pluripotent stem cells (page 51 – 70). The specification provides Example 2 that following bio-printing the bio-printed organoid is cultured for 1 hour in the presence of CHIR99021, and subsequently cultured in FGF9, and Heparin (page 54, para. 000160). The specification provides sufficient teachings only for the enablement of a bio-ink comprising kidney organoids containing kidney progenitor cells and differentiated kidney cells and inducing the bioprinted organoids into bioprinted kidney tissue by culturing with CHIR990021, FGF9, and Heparin. The prior art provides no compensatory guidance, since bioprinted kidney tissue has been printed only with fully differentiated cells not in aggregate and cultured in standard renal cell culture media. The specification’s guidance and working examples provide markedly narrower guidance than encompassed by the breadth of the claims. They fail to provide specific guidance to any other cell type that can be bioprinted and induced to form bioprinted kidney tissue because the described methodology and working example only teaches bioprinting kidney organoids.
(h) The quantity of experimentation needed to make use of the invention based on the content of the disclosure:
The amount of experimentation would be undue because it would require determining the parameters for a given bioink and a given cell to bioprint kidney tissue. Since, as discussed above, it is not routine to determine these parameters for printability and cell viability for bioprinting, significant experimentation would be required to determine which other bioinks and cells could be bioprinted and induced to form bioprinted kidney tissue. This is because one cannot extrapolate between the working example in the specification and the breadth of bioinks and cells claimed since there is little guidance with respect to the use of any other cells or bioinks producing bioprinted kidney tissue. Thus, the full scope of the claims is not enabled by the disclosure.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
19. Claim 4 – 6 and 59 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
20. Regarding claim 4, it is unclear what “high levels” “relative to a manually aggregated or bio-printed kidney organoid generated as a dot or blob of cells” means. An expression level of a manually aggregated or bio-printed kidney organoid generated as a dot or blob of cells is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Thus, recitation of “high levels” is rendered indefinite. Claims 5 and 6 are also rejected as they depend from claim 4 and do not clarify the grounds of rejection.
21. Regarding claim 5, the metes and bounds of “including” are unclear because it raises confusion as to whether the tissue is required to express HNF4A and SLC12A1 or the tissue expresses other markers known in the art to be expressed by proximal tubule and distal tubule segments in nephrons.
22. Regarding claim 59, the scope of the claim is unclear because no active method step is recited and instead an intended result of claim 41 is recited. It is unclear if the claim is further defining the method step of bio-printing (“10,000 cells printed”) or the inducing step (“about 5 to about 100 nephrons/10,000 cells printed”). For the purpose of applying prior art, claim 59 is interpreted as the method of claim 41.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
23. Claims 1, 3 – 6, 8, 9, 15, 34, and 73 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a naturally occurring kidney tissue without significantly more. The claim(s) recite(s) kidney tissue which is not shown to differ from that in nature.
The Office published Office's new guidance document entitled 2019 Revised Patent Subject Matter Eligibility Guidance, published January 7, 2019. Applicant is directed to the Federal Register, Volume 4, No. 4, pages 50-57 at page 74621.
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Step 1 of the USPTO' s eligibility analysis entails considering whether the claimed subject matter falls within the four statutory categories of patentable subject matter identified by 35 U.S.C. 101: Process, machine, manufacture, or composition of matter. The claims are directed to a composition of matter (step 1, Yes).
Step 2A of the 2019 Revised Patent Subject Matter Eligibility Guidance is a two-prong inquiry. In Step 2A Prong One, examiners evaluate whether the claim recites a judicial exception. The composition of matter (kidney tissue) is directed to a natural phenomenon (Step 2A, prong 1, Yes). The markedly different characteristics analysis is used to determine if the nature-based product limitation is a product of nature exception. The claim also recites “bio-printed”, “layer of bio-printed tissue comprising a surface area of nephron tissue of greater than 0.2 mm2 per 10,000 cells printed”, “wherein the nephrons are distributed throughout the tissue”, and “the height of the bio-printed kidney tissue is about 50 µm or less when printed”. The markedly different characteristics analysis is performed by comparing the nature-based product limitation in the claim to its naturally occurring counterpart to determine if it has markedly different characteristics from the counterpart. Here, the closest natural counterpart is naturally occurring kidney tissue. When the claimed kidney tissue is compared to this counterpart, the comparison indicates that there are no differences in structure, function or other characteristics. The claimed kidney tissue is not markedly different from the natural counterpart as a result of bioprinting or bioprinting to specific dimensions or surface area of nephron tissue because naturally occurring kidney tissue contains nephron tissue and nephrons distributed throughout as evidenced by Combes (Combes, Alexander N., et al. BioRXIV (2017): 235499) (Figure 1c; page 28, para. 1; page 3, para. 2) and naturally occurring kidney tissue at height about 50 µm or less is metabolically active as evidenced by Besso (Bessho R, et. al. Am J Physiol Renal Physiol. 2025 Dec 1;329(6):F796-F808) (page 2, last para.; page 3, last para.; page 6, last para.; Figure 2). Therefore, the claimed kidney tissue is a product of nature exception and recites a judicial exception.
In Step 2A Prong Two, examiners evaluate whether the claim recites additional elements that integrate the exception into a practical application of that exception. This evaluation is performed by (a) identifying whether there are any additional elements recited in the claim beyond the judicial exception, and (b) evaluating those additional elements individually and in combination to determine whether the claim as a whole integrates the exception into a practical application. Besides the judicial exception, the claim recites the judicial exception is “bio-printed”. An evaluation of whether this limitation is insignificant extra-solution activity is then performed. Note that because Step 2A Prong Two analysis excludes consideration of whether a limitation is well-understood, routine, conventional activity, this evaluation does not take into account whether or not the limitation is well-known. When so evaluated, this additional element is insignificant extra-solution activity because “bio-printed” is recited so generically. The claim does not recite features of the kidney tissue that distinguish it from naturally occurring kidney tissue as a result of being bioprinted. Therefore, “bio-printed” fails to meaningfully limit the claim because it is at best the equivalent of merely adding the words “apply it” to the judicial exception. Accordingly, “bio-printed” does not integrate the recited judicial exception into a practical application and the claim is therefore directed to the judicial exception (Step 2A, prong 2, No).
In Step 2B, the eligibility analysis evaluates whether the claim as a whole amounts to significantly more than the recited exception, i.e., whether any additional element, or combination of additional elements adds an inventive concept into the claim. As discussed with respect to Step 2A Prong Two, the claim recites “bio-printed”, which is at best the equivalent of merely adding the words “apply it” to the judicial exception. Mere instructions to apply an exception cannot provide an inventive concept. At Step 2B, the evaluation of the insignificant extra-solution activity consideration takes into account whether or not the extra-solution activity is well-known. Here, recitation of the kidney tissue being bio-printed is recited at a high level of generality. Thus, recitation of “bio-printed” does not amount to significantly more and does not provide an inventive concept (Step 2B: No).
Markedly different characteristics can be expressed as the product' s structure, function, and/or other properties. In accordance with this analysis, a product that is purified or isolated, for example, will be eligible when there is a resultant change in characteristics sufficient to show a marked difference from the product' s naturally occurring counterpart. If the claim recites a nature-based product limitation that does not exhibit markedly different characteristics, the claim is directed to a ‘‘product of nature” exception (a law of nature or naturally occurring phenomenon), and the claim will require further analysis to determine eligibility based on whether additional elements add significantly more to the exception.
Limitations that were found not to be enough to qualify as ‘‘significantly more” when recited in a claim with a judicial exception include: Adding the words ‘‘apply it” (or an equivalent) with the judicial exception, or mere instructions to implement an abstract idea on a computer; simply appending well-understood, routine and conventional activities previously known to the industry, specified at a high level of generality, to the judicial exception, e.g., a claim to an abstract idea requiring no more than a generic computer to perform generic computer functions that are well-understood, routine and conventional activities previously known to the industry; adding insignificant extrasolution activity to the judicial exception, e.g., mere data gathering in conjunction with a law of nature or abstract idea; or generally linking the use of the judicial exception to a particular technological environment or field of use.
In the instant case, the limitations of the claim do not impose limits on the claim scope such that they are not markedly different in structure from a naturally occurring product.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
24. Claim(s) 73 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by King (King, Shelby M., et al. in physiology 8 (2017): 123), hereinafter King.
Claim 73 is drawn to Bio-printed kidney tissue produced according to claim 41.
King teaches bioprinted kidney tissue (page 3, left col. para. 2 – 3; page 6, left col. para. 2 – 3 Figure 1 and 2).
Therefore, King anticipates claim 73.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
25. Claim(s) 1, 3 – 6, 8, 9, 15, and 34 is/are rejected under 35 U.S.C. 103 as being unpatentable over Little (WO2016094948A1; Filed 12/15/2015, Published 06/23/2016; previously cited), hereinafter Little as evidenced by Combes (Combes, Alexander N., et al. BioRXIV (2017): 235499.), hereinafter Combes in view of King (King, Shelby M., et al. in physiology 8 (2017): 123.), hereinafter King in view of Nguyen (US94818868; Filed 10/06/2015; Published11/01/2016; previously cited), hereinafter Nguyen.
Regarding claims 1, Little teaches kidney organoids (“kidney tissue”) comprising maximal nephron number per organoid (“wherein nephrons are distributed throughout the tissue” of claim 1) where the kidney organoids are equivalent to the human fetal kidney contemplates a bioprinted renal structure (page 5, lines 1 – 5 and 16 – 19; page7, lines 30 – 33; page 8; page 9, lines 1 – 24; page 27, lines 23 – 26; page 45, lines 5 – 30; page 46, lines 9 – 34; Figure 8 – 11). Little teaches the method of bioprinting a renal structure includes depositing a plurality of nephron progenitor cells and ureteric epithelial progenitor cells to form a renal structure that is a three-dimensional structure that may be constructed or formed from a plurality of bioprinted layers and comprise a glomerulus, collecting ducts, Bowman’s capsule, proximal and/or distal convoluted tubules (“bio-printed kidney tissue” and “layer of bio-printed tissue comprising a surface area of nephron tissue”) (page 28, lines 1 – 29). Little teaches each kidney organoid had >500 fully segmented nephrons per organoid, a number equivalent to a mouse kidney at 14.5 dpc that were surrounded by endothelial and renal interstitium (page 47, lines 22 – 29; page 38, lines 13 – 18). Little teaches the presence of all segments of a normal developing nephron connected to each other in the organoid (page 46, lines 7 – 21). Little does not teach “a surface area of nephron tissue of greater than 0.2 mm2 per 10,000 cells printed” or “the height of the kidney tissue is about 50 µm or less when printed”. However, Little teaches “bioprinting” includes and encompasses utilizing three-dimensional, precise deposition of cells (e.g., organoids, multicellular aggregates, multicellular bodies) via methodology that is compatible with a bioprinter (page 29, lines 1 – 9). Little contemplates bioprinting kidney organoids for nephrotoxicity screening, disease modeling, drug screening for the therapeutic treatment of kidney disease (page 5; page 6, lines 9 – 18; page 27, lines 2 – 9 and 23 – 31; page 28 – 30; page 31, lines 1 – 29; page 33, lines 1 – 2; page 48 – 49). Little teaches generation of bioprinted nephron-forming mesenchyme for creation of a specifically patterned collecting duct network around which nephrons could differentiate such that the final product could act as a transplantable organ capable of directing urine to a single exit (page 49, lines 12 – 19).
Regarding claim 4, Little teaches the kidney tissue expresses HNF4A and CUBN (page 8, lines 22 – 33; Figure 12).
Regarding claim 5, Little teaches the kidney tissue comprises nephrons where the proximal tubule and distal tubule express LTL, HNF4a and SLC12A1 (page 8, lines 22 – 33; page 9, lines 9 – 18; Figure 9, 10, and 12b; page 39, lines 27 – 31; page 47, lines 22 – 29).
Regarding claim 6, Little teaches the kidney tissue expresses HNF4A and CUBN (page 8, lines 22 – 33; Figure 9 and 12b). Kidney tissue produced by the method of Little expresses EPCAM, MAFB, CUBN, and HNF4A as evidenced by Combes (Figure 2, 4J, and Supplementary Figure 3; page 4, para. 2; page 6, para. 2; page 7, para. 1; page 11, para. 3; page 19, para. 3; page 28, para. 2; page 30, para. 1; page 32, para. 3). Little does not teach “LRP2”.
Regarding claim 15, Little teaches intermediate mesoderm cells, metanephric mesenchyme cells, and ureteric epithelial progenitor cells (page 2, lines 14 – 17; page 6, lines 16 – 18; page 33, lines 16 – 33; page 44, lines 15 – 33; page 45, lines 1 – 15).
Regarding claim 34, Little teaches each kidney organoid had >500 nephrons per organoid, a number equivalent to a mouse kidney at 14.5 dpc (page 47, lines 22 – 29; page 38, lines 13 – 18). Little teaches the organoid contains podocytes (Figure 3 and 9, page 8, lines 22 – 33; page 46, lines 15 – 24). Kidney tissue produced by the method of Little expresses MAFB throughout the organoid as evidenced by Combes (Figure 2, 4J, and Supplementary Figure 3; page 4, para. 2; page 6, para. 2; page 7, para. 1; page 11, para. 3; page 19, para. 3; page 28, para. 2; page 30, para. 1; page 32, para. 3). Therefore, the kidney tissue of Little meets the limitations of claim 34.
Little does not teach “a surface area of nephron tissue of greater than 0.2 mm2 per 10,000 cells printed” or “the height of the kidney tissue is about 50 µm or less when printed” of claim 1 or “a layer of bio-printed kidney tissue comprising about 30,000 cell per mm2 or less when printed” of claim 3 or “high levels of” “CUBN” “relative to a manually aggregated or bio-printed organoid generated as a dot or blob of cells of claim 4 or “LRP2” of claim 6 or the dimensions of a bioprinted kidney tissue of claim 8. However, Little teaches the kidney organoids comprise collecting ducts, proximal tubules, distal tubules, proximal tubular epithelium displaying an apical brush border with tight junctions, loops of Henle, glomeruli comprising a Bowman’s capsule, a CD31+ endothelial network with lumen formation, and nephrons (page 28, lines 25 – 29; page 46, lines 10 – 34). Little teaches the kidney organoids comprise nephrons with distinct epithelial subtypes, renal interstitium and endothelia (page 8, lines 22 – 33; page 9, lines 1 – 8; Figure 9; page 46, lines 11 – 34; page 47, lines 22 – 27). Little teaches the utility of stem-cell derived kidney organoids for disease modelling or drug screening will be dependent upon the functional maturation of the nephrons within these organoids (page 47, lines 6 – 8). Little teaches demonstrating the capacity of cubilin-mediated proximal tubule specific endocytosis by the selective uptake of Dextran from the media by LTL+ tubules (page 47 lines 5 – 12; Figure 10a). Little teaches the mature proximal tubular cells were sensitive to cisplatin treatment (page 47, lines 15 – 21; Figure 10b, c). Little teaches there is an urgent need for renal regenerative strategies with the prevalence of end stage renal disease rising 8% per annum globally (page 1, lines 10 – 11). Little teaches nephron formation in the human kidney is completed before birth and there is no postnatal stem cell able to replace lost nephrons (page 1, lines 18 – 20). Little teaches the bio-printed kidney or bio-printed kidney organoid may be used for nephrotoxicity screening (page 33, lines 1 – 2). Little teaches the development of interventions aimed at preventing disease, including drug and cellular-based therapies, is made difficult by the lack of availability of primary human kidney cells for in vitro drug testing (page 32, lines 14 – 16).
Regarding “bio-printed kidney tissue” of claim 1, King teaches a bioprinted kidney tissue comprising a surface area of nephron tissue that is proximal tubule tissue (Abstract; page 3, left col. para. 3; Figure 1; page 6, left col. para. 2). King teaches exposing the bioprinted kidney tissue to cisplatin and measurement of viability where cisplatin induced a reduction in viability demonstrating that the bioprinted kidney tissue were able to recapitulate nephrotoxicity after exposure to clinically-relevant doses of cisplatin (page 9, right col.; page 10; page 11, left col.; Figure 7 and 8). King teaches bioprinting the cells for development of the renal interstitium and adding primary renal epithelial cells to bioprinted cells (page 3, left col. para. 2 – 3; page 6, left col. para. 2 – 3). King teaches in Figure 2B the height of the bioprinted interstitium is greater than 50 µm which is thicker than the native human renal interstitium and the height of the renal epithelial cells is less than 50 µm (page14, left col. para. 2). King teaches very few systems have been developed to study the human renal tubulointerstitial interface in vitro and those that have lack direct contact between the epithelium and relevant interstitial cell types that play both a structural role in orienting the epithelium as well as providing a source of growth factors critical for the continued health and organization of the epithelium (page 14, left col. para. 1). King teaches without these supportive cell types, primary renal epithelial cells rapidly lose their native phenotype in culture, thus preventing the ability to perform the chronic, low dose exposure studies necessary to predict how a molecule will perform in the clinic and precludes the ability to model drug-induced and disease relevant states that require an interaction between the epithelium and interstitium (page 14, left col. para. 1). King teaches primary human renal epithelial cells can be cultured for a limited time (<14 days) before undergoing senescence or epithelial to mesenchymal transition and concomitant loss of renal transporter expression and function but culturing in a 3D context on an interstitial layer enabled retention of epithelial cell viability and function for at least 30 days in culture while retaining gene expression of many renal transporters (page 14, right col. para. 2). King teaches a human 3D multi-cellular renal tissue composed of distinct epithelial and interstitial cell compartments provides a unique test platform for evaluating new drug entities for potential nephrotoxicity, allowing for the assessment of biochemical, transcriptional, and histological endpoints across multiple cell types and anatomical locations ex vivo (page 15, left col. para. 2).
Regarding claim 4 and 6, King teaches a bioprinted kidney tissue that expresses high levels of CUBN and LRP2 (Supplemental Table 1).
King does not teach “a surface area of nephron tissue of greater than 0.2 mm2 per 10,000 cells printed” or “the height of the kidney tissue is about 50 µm or less when printed” of claim 1 or “a layer of bio-printed kidney tissue comprising about 30,000 cell per mm2 or less when printed” of claim 3 or the dimensions of the bioprinted kidney tissue of claim 8. One would have been motivated to combine the teachings of Little and King because Little contemplates bioprinted kidney tissue and teaches the kidney organoids containing proximal tubules are sensitive to cisplatin and King teaches the bioprinted proximal tubule is also sensitive to cisplatin. Additionally, one would have been motivated to combine the teachings of Little and King and substitute the primary renal epithelial cells and interstitium of King with the kidney organoids of Little because Little teaches the organoids contain all of the components of King’s bioprinted kidney tissue including nephrons while King teaches primary renal epithelial cells had to be added to the bioprinted tissue.
Regarding “a surface area of nephron tissue of greater than 0.2 mm2 per 10,000 cells printed” of claim 1, Nguyen’s Example 2 teaches a surface area of 9 mm2 and renal fibroblasts and HUVECs at 150 million cells/ml were printed followed by printing epithelial cells at 1 million cells/ml of cells on the surface of the structure (col. 27, lines 59; col. 28, lines 1 – 27). Nguyen teaches the bioprinted kidney tissue comprises a renal tubule cell model (“nephron tissue”) of 9 mm2 with 2250 epithelial cells and 337,500 fibroblasts/HUVECs printed which is the same as 0.0000264 mm2/cell which is greater than 0.2 mm2 per 10,000 cells printed which is the same as 0.00002 mm2/cell (1 million cells/mL x 0.00225 mL volume of sheet = 2250 cells per 9 mm2; 150 million cells/mL x 0.00225 mL = 337,500 per 9 mm2; 9mm2/339,750 cells = 0.0000264 mm2/cell).
Regarding “wherein the height of the bio-printed kidney tissue is about 50 µm or less when printed” of claim 1, Nguyen teaches bioprinted kidney tissue where renal epithelial cells were bioprinted using an inkjet spray module on top of interstitial tissues (col. 28, lines 1 – 12). Nguyen teaches the printed cells have a height of about 50 µm or less in Figure 7 (col. 6, lines 1 – 3). Nguyen teaches the inkjet spray module facilitated attachment of epithelial cells to the surface of the interstitial layer and resulted in a much lower cell density on the surface of the tissue, which is necessary for forming a polarized monolayer and that following inkjet spraying cells retained high viability (col. 28, lines 13 – 27).
Regarding claim 3, Nguyen’s Example 2 teaches a surface area of 9 mm2 and 1 million cells/ml of cells was bioprinted on the surface of the structure and that it resulted in a much lower cell density on the surface of the tissue (col. 27, lines 59; col. 28, lines 1 – 27). Therefore, Nguyen teaches the bioprinted kidney tissue comprises 250 cells per mm2 which is about 30,000 cells per mm2 or less when printed when the concentration of epithelial cells is 1 million cells/ ml (1 million cells/mL x 0.00225 mL volume of sheet = 2250 cells per 9 mm2 or 250 cells per mm2).
Regarding claim 8, Nguyen teaches in Example 2 the bio-printed sheet has dimensions of 3 mm x 3 mm (col. 27, lines 59 – 65).
Regarding claim 9, Nguyen’s Example 2 teaches a surface area of 9 mm2 and 1 million cells/ml of cells was bioprinted in the surface of the structure and that it resulted in a much lower cell density on the surface of the tissue (col. 27, lines 59; col. 28, lines 1 – 27). As Little teaches each kidney organoid had >500 nephrons per organoid (page 47, lines 27 – 28) and Little teaches bioprinting a plurality of cells, bioprinting 2 organoids on a surface area of 9 mm2 of Nguyen would result in about 100 nephrons/mm2.
Nguyen teaches the bioprinted renal tubule models are suitable for in vitro toxicity assays (col. 30, lines 5 – 49). Nguyen teaches what is needed is an engineered renal tissue model with native-like tissue architecture to predict a drug’s effects, toxicity or metabolism in humans because it would be more predictive of human in vivo response and would be useful for modeling kidney disease and transport (col. 1, lines 28 – 49).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Little regarding kidney tissue comprising nephrons with distinct epithelial subtypes, renal interstitium and endothelia and bioprinted renal structures with the teachings of King regarding a bioprinted kidney tissue with the teachings of Nguyen regarding a bioprinted kidney tissue produced by inkjet spraying to arrive at the claimed bio-printed kidney tissue, wherein the bio-printed kidney tissue is a layer of bio-printed tissue comprising a surface area of nephron tissue of greater than 0.2 mm2 per 10,000 cells printed and wherein nephrons are distributed throughout the tissue, and wherein the height of the bio-printed kidney tissue is about 50 μm or less when printed. One would have been motivated to combine the teachings of Little, King, and Nguyen for bioprinted kidney tissue to test the nephrotoxicity of drugs as Little teaches nephron formation in the human kidney is completed before birth and there is no postnatal stem cell able to replace lost nephrons and Little teaches the development of interventions aimed at preventing disease, including drug and cellular-based therapies, is made difficult by the lack of availability of primary human kidney cells for in vitro drug testing and King teaches a human 3D multi-cellular renal tissue composed of distinct epithelial and interstitial cell compartments provides a unique test platform for evaluating new drug entities for potential nephrotoxicity, allowing for the assessment of biochemical, transcriptional, and histological endpoints across multiple cell types and anatomical locations ex vivo and Nguyen teaches an engineered renal tissue model with native-like tissue architecture to predict a drug’s effects, toxicity or metabolism in humans is needed because it would be more predictive of human in vivo response and would be useful for modeling kidney disease and transport. One would have a reasonable expectation of success in combining the teachings as Little teaches the mature proximal tubular cells in the kidney organoids were sensitive to cisplatin treatment and King and Nguyen’s bioprinted proximal tubule tissue also showed sensitivity to cisplatin and Little teaches the kidney tissue comprises fully segmented nephrons surrounded by endothelial and renal interstitium that includes the bioprinted cells of King and Nguyen.
26. Claim(s) 41, 45, 47, 59, 62, 67, and 69 remain rejected under 35 U.S.C. 103 as being unpatentable over Little (WO2016094948A1; previously cited), hereinafter Little in view of King (King, Shelby M., et al. in physiology 8 (2017): 123), hereinafter King in view of Nguyen (US94818868; Filed 10/06/2015; Published11/01/2016; previously recited), hereinafter Nguyen.
Regarding claim 41, 59, 67, and 69, Little a method comprising bioprinting bioink comprising one or more differentiated cell types and/or intermediate progenitor cell types from kidney organoids in various volumes in a layer to form a bioprinted human kidney organoid or kidney (“bio-printing a pre-determined amount of a bio-ink onto a surface, wherein the bio-ink comprises a plurality of cells” of claim 41 and 59) (page 27, lines 2 – 9; page 29, lines 1 – 28; page 30, lines 3 – 15 and 29 – 34; page 31, lines 1 – 16). Little teaches stimulating organoids with CHIR99021 (claim 69) and FGF9 (claim 67) promoted nephrogenesis (“inducing the bio-printed, pre-determined amount of the bio-ink to form bio-printed kidney tissue”) (page 9, lines 19 – 24). Little does not teach “about 50 µm high or less” and the surface area of claim 41 and 59.
Regarding claim 47, Little teaches the organoids comprise fully differentiated cells and renal progenitors (page 47, lines 22 – 26; page 31, lines 10 – 16 and 30 – 31; page 39, lines 23 – 31; Figure 3).
Regarding claim 62, Little teaches the ECM from a kidney scaffold may be used as a matrix in which to bioprint the one or more differentiated cell types and/or intermediate progenitor cell types (page 27, lines 5 – 9).
Little does not teach “about 50 µm high or less” and the surface area of claim 41 and 59 or “the predetermined amount of bio-ink comprises between approximately 10,000 cells/µl and approximately 400,000 cells/µl” of claim 45. However, Little teaches the bioprinted kidney tissue may comprise a glomerulus, interstitial tissue, proximal convoluted tubules, and vasculature (page 28, lines 25 – 29). Little teaches a bioprinter is advantageous for arranging cells, multicellular aggregates, and/or layers on a biocompatible surface because the technology includes rapid, accurate, and reproducible placement of cells or multicellular bodies (page 29, lines 29 – 34). Little teaches the bioprinted kidney tissue may be used for nephrotoxicity screening (page 32, lines 12 – 13; page 22, lines 1 – 2; page 48, lines 3 – 16). Little teaches the nephrons appropriately segmented into 4 components including the collecting duct, the early distal tubule, early proximal tubule and the glomerulus, and the organoids showed complex morphogenetic patterning mimicking in vivo tissue organization(page 45, lines 20 – 28). Little teaches it is possible to direct human pluripotent stem cells to form a kidney organoid that comprises fully segmented nephrons surrounded by endothelia and renal interstitium and is transcriptionally similar to a human fetal kidney that will help improve understanding of human kidney development (page 47, lines 22 – 27). Little teaches generating mini-kidney organoids for these purposes after bioprinting into a larger format (page 48, lines 8 – 29). Little teaches the mature proximal tubular cells were sensitive to cisplatin treatment (page 47, lines 15 – 21; Figure 10b, c). Little teaches there is an urgent need for renal regenerative strategies with the prevalence of end stage renal disease rising 8% per annum globally (page 1, lines 10 – 11). Little teaches nephron formation in the human kidney is completed before birth and there is no postnatal stem cell able to replace lost nephrons (page 1, lines 18 – 20). Little teaches the development of interventions aimed at preventing disease, including drug and cellular-based therapies, is made difficult by the lack of availability of primary human kidney cells for in vitro drug testing (page 32, lines 14 – 16).
Regarding “bio-printing” of claim 41, King teaches bioprinting a kidney tissue comprising a surface area of nephron tissue that is proximal tubule tissue (Abstract; page 3, left col. para. 3; Figure 1; page 6, left col. para. 2). King teaches bioprinting the cells for development of the renal interstitium and adding primary renal epithelial cells to bioprinted cells (page 3, left col. para. 2 – 3; page 6, left col. para. 2 – 3). King teaches in Figure 2B the height of the bioprinted interstitium is greater than 50 µm which is thicker than the native human renal interstitium and the height of the renal epithelial cells is less than 50 µm (page14, left col. para. 2). King teaches exposing the bioprinted kidney tissue to cisplatin and measurement of viability where cisplatin induced a reduction in viability demonstrating that the bioprinted kidney tissue were able to recapitulate nephrotoxicity after exposure to clinically-relevant doses of cisplatin (page 9, right col.; page 10; page 11, left col.; Figure 7 and 8). King teaches very few systems have been developed to study the human renal tubulointerstitial interface in vitro and those that have lack direct contact between the epithelium and relevant interstitial cell types that play both a structural role in orienting the epithelium as well as providing a source of growth factors critical for the continued health and organization of the epithelium (page 14, left col. para. 1). King teaches without these supportive cell types, primary renal epithelial cells rapidly lose their native phenotype in culture, thus preventing the ability to perform the chronic, low dose exposure studies necessary to predict how a molecule will perform in the clinic and precludes the ability to model drug-induced and disease relevant states that require an interaction between the epithelium and interstitium (page 14, left col. para. 1). King teaches a human 3D multi-cellular renal tissue composed of distinct epithelial and interstitial cell compartments provides a unique test platform for evaluating new drug entities for potential nephrotoxicity, allowing for the assessment of biochemical, transcriptional, and histological endpoints across multiple cell types and anatomical locations ex vivo (page 15, left col. para. 2).
King does not teach “a surface area of nephron tissue of greater than 0.2 mm2 per 10,000 cells printed” or “the height of the kidney tissue is about 50 µm or less when printed” of claim 41 and 59 or “the predetermined amount of bio-ink comprises between approximately 10,000 cells/µl and approximately 400,000 cells/µl” of claim 45. One would have been motivated to combine the teachings of Little and King because Little contemplates bioprinted kidney tissue and teaches the kidney organoids containing proximal tubules are sensitive to cisplatin and King teaches the bioprinted proximal tubule is also sensitive to cisplatin. Additionally, one would have been motivated to combine the teachings of Little and King and substitute the primary renal epithelial cells and interstitium of King with the kidney organoids of Little because Little teaches the organoids contain all of the components of King’s bioprinted kidney tissue including nephrons while King teaches primary renal epithelial cells had to be added to the bioprinted tissue.
Regarding “a surface area of nephron tissue of greater than 0.2 mm2 per 10,000 cells printed” of claim 41 and 59, Nguyen’s Example 2 teaches a surface area of 9 mm2 and renal fibroblasts and HUVECs at 150 million cells/ml were printed followed by printing epithelial cells at 1 million cells/ml of cells on the surface of the structure (col. 27, lines 59; col. 28, lines 1 – 27). Nguyen teaches the bioprinted kidney tissue comprises a renal tubule cell model (“nephron tissue”) of 9 mm2 with 2250 epithelial cells and 337,500 fibroblasts/HUVECs printed which is the same as 0.0000264 mm2/cell which is greater than 0.2 mm2 per 10,000 cells printed which is the same as 0.00002 mm2/cell (1 million cells/mL x 0.00225 mL volume of sheet = 2250 cells per 9 mm2; 150 million cells/mL x 0.00225 mL = 337,500 per 9 mm2; 9mm2/339,750 cells = 0.0000264 mm2/cell).
Regarding “wherein the height of the bio-printed kidney tissue is about 50 µm or less when printed” of claim 41 and 59, Nguyen teaches bioprinted kidney tissue where renal epithelial cells were bioprinted using an inkjet spray module on top of interstitial tissues (col. 28, lines 1 – 12). Nguyen teaches the printed cells have a height of about 50 µm or less in Figure 7 (col. 6, lines 1 – 3). Nguyen teaches the inkjet spray module facilitated attachment of epithelial cells to the surface of the interstitial layer and resulted in a much lower cell density on the surface of the tissue, which is necessary for forming a polarized monolayer and that following inkjet spraying cells retained high viability (col. 28, lines 13 – 27).
Regarding claim 45, Nguyen teaches printing the kidney tissue with renal fibroblasts and HUVECs at 150 million cells/ml which is 150,000 cells/µl and printing epithelial cells at 1 million cells/ml which is the same as 1000 cells/µl (col. 28, lines 1 – 12).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Little regarding bioprinting kidney tissue comprising nephrons with distinct epithelial subtypes, renal interstitium and endothelia and inducing nephrogenesis with CHIR99021, FGF9, and Heparin with the teachings of King regarding bioprinting kidney tissue with the teachings of Nguyen regarding bioprinting kidney tissue using inkjet spraying to print tissue at a height of less than 50 µm to arrive at the claimed method for producing bio-printed kidney tissue comprising the steps of: bio-printing a pre-determined amount of a bio-ink onto a surface, wherein the bio- ink comprises a plurality of cells, and wherein the bio-ink is bio-printed in a layer that is about 50 μm high or less; and inducing the bio-printed, pre-determined amount of the bio-ink to form bio-printed kidney tissue, wherein the bio-printed kidney tissue comprises a surface area of nephron tissue of greater than 0.2 mm2 per 10,000 cells printed. One would have been motivated to combine the teachings of Little, King, and Nguyen in a method of bioprinting kidney tissue to test the nephrotoxicity of drugs as Little teaches nephron formation in the human kidney is completed before birth and there is no postnatal stem cell able to replace lost nephrons and Little teaches the development of interventions aimed at preventing disease, including drug and cellular-based therapies, is made difficult by the lack of availability of primary human kidney cells for in vitro drug testing and King teaches a human 3D multi-cellular renal tissue composed of distinct epithelial and interstitial cell compartments provides a unique test platform for evaluating new drug entities for potential nephrotoxicity, allowing for the assessment of biochemical, transcriptional, and histological endpoints across multiple cell types and anatomical locations ex vivo and Nguyen teaches an engineered renal tissue model with native-like tissue architecture to predict a drug’s effects, toxicity or metabolism in humans is needed because it would be more predictive of human in vivo response and would be useful for modeling kidney disease and transport. One would have a reasonable expectation of success in combining the teachings as Little teaches the mature proximal tubular cells in the kidney organoids were sensitive to cisplatin treatment and King and Nguyen’s bioprinted proximal tubule tissue also showed sensitivity to cisplatin and Little teaches the kidney tissue comprises fully segmented nephrons surrounded by endothelial and renal interstitium that includes the bioprinted cells of King and Nguyen.
Applicant’s Arguments/ Response to Arguments
27. Applicant Argues: On page 6 and page 8, para. 1, Applicant asserts that the cited references fail to teach the recited surface area of nephron tissue because the epithelial cells of Nguyen cannot result in nephrons that must contain ureteric epithelium and podocytes and one cannot simply re-classify the entirety of Nguyen’s fibroblast-endothelial layers as “nephron tissue”.
Response to Arguments: In response, the claim broadly recites “nephron tissue” and proximal tubule is a segment of nephrons and is therefore nephron tissue. In the new rejection set forth above, Nguyen teaches the bioprinted kidney tissue comprises a renal tubule cell model (“nephron tissue”) of 9 mm2 with 2250 epithelial cells and 337,500 fibroblasts/HUVECs printed which is the same as 0.0000264 mm2/cell which is greater than 0.2 mm2 per 10,000 cells printed which is the same as 0.00002 mm2/cell (1 million cells/mL x 0.00225 mL volume of sheet = 2250 cells per 9 mm2; 150 million cells/mL x 0.00225 mL = 337,500 per 9 mm2; 9mm2/339,750 cells = 0.0000264 mm2/cell). Because Little teaches Little teaches the nephrons in the organoids appropriately segmented into 4 components including the collecting duct, the early distal tubule, early proximal tubule and the glomerulus, and the organoids showed complex morphogenetic patterning mimicking in vivo tissue organization(page 45, lines 20 – 28), bioprinting Little’s organoids according to the inkjet method of Nguyen would produce nephron tissue greater than the claimed surface area.
Applicant Argues: On page 7, Applicant asserts that the 20 um thick bioprinted regions of Nguyen are anomalies and Nguyen describes a fundamentally different method that employs ink-jet or extrusion-based bioprinting to deposit distinct monolayer sheets and therefore a POSITA would not have been motivated to combine these references or have expected any success.
Response to Arguments: In response, Little contemplates bioprinting and bioprinted kidney tissue. King and Nguyen teach bioprinting and bioprinted kidney tissue by printing multiple layers of interstitium and epithelial cells to yield a proximal tubule. Little teaches the organoids contain the multiple cell types that are separately printed by King and Nguyen. Therefore, one would have been motivated to combine the teachings of Little and King and substitute the primary renal epithelial cells and interstitium of King with the kidney organoids of Little because Little teaches the organoids contain all of the components of King’s bioprinted kidney tissue including nephrons while King teaches primary renal epithelial cells had to be added to the bioprinted tissue. One would have a reasonable expectation of success because Little’s organoids show sensitivity to cisplatin and the proximal tubules produced by bioprinting 2 layers of cells also show sensitivity to cisplatin.
Regarding the “anomalies in thickness”, Nguyen teaches using inkjet bioprinting to print the epithelial cells at a height of less than 50 µm (Figure 7). Therefore, printing a height of less than 50 µm using the inkjet printer is not an anomaly.
Applicant Argues: On page 8, para. 2, Applicant asserts that the claimed quantitative parameters yielded unexpectedly superior biological outcomes including significantly enhanced nephron maturation, uniform glomerular distribution, and robust expression of key markers such as HNG4A, SLC12A1, and MAFB.
Response to Arguments: In the new rejection set forth above, Little teaches the kidney tissue expresses HNF4A and CUBN (page 8, lines 22 – 33; Figure 12) and Little teaches the kidney tissue comprises nephrons where the proximal tubule and distal tubule express LTL, HNF4a and SLC12A1 (page 8, lines 22 – 33; page 9, lines 9 – 18; Figure 9, 10, and 12b; page 39, lines 27 – 31; page 47, lines 22 – 29). Little teaches bioprinting is advantageous for forming kidney tissue because of the method’s rapid, accurate, and reproducible placement of cells exhibiting planned or pre-determined orientations or patterns of cells with various compositions with assured high cell density, while minimizing cell damage (page 29, lines 29 – 34; page 30, lines 1 – 3). Therefore, the quantitative parameters would be expected in view of the combined teachings of Little, King, and Nguyen.
Applicant Argues: On page 8, last para. and page 9, para. 1, Applicant asserts that claim 4 describes relative increase in marker expression compared to manually aggregated or bio-printed kidney organoid generated as a dot or blob of cells.
Response to Arguments: It is unclear what the level is to be compared to. The claim does not define any value let alone a method of measuring expression. Further, the claim is to a composition and not a method of measuring expression of markers in any tissue aggregated or bioprinted. In the new rejection set forth above, King teaches a bioprinted kidney tissue that expresses high levels of CUBN and LRP2 (Supplemental Table 1). Should Applicant amend the claims to recite a specific value of marker expression, the rejection may be overcome upon further search and consideration.
Applicant Argues: On page 9, para. 2, Applicant asserts that Freedman fails to remedy deficiencies of Little and Nguyen.
Response to Arguments: The previous rejections using the teachings of Freedman have been withdrawn and therefore arguments addressing Freedman are moot.
Applicant Argues: On page 8, para. 4 – 5, Applicant disagrees with the rejection of claim 34 using the teachings of Moriguichi.
Response to Arguments: The previous rejections using the teachings of Moriguichi have been withdrawn and therefore arguments addressing Moriguichi are moot. In the new rejection set forth above, Little teaches each kidney organoid had >500 nephrons per organoid, a number equivalent to a mouse kidney at 14.5 dpc (page 47, lines 22 – 29; page 38, lines 13 – 18). Little teaches the organoid contains podocytes (Figure 3 and 9, page 8, lines 22 – 33; page 46, lines 15 – 24). Kidney tissue produced by the method of Little expresses MAFB throughout the organoid as evidenced by Combes (Figure 2, 4J, and Supplementary Figure 3; page 4, para. 2; page 6, para. 2; page 7, para. 1; page 11, para. 3; page 19, para. 3; page 28, para. 2; page 30, para. 1; page 32, para. 3). Specifically, Supplementary Figure 3 of Combes shows the expression of MAFB throughout the organoid. Therefore, bioprinting the organoid of Little would be expected to produce bioprinted kidney tissue with MAFB expression throughout.
Applicant Argues: On page 10, para. 2, Applicant disagrees with the rejection of claim 41 for the same reasons cited for claim 1.
Response to Arguments: These arguments are addressed above.
Applicant Argues: On page 10, para. 3, Applicant asserts that the previously cited teachings of Nguyen do not show any relevance to bioprinting a pre-determined amount of a bioink onto a surface.
Response to Arguments: The claim broadly recites any amount of bioink and Little contemplates various volumes of bioink for dispensing onto a surface and Nguyen teaches specific amounts of bioink bioprinted for producing the bioprinted kidney tissue.
Applicant Argues: On page 10, para. 5, Applicant asserts Qu fails to remedy the deficiency in the combination of Little and Nguyen.
Response to Arguments: The previous rejections using the teachings of Qu have been withdrawn and therefore arguments addressing Qu are moot.
Applicant Argues: On page 10, para. 6, Applicant asserts that there is no teaching that greater surface area per cell is a result-effective variable for enhanced nephron yield in a bio-printed organoid.
Response to Arguments: Neither the previous rejections or the new rejections set forth above discuss “result-effective variable”.
Conclusion
No claims allowed.
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/ZANNA MARIA BEHARRY/Examiner, Art Unit 1632