DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 06-20-2025 has been entered.
Applicant's amendments to the claims and arguments filed on 06-20-2025 have been received and entered. Claims 26-28, 32-36, 40-47 have been amended, while claims 1-25, 29-31, 37-39 have been canceled. Claims 48-53 are newly added. Claims 26-28, 32-36, 40-53 are pending.
Election/Restrictions
Applicant's election with traverse of Group I (claims 26 to 34) in the reply filed on 06-07-2024 is acknowledged. The response for the traversal can be found in the preceding office action filed on 07-18-2024 and the 35 USC§ 103 rejection below.
Claims 35-36, 40 and 46-47 were previously withdrawn from further consideration pursuant to 37 CPR 1.142(b), as being drawn to a nonelected subject matter, there being no allowable generic or linking claim. Newly submitted claim 53 is directed to a kit comprising a differentiation medium which is non-elected invention. Accordingly, claims 46-47 are withdrawn from consideration as being directed to a non-elected invention. See 37 CFR l.142(b) and MPEP § 821.03.
Claims 26-28, 32-34, 41-45, 48-52 are under consideration.
Priority
This application is a 371 of PCT/EP2020/053237 filed on 02/10/2020 which claims priority from foreign application EP 19156450.9 filed on 02/11/2019.
Receipt is acknowledged of certified copies of papers required by 37 CPR 1.55.
New-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.
Claims 26-28, 32-34, 41-45, 48-52 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.
Regarding to claim 26, the phrase “preliminary brain organoids” is vague and render the claims indefinite. There is no guidance of “preliminary brain organoids” in the specification of the claimed invention. Since there are various brain organoids expressing various cellular markers with structural and functional differences, an Artisan of skill in the art would not know what brain organoids can be included or excluded by the phrase “preliminary brain organoids”. Artisan of skill would not know the difference between “preliminary brain organoids” in step (b) and “differentiated brain organoids” in step (c) of base claim 26.
Regarding to claim 27, the term “solid” is vague and render the claims indefinite. There is no guidance of how “solid” the three-dimensional matrix can be. It is unclear if gel-like or semisolid three-dimensional matrix can also be within the scope of the term “solid”. It is unclear for the degree of “solid” three-dimensional matrix can be to satisfy the scope of the claim. The specification of the claimed invention mentioned once on page 24 line 14 that “Suitable synthetic materials for said scaffold comprise polymers selected from porous solids….”; however, there is no definition for how porous can it be and still be considered as a “solid three-dimensional matrix”.
Claims 28, 32-34, 41-45, 48-52 are included in the rejection because they directly or indirectly depend from base claim. Appropriate correction is required.
New-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.
Claims 26-28, 32-34, 41-45, 48-52 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Regarding to base claim 26, the recitation of “preliminary brain organoids” is considered new matter. Thorough word search for the term “preliminary” in the specification of the claimed invention does not provide any result. There is no definition of “preliminary brain organoids”. Since there are various brain organoids with structurally and functionally differences, Artisan of skill would not recognize from the instant disclosure that Applicant was in possession of any “preliminary brain organoids”. Also, Artisan of skill would not know what can be classified/ categorized as preliminary brain organoids”, and Artisan of skill would not know the difference between “preliminary brain organoids” in step (b) and “differentiated brain organoids” in step (c) of base claim 26.
Regarding to claim 27, the recitation of “no solid three-dimensional matrix already presents in the differentiation medium” is considered new matter. Applicants do not explicitly point for the specific support of the claimed amendment. Upon further review of the instant specification, examiner could not find support for the recited limitations. There is no explicit or implicit support for “no solid three-dimensional matrix already presents in the differentiation medium”. There is no definition for “solid three-dimensional matrix” and no guidance for the degree of “solid” for the three-dimensional matrix to be able to fit in the category of being solid three-dimensional matrix in the instant disclosure. Thus, before the effective filing date of the application was filed, an Artisan of skill would not recognize from the disclosure that Applicant was in possession of “no solid three-dimensional matrix already presents in the differentiation medium” as claimed.
In case if applicants have evidence to support otherwise, applicants are invited to indicate page and line number for the written support specifically for said limitations. MPEP 2163 .06 notes "If new matter is added to the claims, the examiner should reject the claims under 35 U.S.C. 112, first paragraph-written description requirement". In re Rasmussen, 650 F.2d 1212, 211 USPQ 323 (CCPA 1981)". Claims 28, 32-34, 41-45, 48-52 are directly or indirectly depends from the rejected base claim. This is a new matter rejection.
Maintained in modified form and New & 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.
Claim 26, 28, 32-34, 45, 48, 51 and 52 are rejected under 35 U.S.C. 103 as being unpatentable over Jo et al (herein Jo, Pub. No.: US 2019/0169576 Al, Foreign Application Priority Data: Mar. 14, 2016) in view of Hayashi et al (herein Hayashi, Pub. No.: US 2017/0009201 Al, Pub. Date: Jan. 12, 2017).
Claims interpretation:
The term “standardized” is not defined in a limiting way in the specification of the claimed invention. The instant disclosure only mentioned the term “standardized” once : “The omission of a complex three-dimensional matrix such as a gel facilitates the generation of an artificial tissue structure such as brain organoids in a standardized, e.g. automated manner, enabling as well for scale up and large-scale manufacturing processes” (Page 3, lines 24-26) . There is only 1 example without limiting definition or limiting instruction for the term “standardized”. Thus, “a standardized method” in the claims is interpreted broadly as encompassing any published/issued method from the prior art.
According to the specification of the claimed invention, the viscosity enhancer may be for example selected from the group consisting of non-gelling, biocompatible rheology modifiers such as carrageenans, xanthan gum, and cellulose ether derivates such as methyl cellulose, carboxymethyl cellulose, and hydroxy ethyl cellulose, and mixtures thereof (Page 3, lines 17- 20). Therefore, carrageenans, xanthan gum, and cellulose ether derivates such as methyl cellulose, carboxymethyl cellulose, and hydroxy ethyl cellulose are interpreted as biocompatible viscosity enhancers for cell culture medium.
Regarding to claim 26, Jo et al teach a method of deriving and maintaining a midbrain-like organoid in culture (Abstract). The material(s) from which comprise the extracellular matrix is/are but is/are not limited to methylcellulose etc. ([0135], page 16) (For claim 26, the Preamble).
Regarding to claim 26 step a, b, Jo et al teach a) culturing pluripotent stem cells to obtain neuronal lineage embryoid bodies; (b) culturing the neuronal lineage embryoid bodies from (a) to obtain midbrain regionalized tissues (Abstract). Jo et al teaches “Generation of Human Midbrain-Like Organoid (hMLOs): For generation of human midbrain-like organoids, human embryonic stem cell lines were used at less than 40 passages. The human embryonic stem cells were dissociated from intact colonies to single cells with TrypLE Express (Life Technologies), and 10,000 cells were plated in each well of low-cell-adhesion 96-well culture plate with V-bottomed conical wells (Sumitomo Bakelite) to form uniform EBs in neuronal induction medium …… After 3 days, the human midbrain-like organoids started to extrude neuroectodermal buds” ([0158]-[0159], page 20). After neural induction and midbrain patterning, neural organoids gave rise midbrain dopaminergic progenitors” ([0146], page 17). Additionally, Jo et al teach the cells can be grown in suspension or scaffolds ([0145], page 17) (For claim 26, step a, b). Note: the 96 well low attachment plates for culturing pluripotent stem cells with induction medium were also used in similar manner by the claimed invention (see the specification, page 4, lines 1-6).
Regarding to claim 26 step c, Jo et al teach “to promote growth and differentiation, the human midbrain-like organoids (hMLOs) were transferred onto ultra-low attachment 6 well-plate (Costar) by pipetting using a cut 1000 µl pipette tip in final differentiation media containing Neurobasal, 1:100 N2 supplement (Invitrogen) …… Human midbrain-like organoids were cultured using an orbital shaker to enhance exchanges of nutrients and oxygen” ([0159], page 20). Jo et al teach methods for cell culture can require agitation of the cells, for example, when the cells are grown in suspension or when scaffolds are used ([0145], page 17) (For claim 26, step c).
Jo et al teaches that the material(s) from which comprise the extracellular matrix is/are but is/are not limited to matrigel, gelatine, methylcellulose (viscosity enhancer) …. ([0135], page 16). Thus, one of ordinary skill in the art would be motivated to use methylcellulose (viscosity enhancer) for cell culture. However, Jo et al do not specifically teach “differentiation medium with a viscosity between l.7 and 1500 mPa s” and “at a total concentration in the standardized differentiation medium between 0.1% and 2%”. Hayashi et al cures the deficiency
Hayashi et al teach a medium composition for culturing animal and plant cells and/or tissues particularly in a three dimensional or suspended state by using a nanofiber such as polysaccharides and the like having enhanced dispersibility in water, and use thereof ([0001], page 1). One or more kinds of polysaccharides such as methylcellulose and the like can also be mixed with a medium to be used for culture of a carrier supporting the cells and/or tissues on the surface ([0171], page 15). Animal-derived cells in the invention include artificial pluripotent stem cells (iPS cell), neural stem cells etc. ([0078], page 5). Suspending of cells and/or tissues in the invention refers to a state where cells and/or tissues do not adhere to a culture container (non-adhesive). Furthermore, in the present invention, when the cells and/or tissues are proliferated, differentiated or maintained , the state where the cells and/or tissues are uniformly dispersed and suspended in the liquid medium composition in the absence of a pressure on ([0082], page 5). Additionally, Hayashi et al teaches “preparation of Methylcellulose-Containing Medium: DMEM/F-12 medium (manufactured by Aldrich, 100 mL) was placed in a 200 mL recovery flask, and methylcellulose (MO387, manufactured by Aldrich, 0.1 g) was added……Using this solution, medium compositions added with the aqueous methylcellulose solution at a final concentration of 0.1, 0.3, 0.6 or 1.0% (w/v) were prepared” ([0201] and table 2, page 19) (For claim 26, step c).
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Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of Jo et al to generate human midbrain-like organoids from human pluripotent stem cells with the use of methylcellulose in cell culture by using appropriate amount of viscosity enhancer such as 0.1- 1.0% methylcellulose to achieve a viscosity between 1.7 and 1500 mPa.s as suggested by Hayashi et al. Said modifications of using methylcellulose to increase viscosity in cell culture medium are known methods and routinely used in the art to yield predictable results with measurable level of viscosity of the medium, with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to do so because Hayashi et al teaches nanofiber dispersion is useful as a medium additive for preparing the medium composition ([0150], page 11), and Hayashi et al provide medium composition that can maintain cells or tissues in an environment close to the biological environment ([0035], page 3), and the medium composition of Hayashi et al shows a superior effect of suspending cells and/or tissues, and is extremely useful for large-scale cultivation of cells ([0319], page 42). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Hayashi et al were successful in culturing cel in polysaccharides containing medium such as methylcellulose-containing medium with detailed instruction and working examples.
Regarding to claim 28, Jo et al teach the use of scaffold-free systems such as low-adhesion plates, nanoparticle facilitated magnetic levitation, and hanging drop plates ([0045], page 7). Jo et al teaches the cells are grown in suspension ([0145], page 17). Jo et al also teaches the use of methylcellulose ([0135], page 16). Additionally, Hayashi et al teaches “culture method of cells and/or tissues including culturing cells and/or tissues in a suspended state by using a medium composition having an effect of preventing sedimentation of cells and/or tissues…. uniformly dispersed in the liquid medium” (Abstract). Hayashi et al also teaches medium composition can be used for cell differentiation ([0162], page 13). Thus, one of ordinary skill in the art would be motivated to modify Jo et al teachings with Hayashi et al to grow cell in suspension culture with methylcellulose-containing medium without scaffold such as a three-dimensional matrix.
Regarding to claim 32, Jo et al teach “ differentiation media containing Neurobasal, 1:100 N2 supplement (Invitrogen)” ([0159], page 20) and the use of methylcellulose ([0135], page 16). Jo et al also teaches the WNT-signalling activator ([0118], page 14), inhibition of TGF- β type I receptor ALK5 kinase; inhibition of type I Activin/Nodal receptor ALK4; inhibition of type I Nodal receptor ALK7; inhibition of SMAD2/3 phosphorylation; and/or inhibition of the Activin/TGF β /SMAD signalling pathway ([0108] - [0109], page 13).
Regarding to claim 33, Jo et al teaches “human midbrain-like organoids (hMLOs) were transferred onto ultra-low attachment 6 well-plate (Costar) by pipetting using a cut 1000 µl pipette tip in final differentiation media containing Neurobasal, 1:100 N2 supplement (Invitrogen) …… Human midbrain-like organoids were cultured using an orbital shaker to enhance exchanges of nutrients and oxygen ([0159], page 20) (for step d and e). Jo et al also teaches cell culture can require agitation of the cells when the cells are grown in suspension ([0145], page 17).
Regarding to claim 34, Jo et al teaches FIG. 1 provides data showing the generation and characterization of human midbrain-like organoids (hMLOs) from human pluripotent stem cells (hPSCs) ([0013], page 1). Jo et al teaches that to promote growth and differentiation, the hMLOs were transferred onto ultra-low attachment 6 well-plate …… ([0159], page 20). As mentioned above, Jo et al teaches that the material for cell culture can be methylcellulose (viscosity enhancer) …. ([0135], page 16).
Regarding to claim 45, Hayashi et al teach medium compositions added with the aqueous methylcellulose solution at a final concentration of 0.1, 0.3, 0.6 or 1.0% (w/v) were prepared with a viscosity between 4 mPa sand 100 mPa s ([0201] and table 2, page 19) (the table 2 can be found above).
Regarding to claim 48, Hayashi et al teach “the present inventors have conducted intensive studies and found that suspension culture of animal and plant cells and/or tissues can be performed while keeping them still by mixing a nanofiber composed of polysaccharides such as cellulose, chitin and the like in a liquid medium, without Substantially increasing the Viscosity of the liquid medium” ([0032], page 2).
Regarding to claim 51, Jo et al teaches “as defined in the art, a "defined medium" (also known as "chemically defined medium" or "synthetic medium") is a cell culture medium in which all the chemicals used are known and no yeast, animal, or plant tissue is present” ([0040], page 6), and “differentiation media containing Neurobasal, 1:100 N2 supplement (Invitrogen), 1:50 B27 without vitamin A (Invitrogen), 1% GlutaMAX (Invitrogen), 1% minimum essential media-nonessential amino acid (Invitrogen), 0.1 % ~-mercaptoethanol (Invitrogen), 10 ng/ml BDNF (Peprotech), 10 ng/ml GDNF (Peprotech), 100 µM Ascorbic acid (Sigma-Aldrich), and 125 µM db-cAMP (Sigma-Aldrich)” ([0159], page 20)
Regarding to claim 52, Hayashi et al teach the cells and/or tissues can also be cultured by automatically conducting cell seeding, medium exchange, cell image obtainment, and recovery of cultured cells, under a mechanical control and under a closed environment while controlling pH, temperature, oxygen concentration and the like and using a bioreactor and an automatic incubator capable of high-density culture ([0169], page 14).
Claims 27, 41-44, 49-50 are rejected under 35 U.S.C. 103 as being unpatentable over Jo et al (herein Jo, Pub. No.: US 2019/0169576 Al, Foreign Application Priority Data: Mar. 14, 2016) in view of Hayashi et al (herein Hayashi, Pub. No.: US 2017/0009201 Al, Pub. Date: Jan. 12, 2017) as applied to claims 26, 28, 32-34, 45, 48, 51 and 52 above, and further in view of Xiang et al (Cell Stem Cell 21, 383–398 , September 7, 2017, Doi: 10.1016/j.stem.2017.07.007).
The teachings of Jo et al and Hayashi et al above are incorporated herein in their entirety.
Jo et al and Hayashi et al do not specifically teach (i) no solid three-dimensional matrix; (ii) organoid is characterized microscopically by immunocytochemistry.
Jo et al teaches the use of scaffold-free systems such as low-adhesion plates, nanoparticle facilitated magnetic levitation, and hanging drop plates ([0045], page 7). Jo et al teaches the cells are grown in suspension ([0145], page 17). However, Jo et al do not specifically point out there is no solid three-dimensional matrix in the differentiation medium. Xiang et al cure the deficiency.
Regarding to claim 27, Xiang et al teaches organoid techniques provide unique platforms to model brain development and neurological disorders (Abstract). Xiang et al stated that “in forming organoids, we and others do not include Matrigel as supportive matrix, but there are reports suggesting the protective effects of Matrigel (a three-dimensional matrix) in organoid culture …. We found that Matrigel embedding does not improve the formation of hMGEOs (human MGE organoids) and hCOs (human cortical organoids) in our system” (page 395, right column, last para) and Xiang et al teaches generation of hMGEOs and hCOs in neural differentiation media without Matrigel (see “Organoids Culture Conditions” in page e3, Method ). Thus, a person of ordinary skill in the art would follow Xiang et al ’s teachings for forming organoids without including Matrigel as supportive matrix. Since a three-dimensional matrix such as Matrigel can be included or excluded during formation of the organoid according to the teachings of Jo et al and Xiang et al, thereby indicating that the use of three-dimensional matrix was recognized in the prior art to be a result-effective variable, a person of ordinary skill in the art would have been motivated to perform organoid differentiation with or without three-dimensional matrix out of the course of routine optimization.
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of Jo et al by using teachings of Xiang et al to form organoids without three-dimensional matrix such as Matrigel as suggested by Xiang et al. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Xiang et al teach “3D culture system for generating human brain organoids resembling the MGE domain (human MGE organoids [hMGEOs]) and cortical domain (human cortical organoids [hCOs]). hMGEOs and hCOs displayed cellular organizations similar with developing MGE and cortex, respectively. Cells interior of hMGEOs and hCOs efficiently developed neuronal activity” (Page 384, left column, 2nd para). Xiang et al stated that “ these results demonstrate that functional neurons were generated and neuronal networks were efficiently established in hMGEOs and hCOs” (Page 393, right column, 2nd para.). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Xiang et al successfully generated hMGEOs and hCOs organoid without the use of Matrigel embedding with detailed instructions and examples.
Regarding to claim 41 and 43 and 49, Xiang et al teaches Figure 1. Generation of hMGEOs and hCOs with Figure 1F showing typical interior cellular organization of hMGEOs (3 weeks old) and hCOs (4 weeks old), and Figure 1G showing morphology and size of hMGEOs and hCOs after 30 days and 70 days of culture (Page 385). Histological analysis revealed neural rosette-like structures that resemble the proliferative regions of the human ventricular zone (VZ) in both hMGEOs and hCOs (on day 21 for hMGEOs and day 28 for hCOs in Figure 1F) (page 384, right column, 1st para.)
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Regarding to claims 42 and 44, Xiang et al teaches that as observed in developing cortex of human brain, SOX2+ RGs inside hCOs were organized into radial structures, with the apical surfaces marked by the expression of neural specific N-cadherin (Figure 3A) (Page 387, left column, 2nd para.). Figure 3: hCOs Recapitulate Human Dorsal Cortical Organization (A) Immunostaining for SOX2 and N-cadherin in hCO section (40 days old). Arrows show potential oRGs outside of VZ-like area (B) Immunostaining for SOX2 and Tuj1 (B) and PAX6 and NeuN (C) in hCO sections (40 days old). Arrows show potential outer radial glial cells (oRGs) outside of VZ-like areas (ventricular zone (VZ)) (Page 388).
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Regarding to claim 50, Xiang et al “examined the presence of astrocytes in the CP-like area of hCOs after longer culture (day 105) …. These data indicate astrocytes were generated and intertwined with differentiated neurons as hCOs developed” (Page 387, left column, last para). Figure 3: hCOs Recapitulate Human Dorsal Cortical Organization: (M and N) Immunostaining for GFAP and NeuN (M) and GFAP and MAP2 (N) in hCO sections (105 days old) (Page 388-389).
Response to Arguments
Applicant's arguments filed on 06-20-2025 have been fully considered but they are not persuasive.
1. Applicant argues that:
before this application was filed, successful methods for making brain organoids from pluripotent stem cells usually comprised the steps of: (l) partially differentiating the cells; (2) embedding the partially differentiated organoids in a pre-formed extracellular matrix, usually Matrigel and then (3) further differentiating the cells suspended in the extracellular matrix using some kind of differentiation medium. The Jo reference cited in the office action is typical. This approach was an advancement over previous methods, because in standard tissue culture without the extracellular matrix, brain organoids don't form properly. Only loose cellular structures form, which do not have a fully developed internal structure. These type of aggregates ultimately come apart as the culture continues.
The technology described and claimed in this application represents a further advancement of the
art, because it shows that fully differentiated and stable brain organoids can be made reliably without
using Matrigel or any other pre-formed extracellular matrix. Instead, the technology uses a viscosity
enhancer in the differentiation medium.
Thus, the technology described and claimed in this application creates an environment in which
differentiated brain organoids can be prepared \Vith a smooth outer surface and desirable internal
architecture - independently of whether or not an extracellular matrix is present (remarks, page 8-9).
The Jo reference is fundamentally about differentiating cells that have been embedded in an extracellular matrix. According to the abstract, ¶ [0006], and ¶ [0034] of the specification, the method explicitly includes embedding midbrain regionalized tissues in an extracellular matrix. In the top claim, step (c) comprises "embedding and culturing the midbrain regionalized tissues from (b) in an extracellular matrix." (italics added). Jo reiterates the importance of the solid extracellular matrix in ¶¶ [0133], [0159]. [0160], [0168], [0169], and elsewhere.
Paragraph [0145] says that methods for cell culture "can require agitation of the cells, for example, when the cells are grown in suspension or when scaffolds are used. Thus, in one example, an orbital shaker is used in the culturing of the neuroepithelial tissues ...". As depicted in Fig. IA (above), the first step is to culture cells in suspension to make EBs (not yet brain organoids). To obtain mature midrain regionalized tissues, the predifferentiated cells are explicitly embedded in Matrigel and cultured using an orbital shaker.
Paragraph [0135] refers to polymers such as methyl cellulose, but not as a viscosity enhancer: "the material(s) from which comprise the extracellular matrix is/are but is/are not limited to ... methylcellulose ..." amongst about 15 other choices (italics added).
Paragraph [0045] refers to a "variety of platfom1s that can be used to facilitate the growth of three-dimensional cellular structures. It follows very general discussions of "cell culture" [0039], "cell culture media" [0040], "minimal media" [0041 ], "selective media" [0042], "standard ambient growth conditions" [0043], and various types of cell culture vessels [0044]. This is all patent-drafter's boilerplate. There is no indication which if any of these techniques should be used in the various stages of the scheme shov.711 in Fig. 1A. (remarks, page 11-12)
Response to Arguments:
It is noted that as applicant admitted in the instant remark on page 12 last paragraph, “An extracellular matrix is not explicitly excluded in claim 26”. The instant version of the amended claims does not require exclusion of extracellular matrix in the base claim 26. Nevertheless, in response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In the instant case, it is apparent that Jo et al teach the use of methylcellulose for cell culture. ([0135], page 16) which is according to the claimed invention a viscosity enhancer (see the instant disclosure on Page 3, lines 17- 20). Jo et al also teach that “There are a variety of platforms that can be used to facilitate the growth of three-dimensional cellular structures including, but not limited to, scaffold systems such as hydrogel matrices and solid scaffolds, and scaffold-free systems such as low-adhesion plates, nanoparticle facilitated magnetic levitation, and hanging drop plates.” ([0045], page 7). Thus, Jo et al suggested variety platforms and do not exclude the platform that does not require extracellular matrix. Additionally, Hayashi et al teach a medium composition for culturing animal and plant cells and/or tissues particularly in a three dimensional or suspended state by using a nanofiber such as polysaccharides and the like having enhanced dispersibility in water, and use thereof ([0001], page 1). One or more kinds of polysaccharides such as methylcellulose and the like can also be mixed with a medium to be used for culture of a carrier supporting the cells and/or tissues on the surface ([0171], page 15). Finally, Xiang et al teaches organoid techniques provide unique platforms to model brain development and neurological disorders (Abstract). Xiang et al stated that “in forming organoids, we and others do not include Matrigel as supportive matrix, …. We found that Matrigel embedding does not improve the formation of hMGEOs (human MGE organoids) and hCOs (human cortical organoids) in our system” (page 395, right column, last para) and Xiang et al teaches generation of hMGEOs and hCOs in neural differentiation media without Matrigel (see “Organoids Culture Conditions” in page e3, Method ). Thus, a person of ordinary skill in the art would follow Xiang et al ’s teachings for forming organoids without including Matrigel as supportive matrix. Since a three-dimensional matrix such as Matrigel can be included or excluded during formation of the organoid according to the teachings of Jo et al and Xiang et al, thereby indicating that the use of three-dimensional matrix was recognized in the prior art to be a result-effective variable, a person of ordinary skill in the art would have been motivated to perform organoid differentiation with or without three-dimensional matrix out of the course of routine optimization.
One of ordinary skill in the art would have been motivated to combine the above references because Hayashi et al teaches nanofiber dispersion is useful as a medium additive for preparing the medium composition ([0150], page 11), and Hayashi et al provide medium composition that can maintain cells or tissues in an environment close to the biological environment ([0035], page 3), and the medium composition of Hayashi et al shows a superior effect of suspending cells and/or tissues, and is extremely useful for large-scale cultivation of cells ([0319], page 42). Additionally, Xiang et al teach “3D culture system for generating human brain organoids resembling the MGE domain (human MGE organoids [hMGEOs]) and cortical domain (human cortical organoids [hCOs]). hMGEOs and hCOs displayed cellular organizations similar with developing MGE and cortex, respectively. Cells interior of hMGEOs and hCOs efficiently developed neuronal activity” (Page 384, left column, 2nd para). Xiang et al stated that “ these results demonstrate that functional neurons were generated and neuronal networks were efficiently established in hMGEOs and hCOs” (Page 393, right column, 2nd para.).
2. Applicant argues that
a person of ordinary skill in the art would think to combine Hayashi with Jo only by way of hindsight from the current application. Hayashi's technology is a medium containing a nanofiber that can be used for culturing almost anything. Paragraph [0078] of Hayashi lists over 80 different types of cells that can be cultured in their medium - only two of which are neurons or neural stem cells. On the other hand, there are hundreds of references about improved culture techniques that could be incorporated with Jo for various purposes. There is no reason to select and combine these two references in particular, and no expectation that they would successfully achieve the results shown in this application.
Nevertheless, supposing someone reading both references attempted to combine them for some reason, they would still not obtain the invention claimed here. Since Jo requires the use of an extracellular matrix, the extracellular matrix must be present in the combined embodiment. The proposed modification cannot render the prior art unsatisfactory for its intended purpose. MPEP § 2143.0l(V). To remove the extracellular matrix would be contrary to the explicit teachings of Jo. Instead, the combining the two references yields an embodiment in which cells are embedded in an extracellular matrix according to Jo, which is then cultured in a medium that contains microfibers according to Hayashi.
This is not the invention claimed here: a viscosity enhanced medium that is sufficient to differentiate and maintain brain organoids in long term culture. An extracellular matrix is not explicitly excluded in claim 26, but it is not needed to differentiate and maintain the organoids (Remarks, page 12).
Response to Arguments:
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).
Applicant has not explained why “There is no reason to select and combine these two references in particular, and no expectation that they would successfully achieve the results shown in this application” and “To remove the extracellular matrix would be contrary to the explicit teachings of Jo” . There is no evidence in record provided by applicant to show that combining Jo et al and Hayashi et al references and remove the extracellular matrix would result in negative effects on brain organoids differentiation.
In fact, as applicant mentioned above Paragraph [0078] of Hayashi et al lists neurons or neural stem cells as examples that the medium containing a nanofiber that can be used for culturing. This is consistent with the claimed invention because the instant disclosure teaches that “Generated neurons stay within the organoid, this might improve neural differentiation and cortical plate development neural layering” (see page 18 , lines 5-6)
Hayashi et al teach a medium composition for culturing animal and plant cells and/or tissues particularly in a three dimensional or suspended state by using a nanofiber such as polysaccharides and the like having enhanced dispersibility in water, and use thereof ([0001], page 1). One or more kinds of polysaccharides such as methylcellulose and the like can also be mixed with a medium to be used for culture of a carrier supporting the cells and/or tissues on the surface ([0171], page 15). Animal-derived cells in the invention include artificial pluripotent stem cells (iPS cell), neural stem cells etc. ([0078], page 5). One of ordinary skill in the art would have been motivated to combine the above references because Hayashi et al teaches nanofiber dispersion is useful as a medium additive for preparing the medium composition ([0150], page 11), and Hayashi et al provide medium composition that can maintain cells or tissues in an environment close to the biological environment ([0035], page 3), and the medium composition of Hayashi et al shows a superior effect of suspending cells and/or tissues, and is extremely useful for large-scale cultivation of cells ([0319], page 42).
3. Applicant argues that
The Xiang reference teaches how to make brain organoids by plating predifferentiated cells at high density. Organoid culture conditions are described in Xiang on page e3. A population of pluripotent stem cells was predifferentiated, then separated into individual cells, and replated in neural induction medium in a wells of a 96-well plate at a density of 9,000 cells in each well (100-200 µL per well). The cells were cultured at this density for 10 days through several steps until neural organoids were formed. Because of the physics of the culture chamber, the individual cells were necessarily pressed into very close proximity. Perhaps the high-density microenvironment was able to substitute for an extracellular matrix for purposes described in Xiang.
Again, supposing that someone would read both references and attempt to combine them, they would not obtain the invention claimed here. To remove the extracellular matrix would be contrary to the explicit teachings of Jo. Neither reference suggests including a viscosity enhancer as part of the culture environment. Instead, adapting Jo with Xiang would yield an embodiment in which predifferentiated cells are cultured at high density for 10 days according to Xiang with the cells being embedded partway through the process into an extracellular matrix according to Jo.
This is not the invention claimed here: a viscosity enhanced medium that is sufficient unto itself to differentiate and maintain brain organoids and maintain them in long term culture (Remarks, page 13).
Response to Arguments:
It appears that Applicant is arguing that the cited references do not expressly suggest the claimed invention. However, it is well established in case law that a reference must be considered not only for what it expressly teaches, but also for what it fairly suggests. In re Burkel, 201 USPQ 67 (CCPA 1979). Furthermore, in the determination of obviousness, the state of the art as well as the level of skill of those in the art are important factors to be considered. The teaching of the cited references must be viewed in light of these factors. It also appears that applicant is attempting to attack each reference individually. However, in a 103 rejection the references must be considered as a whole.
In the instant case, Xiang et al stated that “in forming organoids, we and others do not include Matrigel as supportive matrix, …. We found that Matrigel embedding does not improve the formation of hMGEOs (human MGE organoids) and hCOs (human cortical organoids) in our system” (page 395, right column, last para) and Xiang et al teaches generation of hMGEOs and hCOs in neural differentiation media without Matrigel (see “Organoids Culture Conditions” in page e3, Method ). Thus, a person of ordinary skill in the art would follow Xiang et al ’s teachings for forming organoids without including Matrigel as supportive matrix. Since a three-dimensional matrix such as Matrigel can be included or excluded during formation of the organoid according to the teachings of Jo et al and Xiang et al, thereby indicating that the use of three-dimensional matrix was recognized in the prior art to be a result-effective variable, a person of ordinary skill in the art would have been motivated to perform organoid differentiation with or without three-dimensional matrix out of the course of routine optimization.
4. Applicant argues that
The invention claimed here provides surprising results that overcome obviousness: As explained above, the invention claimed here solves the problem of how to simplify and standardize the making of differentiated brain organoids, so that the process can be done without variation at an industrial scale. Before the making of this invention, there is no general perception in the art that a viscosity enhancer can be used to replace the structural benefits and protective function of an extracellular matrix. If there were, Corning would lose much of its market for Matrigel (Remarks, page 13).
Response to Arguments:
As per MPEP 716.02, Allegations of Unexpected Results: Any differences between the claimed invention and the prior art may be expected to result in some differences in properties. The issue is whether the properties differ to such an extent that the difference is really unexpected. In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986) (differences in sedative and anticholinergic effects between prior art and claimed antidepressants were not unexpected). In the instant case, there is no evidence in record to show using viscosity enhancer such as methyl cellulose as taught by the claimed invention would lead to unexpected/superior results over viscosity enhancer such as methyl cellulose taught by the cited prior arts. The use of viscosity enhancer is not new according to the cited prior art.
Conclusion
No claim is allowed.
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/KHOA NHAT TRAN/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632