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 .
Applicant’s response filed 3/3/2026 has been received and entered into the case. Claims 1-15 are pending. Claims 1-8 are withdrawn. Claims 9-15 have been considered on the merits herein.
All arguments and amendments have been considered.
The previous rejections of record are withdrawn in light of applicants claim amendments.
Claim Objections
Claim 10 is objected to because of the following informalities: at line 2-the word “data” was deleted; however, it appears the term is necessary in the claim and should read “the evaluation model is generated by training data using…”. Appropriate correction is required.
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.
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.
Claim(s) 9-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Saito et al. (US20210301248, IDS) in view of Gualerzi et al. (Sci. Reports, 2017, IDS), Zhang et al. (PLOS ONE, 2020, IDS), and Braeckmans et al. (WO2017103245).
Regarding claim 9, Saito teaches a method for evaluating the state of cells, particularly differentiation levels on extracellular vesicles (EV), i.e., exosomes (0013, 0021, 0027, 0028) isolated from cell culture supernatant (abstract, 0005, 0006, 0021, Ex. 1-3). Saito teaches collecting culture supernatant from a cell culture, isolating extracellular vesicles from the supernatant and evaluating a differentiation level of a cultured cell based on contents of a component in the supernatant on days 0, 8 and 12 after passage (Ex. 1-3, 0041, 0050, 0055), specifically monitoring exosome changes and exosome markers in the supernatant in a differentiation induction process from iPS cells to dopamine neural progenitor cells (abstract, 0006, 0013-0017, 0021). Saito suggests that the measurement unit includes an instrument capable of detecting exosomes or exosome markers (0024) and at (0025)“The storage unit 6 stores correlation information including a correlation between the content of the component and the differentiation level of the cultured cells. If the component is exosomes, it may be correlation information between a number of culture days for maximum differentiation and an exosome density in the culture supernatant, or may be correlation information between a signal intensity of differentiation markers and the exosome density in the culture supernatant. When the component is exosome markers, it may be correlation information between a signal intensity of the differentiation markers and a signal intensity when the exosome markers are detected. A format of the correlation information is not particularly limited, but the correlation information preferably indicates that the content of the component and the differentiation level of the cultured cells continuously correspond to each other.” Saito also teaches that “When the component is exosomes, a correlation between a time after the differentiation induction of the cultured cells and the content of the exosomes in the culture supernatant is found. For example, it is conceivable that after the differentiation induction of the cultured cell, the culture supernatant is collected at predetermined time intervals, the content of the exosomes in the culture supernatant is measured, and the content of the exosomes is defined as 0% at the start of the differentiation induction, and is defined as 100% at a maximum differentiation time to create a regression line or a regression curve indicating the relationship between the content of the exosomes and the differentiation level. For example, in the case of dopamine neural progenitor cells, since it differentiates at the maximum on the 9th to 15th days, or the 10th to 14th days, or the 11th to 13th days, or the 12th day, 100% may be set on that day, 0% may be set on a differentiation induction start day, and the correlation between the content of the exosomes and the differentiation level (%) may be found. Alternatively, the correlation between the signal intensity of the differentiation markers and the content of the exosomes may be found at predetermined time intervals, a minimum value and a maximum value of the signal intensity of the differentiation markers may be defined as 0% and 100%, respectively, and the correlation of the content of the exosomes and the signal intensity (%) of the differentiation markers may be found. It is preferable to perform an experiment a plurality of times under the same differentiation condition… A method for measuring the content of exosomes is not particularly limited, but as an example in which the measurement can be easily performed while maintaining a closed space…a method using infrared spectroscopy or Raman spectroscopy can be exemplified (0027)”.
Therefore, the Saito suggests that Raman spectroscopy can be used to acquire intensity information analysis on the isolated extracellular vesicles to evaluate the differentiation state of the cells.
Saito teaches an evaluator for evaluating the differentiation level of cultured cells using an analysis unit 5, a measurement unit 4, a storage unit 6 which stores data obtained by the analysis, a control unit 7 (Fig. 1, 0020). Data is obtained based upon measurement data and differentiation level data is stored in the storage unit in advance, if the differentiation level does not reach a predetermined reference value (S2), data of the differentiation level calculated by the analysis unit of the evaluator is transmitted to the automatic cell culture device, and the cell culture is continued. If the differentiation level has reached the predetermined reference value (S2), the cells are subjected to passage as they are, or the data of the differentiation level is transmitted to the automatic culture device, and the cell culture ends. Therefore, it is taken that an evaluation model is generated by training data and corresponding data of known cell differentiation states according to the evaluating step of claims 9 and 10.
Regarding claim 11, the cells are pluripotent stem cells and dopamine neural progenitor cells, for example (0022, 0023, 0041, Ex. 1-5).
Regarding claim 12, the evaluation method is performed during culture of the cells to monitor a change the state of the cells (0013, 0027, 0028, 0038, 0039, Ex. 1-5).
Regarding claim 13, the EV’s are exosomes (0021, 0024, Ex. 1-5).
Regarding claim 15, Saito teaches that the evaluation results is used to determine whether to continue, stop or change culture conditions of the cell culture, i.e. “(0038) First, the cell culture is started and the culture is continued (S0). After culturing in a predetermined period, the content of the exosomes or the exosome markers is measured by the measurement unit 4 for the exosomes in the culture supernatant, using the methods as described above (S1). The analysis unit 5 evaluates the differentiation level, using the relative information of the content of the exosomes or the exosome markers, and the differentiation level stored in the storage unit 6 in advance. (0039) If the differentiation level does not reach a predetermined reference value (S2), data of the differentiation level calculated by the analysis unit of the evaluator is transmitted to the automatic cell culture device, for example, within 12 hours after the calculation, preferably within 6 hours, more preferably within 3 hours, still more preferably within 1 hour, and the cell culture is continued. If the differentiation level has reached the predetermined reference value (S2), the cells are subjected to passage as they are, or the data of the differentiation level is transmitted to the automatic culture device, and the cell culture ends (see also Fig. 3).
Saito differs from the claimed invention in that while Raman spectroscopy is suggested as a measurement method to be used to evaluate the differentiation state of cells in their method, Raman spectroscopy is not specifically exemplified and thus, Raman spectrum data is not acquired according to claims 9, 10, 14.
However, before the effective filing date of the claimed invention, Raman spectroscopy was routinely used to evaluate extracellular vesicles to qualitatively and quantitatively describe the chemical composition of the EV’s. See each of Gualerzi et al. (Sci. Reports, 2017, IDS), Zhang et al. (PLOS ONE, 2020, IDS), and Braeckmans et al. (WO2017103245) below.
Gualerzi teaches an evaluation method comprising performing Raman spectroscopic analysis on extracellular vesicles to qualitatively and quantitatively describe the chemical composition of the EV’s (p. 2, 2nd and 3rd full parag.). Raman spectroscopy (RS) offers a label-free, non-destructive, sensitive, rapid and automatable means of carrying out EV characterization, avoiding the need for specific protein biomarkers, and useful in analyzing undifferentiated and differentiated stem cells. RS is disclosed to be more suitable than widely used techniques including immunoblotting, fluorimetry and spectrometry because it provides reproducible quick label-free results (p. 2, 2nd full parag.). Gualerzi uses RS to distinguish vesicles from differentiated and undifferentiated cells.
The EV’s are isolated via ultracentrifugation, i.e. isolated from culture supernatant of cells (p. 2, Results section EV characterization and Raman spect. Biochem overview Section, 2nd parag.p. 8, Extrac. Ves. Isolation section) from mesenchymal stromal cells from bone marrow and adipose tissue (p. 2, 3rd full parag., Results section, EV charact. Section). The EV’s include exosomes and microvesicles (introduct, p. 1, first 2 parag.).
Regarding claim 10, the claim is interpreted to mean that the data evaluated in the method is compared to training data/database or known reference data sets for cells previously analyzed by Raman spectroscopy. Gualerzi teaches comparing the spectral data to EV’s released from dermal fibroblasts (DF) in order to distinguish EV’s from differentiated and undifferentiation cells. Multivariate analysis was used to assess the spectral differences and distinguish between the different cell groups of DF, BM-MSC’s and ASC’s ( p. 2, 3rd full parag.-p. 5, p. 5, Discussion section 2nd parag-p. 7, whole page, p. 9, Raman spect., data analysis, and CLS fitting sections). The reference teaches that “our data attribute a Raman fingerprint to EV’s from undifferentiated to differentiated cells of diverse tissue origin (abstract). Thus, one is evaluating undifferentiated and differentiated cells based upon Raman spectral data.
Regarding claim 11, the cells are mesenchymal cells (MSC’s) and thus are mesoderm cells.
Regarding claim 14, the EV’s were analyzed by Raman spectroscopy to obtain intensity information (p. 4, Fig. 2) and measurement results in the spectral ranges of 500-1800 cm-1 and 2600-3200 cm-1 (the most significant regions of Raman spectrum for biological specimens) and specifically showing bands of nucleic acids (720-820 cm-1), phenylalanine (1003 cm-1), lipid and protein markers (1450and 2940 cm-1), cholesterol and cholesterol esters (1130, 1442 cm-1), C-C stretches (around 1100 cm-1) and CH, CH2 and CH3 bonds (2600-3200 cm-1, specifically 2850-2950 cm-1)(p. 2, Raman spec. biochem section-p.3, 1st 2 parag., p. 4, Fig. 2), thus corresponding to the at least one range of claim 14 including 713±10, 830±10, 997±10, 1133±10, 1443±10, 2850±10, and 2936±10.
Zhang teaches an evaluation method comprising performing Raman spectroscopic analysis on extracellular vesicles to characterize bovine placental EV’s and peripheral blood mononuclear cell (PBMC’s) EV’s (p. 3, 1st parag.) and obtain biochemical fingerprints of the EV’s (section 3.3) at different stages of pregnancy and gestational age (p. 13, 1st full parag.). Zhang teaches that there is a need for a simpler and faster method for analysis of molecular components of EV’s as the conventional methods have limitations (p. 2 last 2 parag.). Raman spectroscopy (RS) offers a label-free, non-invasive means of discriminating EV’s from different tissues and providing a Raman spectra which is a fingerprint of the EV’s cargo (p. 3, 1st parag.).
The EV’s are isolated via ultracentrifugation, i.e. isolated from culture supernatant of cells (p. 3, section 2.3, section 3.1).
Regarding claim 11, the cells are PBMC’s and thus are mesoderm cells.
Regarding claim 13, the reference teaches that the EV’s are exosomes (p. 6, section 3.1 and Fig. 3).
Regarding claim 14, the EV’s were analyzed by Raman spectroscopy and specifically showing bands between 828 cm-1 to 1663 cm-1 (p. 7, last parag.-p. 8, whole page, Fig. 5A and B), including C-C backbone at 934 cm-1 , phenylalanine at 1003 cm-1, and peaks at 828, 852, 883, 1447 cm-1, thus corresponding to the at least one range of claim 14 including 830±10, 858±10, 885±10, 942±10, 997±10, 1443±10.
Braeckmans (WO’245) teaches a method for evaluating extracellular vesicles, including exosomes comprising performing Raman spectroscopic analysis on exosomes isolated from cell supernatants (p. 2, lines 14-20, p. 3, lines 14-26, p. 10, lines 17-19, p. 13, lines 31-p. 14, lines 1-26). The EV’s are isolated via ultracentrifugation, i.e. isolated from culture supernatant of cells (p. 14, lines 1-26, Fig. 24) and include exosomes (p. 5, line 21).
Regarding claim 10, the claim is interpreted to mean that the data evaluated in the method is compared to training data/database or known reference data sets for cells previously analyzed by Raman spectroscopy. WO245 teaches comparing the spectral data/signal to a library of Raman spectroscopy signals to identify the EV ( p. 5, lines 22-26, p. 13, lines 3-8).
Regarding claim 14, the EV’s were analyzed by Raman spectroscopy in the spectral ranges of 750-1572 cm-1 (p. 18, lines 22-p. 19, lines 1-2, Fig. 10 and 11) including 949, 1065, 1123, 1172, 1307, 1366-1370, 1445 cm-1, thus corresponding to the at least one range of claim 14 including 942±10, 1065±10, 1133±10, 1175±10, 1299±10, 1372±10, 1443±10 .
Therefore, before the effective filing date of the claimed invention, isolating EV’s from cell culture to evaluate the differentiation stage of the cells was disclosed by Saito. Saito suggests that Raman spectroscopy can be used to evaluate and measure EV data (although not specifically used to provide spectral data). Each of the secondary references teach the use of Raman spectroscopy to qualitatively and quantitatively describe the chemical composition of the EV’s, and teach that Raman spectroscopy (RS) offers a label-free, non-destructive, sensitive, rapid and automatable means of carrying out EV characterization, avoiding the need for specific protein biomarkers, and useful in analyzing undifferentiated and differentiated stem cells. RS is disclosed to be more suitable than widely used techniques including immunoblotting, fluorimetry and spectrometry because it provides reproducible quick label-free results. The art recognizes that there is a need for a simpler and faster method for analysis of molecular components of EV’s as the conventional methods have limitations. Raman spectroscopy (RS) offers a label-free, non-invasive means of discriminating EV’s and providing a Raman spectra which is a fingerprint of the EV’s cargo. Thus, Raman spectroscopy was recognized as an improved technique for analysis of EV’s and one of ordinary skill in the art would have been capable of applying this known technique to the evaluation method of Saito (who suggests using Raman spectroscopy in their method) and there would have been a reasonable expectation of successfully evaluating the differentiation state of cells and acquiring a Raman spectrum comprising intensity information and measurement results within the claimed range as claimed.
Response to Arguments
Applicant's arguments filed 3/3/2026 have been fully considered but they are not persuasive. While the 102 rejections of record have been withdrawn, the same prior art references are relied upon to address the claim amendments and are now relied upon in the pending 103 rejection of record. While the 103 rejection was not previously presented, applicants present arguments in anticipation of a 103 rejection, so the arguments will be addressed.
Applicants argue that even if the references were combined, Saito teaches evaluating cell differentiation based on changes in components or markers and relies on regression analysis of data, while the secondary references are directed to characterizing the vesicles themselves by source or cell type, rather than for evaluating the state of the producing cells. None of the references are concerned with generating or using a trained evaluation model correlating spectral features of EV’s with differentiation stage. Applicants argue that combining the references would require selectively extracting disparate concepts from unrelated contexts and assembling with hindsight knowledge. While the references demonstrate EV’s have distinguishable Raman signatures, they do not teach how to evaluate them to evaluate differentiation stage.
It is the Examiner’s position that before the effective filing date of the claimed invention, isolating EV’s from cell culture to evaluate the differentiation stage of the cells was disclosed by Saito. Saito suggests that Raman spectroscopy can be used to evaluate and measure EV data (although not specifically used to provide spectral data). Each of the secondary references teach the use of Raman spectroscopy to qualitatively and quantitatively describe the chemical composition of the EV’s, and teach that Raman spectroscopy (RS) offers a label-free, non-destructive, sensitive, rapid and automatable means of carrying out EV characterization, avoiding the need for specific protein biomarkers, and useful in analyzing undifferentiated and differentiated stem cells. RS is disclosed to be more suitable than widely used techniques including immunoblotting, fluorimetry and spectrometry because it provides reproducible quick label-free results. The art recognizes that there is a need for a simpler and faster method for analysis of molecular components of EV’s as the conventional methods have limitations. Raman spectroscopy (RS) offers a label-free, non-invasive means of discriminating EV’s and providing a Raman spectra which is a fingerprint of the EV’s cargo. Thus, Raman spectroscopy was recognized as an improved technique for analysis of EV’s and one of ordinary skill in the art would have been capable of applying this known technique to the evaluation method of Saito (who suggests using Raman spectroscopy in their method) and there would have been a reasonable expectation of successfully evaluating the differentiation state of cells and acquiring a Raman spectrum comprising intensity information and measurement results within the claimed range as claimed.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TIFFANY MAUREEN GOUGH whose telephone number is (571)272-0697. The examiner can normally be reached M-Thu 8-5.
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/TIFFANY M GOUGH/ Examiner, Art Unit 1651
/MELENIE L GORDON/Supervisory Patent Examiner, Art Unit 1651
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