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 .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 02/02/2026 has been entered.
Claims 1, 2, 6, 7, 9-18 and 20-27 are pending in this application, Claims 6, 7 and 9 are acknowledged as withdrawn, Claims 1, 2, 10-18 and 20-27 were examined on their merits.
The rejection of Claims 1, 2, 5, 8, 10-18 and 20-27 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the invention, has been withdrawn due to the Applicant’s amendment to the claims filed 02/02/2026.
The rejection of Claim(s) 1, 2, 5, 8, 10, 12-14, 16, 18, 20-23, 25 and 26 under 35 U.S.C. § 103 as being unpatentable over Sokol et al. (2016) in view of Robertson et al. (2010), Enderle et al. (2021), and Hof et al. (2021), as evidenced by Robinson et al. (2008) and Invitrogen (2017), all of record, has been withdrawn due to the Applicant’s amendments to the claims filed 02/02/2026.
Claim Interpretation
The Examiner notes the phrase "directly after isolating' in Claim 1. This is
supported at least by Figs. 2 and 18. However, the phrase is not defined by the
disclosure, or limited to any particular time period. For example, Fig. 2 merely depicts that isolation and staining occur on the same day. Therefore, the Examiner has given the phrase its' broadest, reasonable interpretation as isolation and staining occurring
sequentially.
Claim Objections
Claim 1 is objected to because of the following informalities: Claim 1 has been amended to remove subject matter which is not indicated as removed by strike through. Appropriate correction is required.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, 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(s) 1, 2, 8, 10, 12-14, 16, 18, 20-23, 25 and 26 are rejected
under 35 U.S.C. § 103 as being unpatentable over Sokol et al. (2016) in view of
Robertson et al. (2010), Enderle et al. (2021), Hof et al. (2021), Fiorini et al. (2020) and Wang et al. (2019), as evidenced by Robinson et al. (2008) and Invitrogen (2017), all of record.
Sokol et al. teaches a method comprising: obtaining surgical resections from
patients (reduction mammoplasty tissue samples), mechanically and enzymatically
dissociating the samples, isolating individual target cells (from normal epithelial cell
clusters) (Pg. 2, Column 1, Lines 48-52 and Column 2, Lines 1-3 and Pg. 4, Column 2,
Lines 18-21);
thereafter forming stained cells from the isolated, individual cells by stably
transfecting/labeling the primary cell clusters with fluorescent proteins (light responsive
dye) before seeding (encapsulating) into 3D hydrogel scaffolds and culturing (Pg. 6,
Column 2, Lines 40-41 and Pg. 8, Column 1, Lines 1-2 and Pg. 2, Lines 42-48);
wherein the hydrogel comprises hyaluronic acid and collagen (Pg. 2, Column 2,
Lines 5-6);
wherein the cultured cells form an organoid in the hydrogel (Pgs. 5-6, Fig. 2);
and wherein the fluorescent proteins are visible with fluorescence microscopy
(Pgs. 5-6, Fig. 2e), reading on Claims 1, 8, 10, 12, 13, 16, 18, 25 and 26.
With regard to the limitation of Claim 1 of a "light-responsive dye", the stably
transfected fluorescent proteins of Sokol et al. would meet the limitation of being a "dye"
lacking any definition for the term in the Specification and giving the term its' broadest,
reasonable interpretation.
The teachings of Sokol et al. were discussed above.
Sokol et al. did not teach a method wherein at least one of a mitochondria or
nucleus per individual isolated dissociated cell is stained,
wherein the culturing comprises forming a co-culture of cancer, normal or non-transformed, stromal, stem and immune cells,
or wherein while culturing the encapsulated stained cells, tracking, over time and
migratory distance at least one of live or dead encapsulated stained cells via
fluorescence microscopy of the at least one light-responsive dye wherein the migratory
distance is measured in at least three dimensions, wherein the tracking detects at least
one live or dead encapsulated stained cell(s) throughout the cell culture with an X
coordinate, a Y coordinate and a Z coordinate, as required by Claim 1;
wherein the culturing of (and the organoid) comprises a co-culture of cancer, normal, stem, stromal and immune cells, as required by Claims 14 and 15;
wherein mitochondria are stained and the tracking is of live cells, as required by
Claim 20;
wherein nuclei are stained and the tracking is of dead cells, as required by Claim
21;
wherein mitochondria are stained and the tracking is of live cells and wherein
nuclei are stained and the tracking is of dead cells, as required by Claim 22;
wherein the light-responsive dye is configured to cause a color change in the
stained cell when the stained cell transitions from a living cell to a dead cell, as required
by Claim 23.
Robertson et al. teaches staining tumor spheroids stably transfected with a
fluorescent protein with a light responsive dye configured to stain mitochondria of the
target cells (MitoSOX® Red) and a nuclear stain configured to stain the nuclei of target
cells (DRAQ5®) which are visualized by fluorescent microscopy (Pg. 821, Column 1,
Lines 39-43 and Fig. 1C). The reference also teaches that for longer term imaging of
3D spheroids, DRAQ5® can be replaced with DNA dyes that do not alter turnover of
DNA and the ability to immobilize 3D tumor spheroids using CyGEL™ provides the
opportunity to perform live-cell imaging of 3D structures in real-time (Pg. 825, Lines 35-
41).
Robinson et al. evidences that MitoSOX® Red can be used for imaging
superoxide formation in live cells (Pg. 941, Abstract).
Invitrogen evidences that DRAQ5™ is a membrane permeable dye that can label
live or dead cells (Pg. 1, Lines 1-2).
Enderle et al. teaches a method of 2D imaging cell migration in intestinal
organoids comprising imaging fluorescently stained cells over time and tracking their
migratory distance (Pg. 13, Paragraphs 4.9-4.10 and Pg. 8, Fig. 4E) and that live cell
imaging was established allowing for real-time assessment of IEL localization, mobility,
and overall migration pattern in a living IEL-IEC co-culture setting (Pg. 9, Lines 53-54).
Hof et al. teaches a method of 3D imaging of cell migration in human tissue
organoids in an extracellular matrix (ECM) (e.g. a solid cell culture) comprising imaging
individual fluorescently labeled cells over time and tracking their migration distance in at
least three dimensions throughout the solid cell culture, as measured in X, Y and Z coordinate locations (Pg. 5, Fig. 2 and Pg. 6, Column 1, Lines 22-29 and Pg. 17, Column 1, Lines 43-52 and Column 2, Lines 1-5 and Pg. 20, Supplementary Information, Additional file 21: Video 7).
Fiorini et al. teaches that organoids can model the interplay between cancer and
non-cancer cells (tumor microenvironment) in order to unveil biological mechanisms
involved in cancers initiation and progression which might ultimately lead to the
identification of novel intervention strategy for those diseases (Pg. 1, Abstract) and
teaches an organoid co-culture with cancer cells, normal/stromal cells (fibroblasts) and
immune cells (Pg. 4, Fig. 1G).
Wang et al. teaches that stem cells can be used to form organoids (Pg. 4045,
Column 2, Lines 25-27).
It would have been obvious to those of ordinary skill in the art before the effective
filing date of the claimed invention to modify the method of Sokol et al. of preparing a
hydrogel encapsulated breast tissue organoid comprising a transfected light-responsive
fluorescent protein dye with the method of Robertson et al. of further staining the target
cells with a light responsive dye configured to stain mitochondria of living target cells (as
evidenced by Robinson) and a nuclear stain configured to stain the nuclei of living and
dead target cells (as evidenced by Invitrogen) because this is no more than the
application of a known technique (staining individual cell tissue structure transfected
with protein light-responsive dye with light-responsive dyes for mitochondria and nuclei),
to a known product (cell tissue structure transfected with protein light-responsive
dye) ready for improvement to yield predictable results (cell tissue structure transfected
with protein light-responsive dye and stained with light-responsive dyes for mitochondria
and nuclei). See the MPEP at 2141, C. III. Those of ordinary skill in the art would have been motivated to make this modification in order to stain live and/or dead cells in organoids for real-time imaging. There would have been a reasonable expectation of success in making this modification because at least both Sokol et al. and Robertson et al. are drawn to the staining and visualization of cells in solid 3D cell culture.
It would have been further obvious to those of ordinary skill to modify the method
of Sokol and Robertson to track encapsulated, stained live cells over time via
fluorescence microscopy because Robertson suggests the use of dyes for longer term
imaging and imaging of gel-immobilized cells in real time.
Those of ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to make this modification in order to stain and monitor multiple organelles in the same living tissue structure simultaneously over time. Those of ordinary skill in the art would have had a reasonable expectation of success in making this modification because both references are drawn to the in vitro labeling and imaging of living cell structures (spheroids/organoids).
It would have been further obvious to those of ordinary skill in the art to modify
the method of Sokol and Robertson of tracking encapsulated, stained live cells in
organoids/spheroids over time via fluorescence microscopy to track the migration
distance of those cultured fluorescently stained organoid cells throughout a solid cell culture over time in 3D in at least X, Y and Z coordinate locations as taught by Hof because this would allow the artisan to track not just the live/dead status of cells over time in the organoid/spheroid but their movements as well. Those of ordinary skill in the art would have been motivated to make this modification because Enderle teaches that live cell imaging allows for real-time assessment of cell localization, mobility, and overall migration pattern in a living co-culture setting. Those of ordinary skill in the art would have had a reasonable expectation of success in making this modification because all of the references are drawn to the in vitro labeling and imaging of living cell structures (spheroids/organoids).
It would have been obvious to those of ordinary skill in the art before the effective
filing date of the claimed invention to modify the method of Sokol et al. and Robertson et
al. of preparing a breast tissue organoid comprising a light-responsive dye to further utilize a co-culture of cancer cells, stromal/normal cells and immune cells to form the organoid, as taught by Fiorini et al., and further including stem cells (which also form organoids) as taught by Wang et al. to form and culture an organoid because this would provide a more realistic model of the tumor microenvironment. Those of ordinary skill in the art would have been motivated to make this modification in order to provide a more realistic in vitro model of in vivo conditions of a cancer to develop novel treatments. There would have been a reasonable expectation of success in making this modification because all of the references are reasonably drawn to the same field of endeavor, that is, characterization of tissue spheroids/organoids.
With regard to the limitation of Claim 2 of: "configured to mimic core components
of human tissue extracellular matrices and disease-specific niches", this is a recitation
of, an inherent characteristic of the claimed hydrogel. As the hydrogel of the cited prior
art meets the structural limitations of the claimed hydrogel, it would be expected to
function in an equivalent manner.
With regard to the limitation of Claim 23 of; "is configured to cause a color
change in the stained cell when the stained cell transitions from a living cell to a dead
cell", this is a recitation of an inherent characteristic of the claimed light- responsive dye.
The light-responsive MitoSOX™ dye of the cited prior art will cease to fluorescently label (a color change) the superoxide formation characteristic of living cells when they transition to being a dead cell and thus meets the structural limitation of the claimed light-responsive dye.
Claim(s) 1, 2, 8, 10, 11, 12-14, 16, 17, 18, 20-23, 24, 25 and 26 are rejected
under 35 U.S.C. § 103 as being unpatentable over Sokol et al. (2016) in view of
Robertson et al. (2010), Enderle et al. (2021), Hof et al. (2021), Fiorini et al. (2020) and Wang et al. (2019), as evidenced by Robinson et al. (2008) and Invitrogen (2017), as applied to Claims 1, 2, 8, 10, 12-14, 16, 18, 20-23, 25 and 26 above, and further in view of Walsh et al. (2017), all of record.
The teachings of Sokol et al., Robertson et al., Enderle et al., Hof et al., Fiorini et al. and Wang et al. were discussed above.
None of the above references taught a method wherein the dissociated cells are
isolated from a patient-derived tumor sample, as required by Claims 11 and 17;
or wherein the target cells are breast cancer tumor cells, as required by Claim
24.
Walsh et al. teaches that organoids grown from primary tumor tissues provide a
patient-specific model of solid tumors and retain the organ structure, morphology,
stromal composition, genetic mutations and heterogeneity of the original tumor (Pg.
1367, Column 2, Lines 17-18 and 30-32), teaches that organoids are prepared by
mechanical or enzymatic dissociation of the original tumor sample, embedding the
tissue in an ECM (e.g. a hydrogel), such as MATRIGEL® or collagen (Pg. 13867,
Column 2, Lines 18-22), suggests the use of organoids for patient specific drug-testing
(Pg. 1367, Column 2, Lines 3-8) and teaches an organoid derived from a human breast
cancer biopsy sample (Pg. 1371, Fig. 4).
It would have been obvious to those of ordinary skill in the art before the effective
filing date of the claimed invention to modify the method of at least Sokol et al. and Robertson et al. of preparing a breast tissue organoid comprising a light-responsive mitochondrial and nucleus dyes to utilize cells isolated from a patient-derived breast tumor sample as taught by Walsh et al. because this is no more than the application of a known technique (organoid culture derived from breast cancer tumor) to a known method (fluorescent dye labeled organoid culture from normal breast tissue) ready for
improvement to yield predictable results (fluorescent dye labeled organoid culture
derived from breast cancer tumor). Those of ordinary skill in the art before the effective
filing date of the claimed invention would have been motivated to make this modification
in order to prepare an organoid derived from a human breast cancer biopsy sample for
patient specific drug-testing.
There would have been a reasonable expectation of success in making this modification because both references are drawn to the same field of endeavor, that is, the preparation of breast tissue organoids.
Claim(s) 1, 2, 8, 10, 12-14, 16, 18, 20-23, 25, 26 and 27 are rejected
under 35 U.S.C. § 103 as being unpatentable over Sokol et al. (2016) in view of
Robertson et al. (2010), Enderle et al. (2021), Hof et al. (2021), Fiorini et al. (2020) and Wang et al. (2019), as evidenced by Robinson et al. (2008) and Invitrogen (2017), as applied to Claims 1, 2, 8, 10, 12-14, 16, 18, 20-23, 25 and 26 above, and further in view of Fang et al. (2016), all of record.
The teachings of Sokol et al., Robertson et al., Enderle et al., Hof et al., Fiorini et al. and Wang et al. were discussed above.
None of the above references taught a method wherein the tracking is performed
over more than a day, as required by Claim 27.
Fang et al. teaches that neuronal cells stained with MitoSOX® were assessed up
to 20 days in culture (Pg. 682, Column 2, Lines 17-21 and Figs. 3F and G).
It would have been obvious to those of ordinary skill in the art before the effective
filing date of the claimed invention to modify the method of Sokol, Robertson, Enderle
Hof, Fiorini and Wang of culturing and tracking the migration distance over time of MitoSOX™ and DRAQ5® stained, encapsulated cells of organoids in 3D via
fluorescence microscopy to perform the tracking over several days as taught by Fang et
al. because this would allow long-term monitoring of the cells in real time. Those of
ordinary skill in the art would have been motivated to make this modification in order to
track encapsulated living cells for more than a short period of time. There would have
been a reasonable expectation of success in making this modification because
Robertson suggests that stained, immobilized cells are suitable for long-term tracking in
real time and Fang et al. teaches that MitoSOX® stained cells can be monitored over a
multiple day period.
Response to Arguments
Applicant’s arguments, see Remarks, filed 02/02/2026, with respect to the above withdrawn rejections have been fully considered and are persuasive.
Applicant's remaining arguments filed 02/02/2026 have been fully considered but they are not persuasive insofar as they apply to the pending rejections.
The Applicant argues that the cited prior art does not teach or suggest the claimed invention. Applicant asserts that Sokol is silent with regard to a 3D culture of dissociated cells (Remarks, Pg. 7, Lines 6-16).
This is not found to be persuasive for the reasoning provided in the above rejections, the Examiner notes that Sokol et al. teaches a method comprising: obtaining surgical resections from patients (reduction mammoplasty tissue samples), mechanically and enzymatically dissociating the samples, isolating individual target cells (from normal epithelial cell clusters) (Pg. 2, Column 1, Lines 48-52 and Column 2, Lines 1-3 and Pg. 4, Column 2, Lines 18-21); thereafter forming stained cells from the isolated, individual cells by stably transfecting/labeling the primary cell clusters with fluorescent proteins (light responsive dye) before seeding (encapsulating) into 3D hydrogel scaffolds and culturing (Pg. 6, Column 2, Lines 40-41 and Pg. 8, Column 1, Lines 1-2 and Pg. 2, Lines 42-48). Thus, the reference teaches the 3D culture of dissociated cells.
The Applicant argues that the Examiner concedes that Fiorini teaches an organoid co-culture and asserts that the model has utility in modeling the interplay between cancer and non-cancer cells. Applicant notes that Wang and Fiorini are allegedly silent with regard to the three dimensional culture of dissociated cells (Remarks, Pg. 7, Lines 17-27).
This is not found to be persuasive for the following reasons, as discussed above, the Sokol reference teaches the 3D culture of dissociated cells. Fiorini et al. teaches that organoids can model the interplay between cancer and non-cancer cells (tumor microenvironment) in order to unveil biological mechanisms involved in cancers initiation and progression which might ultimately lead to the identification of novel intervention strategy for those diseases (Pg. 1, Abstract) and teaches an organoid co-culture with cancer cells, normal/stromal cells (fibroblasts) and immune cells (Pg. 4, Fig. 1G) while Wang et al. teaches that stem cells can be used to form organoids (Pg. 4045,
Column 2, Lines 25-27). Therefore, it would have been obvious to those of ordinary skill in the art to modify the method of Sokol et al. and Robertson et al. of preparing a breast tissue organoid comprising a light-responsive dye to further utilize a co-culture of cancer cells, stromal/normal cells and immune cells, as taught by Fiorini et al., and stem cells (which form organoids) as taught by Wang et al. to form and culture an organoid because this would provide a more realistic model of the tumor microenvironment. Those of ordinary skill in the art would have been motivated to make this modification in order to provide a more realistic in vitro model of in vivo conditions of a cancer to develop novel treatments. There would have been a reasonable expectation of success in making this modification because all of the references are reasonably drawn to the same field of endeavor, that is, characterization of tissue spheroids/organoids.
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the Examiner should be directed to PAUL C MARTIN whose telephone number is (571)272-3348. The Examiner can normally be reached Monday-Friday 12pm-8pm EST.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, Applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the Examiner’s supervisor, Sharmila G Landau can be reached at (571) 272-0614. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/PAUL C MARTIN/Examiner, Art Unit 1653 02/27/2026