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 .
Status of the Claims
Upon entry of the response submitted on 2/10/26:
Claims 27, 31 and 33 were amended.
Claim 34 was cancelled.
Claims 17-22, 24-33 and 42 are pending and under current consideration.
Claim Rejections/Objections
Applicant’s arguments with respect to the wherein clause in claim 17, “wherein a ratio of a convective volume of the culture medium circulating outside the microcompartments in the closed chamber to a diffusive volume of the culture medium diffusing inside the microcompartments is between 1 and 10,000” were persuasive. The passages from Lu cited as teaching the recited ratio were not correct as pointed out in Applicant’s arguments. The examiner apologizes for the confusion.
The rejection of claims 27 and 34 under 112(b) is withdrawn in light of the amendments to claim 27 and cancellation of claim 34.
The objection to claim 31 is withdrawn in light of the claim amendment.
A new grounds of rejection is set forth below.
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) 17-22, 24-27 and 30-33 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lu et al (of record) as evidenced by Hernandez (of record).
Regarding claim 17, Lu teaches compartmentalized hydrogel microparticles (reads on microcompartments) with a tunable extracellular matrix (ECM) support for cell encapsulation and scalable 3D cell culture (Abstract). Lu teaches that the microparticles, which have a uniform spherical shape, have a larger surface-to-volume ratio for mass transfer and can be potentially cultured in suspensions in stirred bioreactors for scale up (p 353, col 2, para 2). Lu teaches hydrogel microparticles with a double or triple layer of alginate hydrogel (Fig 1), and replacing one or more of the inner alginate fluids with ECM hydrogel precursor solutions (p 354, col 2, para 1; Fig 2a). The resulting microparticles comprise an outer layer of alginate hydrogel and an inner core (reads on cavity) encapsulating cells and an ECM substitute, such as collagen or MatrigelTM (p 354, col 2, para 1; Fig 2b-c). After encapsulation, the microparticles comprising cells are cultured in appropriate growth medium (p 359, col 1, para 1). Lu teaches culturing small intestinal crypts containing Lgr5+ intestine stem cells (mammalian progenitor cells in claim 27) in the microparticles, wherein the crypts grew into structured organoids with an enriched Lgr5+ stem cell population (p 354, col 2 – p 355, col 1, para 1; Fig 2f) (claims 24-26). Lu further teaches culturing two different breast epithelial cells, MCF-10A (non-malignant) and MDA-MB-231(invasive), in the microparticles (p 355, col 1, para 2 – col 2, para 1; Fig 3), where the MDA-MB-231 cells enclose the MCF-10A cells to form a boundary around them (form a cyst-claim 24); culturing of two cell types reads on claim 32. Lu also taught culturing of human umbilical vein endothelial cells in microparticles (claims 31 and 33). See page 356, column 2, last paragraph.
Fig 3 shows MCF-10A and MDA-MB-231 cells in the alginate/MatrigelTM double layer microparticles. Lu teaches that the cells grow in the inner layer comprising MatrigelTM and stop growing, or grow at a much slower rate, after filling the inner layer and reaching the alginate/MatrigelTM interface (p 355, col 2, para 1; Fig 3c). This suggests that the size of microtissues cultured in the microparticle serves as an approximate proxy for the size of the MatrigelTM layer. Fig 3(a3) and Fig 3(b3) show the microcompartments comprising MCF-10A cells (a3) and MDA-MB-231 (b3) cells before recovery, and both panels appear to show microcompartments with thickness of the outer alginate layer representing 5 to 40% of the radius of said microcompartment, and thickness of the microtissues (proxy for the MatrigelTM layer, which reads on extracellular matrix substitute layer) representing 5 to 80% of the radius of said compartment (claims 18-20). Panels (a1) and (b1) in Figure 3 show MCF-10A cells (a1) and MDA-MB-231 cells (b1) in MatrigelTM in the microparticles on day 0. Both Fig 3(a1) and Fig 3(b1) appear to show compartments with a cell density of less than 10% occupancy of the internal volume of the compartments (cell density on day 0 reads on initial cell density). Fig 3(a3) and Fig 3(b3) show the microcompartments comprising MCF-10A cells (a3) and MDA-MB-231 (b3) cells before recovery, and both panels show between 10% and 98% by volume of cells (claim 30). Lu teaches recovering microtissues grown in the microparticles by using an EDTA solution to dissolve the alginate outer layer (Fig 3(a4), (b4)).
Lu further teaches that the microparticles can be suspended in stirred bioreactors, which Hernandez shows is a closed system (p 15, ln 17-19), for large-scale cell culture and expansion (p 354, col 2, para 1). At page 358, column 2, in the last three lines of the first paragraph, Lu teaches that the average diameter of the spherical microparticles is 420 [Symbol font/0x6D]m, thus meeting the limitations of claim 21. Ovoid is interpreted to read on spherical and thus Lu would also meet the limitations of claim 22.
Regarding the wherein clause in claim 17, “wherein a ratio of convective volume of the culture medium circulating outside the microcompartments in the closed chamber to a diffusive volume of the culture medium diffusing inside the microcompartments is between 1 and 10,000”, applicant is reminded that claim scope is not limited by language that suggests or makes optional but does not require steps to be performed, or by claim language that does not limit a claim to a particular structure. See MPEP 2111.04 (I). In this case there is no recited structural feature of the claimed bioreactor cell culture system that relates to the ratio of convective volume to diffusive volume being between 1 and 10,000. The claim merely requires a closed chamber containing microcompartments (comprising hydrogels and cells) suspended in a culture medium. In this case the wherein clause is interpreted to be optional and/or an intended use, with no patentable weight.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Lu by introducing the microparticles into stirred bioreactors. One of ordinary skill in the art would have been motivated to make this modification because Lu teaches that the use of a bioreactor facilitates large-scale cell culture and expansion. One of ordinary skill in the art would have had a reasonable expectation of making this modification because Lu teaches that the microparticles taught therein can be suspended in stirred bioreactors, which Hernandez shows is a closed system (page 15, lines 17-19).
Therefore, the invention as a whole as prima facie obvious before the effective filing date of the invention.
Response to Arguments
As stated above applicant’s arguments directed to the passages of Lu cited as teaching a ratio of between 1 and 10,000 of convective volume to diffusive volume were found persuasive. Applicant also argued that Hernandez only provided a general statement with respect to bioreactors and the preferred embodiment of Hernandez is a conventional cell culture plate.
In response, applicant is directed to the new grounds of rejection above addressing the ratio. Applicant’s arguments with respect to the bioreactor of Hernandez are not persuasive. Lu in fact suggested that a bioreactor could be used and Hernandez provided an evidentiary statement that a stirred bioreactor is in fact a closed system. The suggestion to use a bioreactor was made by Lu.
Claim(s) 17-22, 24-33 and 42 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lu as evidenced by Hernandez taken with Dang et al (of record) and Galliher (of record).
The teachings of Lu as evidenced by Hernandez are relied on above with respect to claims 17-22, 24-27 and 30-33.
The wherein clause in claim 17, “wherein a ratio of convective volume of the culture medium circulating outside the microcompartments in the closed chamber to a diffusive volume of the culture medium diffusing inside the microcompartments is between 1 and 10,000”, is not taught by Lu.
However, at the before the effective filing date of the claimed invention bioreactors having a ratio of convective volume of the culture medium circulating outside the microcompartments in the closed chamber to a diffusive volume of the culture medium diffusing inside the microcompartments is between 1 and 10,000 were known as taught by Dang et al.
In particular, Dang teaches that human ES cells (mammalian pluripotent stem cells-claims 26 and 27) at 2 x 106 cells/ml were encapsulated in agarose hydrogel capsules. See, encapsulation process at page 276, at the second column, last paragraph bridging to page 277. Dang further teaches inoculating a bioreactor, containing 200 ml of ES cell media with 5 x 105 ES cells (12,500 ES cell-containing capsules). See Bioreactor Culture at page 277, first column at the top. This equals about 40 ES cells/capsule. Given the concentration of cells, at 2 x 106 cells/ml during the encapsulation process, the collective volume of the microcapsules (12,500) would be 0.25 ml. Therefore, a ratio of convective volume (200ml) to diffusive volume (0.25ml) of the microcapsules is 800, which falls within the range of 1 to 10,000 as recited in the claim.
The teachings of Lu et al as evidenced by Hernandez also do not teach: culturing of human pluripotent stem cells in microcarriers in a bioreactor; that the bioreactor is selected from a batch mode, fed-batch or continuous mode bioreactor; and that the bioreactor has a volume of between 1 ml and 10,000 L.
However, these deficiencies are remedied by Dang and Galliher. Regarding claim 27 as discussed above, Dang teaches encapsulation of human ES cells in agarose capsules which are then placed in stirred-suspension bioreactors for embryoid body formation (see abstract). Dang teaches that culturing encapsulated human ES cells in stirred suspension bioreactors can be used to produce scalable quantities of ES-derived cells in a controlled environment (abstract, page 276, left col, paragraph 2). Regarding claims 28 and 29, Dang teaches using 400 ml vessels in batch culture mode and further teaches that stirred-suspension bioreactors are readily scaled in size to match production needs (page 277, left column, paragraph 2). At page 281, second column Dang suggests that cell encapsulation may prove useful by providing a scaffold onto which cytokines or extracellular matrix proteins can be attached. Delivery of bioactive molecules in this highly localized manner may allow for the provision of developmentally relevant gradients of molecules in scalable culture and will certainly provide a cost-effective alternative to maintaining the entire bulk media at a specific concentration. Ultimately, encapsulated stirred culture may provide the leverage to control most exogenous factors that affect ES cell growth and differentiation including cell-cell interactions, physicochemical factors, and cytokine delivery.
Regarding claim 42, Galliher teaches continuous perfusion bioreactors [0002]. In some cases, as taught by Galliher, increased efficiency may be achieved using continuous bioprocessing as compared to batch or fed-batch operations [0004]. Galliher teaches that a variety of components, suspended or contained in a liquid can be contained in systems and apparatuses of the invention, such as polymer spheres, solid spheres, gelatinous particles, 3D carriers such as semi-permeable membranes comprising porous fibers and other solid objects that can be used for culturing cells [0062-0063].
Therefore, before the effective filing date of the invention, it would have been obvious to one of ordinary skill in the art to substitute the microcapsules of Dang with the microparticles of Lu, which contain an ECM component as well as to use a batch culture bioreactor as taught by Dang or a continuous mode bioreactor as taught by Galliher in the culturing method of Lu as evidenced by Hernandez. One of ordinary skill in the art would have been motivated to do this because of the scale up and efficiency potentials as discussed by Dang and Galliher above, and particularly with respect to the culturing and differentiation of ES cells as suggested by Dang. One of ordinary skill would have had a reasonable expectation of success since modifying the methods of Lu as evidenced by Hernandez and Dang would require substituting one bioreactor for another and one microparticle for another. The methods of Lu evidenced by Hernandez and Dang were both successful in culturing cells, are optimizable and yielded predictable results. Thus, combining/modifying these references satisfies KSR rationales (A) Combining prior art elements according to known methods to yield predictable results; and (B) Simple substitution of one known element for another to obtain predictable results. See MPEP 2141 (III).
In addition, regarding the wherein clause in claim 17, “wherein a ratio of convective volume of the culture medium circulating outside the microcompartments in the closed chamber to a diffusive volume of the culture medium diffusing inside the microcompartments is between 1 and 10,000”, applicant is reminded that claim scope is not limited by language that suggests or makes optional but does not require steps to be performed, or by claim language that does not limit a claim to a particular structure. See MPEP 2111.04 (I). In this case there is no recited structural feature of the claimed bioreactor cell culture system that relates to the ratio of convective volume to diffusive volume being between 1 and 10,000. The claim merely requires a closed chamber containing microcompartments (comprising hydrogels and cells) suspended in a culture medium. The wherein clause is interpreted to be optional and/or an intended use, with no patentable weight.
Therefore, the invention as a whole as prima facie obvious before the effective filing date of the invention.
Response to Arguments
Applicant’s arguments with respect to a ratio of between 1 and 10,000 of convective volume to diffusive volume not taught by the cited references are reiterated. Applicant further argues that claimed bioreactor cell culture system is an improvement over the art of mass bioproduction methods which had little or no applicability to fragile cells. Finally, applicant argues that the claimed bioreactor system is an unexpected result over the cited art as combined.
In response, applicant’s arguments have been considered but are not found persuasive. Two new grounds of rejection addressing the ratio have been presented above.
Next, regarding the argument that the claimed bioreactor cell culture system is an improvement over the art, it is unclear where the improvement lies since the recited ratio does not correlate to any particular structural limitation recited in the claims. Independent claim 17 merely requires, a bioreactor comprising a closed chamber and a plurality of microcompartments suspended in culture medium. These components are known or suggested in the art of record. See the rejections above. Regarding the recited ratio, claim scope is not limited by language that suggests or makes optional but does not require steps to be performed, or by claim language that does not limit a claim to a particular structure. See MPEP 2111.04 (I). In this case there is no recited structural feature of the claimed bioreactor cell culture system that relates to the ratio of convective volume to diffusive volume being between 1 and 10,000. The wherein clause is interpreted to be optional and/or an intended use, with no patentable weight. Further even if the recited ratio was given patentable weight, it was suggested by Dang. See the rejection above.
Finally, regarding the argument that the claimed bioreactor system is an unexpected result, it is not understood what exactly the unexpected result is. The structural limitations of the claimed bioreactor system are known in the art as discussed above. The recited ratio does not appear to be associated with any structural limitation recited in the claims and is not given patentable weight. Even if the ratio were given patentable weight, it was suggested by Dang as discussed above. Applicant is encouraged to submit evidence of unexpected results in the form of an affidavit. Further, the claims under examination are to a bioreactor cell culture system and not methods of culturing cells. Any evidence of unexpected results presented must be commensurate in scope with the claimed bioreactor system.
Conclusion
No claim is allowed.
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/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632
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