DETAILED ACTION
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 to Election/Restriction Filed, Amendment, and Arguments/Remarks, filed 27 March 2026, have been entered. Applicant added new claim 26. Claims 1-14, 18, and 21-26 are currently pending. Claims 1, 13, and 18 are independent claims. Applicant’s election with traverse of the invention of Group I, drawn to a method for preparing stem cells for transplantation into a patient in need thereof and a method for preparing stem cells for transfusion, is acknowledged.
Applicant argues that Chapman does not teach, suggest, or otherwise provide for all the elements of independent claims 1 and 13, and so Applicant’s assertion that the claims do not make a contribution over the prior art in view of Chapman is unfounded. However, this is not agreed.
Note that the technical feature shared between Groups I and II is the technical feature of hypoxic stored stem cells and not every limitation recited in independent claims 1 and 13. Chapman was cited for teaching storage of stem cells under low oxygen concentration conditions [abstract, 0003, 0006, 0013]. Therefore, the technical feature of hypoxic stored stem cells is not a special technical feature and a unity of invention does not exist between Group I and Group II. Therefore, the restriction requirement is still deemed proper and is therefore made FINAL.
Claims 18 and 21 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 1-14 and 22-26 are currently pending in the application and under examination to which the following grounds of rejection are applicable. An action on the merits follows.
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/US2022/019140, filed 07 March 2022, which claims priority to U.S. Provisional Application No. 63/158,267, filed 08 March 2021.
Thus, the earliest possible priority for the instant application is 08 March 2021.
Information Disclosure Statement
The information disclosure statements filed 19 December 2023 and 22 April 2026 have been considered by the Examiner.
Examiner notes the filing of IDS Size Fee assertions for the IDS filed 22 April 2026, as required under 37 CFR 1.98, indicating that no IDS size fee is required under 37 CFR 1.17(v) at this time.
Specification
The use of the terms “Plamsa-Lyte” in [0078], “HypoThermosol” in [0078], “trolox” in [0078, 00160], which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Note that the specification has not been inspected sufficiently to identify all instances of trade names and/or marks used in commerce. It is Applicant’s responsibility to ensure complete compliance.
Claim Rejections - 35 USC § 112(b)
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 1-12 and 22 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. Dependent claims 3, 5, 9-10, and 12 are included in this rejection due to their dependence on independent claims 1 and/or 2.
Independent claim 1 has multiple issues of indefiniteness.
Claim 1 recites, “initial partial pressure”, which is indefinite because it is unclear what is considered the initial timepoint or condition. For example, it is unclear whether the initial time point/ condition of the blood product is the state of the blood in the subject prior to being drawn from the subject (e.g., arterial blood or venous blood) or at some point following collection from the subject, such as the blood product prior to, at the point of, or following collection into an oxygen absorbing environment comprising a hypoxic collection container.
Claim 1 additionally recites, “the oxygen reduced blood product” in lines 10 and 11. There is insufficient antecedent basis for this limitation in the claim. Claim 1 has no prior recitation of any “oxygen reduced blood product”.
Claim 1 recites “the stem cell” in lines 11-12, 14, and 15. There is insufficient antecedent basis for this limitation in the claim. Claim 1 has prior recitations of “stem cells” in lines 1 and 3, “leukocytes and stem cells” in line 10 and 12. Therefore, it is unclear which stem cells “the stem cells” of lines 11-12, 14, and 15 are referring.
Claim 1 also recite “the cells” in line 17. There is insufficient antecedent basis for this limitation in the claim. Claim 1 has multiple recitations of cells including “stem cells” in lines 1 and 3, “leukocytes and stem cells” in line 10 and 12, “stem cells and leukocytes” in lines 10-11, and “the stem cells” in lines 14 and 15. Therefore, it is unclear which cells “the cells” of line 17 is referring.
The term “isotonic” in claim 1 line 14 is a relative term which renders the claim indefinite. The term “isotonic” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Therefore, it is unclear whether the isotonic media is meant to be isotonic with respect to blood, an environment internal to a cell, or some other solution or reference.
As such, the metes and bounds of the claim cannot be determined.
Claims 2 and 8 recite “the stem cells” in lines 1 and 2 of claim 2 and line 2 of claim 8. Claim 1 has prior recitations of “stem cells” in lines 1 and 3, “leukocytes and stem cells” in line 10 and 12, and “the stem cell” in lines 11-12, 14, and 15. Therefore, it is unclear which stem cells “the stem cells” of claim 2 are referring.
As such, the metes and bounds of the claim cannot be determined.
Claim 4 recites, “under a pO2”, which is indefinite because it is unclear in what way the reducing, separating, eluting, and transferring are performed “under” the specified range of pO2. For example, it is unclear whether the pO2 recited is meant to be the pO2 of the blood product/cells in solution, the pO2 of the air surrounding the blood product/cells solution within the containers comprising the blood product/cells, the pO2 of the environment in which the procedures are being performed outside of the containers comprising the blood product/cells, the headspace pO2 of the container comprising the blood product/cells, and/or any other solution/air/environment involved in the recited method steps.
Additionally, the term “under” is a relative term which renders the claim indefinite. The term “under” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
As such, the metes and bounds of the claim cannot be determined.
Claim 6 recites, “wherein the mixing is until the initial pO2 of the blood product is reduced by at least 50%”, which conflicts with the limitations of independent claim 1, upon which it depends, because claim 1 recites, “mixing the blood product until the initial partial pressure of oxygen (pO2) of the blood product is reduced by between 80 and 95%”.
As such, the metes and bounds of the claim cannot be determined.
Claim 7 recites “wherein the isotonic media is deoxygenated isotonic media comprising a pO2 of at least 5333 Pascals”, which is indefinite because “a pO2 of at least 5333 Pascals” encompasses media which is not deoxygenated as well as media which has increased oxygen levels compared to media which is equilibrated with ambient air.
Additionally, the term “deoxygenated” is a relative term which renders the claim indefinite. The term “deoxygenated” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The claim does not indicate what the isotonic media needs to be deoxygenated relative to, such as relative to isotonic media equilibrated with ambient air.
As such, the metes and bounds of the claim cannot be determined.
Claim 11 recites, “the inner collapsible blood container” in line 4. There is insufficient antecedent basis for this limitation in the claim. Claim 11 has a prior recitation of “a collapsible inner blood container” but no prior recitation of any “inner collapsible blood container”.
Claim 11 also recites “hypoxic storage container” in line 7, which is indefinite because it is unclear whether the “hypoxic storage container” in line 7 is meant to be the same hypoxic storage container recited in claim 11 line 1 and claim 1 line 15.
As such, the metes and bounds of the claim cannot be determined.
Claim 22 recites, “selected from” in lines 1-2, which is indefinite because it is unclear whether the list recited is an open or closed list.
As such, the metes and bounds of the claim cannot be determined.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1-14 and 22-26 are rejected under 35 U.S.C. 103 as being unpatentable over Yoshida [US20170202740A1, published 20 July 2017]; in view of Bitensky [US6162396A, published 19 December 2000]; Collins et al. [2015, Breathe, 11(3), 194-201]; Wolf [US20170181426A1, published 29 June 2017] Peytour et al. [2010, Transfusion, 50, 2151-2157]; Yu et al. [2013, Translational Stroke Research, 4, 76-88]; Dos Santos et al. [2010, Journal of Cellular Physiology, 223, 27-35]; and Peytour et al. [2013, Stem Cell Research, 11, 736-742].
Regarding claim 1, Yoshida teaches a method for improved preservation of blood components for transfusion into a patient in need thereof comprising collecting a blood product containing stem cells (e.g., whole blood) into an oxygen absorbing environment comprising a hypoxic collection container comprising an oxygen (O2) barrier characterized by an O2 permeability of less than 0.5 cc of oxygen per square meter per day and an oxygen sorbent [0002, 0008-0009, 0013-0014, 0016-0017, 0042-0043, 0057, 0060, 0168]; reducing the oxygen level in the blood product until the initial saturation (SO2) of oxygen (pO2) of the blood product is reduced by at least 75% (i.e., from about 20% SO2 to less than 5%) [0090, 0106-0107, 0208, 0216, Figure 14].
Yoshida also teaches leukocyte filters and leukocyte depleted blood products [0043, 0045, 0073, 201].
Yoshida further teaches transferring blood cells into a hypoxic storage container comprising an O2 barrier characterized by an O2 permeability of less than 0.5 cc of oxygen per square meter per day and storing the cells to prepare hypoxic stored blood cells [0016-0017, 0057, 0060].
Yoshida does not teach wherein the initial partial pressure of oxygen (pO2) of the blood product is reduced by between 80 and 95%.
However, Bitensky teaches that to removal of oxygen from stored blood cells is preferably reduction of oxygen content to levels below about 15% of full saturation levels, more preferably to levels no greater than about 5-10%, wherein such reduction in the oxygen content of the red blood cells can achieve about 75% survival rate of the red blood cells at 24 hours post-transfusion when the cells have been stored for periods greater than 5 weeks or for 10-15 weeks [column 2 line 10-12, 33-43, column 4 lines 11-26]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to reduce the initial oxygen saturation of the blood product to no greater than about 5-10%.
Further, Collins teaches wherein the initial oxygen saturation of venous blood is 75% [page 196 column 1 ¶ 4], which corresponds to an initial pO2 of collected venous blood of about 40 mmHg (about 5333 kPa) [Figure 2, 3, 4]. Collins also teaches that 5-10% oxygen saturation is equivalent to about 5-10 mmHg (about 0.6-1.3 kPa) [Figure 2]. As such, a reduction to about 5-10% oxygen saturation is a reduction to about 5-10 mmHg, which is a reduction of about 30-35 mmHg from the initial pO2 of the collected venous blood product, such that the initial pO2 of the blood product is reduced by between about 75% and 88%, which overlaps with the claimed reduction of between 80% and 95%.
Yoshida additionally does not teach wherein the oxygen level is reduced by mixing.
However, Wolf teaches that an advantage of agitation and mixing of an oxygen depletion device is that the movement of the blood or blood component caused by the agitator also moves the sorbent sachet such that the active ingredient that absorbs the oxygen in the headspace is constantly settling, which moves the oxidized iron particles out of the way of non-oxidized iron particles, speeding up the oxygen absorption potential of the sorbent [0251]. Wolf also teaches that there is a need to begin the reduction of oxygen from blood as early as possible, preferably at collection before the temperature of the collected blood has been significantly reduced [0012]. Therefore, and ordinarily skilled artisan at the time of filing the instant application would have been motivated to reduce the oxygen level in a collected blood product by mixing to speed up the oxygen absorption process.
Yoshida also does not teach separating leukocytes and stem cells from the oxygen reduced blood product comprising applying the oxygen reduced blood product to a filter wherein the stem cells and leukocytes are retained on the filter to prepare a leukocyte and stem cells depleted blood product; and eluting the stem cells from the filter with an isotonic media.
Peytour (2010) teaches obtaining CD34+ stem cells from healthy blood donors for regenerative medicine using leukoreduction filters (LRFs) [title, abstract, column 2 ¶ 1], wherein blood product is applied to a filter such that the stem cells and leukocytes are retained on the filter to prepare a leukocyte and stem cells depleted blood product, after which the stem cells are eluted from the filter with an isotonic media (i.e., phosphate-buffered saline supplemented with ACD-A and human serum) [abstract, column 2 ¶ 1, column 3 ¶ 2]. Peytour (2010) further teaches that LRFs are a convenient and abundant source of CD34+ cells since they contain a high number of white blood cells (WBCs) and are discarded after the preparation of red blood cells (RBCs) [column 10 ¶ 3]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to separate leukocytes and stem cells from a blood product by applying the blood product to a leukocyte filter, wherein the stem cells and leukocytes are retained on the filter, and then to elute the stem cells from the filter with an isotonic media to recover the stem cells for regenerative medicine.
Yoshida teaches that among the benefits of storing blood under oxygen depleted conditions are improved levels of ATP and 2,3-DPG; and that storing blood under oxygen depleted conditions can also result in reduced microparticle levels, reductions in the loss of deformability, reduced lipid and protein oxidation and higher post transfusion survival when compared to blood stored under conventional conditions [0006]. Bitensky teaches that removing oxygen from the stored blood prolongs the storage life of the deoxygenated blood [abstract].
However, Yoshida and Bitensky do not teach specifically transferring the blood-product derived stem cells into the hypoxic storage container to prepare hypoxic stored stem cells.
Yu teaches that a sublethal hypoxic exposure significantly increases the tolerance and regenerative properties of stem cells and progenitor cells, such that preconditioned stem cells and progenitors generally show much better cell survival, increased neuronal differentiation, enhanced paracrine effects leading to increased trophic support, and improved homing to the lesion site [abstract, Table 1, 2]. Yu also teaches that transplantation of preconditioned cells helps to suppress inflammatory factors and immune responses, and promote functional recovery [abstract]. Yu further teaches that accumulating information from reports over the years indicates that hypoxic preconditioning is an attractive, if not essential, prerequisite for transplanted cell [abstract]. Therefore, an ordinarily skilled artisan would have been motived to expose stem cells to hypoxic preconditioning prior to transplantation for improved cellular survival.
Given the motivation taught by Bitensky to reduce the initial oxygen saturation of the blood product to no greater than about 5-10%; the motivation taught by Wolf to reduce the oxygen level in a collected blood product by mixing to speed up the oxygen absorption process; the motivation taught by Peytour (2010) to separate leukocytes and stem cells from a blood product by applying the blood product to a leukocyte filter, wherein the stem cells and leukocytes are retained on the filter, and then to elute the stem cells from the filter with an isotonic media to recover the stem cells for regenerative medicine; and the motivation taught by Yu to expose stem cells to hypoxic preconditioning prior to transplantation for improved cellular survival; it would have been prima facie obvious to an ordinarily skilled artisan at the time of filing the instant application to modify the method of Yoshida to reduce the pO2 level in the blood product by between 80 and 95% by mixing, to subsequently to recover stem cells from the leukocyte filter used to prepare a leukocyte and stem cell reduced red blood cell product by eluting the stem cells from the filter with an isotonic media, and to transfer the stem cells into a hypoxic storage container as taught by Yoshida with a reasonable expectation of success.
Regarding claim 2, Peytour (2010) teaches that CD43+ cells obtained with this convenient, rapid, and efficient procedure have satisfying functional properties as evidenced in part by their ex vivo expansion [column 10 ¶ 3], indicating that ex vivo expansion of stem cells is a desirable functional property.
Dos Santos teaches that human mesenchymal stem cells (MSCs) have become one of the most promising candidates for tissue engineering and regenerative medicine, and that an efficient and Good Manufacturing Practices-compliant ex vivo expansion process is required to achieve MSC clinically relevant numbers [column 1 ¶ 1-2]. Dos Santos additionally teaches a more efficient BM MSC expansion at 2% O2 compared to normoxic conditions is associated to an earlier start of cellular division as supported by an increase in cellular metabolism efficiency towards the maximization of cell yield for application in clinical settings [abstract]. Dos Santos further teaches that the potential reduction of culture time associated with the differences observed in cell proliferation might be relevant to accelerate significantly the clinical cell expansion process [column 13 ¶ 1]. Dos Santos also teaches transferring the stem cells to a hypoxic stem cell expansion container (i.e., 12-well plates under hypoxia) to expand the stem cells [column 3 ¶ 3, column 14 ¶ 3]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to expand stem cells in a hypoxic stem cell expansion container to form an expanded stem cell population to increase cell numbers in a shorter amount of time prior to transplantation of cells in a clinical setting.
Regarding claims 3 and 8, Peytour (2013) teaches expanding CD34+ stem cells prior to and/or after banking of the cells for hematopoietic transplantation [Figure 2]. Therefore, expanding the stem cell prior to transferring them to the hypoxic storage container represents one of a limited number of two options taught by Peytour (2013). Accordingly, the other alternative of the two options taught by Peytour (2013) comprises banking the CD34+ stem cells right after elution. As such, it would have been obvious to an ordinarily skilled artisan to select either option for stem cell expansion, including transferring the eluted stem cells to an expansion container or eluting into a storage container.
Regarding claim 4, Yoshida, Bitensky, Collins, Wolf, Peytour (2010), and Yu teach the limitations of claim 1. As described above, Bitensky teaches that to removal of oxygen from stored blood cells is preferably reduction of oxygen content to levels below about 15% of full saturation levels, more preferably to levels no greater than about 5-10%. Further, Collins also teaches that 5-10% oxygen saturation is equivalent to about 5-10 mmHg (about 0.6-1.3 kPa) [Figure 2], which falls within the claimed range for 400 and 2000 Pascals. Additionally, as discussed above, Dos Santos teaches expanding stem cells at 2% O2 conditions, which corresponds to a pO2 in the hypoxic environment of about 2000 Pascals. Dos Santos also teaches that the bone marrow microenvironment has oxygen tension which varies between 1% and 6% [column 12 ¶ 4], which corresponds to a range of about 1-6 kPa and overlaps with the claimed range of between 400 and 2000 Pascals. Therefore, Yoshida, Bitensky, Collins, Wolf, Peytour (2010), and Yu teach to perform the method steps of claim 1 under pO2 between 400 and 2000 Pascals and/or it would have been obvious to an ordinarily skilled artisan to perform the method steps of claim 1 as taught by Yoshida, Bitensky, Collins, Wolf, Peytour (2010), and Yu under pO2 between 400 and 2000 Pascals given the teachings of Dos Santos to expose stem cells to 2% O2 and that the bone marrow microenvironment, which is the natural reservoir for BM stem cells, has oxygen levels which vary between 1-6%.
Regarding claims 5 and 6, Wolf teaches agitating/mixing the oxygen depletion device for up to 3 hours or between 1 and 3 hours to produce oxygen-reduced blood having less than 20% oxygen saturation [0020, 0238], which Collins teaches corresponds to a pO2 of about 18 mmHg (2400 Pa) [Figure 2]. Therefore, a reduction to less than 20% oxygen saturation (pO2 of about 2400 Pa) is at least a 50% reduction in pO2 from an initial oxygen saturation of about 75% (pO2 of about 5333 Pa) for deoxygenated venous blood.
Regarding claim 7, note that “at least 5333 Pascals” encompasses an isotonic media which comprises oxygen levels up to full saturation with oxygen. As discussed above, Collins teaches wherein the initial oxygen saturation of venous blood is about 75% in healthy individuals at rest [page 196 column 1 ¶ 4], which corresponds to an initial pO2 of collected venous blood of about 40 mmHg (about 5.333 kPa) [Figure 2, 3, 4]. Collins also teaches that the oxygen saturation of arterial blood is between 96% and 98% [page 196 column 1 ¶ 3], which corresponds to a pO2 of about 85 mmHg (about 11.3 kPa) [Figure 2, 3, 4]. Therefore, an ordinarily skilled artisan would be motivated to process blood cells in a media that comprises a reduced/deoxygenated pO2 level comparable to the oxygen level in arterial (about 11.3 kPa) or venous (about 5.333 kPa) blood of healthy individuals to mimic the natural environment of the peripheral blood cells, and in so doing would use a deoxygenated isotonic media comprising a pO2 of at least 53333 Pa.
Regarding claim 9, Peytour (2010) teaches that two sequential 50 mL elutions (100 mL total) resulted in a higher yield of CD34+ cells compared to two sequential 20 mL elutions or a single 500 ml continuous elution [Table 1]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to elute the stem cells from the leukocyte filter using 50 or 100 ml of the isotonic media.
Regarding claim 10, Peytour (2010) teaches elution with phosphate buffered saline-based solutions without indicating that the oxygen content of the solutions has been deliberately altered. As such, the isotonic solution of Peytour (2010) is presumed to be equilibrated with oxygen in the air.
Regarding claim 11, Yoshida teaches wherein the hypoxic storage container comprises an outer receptable substantially impermeable to oxygen, a collapsible inner blood container, an oxygen or oxygen and carbon dioxide sorbent situated between the outer receptacle and the inner collapsible blood container, and at least one inlet/outlet that is substantially impermeable and passing through the outer receptable and that is in hypoxic fluid communication with the collapsible inner blood container, wherein the hypoxic storage container maintains a pO2 of 1 mmHg (133 Pascals) or less, which is less than 1,400 Pascals [0012-0013, 0054, 0058, 0084, 202, Figure 1].
Regarding claim 12 and 22, Yoshida teaches wherein the blood product can be whole blood collected from a blood donor (i.e., peripheral blood) [0043-0044].
Regarding claim 13, as described above, Yoshida, Bitensky, Collins, Wolf, Peytour (2010), and Yu teach the limitations of claim 1, including collecting a blood product comprising stem cells, separating leukocytes and the stem cells from the blood product, transferring the stem cells into a hypoxic storage container comprising an oxygen (O2) barrier characterized by an O2 permeability of less than 0.5 cc of oxygen per square meter per day, and storing the stem cells in the hypoxic storage container to prepare stored stem cells. Additionally, Yoshida teaches storing the cells in the hypoxic storage container at a pO2 of 1 mmHg (133 Pascals) or less, which is less than the claimed less than 3500 Pascals for a period of time at a temperature of between 2 and 6 oC, which is less than 37 oC [0202].
Regarding claim 14, Yoshida teaches wherein the hypoxic storage container comprises an outer receptable substantially impermeable to oxygen, a collapsible inner blood container, an oxygen or oxygen and carbon dioxide sorbent situated between the outer receptacle and the inner collapsible blood container, and at least one inlet/outlet that is substantially impermeable and passing through the outer receptable and that is in hypoxic fluid communication with the collapsible inner blood container, wherein the hypoxic storage container maintains a pO2 of 1 mmHg (133 Pascals) or less, which is less than 1,400 Pascals [0012-0013, 0054, 0058, 0084, 202, Figure 1].
Regarding claim 23, Peytour (2010) teaches wherein the separating leukocytes and stem cells from the blood product comprises centrifugation to prepare concentrated stem cells [abstract, column 4 ¶ 1].
Regarding claim 24, Peytour (2010) teaches removing supernatant from the concentrated stem cells and resuspending the concentrated stem cells in a selection buffer (i.e., a stem cell suspension medium) [column 4 ¶ 1]. As discussed above, Yu teaches that a sublethal hypoxic exposure significantly increases the tolerance and regenerative properties of stem cells and progenitor cells, such that preconditioned stem cells and progenitors generally show much better cell survival, increased neuronal differentiation, enhanced paracrine effects leading to increased trophic support, and improved homing to the lesion site [abstract, Table 1, 2]. Yu also teaches that transplantation of preconditioned cells helps to suppress inflammatory factors and immune responses, and promote functional recovery [abstract]. Yu further teaches that accumulating information from reports over the years indicates that hypoxic preconditioning is an attractive, if not essential, prerequisite for transplanted cell [abstract]. Therefore, an ordinarily skilled artisan would have been motived to expose stem cells to hypoxic preconditioning prior to transplantation for improved cellular survival.
Regarding claim 25, Peytour (2013) teaches expanding CD34+ stem cells prior to and/or after banking/storing of the cells for hematopoietic transplantation, wherein the banking/storing is in liquid nitrogen [Figure 2]. Wolf teaches freezing at -80 oC for long term storage of blood products [0009]. Therefore, an ordinary skilled artisan would have been motivated to store stem cells at -80 oC for long term storage or in liquid nitrogen for bio-banking.
Regarding claim 26, as discussed above, Peytour (2010) teaches that CD43+ cells obtained with this convenient, rapid, and efficient procedure have satisfying functional properties as evidenced in part by their ex vivo expansion [column 10 ¶ 3], indicating that ex vivo expansion of stem cells is a desirable functional property.
Additionally, Dos Santos teaches that human mesenchymal stem cells (MSCs) have become one of the most promising candidates for tissue engineering and regenerative medicine, and that an efficient and Good Manufacturing Practices-compliant ex vivo expansion process is required to achieve MSC clinically relevant numbers [column 1 ¶ 1-2]. Dos Santos additionally teaches a more efficient BM MSC expansion at 2% O2 compared to normoxic conditions is associated to an earlier start of cellular division as supported by an increase in cellular metabolism efficiency towards the maximization of cell yield for application in clinical settings [abstract]. Dos Santos further teaches that the potential reduction of culture time associated with the differences observed in cell proliferation might be relevant to accelerate significantly the clinical cell expansion process [column 13 ¶ 1]. Dos Santos also teaches transferring the stem cells to a hypoxic stem cell expansion container (i.e., 12-well plates under hypoxia) to expand the stem cells [column 3 ¶ 3, column 14 ¶ 3]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to expand stem cells in a hypoxic stem cell expansion container to form an expanded stem cell population to increase cell numbers in a shorter amount of time prior to transplantation of cells in a clinical setting.
Given the motivation taught by Dos Santos to expand stem cells in a hypoxic stem cell expansion container to form an expanded stem cell population to increase cell numbers in a shorter amount of time prior to transplantation of cells in a clinical setting; the teachings of Peytour (2013) to expand CD34+ stem cells prior to and/or after banking of the cells for hematopoietic transplantation; the teachings of Dos Santos to expose stem cells to 2% O2 and that the bone marrow microenvironment, which is the natural reservoir for BM-derived stem cells, has oxygen levels which vary between 1-6%; the teachings of Wolf to agitate/mix the oxygen depletion device for up to 3 hours or between 1 and 3 hours; the teachings of Collins that the initial oxygen saturation of venous blood in healthy individuals at rest corresponds to a pO2 of venous blood of about 5,333 Pa and that the oxygen saturation of arterial blood corresponds to a pO2 of about 11.3 kPa; the motivation taught by Peytour (2010) to elute the stem cells from the leukocyte filter using 50 or 100 ml of the isotonic media followed by centrifugation and resuspension in a media; the motivation taught by Yu to expose stem cells to hypoxic preconditioning prior to transplantation for improved cellular survival; the motivation taught by Peytour (2013) and Wolf to store stem cells at -80 oC for long term storage or in liquid nitrogen for bio-banking; it would have been prima facie obvious to an ordinarily skilled artisan at the time of filing the instant application to further modify the method of Yoshida to expand the stem cells prior to or following storage to form an expanded stem cell population; to perform the separating, eluting, and transferring steps under a pO2 of between 0.6-2 kPa; to reduce the oxygen level in the blood by mixing for up to 3 hours until the initial pO2 is reduced by at least 50%; to elute the leukocytes and stem cells from the leukocyte filter using 50-200 mL of a deoxygenated isotonic media comprising a pO2 of at least 5333 Pa or isotonic media equilibrated with oxygen in the air; centrifuging the eluted stem cells, removing the resulting supernatant, and resuspending the stem cells in a hypoxic stem cell suspension media; and storing the stem cells at -80 oC or in liquid nitrogen with a reasonable expectation of success.
Conclusion
No claim is allowed.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634
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