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 .
The response on 05/04/2026 has been received and entered. Claims 1, 2, 4, 6-18, and 21-22 remain pending and have been considered on the merits.
Status of Prior Rejections/Response to Arguments
Applicants have presented arguments addressing the invention, in general, in comparison to the collective prior arts. Attempts have been made to correlate the relevant parts of Applicants’ arguments to each individual rejection, and those arguments have been addressed.
Applicants argue the cited references do not teach or suggest determining vitrification CPA cocktail by quantitatively analyzing the specific biophysical parameters. However, the claims recite this step with a high le vel of generality and the step of quantitatively analyzing could be a mental process. As claimed, the step of “quantitatively analyzing” could comprise a mental analysis of the biophysical parameters by a person of ordinary skill in the art who could then use the information to determine the optimal CPA composition based on the analysis. The claims do not specifically require quantitatively measuring the specific parameters nor do they require “computationally determining CPA composition and concentration based on the recited mass transport and toxicity” (argued at pg. 13, last paragraph).
RE: Rejection of claims 1-2 and 4-14 under 35 U.S.C. 112(b)
Amendments to the claims overcome the rejection of record. The rejection is withdrawn.
RE: Rejection of claim 18 under 35 U.S.C. 102 over Nakayama-Iwatsuki et al (Islets, 2020)
Applicants amended claim 18 to require transplanting at least about 100,000 VR islets with greater than about 95% recovery. Nakayama-Iwatsuki et al does not anticipate the claim as amended. The rejection is withdrawn.
RE: Rejection of claims 1, 2, 4- 7, 11 - 14, 18, and 19 under 35 U.S.C. 103 over Nakayama-Iwatsuki et al (Islet, 2020) in view of Best (Rejuvenation Res, 2015)
Applicants traverse the rejection of record on the grounds that Best discuses general cryoprotectant behavior and cryobiology but does not disclose a method where the CPA identity or concentration is determined by analyzing biophysical parameters.
In response the argument has been fully considered but is not persuasive. Best teaches CPA toxicity arises from conditions such as temperature, CPA concentration, CPA exposure time, and CPA carrier solutions (See Introduction). Thus, it would have been obvious to a person of ordinary skill in the art to determine the identity and/ or concentration of CPAs used by analyzing the temperature and exposure time to CPAs to minimize toxicity.
Applicants further traverse the rejection of record on the grounds that the method of Nakayama-Iwatsuki et al is applied to 100 islets per NM device and thus it is not feasible to achieve VR of islets required for clinical transplant using the method of Nakayama-Iwatsuki et al. Additionally, applicants argue scaling up in volume and islet quantities can lead to reduced cooling and warming rates which impact viability and success.
In response, the argument has been fully considered but is not persuasive. Nakayama-Iwatsuki et al teach recovery of 5715 islets and thus teach scaling up of the method is successful. Mere scaling up of a prior art process capable of being scaled up, if such were the case, would not establish patentability in a claim to an old process so scaled (See 531 F.2d at 1053, 189 USPQ at 148 and MPEP2144.04(IV)(A)).
The rejection over claims 1, 2, 4, 6, 7, 11-14 and 18 is updated and maintained.
Claims 5 and 19 have been cancelled rendering their rejection moot.
RE: Rejection of claims 1, 2, 4-7, 11, 12, and 15-17 under 35 U.S.C. 103 over Maehara et al (BMC Biotechnology, 2013) in view of Best (Rejuvenation Res, 2015).
Applicants traverse the rejection of record on the grounds that the method of Maehara et al is designed to work on cell sheets rather than cell clusters. Applicants argue 1) a cell sheet does not fall under the dictionary definition of a cell cluster and 2) cell sheets are not among the examples of cell clusters listed in the specification.
In response, the argument has been fully considered but is not found persuasive. The specification of the instant application does not define a cell cluster. HarperCollins Dictionary defines a “cell cluster” as a number of cells grouped together (See Harper Collins Dictionary definition of “cell cluster”). Furthermore, Efremov et al (Biophysical Reviews, 2021), teaches cell aggregates include cell sheets and spheroids, thus the art recognized cell sheets as cell aggregates (i.e. clusters) (See abstract). Thus while the specification does recite cell sheets as an example of a “cell aggregates or cell cluster”, the specification does not recite a specific definition of a cell cluster which excludes cell sheets or a requirement that a cell cluster be a 3D system. Therefore, the broadest reasonable interpretation of the term “cell cluster” includes a cell sheet.
Applicants further argue that because the cell sheets of Maehara et al are a 1-D system, a person of ordinary skill in the art would have been motivated to use longer loading steps for cell cluster of 100-300 micron diameter. In response, the claims do not require a cell cluster of 100-300 micron diameter; loading length and diameter of cell clusters are not claimed limitations. Only claim 22 requires 3D cell clusters. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Furthermore, a cell sheet is a 2D system, not 1D (See Fig. 1 of Efremov et al (Biophysical Reviews, 2021)).
Applicants further argue Best does not does not provide sufficient disclose to make obvious a method where the CPA identity or concentration is determined by analyzing biophysical parameters.
In response the argument has been fully considered but is not persuasive. Best teaches CPA toxicity arises from conditions such as temperature, CPA concentration, CPA exposure time, and CPA carrier solutions (See Introduction). Thus, it would have been obvious to a person of ordinary skill in the art to determine the identity and/ or concentration of CPAs used by analyzing the temperature and exposure time to CPAs to minimize toxicity.
The rejection over claims 1, 2, 4, 6, 7, 11, 12, and 15-17 is updated and maintained.
Claim 5 has been cancelled rendering its rejection moot.
RE: Rejection of claims 1, 2, 4-8, 11, 12, and 15-17 under 35 U.S.C. 103 over Maehara et al (BMC Biotechnology, 2013) in view of Best (Rejuvenation Res, 2015) and Gao et al (Current Frontiers in Cryobiology, 2011.
Applicants have not provided any arguments specific to this rejection.
The rejection over claims 1, 2, 4, 6-8, 11, 12, and 15-17 is maintained.
Claim 5 has been cancelled rendering its rejection moot.
RE: Rejection of claims 1, 2, 4-7, 9-12, and 15-17 under 35 U.S.C. 103 over Maehara et al (BMC Biotechnology, 2013) in view of Best (Rejuvenation Res, 2015) in view of Khosla et al (Langmuir, 2018).
Applicants traverse the rejection of record on the grounds that it would not have been obvious to substitute the rewarming method of Khosla et al in the method of Maehara et al because the method of Khosla et al results in suboptimal outcomes for islets.
In response, the argument has been fully considered but is not found persuasive. The method of Maehara et al is used to cryopreserve chondrocytes, not islets. Additionally, the claims do not require islets.
The rejection over claims 1, 2, 4, 6, 7, 9-12, and 15-17 is maintained.
Claim 5 has been cancelled rendering its rejection moot.
New/Maintained Rejections
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 2, 4, 6-13, 15-17, 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.
Claims 1 and 15 recite the limitation “wherein the biological material is selected from
the group consisting of pancreatic islets, stem-cell derived islet like cell clusters, and comparable cell cluster.” The terms “islet like cell cluster” and “comparable cell cluster” are indefinite because it is not clear what properties of the cells make cells make the cell clusters “islet like” or “comparable”. Any mammalian cell can be considered comparable to an islet cell in that both cells are mammalian cells and have a cell membrane and nucleus. Additionally, any stem-cell derived cell can be considered comparable to a stem-cell derived islet cell in that both cells were derived from stem-cells. The claim will thus be interpreted as requiring cell clusters of any mammalian cell. Claims 2, 4, 6-13, 15-17, and 22 depend from claims 1 and 15 without further defining the cell clusters and thus inherit the deficiencies of claims 1 and 15.
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.
Claims 18 and 21 are rejected under 35 U.S.C. 103 as being unpatentable over O'Heeron et al (WO2020093047A1).
O’Heeron et al discloses a method of enhancing survival of allogeneic or autologous islet cells and subsequently transplanting the islets to an individual (See claims 1-4). Preparations of islet cells that contain at least 2.2x105 islets are useful as 5000-20000 islet equivalents (IE) can be transplanted/kg recipient body weight (See ¶0074). Preparations in which at least 70% ( e.g. at least 75%, 80%, 85%, 90%, 95%, or 97%) viable cells are particularly useful (See ¶0074). Additionally, O’Heeron teaches islets can be cryopreserved for long-term storage and producing an islet bank (See ¶0072).
Regarding claims 18 and 21: Claims 18 and 21 require the limitation of “VR islets”. VR (i.e. vitrification and rewarming) is a product-by-process limitation. Product-by-process limitations are considered only in so far as the process of production affects the final product. Therefore, if the product as claimed is the same or obvious over a product of the prior art (i.e., is not structurally or chemically distinct), the claim is considered unpatentable over the prior art, even though the prior art product is made by a different process. See MPEP 2113. In the instant case, the process of production involves vitrification and rewarming of islet cells. The methods for producing VR islet cells recited in claims 1 and 15 of the instant application comprise a step of unloading CPAs to eliminate CPAs from the VR islet cells. Thus, the final VR islet cells do not comprise CPAs and are thus indistinct from islet cells. Additionally, the limitation 95% recovery is not an active step.
O’Heeron et al discloses a method of transplanting islets to an individual which reads on a method of therapeutic transplantation of a biological material comprising transplanting the biological material into a patient, wherein the biological material comprises VR islets. The islets of O’Heeron et al are allogeneic or autologous which reads on islets derived from one or more donors and can be cryopreserved. O’Heeron et al teaches 5000-20,000 IE can be transplanted per kg of the recipients body weight. Additionally, O’Heeron et al teaches preparations can have at least 80% viability.
While O’Heeron et al does not specifically disclose administering 100,000 or 300,000 islets, it would have been prima facie obvious to modify the method of O’Heeron et al to administer 100,000 or 300,000 islets based on the recipients weight. One would have been motivated to modify the method of O’Heeron et al based on the recipients weight because O’Heeron et al teaches 5000-20,000 IE can be transplanted per kg of recipients weight. There is a reasonable expectation of success because O’Heeron teaches the number of islets administered depends on the recipients weight.
Claims 18 and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Nakayama-Iwatsuki et al (Islet, 2020).
Nakayama-Iwatsuki et al teaches a method for cryopreserving primary rat islet cells, isolated from a rat pancreas) using a MVC protocol (MVC). The method of Nakayama-Iwatsuki et al compares the use of a nylon mesh vs a silk sponge during the cooling step (See abstract). 100 islets can be cooled per mesh/sponge and a total of 97.1% (5,550/5,715) and 93.8% (4,690/5,000) islets were recovered with a viability of 77.9% and 74.4% using a nylon mesh and a silk sponge respectively (See Introduction and abstract). The recovered islets were transplanted into mice to test their function (See abstract).
The vitrification protocol comprises equilibrating the islets with a first solution (ES) comprising 7.5% DMSO and 7.5% ethylene glycol, placing the cells in the mesh, then transferring the mesh to a Kim wipe to blot off excess ES solution. The mesh was then transferred to a VS solution comprising 15% DMSO, 15% EG, and 0.5M sucrose, transferred to a Kim wipe to remove excess solution, then placed in liquid nitrogen (See Sec. NM vitrification). The rewarming method comprises immersing the mesh in a series of solutions comprising .5, .25, and 0 M sucrose (See Sec. NM vitrification).
Regarding claims 18 and 21: Nakayama-Iwatsuki et al discloses a method for vitrification and rewarming (VR) of primary rat islets (reads on VR islets derived from one or more donors). The VR islets had a viability of the vitrified and rewarmed islets was 77. 9 ± 3%. The standard error is based on 5 replicates which reads on in some experiments the viability was 80%. The VR islets are transplanted into diabetic rats resulting in improvement to the phenotype (reads on a method of therapeutic transplantation of a biological material comprising transplanting the biological material into a patient).
Although Nakayama-Iwatsuki et al does not disclose producing at least about 100,000 or 300,000 islets per batch, it would have been prima facie obvious to a person of ordinary skill in the art to increase the number of islets produced to at least 100,000 or 300,000 in order to produce sufficient islets for commercial use. Mere scaling up of a prior art process capable of being scaled up, if such were the case, would not establish patentability in a claim to an old process so scaled (See 531 F.2d at 1053, 189 USPQ at 148 and MPEP2144.04(IV)(A)).
Claims 1, 2, 4, 6, 7, 11 - 15, 17, and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Nakayama-Iwatsuki et al (Islet, 2020) in view of Best (Rejuvenation Res, 2015).
The teachings of Nakayama-Iwatsuki et al are set forth above.
Regarding claims 1, 5, 6, and 7: Nakayama-Iwatsuki et al teaches a method for cryopreserving primary rat islets (reads on pancreatic islets) by vitrification which reads on a method for cryopreservation of a biological material. The method comprises incubating the islets in a first solution comprising 7.5% DMSO and 7.5% EG then transferring the islets to a second vitrification solution comprising 15% DMSO, 15% EG, and 0.5M sucrose (reads on loading is multi-step and concentration of CPAs is increased in each successive step) which reads on identifying a CPA cocktail for vitrification of a biological material wherein the CPA cocktail comprises one or more CPAs at a vitrification CPA concentration for biological material and loading the biological material with the one or more CPAs to attain the vitrification CPA concentration within the biological material. Following incubation in the first vitrification solution, the cells are transferred to a nylon mesh which reads on transferring the CPA loaded biological material onto a cryomesh. Following each incubation in vitrification solution, excess solution is blotted off with a Kim wipe which reads on removing excess SPA mocktail surrounding the CPA loaded biological material prior to cooling the CPA loaded biological material. The cells are cooled by submerging the mesh comprising the cells into liquid nitrogen which reads on cooling the CPA loaded biological material on the cryomesh to form a vitrified biological material.
Nakayama-Iwatsuki et al further teaches a rewarming method comprising immersing the mesh in a series of solutions comprising .5, .25, and 0 M sucrose (reads on unloading is multi-steps and the concentration of one or more COAs is decreased at each step) which reads on unloading the one or more PCAs from within the rewarmed biological material to eliminate the one or more CPAs from vitrified and rewarmed biological material.
Nakayama-Iwatsuki does not teach the identity and loading protocol of the one or more CPAs and or the vitrification CPA concentration are determined by analyzing the biophysical parameters of the biological material wherein the biophysical parameters comprise inactive volume fraction, hydraulic conductivity and membrane permeability to the one or more CPAs, and/or temperature and rate dependent toxicity of the CPA.
Best teaches CPA toxicity arises from conditions such as temperature, CPA concentration, CPA exposure time, and CPA carrier solutions (See Introduction).
Given that Nakayama-Iwatsuki et al teaches a method of cryopreservation comprising CPAs and Best et al teaches CPAs can be toxic to cells (reads on cell parameters), and the toxicity arises from conditions such as temperature and exposure time, it would have been prima facie obvious to determine the identify, concentration, and loading protocol of the CPAs used in the method of Nakayama-Iwatsuki by analyzing the temperature and rate dependent toxicity of the CPAs. One would have been motivated to identify the CPAs and concentration of CPAs used in the method of Nakayama-Iwatsuki et al based on temperature and rate dependent CPA toxicity because this would result in cells with improved viability. There is a reasonable expectation of success because Best et al teaches the temperature and exposure time to CPAs affect CPA toxicity to cells.
Regarding claim 2: Following the discussion of claim 1 above Nakayama-Iwatsuki et al teaches a method for cryopreserving and rewarming islet cells comprising unloading CPAs in a series of solutions comprising sucrose.
Best teaches high levels of CPAs can eliminate ice formation during cryopreservation of cells, tissues, and organs but CPAs become increasingly toxic as the concentration increases (See abstract). Additionally different cells have different permeability to different CPAs and CPAs with low permeability can cause osmotic stress (See Sec. Osmotic Damage, Cold Shock, and Chilling Injury).
Given that Nakayama-Iwatsuki et al et al teaches a method of cryopreservation comprising CPAs and Best et al teaches CPAs can be toxic to cells (reads on chemical toxicity) and cause osmotic stress, it would have been prima facie obvious to modify the method of Nakayama-Iwatsuki et al et al to minimize the osmotic stress and/or chemical toxicity to the chondrocytes during the loading, cooling, rewarming, and/or unloading steps. One would have been motivated to modify the method because minimizing osmotic stress and cytotoxicity to the cells would result in cells with improved viability. There is a reasonable expectation of success because Best et al teaches osmotic stress and toxicity are affected by concentration and type of CPA which are both optimizable factors.
Regarding claims 4: Following the discussion of claim 1 above Nakayama-Iwatsuki et al teaches a method for cryopreserving islet cells comprising the use of CPAs.
Nakayama-Iwatsuki et al does not teach the biophysical parameters determine the vitrification concentration of the one or more CPAs in the loading step and length of time to load the one or more CPAs to minimize osmotic stress and toxicity to the biological material.
Best teaches CPAs become increasingly toxic as the concentration increases. Additionally different cells have different permeability to different CPAs.
Given that Nakayama-Iwatsuki et al teaches a method of cryopreservation comprising CPAs and Best et al teaches high concentrations of CPAs increase cell toxicity and that cell type affects CPA permeability, it would have been prima facie obvious to determine the concentration of the one or more CPAs and the length of time the cells are exposed to the CPAs in the, during the loading step, method of Nakayama-Iwatsuki et al, to minimize osmotic stress and toxicity. One would have been motivated to determine the concentration and length of exposure to the CPAs because high concentrations of CPAs are toxic and the type of cell used affects how permeable the cell is to the CPAs. There is a reasonable expectation of success because concentration and length of time are optimizable factors.
Regarding claim 11 and 13: Following the discussion of claim 1 above, Nakayama-Iwatsuki et al discloses recovering 97.1% (5,550/5,715) of islets (reads on greater than about 2500 islets with greater than about 95% recovery) Nakayama-Iwatsuki further discloses the viability of the vitrified and rewarmed islets was 77. 9 ± 3%.
Nakayama-Iwatsuki et al does not disclose recovering islets with greater than about 85% viability.
Best teaches CPA concentration can affect cell toxicity.
Given that Nakayama-Iwatsuki et al teaches a method of cryopreservation for islet cells which uses CPAs and Best teaches the concentration of CPAs affects cell toxicity, it would have been prima facie obvious to one of ordinary skill in the art to optimize the concentration of CPAs in the cryopreservation solution of Nakayama-Iwatsuki et al to arrive at a viability of greater than about 85%. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP2144.05(II).
Regarding claim 12: Following the discussion of claim 1 above: Nakayama-Iwatsuki et al discloses transplanting the islet cells into rats.
Regarding claim 14: Nakayama-Iwatsuki et al in view of Best teach a method of cryopreserving and rewarming islets according to claim 1.
Although Nakayama-Iwatsuki et al does not disclose producing at least about 100,000 islets per batch, it would have been prima facie obvious to a person of ordinary skill in the art to increase the number of islets produced to at least 100,000 in order to produce sufficient for commercial use. Mere scaling up of a prior art process capable of being scaled up, if such were the case, would not establish patentability in a claim to an old process so scaled (See 531 F.2d at 1053, 189 USPQ at 148 and MPEP2144.04(IV)(A)).
Regarding claims 15 and 17: Nakayama-Iwatsuki et al teaches a method for cryopreserving primary rat islets (reads on pancreatic islets) by vitrification which reads on a method for cryopreservation of a biological material. The method comprises incubating the islets in a first solution comprising 7.5% DMSO and 7.5% EG then transferring the islets to a second vitrification solution comprising 15% DMSO, 15% EG, and 0.5M sucrose (reads on loading is multi-step and concentration of CPAs is increased in each successive step) which reads on identifying a CPA cocktail for vitrification of a biological material wherein the CPA cocktail comprises one or more CPAs at a vitrification CPA concentration for biological material and loading the biological material with the one or more CPAs to attain the vitrification CPA concentration within the biological material. Following incubation in the first vitrification solution, the cells are transferred to a nylon mesh which reads on transferring the CPA loaded biological material onto a cryomesh. Following each incubation in vitrification solution, excess solution is blotted off with a Kim wipe which reads on removing excess SPA mocktail surrounding the CPA loaded biological material prior to cooling the CPA loaded biological material. The cells are cooled by submerging the mesh comprising the cells into liquid nitrogen which reads on cooling the CPA loaded biological material on the cryomesh to form a vitrified biological material.
Nakayama-Iwatsuki et al further teaches a rewarming method comprising immersing the mesh in a series of solutions comprising .5, .25, and 0 M sucrose (reads on unloading is multi-steps and the concentration of one or more COAs is decreased at each step) which reads on unloading the one or more PCAs from within the rewarmed biological material to eliminate the one or more CPAs from vitrified and rewarmed biological material.
Nakayama-Iwatsuki does not teach the identity and loading protocol of the one or more CPAs and or the vitrification CPA concentration are determined by analyzing the biophysical parameters of the biological material wherein the biophysical parameters comprise inactive volume fraction, hydraulic conductivity and membrane permeability to the one or more CPAs, and/or temperature and rate dependent toxicity of the CPA.
Best teaches CPA toxicity arises from conditions such as temperature, CPA concentration, CPA exposure time, and CPA carrier solutions (See Introduction).
Given that Nakayama-Iwatsuki et al teaches a method of cryopreservation comprising CPAs and Best et al teaches CPAs can be toxic to cells (reads on cell parameters), and the toxicity arises from conditions such as temperature and exposure time, it would have been prima facie obvious to determine the identify, concentration, and loading protocol of the CPAs used in the method of Nakayama-Iwatsuki by analyzing the temperature and rate dependent toxicity of the CPAs. One would have been motivated to identify the CPAs and concentration of CPAs used in the method of Nakayama-Iwatsuki et al based on temperature and rate dependent CPA toxicity because this would result in cells with improved viability. There is a reasonable expectation of success because Best et al teaches the temperature and exposure time to CPAs affect CPA toxicity to cells.
Additionally, Nakayama-Iwatsuki et al does not disclose producing at least about 10,000 or 100,000 islets per batch. However, it would have been prima facie obvious to a person of ordinary skill in the art to increase the number of islets produced to at least 10,000 or 100,000 in order to produce sufficient for commercial use. Mere scaling up of a prior art process capable of being scaled up, if such were the case, would not establish patentability in a claim to an old process so scaled (See 531 F.2d at 1053, 189 USPQ at 148 and MPEP2144.04(IV)(A)).
Regarding claim 22: Following the discussion of claim 15 above, Nakayama-Iwatsuki et al discloses cryopreserving islets which reads on cell clusters that are 3 dimensional.
Claims 1, 2, 4, 6, 7, 11, 12, and 15-17 are rejected under 35 U.S.C. 103 as being unpatentable over Maehara et al (BMC Biotechnology, 2013) in view of Best (Rejuvenation Res, 2015).
Maehara et al teaches a method for cryopreserving chondrocyte cell sheets which allows the chondrocyte cell sheets to retain normal characteristic upon thawing (See abstract). The vitrification method of Maehara et al is based on the minimum volume cooling (MVC) method which has been previously used in embryos (See Introduction). Maehara et al teaches that embryos are susceptible to toxicity of cryoprotectants, osmotic shock, and temperature shock which makes their cryopreservation difficult (See Introduction). The MVC methods uses very small amounts of vitrification solution which helps achieve high rate of survival (See Introduction).
The method of Maehara et al comprises isolating primary chondrocytes from the knee cartilage of rabbits and further culturing the chondrocytes. The cell sheets are made by allowing cells to grow to confluency in a temperature sensitive cell culture dish. When the dish is placed at 20°C, the cells detach from the plate and float as a sheet (See Sec. Preparation of rabbit chondrocyte sheets). The cells are vitrified in solutions compromising permeable cryoprotectants (CPAS) DMSO and ethylene glycol (EG) and non-permeable CPA sucrose. In some experiments non-permeable CPA carboxylated poly-L-lysine (COOH-PLL) was also included (See Sec. Vitrification solutions). The cell sheet is immersed in a first solution (ES) comprising 10% DMSO and 10% EG, then transferred to the same solution to equilibrate. Next, the sheet was transferred to a second solution (VS) comprising 20% DMSO, 20% EG, and 0.5M sucrose, then transferred to fresh VS solution to allow permeation of the permeable CPAs. VS with and without COOH-PLL was compared. The cell sheet is then placed on a stainless-steel mesh and held 1 cm above the surface of liquid nitrogen to expose to liquid nitrogen vapor and vitrify cells (See Sec. Vitrification procedures: Coating method). To rewarm the sheet, the mesh was placed on an electric heating plate. To dilute and remove the CPAs in a stepwise manner, the cell sheet was first placed in a first rewarming solution (RS) comprising 1M sucrose, transferred into a second rewarming solution (DS) comprising .5 M sucrose, then transferred twice into a third solution (WS) which did not comprise cryoprotectants. The cell sheet was gently shaken several times in each solution to help diffusion of the CPAs (See Sec. Vitrification procedures: Coating method).
Maehara et al concludes that using VS which included COOH-PLL prevented cracking of the sheets and resulted in cells with 92.1% viability (See Sec. Maintenance of cell sheet structure and cell viability after vitrification and Fig. 3). Additionally, Maehara et al teaches cell sheets can be applied to injured tissue for example cartilage derived cells sheets can be used to treat knee cartilage injuries (See Background)
Regarding claim 1: Maehara et al teaches method for cryopreserving chondrocyte cell sheets (reads on a method for cryopreservation of a comparable cell cluster). The method is based on MVC methods and comprises incubating chondrocyte sheets in successive vitrification solutions comprising increasing concentrations of DMSO, EG, sucrose, and in some cases COOH-PLL (reads on loading the biological material with the one or more CPAs to attain the vitrification CPA concentration within the biological material). The use of COOH-PLL as a CPA is found to improve viability and prevent cracking of the cell sheets (reads on identifying a CPA cocktail for vitrification of a biological material wherein the CPA cocktail comprises one or more CPAs and the identity of the one or more CPAs is determined by analyzing the biophysical parameters of the biological material). The cell sheets are transferred from the second vitrification solution into a nylon mesh using forceps which reads on transferring the CPA loaded biological material onto a cryomesh and removing excess CPA cocktail surrounding the CPA loaded biological material prior to cooling the CPA loaded biological material. The cryomesh comprising the chondrocyte sheet is held in liquid nitrogen vapor to vitrify the cells which reads on cooling the CPA loaded biological material on the cryomesh to form a vitrified biological material.
To rewarm the material, the mesh is placed on an electric heating plate then the cell sheet is transferred to a series of solutions comprising decreasing concentrations of sucrose which result in dilution and removal of the CPAs from the material (reads on rewarming the vitrified biological material and unloading the one or more CPAs from within the rewarmed biological material to eliminate the one or more CPAs from the vitrified and rewarmed biological material.
Maehara does not teach the identity of the one or more CPAs and or the vitrification CPA concentration and loading protocol are determined by analyzing the biophysical parameters of the biological material wherein the biophysical parameters comprise inactive volume fraction, hydraulic conductivity and membrane permeability to the one or more CPAs, and/or temperature and rate dependent toxicity of the CPA.
Best teaches CPA toxicity arises from conditions such as temperature, CPA concentration, CPA exposure time, and CPA carrier solutions (See Introduction).
Given that Maehara et al teaches a method of cryopreservation comprising CPAs and Best et al teaches CPAs can be toxic to cells (reads on cell parameters), and the toxicity arises from conditions such as temperature and exposure time, it would have been prima facie obvious to determine the identify, concentration, and loading protocol of the CPAs used in the method of Maehara by quantitatively analyzing the temperature and rate dependent toxicity of the CPAs. One would have been motivated to identify the CPAs and concentration of CPAs used in the method of Maehara et al based on temperature and rate dependent CPA toxicity because this would result in cells with improved viability. There is a reasonable expectation of success because Best et al teaches the temperature and exposure time to CPAs affect CPA toxicity to cells.
Regarding claim 2: Following the discussion of claim 1 above, Maehara et al discloses the method of claim 1 comprising unloading the CPAs in a solution comprising sucrose.
Best teaches high levels of CPAs can eliminate ice formation during cryopreservation of cells, tissues, and organs but CPAs become increasingly toxic as the concentration increases (See abstract). Additionally different cells have different permeability to different CPAs and CPAs with low permeability can cause osmotic stress (See Sec. Osmotic Damage, Cold Shock, and Chilling Injury).
Given that Maehara et al teaches a method of cryopreservation comprising CPAs and Best et al teaches CPAs can be toxic to cells (reads on chemical toxicity) and cause osmotic stress, it would have been prima facie obvious to modify the method of Maehara et al to minimize the osmotic stress and/or chemical toxicity to the chondrocytes during the loading, cooling, rewarming, and/or unloading steps. One would have been motivated to modify the method because minimizing osmotic stress and cytotoxicity to the cells would result in cells with improved viability. There is a reasonable expectation of success because Best et al teaches osmotic stress and toxicity are affected by concentration and type of CPA which are both optimizable factors.
Regarding claims 4: Following the discussion of claim 1 above, Maehara et al teaches a method for cryopreserving chondrocyte sheets according to the method of claim 1.
Maehara et al does not teach the biophysical parameters determine the vitrification concentration of the one or more CPAs in the loading step and length of time to load the one or more CPAs to minimize osmotic stress and toxicity to the biological material.
Best teaches CPAs become increasingly toxic as the concentration increases. Additionally different cells have different permeability to different CPAs.
Given that Maehara et al teaches a method of cryopreservation comprising CPAs and Best et al teaches high concentrations of CPAs increase cell toxicity and that cell type affects CPA permeability, it would have been prima facie obvious to determine the concentration of the one or more CPAs and the length of time the cells are exposed to the CPAs in the, during the loading step, in the method of Maehara, to minimize osmotic stress and toxicity. One would have been motivated to determine the concentration and length of exposure to the CPAs because high concentrations of CPAs are toxic and the type of cell used affects how permeable the cell is to the CPAs. There is a reasonable expectation of success because concentration and length of time are optimizable factors.
Regarding claim 6: Maehara et al discloses a CPA cocktail comprising ethylene glycol and DMSO.
Regarding claim 7: The method of Maehara et al comprises a CPA loading method comprising incubating cell sheets in a first vitrification solution, then transferring the cell sheet to a second vitrification solution. The first solution comprises 10% DMSO and 10% EG (read on CPAs) and the second solution comprises 20% DMSO, 20% EG, and 0.5M sucrose (read on CPAs). Therefore the loading method of Maehara et al comprises multi-steps and the concentration of the one or more CPAs is increased in each successive loading step. Additionally, the method of Maehara et al comprises unloading CPAs in three steps comprising three unloading solutions. The first solution comprises 1M sucrose (reads on CPA), the second solution comprises 0.5 M sucrose, and the third solution does not comprise sucrose. Therefore, the unloading method of Maehara et al discloses unloading in multi-steps and decreasing the concentration of the one or more CPAs in each successive unloading step.
Regarding claim 11: Following the discussion of claim 1 above, Maehara et al discloses recovering chondrocyte sheets with a viability of 92.1%.
Regarding claim 12: Following the discussion of claim 1 above, Maehara et al discloses the chondrocyte sheets can be used to treat knee cartilage injuries which reads on transplanting the VR biological material.
Regarding claims 15 and 17: Maehara et al teaches method for cryopreserving chondrocyte cell sheets (reads on a method for cryopreservation of a biological material). The method is based on MVC methods and comprises incubating chondrocyte sheets in successive vitrification solutions comprising increasing concentrations of DMSO, EG, sucrose, and in some cases COOH-PLL (reads on loading the biological material with the one or more CPAs to attain the vitrification CPA concentration within the biological material). The use of COOH-PLL as a CPA is found to improve viability and prevent cracking of the cell sheets (reads on identifying a CPA cocktail for vitrification of a biological material wherein the CPA cocktail comprises one or more CPAs and the identity of the one or more CPAs is determined by analyzing the biophysical parameters of the biological material). The cell sheets are transferred from the second vitrification solution into a nylon mesh using forceps which reads on transferring the CPA loaded biological material onto a cryomesh and removing excess CPA cocktail surrounding the CPA loaded biological material prior to cooling the CPA loaded biological material. The cryomesh comprising the chondrocyte sheet is held in liquid nitrogen vapor to vitrify the cells which reads on cooling the CPA loaded biological material on the cryomesh to form a vitrified biological material.
To rewarm the material, the mesh is placed on an electric heating plate then the cell sheet is transferred to a series of solutions comprising decreasing concentrations of sucrose which result in dilution and removal of the CPAs from the material (reads on rewarming the vitrified biological material and unloading the one or more CPAs from within the rewarmed biological material to eliminate the one or more CPAs from the vitrified and rewarmed biological material.
Maehara does not teach the identity of the one or more CPAs and or the vitrification CPA concentration and loading protocol are determined by quantitatively analyzing the biophysical parameters of the biological material wherein the biophysical parameters comprise inactive volume fraction, hydraulic conductivity and membrane permeability to the one or more CPAs, and/or temperature and rate dependent toxicity of the CPA.
Best teaches CPA toxicity arises from conditions such as temperature, CPA concentration, CPA exposure time, and CPA carrier solutions (See Introduction).
Given that Maehara et al teaches a method of cryopreservation comprising CPAs and Best et al teaches CPAs can be toxic to cells (reads on cell parameters), and the toxicity arises from conditions such as temperature and exposure time, it would have been prima facie obvious to determine the identify and concentration of the CPAs used in the method of Maehara by analyzing the temperature and rate dependent toxicity of the CPAs. One would have been motivated to identify the CPAs, concentration of CPAs, and CPA loading protocol used in the method of Maehara et al based on temperature and rate dependent CPA toxicity because this would result in cells with improved viability. There is a reasonable expectation of success because Best et al teaches the temperature and exposure time to CPAs affect CPA toxicity to cells.
Additionally, Maehara et al does not disclose the method produces at least about 10,000 100,000 cell clusters per batch.
Although Maehara et al does not disclose producing at least about 10,000 or 100,000 cell clusters per batch, it would have been prima facie obvious to a person of ordinary skill in the art to increase the number of cell clusters produced to at least about 10,000 or 100,000 cell sheets (clusters) in order to produce sufficient cell sheets for commercial use. Mere scaling up of a prior art process capable of being scaled up, if such were the case, would not establish patentability in a claim to an old process so scaled (See 531 F.2d at 1053, 189 USPQ at 148 and MPEP2144.04(IV)(A)).
Regarding claim 16: Following the discussion of claim 15 above, Maehara et al teaches a cryopreservation method comprising cooling cell sheets on a mesh.
Maehara et al does not teach the mesh is stacked to increase the number of cell clusters per batch.
Although Maehara et al does not teach stacking the mesh to increase the number of cell clusters that can be cooled per batch, it would have been prima facie obvious to a person of ordinary skill in the art to stack the meshes using a suitable container, in order to cool more cell sheets using a liquid nitrogen tank. Maehara et al discloses cooling in the vapor phase of liquid nitrogen, and a greater area of liquid nitrogen vapor would exist vertically within a liquid nitrogen tank, thus stacking meshes vertically would take advantage of the area of the liquid nitrogen tank comprising vapor.
Claims 1, 2, 4, 6-8, 11, 12, and 15-17 are rejected under 35 U.S.C. 103 as being unpatentable over Maehara et al (BMC Biotechnology, 2013) in view of Best (Rejuvenation Res, 2015) and Gao et al (Current Frontiers in Cryobiology, 2011.
The teachings of Maehara et al and Best et al are set forth above.
Maehara et al and Best et al render claims 1, 2, 4, 6, 7, 11, 12, and 15-17 obvious.
Regarding claim 8: Following the discussion of claims 1 and 6 above, Maehara et al teaches a method for cryopreserving chondrocyte sheets according to the method of claim 1 which comprises a multi-step CPA unloading step.
Maehara et al does not teach the unloading comprises gradually increasing the amount of a non-penetrating CPA in the CPA cocktail.
Gao et al teaches solutions comprising hyperosmotic amounts of sucrose (reads on non-penetrating CPA) can be used to remove cryoprotectants. Gao et al further teaches using multi-step methods for unloading cryoprotectants can minimize osmotic injury (See introduction).
Given that Maehara et al teaches a multi-step method of unloading CPAs from cells and Gao et al teaches a hyperosmotic solution comprising sucrose can be used to remove penetrating CPAs and that performing the method in multiple steps minimizes osmotic injury, it would have been prima facie obvious to substitute the unloading method of Maehara et al comprising diluting the CPAs in multi-steps, with the method of Gao et al which comprises using a hyperosmotic sucrose solution, and gradually increasing the concentration of sucrose at each step to limit osmotic stress on the cells. One would expect the methods to work equivocally because both methods comprise removing CPAs from cells in multiple-steps. Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable is considered to be obvious. See KSR International Co. V Teleflex Inc 82 USPQ2d 1385 (US2007) at page 1395.
Claims 1, 2, 4, 6, 7, 9-12, and 15-17 are rejected under 35 U.S.C. 103 as being unpatentable over Maehara et al (BMC Biotechnology, 2013) in view of Best (Rejuvenation Res, 2015) in view of Khosla et al (Langmuir, 2018).
The teachings of Maehara et al and Best et al are set forth above.
Maehara et al and Best et al render claims 1, 2, 4, 6, 7, 11, 12, and 15-17 obvious.
Regarding claim 9: Following the discussion of claim 1 above, Maehara et al teaches a method for cryopreserving chondrocyte sheets according to the method of claim 1.
Maehara et al does not teach the cooling rate for the biological material is greater than about 50,000°C/min.
Khosla et al teaches a method of vitrification and rewarming of fibroblasts. The method of Khosla uses a modified Cryotop device to cool the fibroblasts at a rate of 90,000°C/min in liquid nitrogen (See abstract).
Given that both Maehara et al and Khosla et al teach methods of cryopreservation by vitrification comprising a step of cooling in liquid nitrogen, it would have been prima facie obvious to substitute the cooling method of Maehara, comprising holding a mesh in the vapor phase of liquid nitrogen, with the cooling step of Khosla et al, comprising the use of a Cryotop device. One would have expected the vapor phase of the liquid nitrogen and the Cryotop device to work equivocally in the method of Maehara et al because both methods use liquid nitrogen to cool cells. Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable is considered to be obvious. See KSR International Co. V Teleflex Inc 82 USPQ2d 1385 (US2007) at page 1395.
Regarding claim 10: Following the discussion of claim 1 above, Maehara et al teaches a method for cryopreserving chondrocyte sheets according to the method of claim 1.
Maehara et al does not teach the rewarming rate for rewarming the vitrified biological composition is greater than about 200,000°C/min.
Khosla et al teaches a method of vitrification and rewarming of fibroblasts. The method of Khosla comprises freezing cells surrounded by nanoparticles, which allow the cells to be rapidly rewarmed at a rate of 1.4 x 10^7°C/min using a laser (See abstract and Fig. 2).
Given that both Maehara et al and Khosla et al teach methods of rewarming cells which have been cryopreserved by vitrification, it would have been prima facie obvious to substitute the rewarming method of Maehara, comprising using a heating plate, with the rewarming step of Khosla et al, comprising surrounding the cells with nanoparticles and using a laser. One would have expected heating plate and the laser to work equivocally in the method of Maehara et al because both methods produce heat to rewarm cells. Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable is considered to be obvious. See KSR International Co. V Teleflex Inc 82 USPQ2d 1385 (US2007) at page 1395.
Conclusion
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/MARISOL ANN O'NEILL/Examiner, Art Unit 1633
/ALLISON M FOX/Primary Examiner, Art Unit 1633
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