Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 10/09/2025 has been entered.
Response to Amendment
The amendment filed 10/09/2025 amending claim(s) 1 and 11 is acknowledged.
Claims 1, 3-11, and 13-22 are pending and under examination.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 9/03/2025, 9/22/2025, and 10/20/2025 are considered by the examiner.
Claim Rejections - 35 USC § 112(a) – Written Description; New, necessitated by amendment
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 3-11, and 13-22 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Newly amended claims 1 and 11 recite “the cryopreservation protocol is conducted such that the trehalose does not enter inside the cellular material.”
In analyzing whether the written description requirement is met for genus claims, it is first determined whether a representative number of species have been described by their complete structure. To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, methods of making the claimed product, or any combination thereof. The disclosure of a single species is rarely, if ever, sufficient to describe a broad genus, particularly when the specification fails to describe the features of that genus, even in passing. (see In re Shokal 113USPQ283(CCPA1957); Purdue Pharma L.P. vs Faulding Inc. 56 USPQ2nd 1481 (CAFC 2000).
The court explained that “reading a claim in light of the specification, to thereby interpret limitations explicitly recited in the claim, is a quite different thing from ‘reading limitations of the specification into a claim,’ to thereby narrow the scope of the claim by implicitly adding disclosed limitations which have no express basis in the claim.” The court found that applicant was advocating the latter, i.e., the impermissible importation of subject matter from the specification into the claim.). See also In re Morris, 127 F.3d 1048, 1054-55, 44 USPQ2d 1023, 1027-28 (Fed. Cir. 1997).
The claim does not recite the structure/method steps necessary to conduct the protocol “such that the trehalose does not enter inside the cellular material.” When searching the specification for intracellular-extracellular trehalose and trehalose entering the cellular material, the following recitations are found:
[00063] Once the cryopreservation composition has been prepared (and trehalose in the absence of any other added conventional cryoprotectants (such as DMSO, glycerin/glycerol, ethylene glycol, propylene glycol or the like) is associated with the cellular material to be preserved), the cooling for cryopreservation may be conducted at the rapid cooling rate described above (e.g., where trehalose alone is used in the media around the cellular material to be preserved, without placing it inside the cells (i.e., extracellular trehalose).
[00074] Prior to the development of the methodology of the present disclosure, the need to have trehalose on both sides of the cell membrane has long been thought to be the best strategy for maximum protection by trehalose during cryopreservation (Stewart et al., Intracellular Delivery of Trehalose for Cell Banking, Langmuir, 2019, 35(23): 7414-7422).
Despite the development of several methods to introduce trehalose into cells, each protocol has drawbacks and the results have been mixed about whether these methods can consistently protect cells. Studies have also been done that utilize trehalose with fast cooling rates (>50C/minute) (Heo et al., "Universal" vitrification of cells by ultra-fast cooling, Technology (Singap World Sci), 2015, 3(1), 64-71; Liebermann et al., Potential Importance of Vitrification in Reproductive Medicine, Biology of Reproduction, Volume 67,Issue 6, 2002, pages 1671-1680). However, the faster cooling rates are used in conjunction with small
volumes (0300 pL), so that these samples are vitrified (no formation of ice). These types of protocols are mainly used for reproductive tissue processes and normally involve the use of cryoprotectant cocktails containing trehalose. Somewhat slower cooling rates (-5 to - 60°C/minute) have also been used, but these studies have involved using trehalose as a supplemental cryoprotectant in a cocktail with DMSO (Barbas et al., Cryopreservation of domestic animal sperm cells, Cell and Tissue Banking, 2008, 10(1), 49-62) while the approach used in the present disclosure uses extracellular trehalose as the sole cryoprotectant.
[00075] Prior to the development of the methodology of the present disclosure, the
inventors of the instant application previously assessed a nanotechnology for intracellular trehalose delivery developed by Professor Xiaoming He, University of Maryland (Zhang et al., Cold-Responsive Nanoparticle Enables Intracellular Delivery and Rapid Release of Trehalose for Organic-Solvent-Free Cryopreservation, Nano Lett. 2019, 19, 9051-9061; Rao
et al., Nanoparticle-Mediated Intracellular Delivery Enables Cryopreservation of Human Adipose-Derived Stem Cells Using Trehalose as the Sole Cryoprotectant, ACS Appl Mater Interfaces, 2015, 7(8), 5017-5028), that he had shown promoted cryopreservation of adipose tissue derived hMSCs, for bone marrow-derived MSCs. Unfortunately, the trehalose nanotechnology failed during these studies, probably due to instability of the trehalose nanoparticles during storage and transport from the University of Maryland to Charleston, SC. During these studies, it was observed that MSCs survived cryopreservation quite well when trehalose was present only extracellularly, which was being used as a negative control. Encouraged by these unanticipated results, the range of exogenous trehalose employed was expanded to 0.2-0.8M and compared -1, -5 and -15C/minute cooling rates.
The specification provides little guidance on how an artisan is to conduct the method in such a manner. Based on [0063] of the specification, using trehalose in the cellular media around the cellular material to be preserved is enough to be considered extracellular trehalose that does not enter the cellular material. In turn, the specification teaches specific, intentional methods to place trehalose inside of cells, such as using nanoparticles to delivery trehalose to cells (see Zhang et al. and Rao et al. cited in [00075] of specification).
Additionally, the specification references Zhang et al. and Rao et al. in regards to extracellular trehalose, and how during these studies, it was observed that MSCs survived cryopreservation quite well when trehalose was present only extracellularly. In Zhang et al., the pretreatment of cells with free trehalose (fTre) prior to cryopreservation for 4 hours, followed by cryopreservation with 0.3 M extracellular trehalose resulted in over 40-60% viability of cells immediately after thawing (pg. 9057, col 1, para 2; Fig. 6). Rao et al. teaches suspending cells in 200 μl hADSC culture medium containing 200 mM free trehalose prior to freezing the sample at approximately -1 °C/min to −80 °C in a −80 °C freezer (pg. 6, “2.8 Cryopreservation of hADSCs”).
Additionally, there is contradictory evidence provided by the Applicant on whether trehalose naturally enters cells after a certain period of incubation. [00014] of the specification states: “Trehalose is not metabolized by mammalian cells and there are no active mammalian transport mechanisms for uptake of trehalose.” However, in the response from the Applicant filed 10/09/2025, the Applicant argues a “1 hour incubation is long enough for internalization of some trehalose so that trehalose would be present on both sides of the cell membrane, as evidenced by the previously attached paper "Loading Human Mesenchymal Stem Cells with Trehalose by Fluid-Phase Endocytosis." In this paper, internalization of trehalose is shown in MSCs by endocytosis during incubation under physiological conditions. Monocytes, such as those used in Kanias, are more actively endocytic (phagocytic) than MSCs so it is likely that they would have internalized more trehalose after 1 hour. Thus, Kanias does not teach that trehalose does not enter inside the cellular material.” How can both of these statements be true?
The prior art in general teaches trehalose to be a non-penetrating cryoprotectant. For example, Whaley et al. teaches trehalose to be a non-penetrating cryoprotective agent, defined as agents that do not permeate intracellularly and therefore exert their protective influence outside of the cell (pg. 3, “Non-permeating agents”).
Currently, the incubation time of cells in trehalose is generic. The working examples of the specification, specifically the preservation method steps, provide minimal guidance. [00077] teaches the 0.2 M trehalose hMSC group cooled at a rate of -15 degrees C/minute had the best outcomes; however, now further details on the method, such as incubation time are provided. [00080] describes the cryopreservation of T cells. Again, cryopreservation details such as how long the cells were incubated in trehalose are not provided. No further guidance on incubation time is provided in the specification either. As such, even the claimed invention reads on incubating the cells for at least 1 hour, which according to the reference cited by the Applicant, may lead to the internalization of trehalose.
In summary, the claims are considered to lack adequate written description for failing to recite the structure that is necessary and sufficient to result in trehalose not entering the cell (and how the claimed method differs structural from those found in the art, such as disclosed by Kanias, or if they do not differ structurally, how the methods would produce different results). The specification fails to disclose what changes to the structure of the claimed composition is necessary and sufficient to result in trehalose not entering the cell, and thus the ordinary artisan would not know what modification(s) must be made in order to fulfill the instant recitation.
Additionally, the claims are broad for reciting a genus of cryoprotectants. The specification provides the following guidance on the term “cryoprotectant”:
[00018] The present disclosure provides improved preservation methods using trehalose in the absence of other added conventional cryoprotectants (such as DMSO, glycerin/glycerol, ethylene glycol, propylene glycol or the like, particularly DMSO) in cryopreservation protocols.
[00019] In some embodiments, the present disclosure is directed to providing cryopreservation methodology that achieves protective effects and low toxicity for cells or tissues by replacing conventional cryoprotectants (e.g., those that are known to be toxic, such as DMSO, and/or those that are designed to be removed after the cells or tissues are cryopreserved at -80 C or below and rewarmed).
[00049] As used herein, the term "substantially free of cryoprotectant other than
trehalose" means a cryoprotectant (other than trehalose) in an amount less than 0.01 w/w %. In some embodiments, the methods of the present disclosure may use and/or achieve a medium/solution and/or cellular material that is substantially free of cryoprotectant (other than trehalose), such as a cellular material that is substantially free of DMSO (i.e., the DMSO is in an amount less than 0.01 w/w %). In some embodiments, the methods of the present disclosure may use and/or achieve a medium/solution and/or cellular material that is substantially free of any added cryoprotectant other than the trehalose. The cryoprotectant other than trehalose that may be excluded in this regard may be one or more cryoprotectant that are conventionally used when the cells are immersed in a cryopreservation liquid and then cryopreserved at -804 C or below, or one or more of the following cryoprotectants (commonly added for that function): DMSO, glycerin, acetamide, agarose, alginate, alanine, albumin, ammonium acetate, anti-freeze proteins, butanediols (such as 2,3-butanediol), chondroitin sulfate, chloroform, choline, cyclohexanediols, cyclohexanediones, cyclohexanetriols, dextrans, diethylene glycol, dirnethyl acetamide, dirnethyl forrnamide (such as n-dimethyl formamide), dimethyl sulfoxide, erythritol, ethanol, ethylene glycol, ethylene glycol monomethyl ether, formamide, glucose, glycerol, glycerophosphate, glycerylmonoacetate, glycine, glycoproteins, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methoxy propanediol, methyl acetamide, methyl formamide, methyl ureas, methyl glucose, methyl glycerol, phenol, pluronic polyols, polyethylene glycol, polyvinylpyrrolidone, proline, propanediols (such as 1,2-propanediol and 1,3-propanediol), pyridine N-oxide, raffinose, ribose, serine, sodium nitrate, sodium nitrite, sodium sulfate, sorbitol, triethylene glycol, trimethylamine acetate, urea, valine and xylose.
While the specification provides examples of cryoprotectants, the instant disclosure does not define what is considered a cryoprotectant.
The prior art provides varied guidance on what may be considered a cryoprotectant and what the genus encompasses. For example, Kanias defines a cryoprotectant agent as “any agent that is added to a solution and which improves the post thaw viability of a biological material cryopreserved in that solution”, including “proteins” and “carbohydrates” generically recited (col 9, para 2). However, in their review of cryopreservation, Whaley et al. divides cryoprotectants into two categories: permeating and non-permeating agents. Permeating agents, such as DMSO and glycerol, protect a cell from mechanical and osmotic effects of freezing and must be highly water soluble at low temperatures and able to easily cross biological membranes (pg. 2, “Permeating Agents”). Non-permeating agents, such as PEG, sucrose, and trehalose, do not permeate intracellularly and therefore exert their protective influence outside of the cell. They are typically larger, and covalently linked as either polymers, dimers, or trimers (pg. 3, “Non-permeating agents”). Whaley et al. does not teach proteins (such as fetal calf serum) being a cryoprotectant.
Wowk refers to cryoprotectants as “chemicals that dissolve in water and lower the melting point of water”, but notes “not all chemicals that dissolve in water are cryoprotectants. In addition to being water soluble, good cryoprotectants are effective at depressing the melting point of water, do not precipitate or form eutectics or hydrates, and are relatively non-toxic to cells at high concentration. All cryoprotectants form hydrogen bonds with water” (pg. 3, “Properties of Cryoprotectants”). Additionally, Wowk describes non-penetrating cryoprotectants as large molecules, usually polymers, added to cryoprotectant solutions. They inhibit ice growth by the same mechanisms as penetrating cryoprotectants, but do not enter cells (pg. 5, “Non-penetrating Cryoprotectants (optional ingredient)”).
As evidenced above, there is no clear, strictly followed definition for “cryoprotectant” in the prior art. While some agents such as DMSO and glycerol are largely considered to be cryoprotectants in the art, inclusion of other agents/molecules such as proteins are less common/clear, and may depend on how an artisan defines “cryoprotectant”. Therefore, how does an Artisan determine what is a cryoprotectant or not in the claimed invention, given the minimal guidance in the specification and varied properties a cryoprotectant must possess in the prior art.
Thus, for the reasons outlined above, it is concluded that the claims do not meet the
requirements for written description under 35 U.S.C. 112, first paragraph.
Claim Rejections - 35 USC § 102 – Maintained, modified due to arguments/amendments
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 3-11, 13-15, and 19-21 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Kanias (US8198085B2, published 06/12/2012).
Regarding claims 1, 3-5, 9-11, 13-15, 19, and 20, Kanias discloses a cryopreservation method that is free of added cryoprotectant other than trehalose, uses an effective amount of trehalose as an extracellular cryoprotectant that is not placed inside the cellular material (i.e., cells are not altered to uptake trehalose/have trehalose enter the cell) in a range of 200-800 mM (e.g., 0.5M), and is free of any other cryoprotectants or additional chemicals listed in claim 11, to form a solution to suspend cells in for cryopreservation (Figure 6; col 22, starting at line 9). Cells were then cooled at a rate of 5.1°C/min (i.e., ranging from -3°C/min to -50°C/min) (i.e., ranging from -5°C/min to -30°C/min- claims 3 and 13) (col 22, line 41). Samples were then frozen and plunged into liquid nitrogen for storage (i.e., storing cellular material at -80°C for greater than one hour – claims 4 and 14) (i.e., storing cellular material at -135°C for greater than one hour, as liquid nitrogen is at a temperature of -196°C – claims 5 and 15) (col 22, line 42-43; col 12, lines 12-31). Cell viability (i.e., survival) of the macrophages was found to be an average of 60%, with the best survival rate being 72% (i.e., at least 60%- claims 9 and 19) (i.e., at least 70%- claims 10 and 20), achieved with a concentration of 0.5M trehalose (col 22, lines 56-61; Fig 6).
Regarding claims 6-8, and 21, Kanias also discloses the cryopreservation protocol above including a polyphenol, such as EGCG (claims 1-7 of Kanias). Kanias discloses that the cryopreserved cells may be lymphocytes (i.e., which include T cells- claim 6), or human mesenchymal stem cells (claims 7 and 8) (col 6, lines 63-67-col 7, lines 1-17).
Claim Rejections - 35 USC § 103 - Maintained
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.
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 16-18 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Kanias as applied to claims 1, 3-11, 13-15, and 19-21 above, and further in view of Wolpe (US-20170121673-A1, published May 4th, 2017).
The teachings of Kanias are recited above and applied as for claims 1, 3-11, 13-15, and 19-21. As shown above, the base claims are anticipated by the base art, and thus, are also obvious over the base art.
Kanias fails to teach a cryopreservation protocol excluding the chemicals listed in claim 11, wherein the cellular material comprises T-cells, mesenchymal stem cells, or human mesenchymal stem cells, where the cell viability after warming is at least 60% (claim 22).
An Artisan, interested in cryopreserving T cells and/or MSCs, would be aware of Wolpe for teaching methods of cryopreserving T cells and MSCs while maintaining cellular properties
Wolpe teaches a method for cryopreserving cells under conditions that retain optimum levels of cell surface fucosylation. In Wolpe, the method for cryopreservation involves freezing cells in a cryopreservation composition comprising a physiologically balanced salt solution and cryoprotectant (Claim 2, Claim 3), where one of the cryoprotectants that can be selected for the method is trehalose (Claim 14). Additionally, Wolpe teaches that this method, with trehalose as a sole cryoprotectant used, can be used for cryopreservation of T cells and mesenchymal stem cells, including human mesenchymal stem cells (Claim 9; para 0084; para 0063).
It would be obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to do a simple substitution of the macrophage cells taught in Kanias for the T cells and mesenchymal stem cells taught in Wolpe. An artisan would have a reasonable expectation of success because the simple substitution of one known element for another would have yielded predictable results to one of ordinary skill in the art at the time of the invention. M.P.E.P. §2144.07 states "The selection of a known material based on its suitability for its intended use supported a prima facie obviousness determination in Sinclair & Carroll Co. v. Interchemical Corp., 325 U.S. 327, 65 USPQ 297 (1945).” “When substituting equivalents known in the prior art for the same purpose, an express suggestion to substitute one equivalent component or process for another is not necessary to render such substitution obvious. In re Fout, 675 F.2d 297, 213 USPQ 532 (CCPA 1982).” M.P.E.P. §2144.06.
Additionally, the use of trehalose as the sole cryoprotectant for preserving diverse cellular materials is well known in the art as demonstrated by Kanias, so one would consider applying the known method to other types of cells and expect similar results to those taught in Kanias. One of ordinary skill would have been motivated to substitute T cells and mesenchymal stem cells taught in Wolpe into the method taught by Kanias because as taught by Kanias, other commonly used cryoprotectants, such as DMSO, are toxic to cells and after thawing, must be removed (col 3, lines 54-61).
Response to Arguments
Applicant's arguments filed 10/09/2025 have been fully considered but they are not persuasive.
The Applicant argues that Kanias does not teach the claim limitation of “the cryopreservation protocol is free of added cryoprotectant other than trehalose” because Kanias teaches administering fetal calf serum before cells were incubated and the dosages in Fig. 6 were applied.
This argument is not found to be persuasive because fetal calf serum is not listed in the instant specification as an example of a cryoprotectant. For example:
[00018] The present disclosure provides improved preservation methods using trehalose in the absence of other added conventional cryoprotectants (such as DMSO, glycerin/glycerol, ethylene glycol, propylene glycol or the like, particularly DMSO) in cryopreservation protocols.
[00019] In some embodiments, the present disclosure is directed to providing cryopreservation methodology that achieves protective effects and low toxicity for cells or tissues by replacing conventional cryoprotectants (e.g., those that are known to be toxic, such as DMSO, and/or those that are designed to be removed after the cells or tissues are cryopreserved at -80 C or below and rewarmed).
[00049] As used herein, the term "substantially free of cryoprotectant other than
trehalose" means a cryoprotectant (other than trehalose) in an amount less than 0.01 w/w %. In some embodiments, the methods of the present disclosure may use and/or achieve a medium/solution and/or cellular material that is substantially free of cryoprotectant (other than trehalose), such as a cellular material that is substantially free of DMSO (i.e., the DMSO is in an amount less than 0.01 w/w %). In some embodiments, the methods of the present disclosure may use and/or achieve a medium/solution and/or cellular material that is substantially free of any added cryoprotectant other than the trehalose. The cryoprotectant other than trehalose that may be excluded in this regard may be one or more cryoprotectant that are conventionally used when the cells are immersed in a cryopreservation liquid and then cryopreserved at -804 C or below, or one or more of the following cryoprotectants (commonly added for that function): DMSO, glycerin, acetamide, agarose, alginate, alanine, albumin, ammonium acetate, anti-freeze proteins, butanediols (such as 2,3-butanediol), chondroitin sulfate, chloroform, choline, cyclohexanediols, cyclohexanediones, cyclohexanetriols, dextrans, diethylene glycol, dirnethyl acetamide, dirnethyl forrnamide (such as n-dimethyl formamide), dimethyl sulfoxide, erythritol, ethanol, ethylene glycol, ethylene glycol monomethyl ether, formamide, glucose, glycerol, glycerophosphate, glycerylmonoacetate, glycine, glycoproteins, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methoxy propanediol, methyl acetamide, methyl formamide, methyl ureas, methyl glucose, methyl glycerol, phenol, pluronic polyols, polyethylene glycol, polyvinylpyrrolidone, proline, propanediols (such as 1,2-propanediol and 1,3-propanediol), pyridine N-oxide, raffinose, ribose, serine, sodium nitrate, sodium nitrite, sodium sulfate, sorbitol, triethylene glycol, trimethylamine acetate, urea, valine and xylose.
As such, the specification provides no guidance for an artisan to conclude fetal calf serum reads on the term “cryoprotectant”. Further, the Applicant’s argument raises the question of what is/is not considered a cryoprotectant. If the Applicant is asserting fetal calf serum to be a cryoprotectant, what else would fall under the Applicant’s interpretation of “cryoprotectant’? The instant specification provides no clear, distinct definition for “cryoprotectant” (listing embodiments such as in the paragraphs cited above do not constitute a definition). The Applicant’s arguments point out how broad and generic the recitation of “cryoprotectant” is in the art. Kanias, for example, broadly recites that a cryoprotectant agent may be “any agent that is added to a solution and which improves the post thaw viability of a biological material cryopreserved in that solution”, which can read on a wide variety of factors and solutions, including fetal calf serum.
Additionally, Kanias (in the section referenced by the Applicant, col 22, lines 330-36) refers to fetal calf serum as part of a culture medium, not as a cryoprotectant in the cited working example.
Further, the Applicant argues that Kanias fails to recognize that the amount of fetal calf serum used in Fig. 6 has other effects outside the preservation process, and that any amount of serum increases the risk of infection. This argument is not persuasive because the Applicant fails to provide evidence for this assertion. Additionally, Kanias teaching an embodiment with fetal calf serum does not negate their teaching of the cell preparation being essentially free of serum proteins.
Additionally, the Applicant argues Kanias does not teach the cryopreservation protocol being conducted such that the trehalose does not enter inside the cellular material because cells are incubated for 1 hour. This argument is not found to be persuasive because the instant claims do not recite a incubation time period. As such, Kanias discloses the methods steps (i.e., structure) of the claimed invention, and absent evidence on the contrary, and artisan would expect the same outcome of trehalose not entering inside the cellular material as recited in the claims.
Further, the assertion that trehalose is internalized in MSCs by the Applicant contradicts their statement in the specification that “Trehalose is not metabolized by mammalian cells and there are no active mammalian transport mechanisms for uptake of trehalose” [00014]. As previously stated in the response to arguments in the Final Office Action filed 06/20/2025, the disclosure of the current invention (e.g., [00063, 00084]) also do not exclude the cells from naturally transporting the trehalose inside the cell.
As discussed in more detail in the 112(a) rejection above, when discussing intracellular vs. extracellular trehalose in the instant specification, intracellular trehalose is discussed as an intentional, active method, such as using nanoparticles to deliver trehalose into cells [00075]. However, extracellular trehalose is referenced as “No attempt was made to introduce trehalose into the cells that is deemed necessary in the literature” [00078]. Further, in the references cited in [00075] of the specification for teaching “that MSCs survived cryopreservation quite well when trehalose was present only extracellularly, which was being used as a negative control”, the “extracellular trehalose” was simply culturing/incubating the cells in trehalose (e.g., “free Trehalose”), the same method taught by Kanias. The Applicant fails to distinctly point out how the teachings of Kanias differ from the instant claims and specification, as well as the cited prior art used to teach “extracellular trehalose” in the specification.
Conclusion
No claims are allowed.
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/ALLISON MARIE JOHNSON/Examiner, Art Unit 1638
/ROBERT M KELLY/Primary Examiner, Art Unit 1638
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