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 .
Priority
This application 18/001,259 filed on 12/08/2022 is a national phase application under 35 U.S.C. § 371 that claims priority to International Application No. PCT/CN2021/099865 field on 06/11/2021, and claims priority of foreign application CN 202010537360 field on 06/12/2020.
A certified copy of foreign application CN 202010537360 field on 06/12/2020, which is not in English, has been submitted of the record by Applicants on 12/08/2022.
In the absence of English translation of foreign application CN 202010537360, the priority date of claim set field on 09/08/2025 is determined to be 06/11/2021, the filing date of PCT/CN2021/099865.
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a) (d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Restriction/Election
Applicant’s election of Group I invention, claims 1-6 and 8-12, in the reply filed on 09/08/2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
For species election (I), Applicant elected methods of (i) preventing and/or treating acute respiratory distress syndrome and/or acute lung injury. For species election (II), Applicant elected growth factor (ii) BDNF.
Claims 3-8 and 13-15 are cancelled, and claims 16-18 are newly added in the claim set filed on 09/08/2025.
Claims 1, 2, 9-12 and 16-18 are pending.
Claim 12 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 09/08/2025.
Claims 1, 2, 9-11, and 16-18 are currently under examination to the extent of elected species.
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.
Claim 1, 2, 9-11, 16-18 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.
(i) Claim 1 recites “A method of preventing and/or treating acute respiratory distress syndrome and/or acute lung injury, comprising administering an effective amount of a composition to a subject in need thereof, wherein the composition comprises a cell-free fat extract: wherein ---”. The metes and bounds of “a subject in need thereof” is unclear in the context of “preventing acute respiratory distress syndrome and/or acute lung injury”. It is unclear what criteria are used for determination who which subject is “in need of” “preventing acute respiratory distress syndrome and/or acute lung injury”.
Additionally, claim 1 recites “(3) for the layered mixture, the upper oil layer and the lower water layer are removed, and collecting the intermediate layer, i. e. the fat layer containing fat cells; (4) emulsifying the intermediate layer to obtain an emulsified fat mixture;(5) centrifuging the emulsified fat mixture, thereby obtaining an intermediate liquid layer, i.e. a fat primary extract; in the step (5), after centrifugation, the emulsified fat mixture is layered into four layers, the first layer is an oil layer, the second layer is a residual fatty tissue layer, the third layer is a liquid layer, i. e., the intermediate liquid layer, and the fourth layer is a cell/tissue debris precipitation layer”. It is unclear whether the limitation recited after “i. e.” is identical to or an example of the limitation recited before “i. e.”.
Claims 2, 9-11, 16-18 depend from claim 1.
(ii) Claim 9 recites the limitation "the composition or preparation" in the context of “The method of claim 1, wherein the composition or preparation is in the form of an oral preparation, an topical preparation or an injection preparation”. There is insufficient antecedent basis for this limitation "the preparation" in the claim because claim 1 recites “the composition comprises a cell-free fat extract”.
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, 2, 9-11, 16-18 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.
Claim 1 recites “A method of preventing and/or treating acute respiratory distress syndrome and/or acute lung injury, comprising administering an effective amount of a composition to a subject in need thereof, wherein the composition comprises a cell-free fat extract: wherein ---”. The specification does not provide any description regarding determination of and selection of “a subject in need thereof” in the context of “preventing acute respiratory distress syndrome and/or acute lung injury”. Does “a subject in need of” “preventing acute respiratory distress syndrome and/or acute lung injury” require any undisclosed physiological evaluation and/or genetic and molecular screening of certain phenotype/markers to be considered “a subject in need of preventing acute respiratory distress syndrome and/or acute lung injury”. The specification does any written description in this regard.
Claims 2, 9-11, 16-18 depend from claim 1.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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 1, 2, 9-11, 16 and 18 are rejected under 35 U.S.C. 103 as being unpatentable Yu et al. (2018) (Yu et al., Fat extract promotes angiogenesis in a murine model of limb ischemia: a novel cell-free therapeutic strategy, Stem Cell Res Ther. 2018 Nov 8;9(1):294. doi: 10.1186/s13287-018-1014-y; NPL #2 of IDS filed by Applicants on 12/08/2022) in view of Harrell et al. (2019) (Harrell et al., Mesenchymal Stem Cell-Based Therapy of Inflammatory Lung Diseases: Current Understanding and Future Perspectives, Stem Cells Int. 2019 May 2:2019:4236973. doi: 10.1155/2019/4236973. eCollection 2019), and Hahn et al., 2006 (Hahn et al., Airway epithelial cells produce neurotrophins and promote the survival of eosinophils during allergic airway inflammation, J Allergy Clin Immunol. 2006 Apr;117(4):787-94. doi: 10.1016/j.jaci.2005.12.1339. Epub 2006 Feb 21) and
Regarding, limitations recited in claims 1, 2, and 9, Yu et al. (2018) teaches that “Background: The proangiogenic capacity of adipose tissue and its derivatives has been demonstrated in a variety of studies. The paracrine mechanism of the cellular component is considered to play a critical role in the regenerative properties of these tissues. However, cell-based therapy for clinical application has been hindered by limitations such as safety, immunogenicity issues, and difficulties in cell preservation, transportation, and phenotype control. In the current study, we aimed to produce a cell-free extract directly from human fat tissue and evaluate its potential therapeutic efficacy. Methods: We developed a novel physical approach to produce a cell-free aqueous extract from human fat tissue (fat extract (FE)). The therapeutic potential of FE was investigated in the ischemic hindlimb model of nude mice. After establishment of hindlimb ischemia with ligation of the left femoral artery and intramuscular injection of FE (which reads on an injection preparation recited in claim 9), blood perfusion was monitored at days 0, 7, 14, 21, and 28. Tissue necrosis and capillary density were evaluated. Enzyme-linked immunosorbent assay was used to analyze the growth factors contained in FE. Moreover, the proliferation, migration, and tube formation ability were tested on human umbilical vein endothelial cells (HUVECs) in vitro when treated with FE. The proangiogenic ability of FE was further assessed in an in-vivo Matrigel plug assay. Results: FE was prepared and characterized. The intramuscular injection of FE into the ischemic hindlimb of mice attenuated severe limb loss and increased blood flow and capillary density of the ischemic tissue. Enzyme-linked immunosorbent assay showed that FE contained high levels of various growth factors. When added as a cell culture supplement, FE promoted HUVEC proliferation, migration, and tube formation ability in a dose-dependent manner. The subcutaneous injection of Matrigel infused with FE enhanced vascular formation (which reads on “increasing blood oxygen level” recited in (i) of claim 2). Conclusions: We developed a novel cell-free therapeutic agent, FE, produced from human adipose tissue. FE was able to attenuate ischemic injury and stimulate angiogenesis in ischemic tissues. This study indicates that FE may represent a novel cell-free therapeutic agent in the treatment of ischemic disorders (See Abstract).
Regarding limitation “the cell-free fat extract is a cell-free fat extract obtained from fat tissue of human; and the cell-free fat extract is prepared by the following method --- recited in claim 1, and limitations recited in claims 16 and 18, Yu et al. teaches that “The detailed procedures for isolating FE are shown in Fig. 1.
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The lipoaspirate was first rinsed with saline to remove red blood cells and then centrifuged at 1200 × g for 3 min. After the first spin, the superior oily and inferior fluid layers were discarded, and the middle fat layer was collected and mechanically emulsified. The emulsification was achieved via 30 passes of shifting the fat between two 10-cm3 syringes (recited in instant claim 16) connected by a female-to-female Luer-Lok connector (B. Braun Medical Inc., Melsungen, Germany). The emulsified fat was then frozen at − 80 °C and thawed at 37 °C for further disruption of the fat tissue. After one cycle of the freeze/thaw process (recited in instant claim 18), the fat was again centrifuged at 1200 × g for 5 min. After a second spin, the fat was separated into four layers. The upper layer of oil was discarded; the second layer of unbroken fat and the fourth layer of debris was discarded; and the third aqueous layer, namely the FE, was carefully aspirated without contamination of the bottom pellet. The final extract was produced by passing it through a 0.22-μm filter (Corning Glass Works, Corning, NY, USA) for sterilization and removal of cell debris. The extract was then stored at − 20 °C for future use. The protein concentrations of FE were measured with a Pierce BCA protein assay kit (Thermofisher Scientific, Waltham, MA, USA (See bridging paragraph from left to right column, page 3 of 14).
Consistent with the steps of obtaining a cell-free fat extract from fat tissue of human described in the preceding paragraph, Yu et al. further teaches that “The procedure for generating FE follows the nanofat processing procedure described by Tonnard et al. with some modifications. To obtain a more condensed fat emulsion, we first centrifuged the lipoaspirate before emulsification to remove the watery content. After the mechanical emulsification, the emulsified fat then underwent a freeze/thaw cycle to further lyse the tissues/cells. This freeze/thaw procedure makes it easier to separate the liquid portion from oil droplets and enhances (approximately 30%) the FE yield, but does not change the concentration of growth factors within the FE (data not shown). Thus, this procedure could be omitted for the urgent use of FE in the clinical setting. The final procedure consisted of passing the extract through a 0.22-μm filter to remove the residual cells and cell debris as well as the unexpected contaminating bacteria during processing, which resulted in a pure cell-free, bacteria free extract that could be safely used in the clinical setting. The whole procedure is relatively simple and safe, as only physical methods were used to break adipose tissue and separate components; no enzymes and chemical reagents were involved during the entire process. For those who later may need multiple injections, cryopreserved FE at − 20 °C did not compromise the proangiogenic effects when used within 6 months, which was evaluated in this study. In addition, no cryoprotectant is required during storage. However, the long-term preservation of FE requires future investigations. In the current study, human liposuction aspirates were obtained from healthy female donors aged 24–36 years who underwent liposuction. Unfortunately, we have not measured the body mass index of each donor. It is possible that the composition of FE maybe different from individuals with different age, gender, and body mass index. It is worth comparing this composition in future studies (See bridging paragraph from left to right column, page 12 of 14).
Regarding claims 10 and 11, Yu et al. teaches mean fat extract growth factor concentration (pg/ml) of BDNF is 1860.99 (See Table 1).
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Yu et al. does not explicitly teach (i) “treating acute respiratory distress syndrome and/or acute lung injury”, recited in claim 1, by administering an effective amount of a composition comprising a cell-free fat extract; (ii) the functional characteristics of growth factor BDNF in the underlying etiology and molecular pathology of “acute respiratory distress syndrome and/or acute lung injury”.
(i) Regarding the limitation “treating acute respiratory distress syndrome and/or acute lung injury”, Harrell et al. (2019) teaches that “During acute or chronic lung injury, inappropriate immune response and/or aberrant repair process causes irreversible damage in lung tissue and most usually results in the development of fibrosis followed by decline in lung function. Inhaled corticosteroids and other anti-inflammatory drugs are very effective in patients with inflammatory lung disorders, but their long-term use is associated with severe side effects. Accordingly, new therapeutic agents that will attenuate ongoing inflammation and, at the same time, promote regeneration of injured alveolar epithelial cells are urgently needed. Mesenchymal stem cells (MSCs) are able to modulate proliferation, activation, and effector function of all immune cells that play an important role in the pathogenesis of acute and chronic inflammatory lung diseases. In addition to the suppression of lung-infiltrated immune cells, MSCs have potential to differentiate into alveolar epithelial cells in vitro and, accordingly, represent new players in cell-based therapy of inflammatory lung disorders. In this review article, we described molecular mechanisms involved in MSC-based therapy of acute and chronic pulmonary diseases and emphasized current knowledge and future perspectives related to the therapeutic application of MSCs in patients suffering from acute respiratory distress syndrome, pneumonia, asthma, chronic obstructive pulmonary diseases, and idiopathic pulmonary fibrosis (See Abstract).
Harrell et al. (2019) further teaches that “The respiratory system is continuously exposed to various irritants such as inhaled toxins, carbon granules, pathogens, and their products. Pulmonary homeostasis is maintained by interaction between alveolar epithelial cells and lung resident immune cells that continually monitor the pulmonary microenvironment, induce tolerance to innocuous inhaled particles, or provide efficient immune reactions against invading microbes. Accordingly, in the lungs, inflammation is the result of the infection, trauma, and hypersensitivity caused by pathogens, airborne irritants, hazardous pollutants, toxins, and allergens. Pathogen-associated molecular patterns (PAMPs) expressed on the lung infiltrated microbes, as well as damage-associated molecular patterns (DAMPs) and alarmins, released from the injured lung parenchymal cells, activate residential macrophages which produce a large amount of inflammatory chemokines and cytokines, attract circulating immune cells in the lungs, and initiate inflammation. Clinically, acute lung injury and inflammation is seen in pneumonia and acute respiratory distress syndrome (ARDS), whereas chronic inflammation is represented by asthma and chronic obstructive pulmonary diseases (COPD). The repair of the airway epithelium after acute or chronic injury is modulated by matrix metalloproteinases (MMPs), cytokines, and growth factors produced by epithelial cells, lung-resident immune cells, fibroblasts, and chondrocytes (See Introduction, bridging paragraph, pages 1-2).
(ii) Relevant to the functional characteristics of growth factor BDNF in the underlying etiology and molecular pathology of “acute respiratory distress syndrome and/or acute lung injury”, Hahn et al. (2006) teaches that “Background: Eosinophil-epithelial cell interactions make a major contribution to asthmatic airway inflammation. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and other members of the neurotrophin family, originally defined as a class of neuronal growth factors, are now recognized to support the survival and activation of immune cells. Neurotrophin levels are increased in bronchoalveolar lavage fluid during allergic asthma. Objective: We sought to investigate the role of neurotrophins as inflammatory mediators in eosinophil-epithelial cell interactions during the allergic immune response. Methods: Neurotrophin expression in the lung was investigated by means of immunohistochemistry and ELISA in a mouse model of chronic experimental asthma. Coculture experiments were performed with airway epithelial cells and bronchoalveolar lavage fluid eosinophils. Results: Neurotrophin levels increased continuously during chronic allergic airway inflammation, and airway epithelial cells were the major source of NGF and BDNF within the
inflamed lung. Epithelial neurotrophin production was upregulated by IL-1b, TNF-a, and TH2 cytokines. Lung eosinophils expressed the BDNF and NGF receptors tropomyosin-related kinase (Trk) A and TrkB, and coculture with airway epithelial cells resulted in enhanced epithelial
neurotrophin production, as well as in prolonged survival of eosinophils. Eosinophil survival was completely abolished in the presence of the neurotrophin receptor Trk antagonist K252a. Conclusion: During allergic inflammation, airway epithelial cells express increased amounts of NGF and BDNF that promote the survival of tissue eosinophils. Controlling epithelial neurotrophin production might be an important therapeutic target to prevent allergic airway eosinophilia. Clinical implications: Attenuating the release of inflammatory mediators from the activated airway epithelium will become an important strategy to disrupt the pathogenesis of chronic allergic asthma. (Hahn et al., J Allergy Clin Immunol 2006;117: 787-94.) (See Abstract).
Hahn et al. (2006) further teaches that “It is well known that eosinophils play an important role in the pathogenesis of allergic airway diseases because they contribute to the initiation and maintenance of the allergic response. They are recruited at allergic inflammatory
sites, and eosinophil accumulation into the inflamed lung is associated with both acute and chronic phases of allergic asthma. The number of eosinophils within the allergic tissue depends primarily on the extent of cell infiltration during the acute inflammatory response (See right column, page 787).
It would have been prima facia obvious for a skilled artisan to combine the teachings of Yu et al. (2018), Harrell et al. (2019), and Hahn et al. (2006) to reach claimed methods with reasonable expectation of success because acute inflammation response is the underlying molecular mechanism that results in the ischemia taught by Yu et al. (2018) (See Fig. 3 for example), acute lung injury and acute respiratory distress syndrome (ARDS) taught by Harrell et al. (2019) (See bridging paragraph, pages 1-2), and allergic airway inflammation taught by Hahn et al. (2006) (See Abstract and Introduction, page 787).
A skilled artisan would have been motivated to combine the teachings of Yu et al. (2018), Harrell et al. (2019), and Hahn et al (2006) because (i) Yu et al. teach that “the intramuscular injection of FE into the ischemic hindlimb of mice attenuated severe limb loss and increased blood flow and capillary density of the ischemic tissue. Enzyme-linked immunosorbent assay showed that FE contained high levels of various growth factors”, including BDNF (See Results, page 1 of 14), (ii) Harrell et al. teach that “Pathogen-associated molecular patterns (PAMPs) expressed on the lung infiltrated microbes, as well as damage-associated molecular pat terns (DAMPs) and alarmins, released from the injured lung parenchymal cells, activate residential macrophages which produce a large amount of inflammatory chemokines and cytokines, attract circulating immune cells in the lungs, and initiate inflammation. Clinically, acute lung injury and inflammation is seen in pneumonia and acute respiratory distress syndrome (ARDS)” (bridging paragraph, pages 1-2), and (iii) Hahn et al. teaches that “During allergic inflammation, airway epithelial cells express increased amounts of NGF and BDNF that promote the survival of tissue eosinophils. Controlling epithelial neurotrophin production might be an important therapeutic target to prevent allergic airway eosinophilia” (See Conclusion, left column, page 787).
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Yu et al. (2018) (Yu et al., Fat extract promotes angiogenesis in a murine model of limb ischemia: a novel cell-free therapeutic strategy, Stem Cell Res Ther. 2018 Nov 8;9(1):294. doi: 10.1186/s13287-018-1014-y) in view of Harrell et al. (2019) (Harrell et al., Mesenchymal Stem Cell-Based Therapy of Inflammatory Lung Diseases: Current Understanding and Future Perspectives, Stem Cells Int. 2019 May 2:2019:4236973. doi: 10.1155/2019/4236973. eCollection 2019), and Hahn et al. (2006) (Hahn et al., Airway epithelial cells produce neurotrophins and promote the survival of eosinophils during allergic airway inflammation, J Allergy Clin Immunol. 2006 Apr;117(4):787-94. doi: 10.1016/j.jaci.2005.12.1339. Epub 2006 Feb 21) as applied to claims 1, 2, 9-11, 16 and 18 above, and further in view of Brasaemle et al. (2016) (Brasaemle et al., Isolation of lipid droplets from cells by density gradient centrifugation. Curr. Protoc. CellBiol. 72:3.15.1-3.15.13. doi: 10.1002/cpcb.10).
The teachings by Yu et al. (2018), Harrell et al. (2019), and Hahn et al (2006) have been documented in the preceding 103 rejection. The combined the teachings of Yu et al. (2018), Harrell et al. (2019), and Hahn et al (2006), and do not explicitly teach the limitation “emulsification is a method of crushing by a tissue homogenizer” recited in claim 17
Brasaemle et al. (2016) teaches that “The isolation of lipid droplets by centrifugation is a relatively simple procedure. A very low protein-to-lipid ratio renders lipid droplets more buoyant than all other subcellular structures; lipid droplets can be separated from more dense subcellular compartments using discontinuous density gradients. The most critical step in the isolation of lipid droplets is making an appropriate choice for the method of cell disruption to keep the droplets intact, particularly when working with cells containing very large lipid droplets. Gentle disruption of cells is required to preserve lipid droplet structure. The use of rotor-stator homogenizers or sonication to lyse cells will disrupt and emulsify lipid droplets. Likewise, the extrusion of cells or isolated lipid droplets through small openings exerts sufficient shear force
to disrupt lipid droplets. Gentle homogenization using a hand-operated homogenizer with
a loose-fitting Teflon pestle, or the use of a nitrogen cavitation bomb, will yield better results (See left column, page 10 of 13).
It would have been prima facia obvious for a skilled artisan to incorporate the teachings of Brasaemle et al. (2016) into the combined teachings of Yu et al. (2018), Harrell et al. (2019), and Hahn et al. (2006), and because mechanical emulsification by passing through syringe taught by Yu et al. and the use of rotor-stator homogenizers or sonication to lyse cells will disrupt and emulsify lipid droplets taught by Brasaemle et al. (2016) are analogous steps for generating fat emulsion from mammalian/human fat tissue.
A skilled artisan would have been motivated to incorporate the teachings of Brasaemle et al. (2016) into the combined teachings of Yu et al. (2018), Harrell et al. (2019), and Hahn et al. (2006), because (i) Yu et al. (2018) teaches that “The lipoaspirate was first rinsed with saline to remove red blood cells and then centrifuged at 1200 × g for 3 min. After the first spin, the superior oily and inferior fluid layers were discarded, and the middle fat layer was collected and mechanically emulsified. The emulsification was achieved via 30 passes of shifting the fat between two 10-cm3 syringes connected by a female-to-female Luer-Lok connector (See left column, page 3 of 14 and Fig. 1), and (ii) Brasaemle et al. (2016) teaches isolation of lipid droplets from cells by density gradient centrifugation (See title), and “Lipid droplet purity may be improved by additional wash steps; transfer the lipid droplet fraction to a microcentrifuge tube, centrifuge 10 min at maximum speed at 4°C, and remove the infranatant from beneath the floating lipid droplet layer using a gel-loading pipet tip or wide-gauge needle attached to a syringe. Resuspend lipid droplets in ice-cold HLM and repeat centrifugation step, as needed” (See step 14, page 3 of 13).
Conclusion
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/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682