Use of emulsifiers and amphiphilic agents to increase yields during cell isolations

Abstract

A method of isolating mature adipocytes from a tissue is disclosed. The disclosed method comprises incubating the tissue in a solution comprising an amphiphilic agent and an enzyme. The amphiphilic agent may be Pluronic F-68 and the enzyme may be collagenase. Methods of culturing mature adipocytes comprising culturing mature adipocytes isolated by one of the methods disclosed herein and methods of differentiating fat cells comprising culturing mature adipocytes according to any one of the methods disclosed herein are also disclosed. A kit comprising an amphiphilic agent and an enzyme is disclosed.

Description

USE OF EMULSIFIERS AND AMPHIPHILIC AGENTS TO INCREASE YIELDS DURING CELL ISOLATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 662,568 that was filed June 21, 2024. The entire contents of which are hereby incorporated by reference.BACKGROUND OF THE INVENTION

[0002] Traditionally, adipose tissue is mechanically broken down to increase its surface area in an attempt to enhance the efficacy of collagenase in degrading the extracellular matrix (ECM) to liberate individual cells from the tissue matrix. These methods frequently result in suboptimal yields when isolating from adipose tissue. Existing technology requires performing repeated isolations until an attempt (by chance) yields sufficient cells because individual isolation attempts vary greatly in cell yield. Therefore, a need in the art exists to provide methods of cell isolation which produces predictable, consistent, and sufficient yield.

[0003] The present disclosure provides methods and uses of isolating cells from tissue through the utilization of emulsifiers and amphiphilic agents during cell isolation processes.BRIEF SUMMARY OF THE INVENTION

[0004] In some aspects, the present disclosure provides a method of isolating mature adipocytes from a tissue, the method comprising incubating the tissue in a solution, wherein the solution comprises an amphiphilic agent and an enzyme. The enzyme may be selected from collagenase, trypsin, and dispase and may be present in the solution at a concentration of about 0.025% to about 1%. The amphiphilic agent may be an emulsifier and may be selected from the group consisting of poloxamers, polysorbates, phospholipids, lecithins, phosphotidylcholine, monoglycerides, diglycerides, esters, succinylated glycerides, ethoxylated glycerides, saponins, sodium dodecyl sulfate, sodium stearoyl lactylate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate), glycerol derivatives, glyceryl monostearate, propylene glycol derivatives, propylene glycol esters, modified fatty acids, octylphenol ethoxylates, emulsan, lauryl fructose, PEG derivatives other than poloxamer, rhamnolipids, sophorolipids, mannosylerythritol lipids,trehalose lipids, alkyl polyglucosides, poloxamer-188 (Pluronic F-68), and polyoxyethyleneorbitan 20. The amphiphilic agent is present in the solution at a concentration of 0.005% to 20% and may be present in the solution at a concentration of 0.1% to 1%.

[0005] In some aspects, the amphiphilic agent is present in the solution at a concentration of at least 1%. The tissue may be incubated in the solution for at least thirty minutes and may be incubated in the solution for at least one hour. The tissue may be incubated in the solution at room temperature or may be incubated in the solution at 37°C. The method may further comprise a filtering step following incubation, and the filtering step may comprise filtering the solution through a 750 micron filter and / or a 300 micron filter. The method may further comprise centrifuging the solution, and the solution may be centrifuged at about 100 x g to about 500 x g.

[0006] In some aspects, the tissue is from an animal selected from the group consisting of bovine, porcine, galline, piscine, hominine, caridean, and homarine.

[0007] In some aspects, the present disclosure provides a method of isolating mature adipocytes from a tissue, the method comprising incubating the tissue in a solution, wherein the solution comprises a poloxamer and collagenase, wherein the poloxamer is present in the solution at a concentration of 1%, and wherein collagenase is present in the solution at a concentration of 0.1%.

[0008] In some aspects, the present disclosure provides a method of culturing mature adipocytes, the method comprising culturing mature adipocytes isolated by a method disclosed herein. The mature adipocytes may be cultured via a ceiling culture technique. The method may comprise culturing the mature adipocytes for at least 4 days. The mature adipocytes may dedifferentiate in the culture.

[0009] In some aspects, the present disclosure provides a kit comprising an amphiphilic agent and an enzyme. The enzyme may be selected from the group consisting of collagenase, trypsin, and dispase. The amphiphilic agent may be an emulsifier, and the emulsifier may be selected from the group consisting of poloxamers, polysorbates, phospholipids, lecithins, phosphotidyl choline, monoglycerides, diglycerides, esters, succinylated glycerides, ethoxylated glycerides, saponins, sodium dodecyl sulfate, sodium stearoyl lactylate, CHAPS (3-[(3- cholamidopropyl)dimethylammonio]-l -propanesulfonate), glycerol derivatives, glyceryl monostearate, propylene glycol derivatives, propylene glycol esters, modified fatty acids, octylphenol ethoxylates, emulsan, lauryl fructose, PEG derivatives other than poloxamer, rhamnolipids, sophorolipids, mannosyl erythritol lipids, trehalose lipids, alkyl polyglucosides,poloxamer-188 (Pluronic F-68), and polyoxyethyleneorbitan 20. The kit may further comprise an antibiotic. The kit may further comprise a 750 micron filter and / or a 300 micron filter.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0011] FIG. 1 shows tubes of fat cell isolates following centrifuge. Tubes 2 and 3 were isolated with a collagenase solution comprising 0.5% and 1%, respectively, of Pluronic F-68 while Tube 1 was isolated without Pluronic F-68. The layer of mature adipocytes obtained at the top of the liquid in tubes 2 and 3 is marked with a black bar to provide an approximation of their height which may be indicative of their overall yield of mature adipocytes.

[0012] FIG. 2 shows tubes of fat cell isolates following centrifuge. From left to right, cells were isolated with a collagenase solution comprising 0.25%, 0.75%, and 1% of Pluronic F-68. The layer of mature adipocytes obtained at the top of the liquid is marked with a black bar to provide an approximation of their height which may be indicative of their overall yield of mature adipocytes. 0.25%, 0.75%, and 1% poloxamer 188 (pluronic F-68) yielded 2.59 mm, 3.17 mm, and 2.88 mm bands of mature adipocytes in a 50 ml conical tube, respectively.

[0013] FIG. 3 shows cell growth of immortalized bovine dedifferentiated fat cells isolated using the disclosed technology. The immortalized bovine dedifferentiated fat (DFAT) cells were isolated using the disclosed technology. The cells were then cultured for 100 days in a growth medium composed of DMEM / F12, 2.5% Fetal Bovine Serum (FBS), bovine Fibroblast Growth Factor 2 (FGF2), and Platelet-Derived Growth Factor BB (PDGF-BB). During this period, cells were seeded at a density between 5,000 to 15,000 cells / cm2and were passaged upon reaching 70-90% confluency. Cell counts were performed during each passage using a Chemometec NC-202 automated cell counter with Via-2 cassettes, which use a DNA stain-based method (DAPI and acridine orange) to quantify the cell population. This long-term culture demonstrated that the isolated cells are vigorous and capable of sustained growth and expansion, achieving approximately 80 cell doublings over the 100-day period.

[0014] FIGs. 4A, 4B, and 4C show (A) Three separate primary cell isolations from bovine adipose tissue. For each tube, 4 - 4.5 g of bovine fat tissue from an anesthetized, live cow biopsywas minced and combined with collagenase (with or without poloxamer 188), then agitated for 1 .5 hours). (B) The results of three primary mature adipocyte isolations after centrifugation and cell straining to isolate cells from residual tissue. A layer of mature adipocytes is visible in the samples treated with an emulsifier, poloxamer 188. 0% poloxamer 188 yielded insufficient mature adipocytes to be detected by the naked eye. In contrast, 0.5% poloxamer 188 yielded a 3.5 mm band of mature adipocytes in a 50 ml conical tube and 1% poloxamer 188 yielded a 4.14 mm band of mature adipocytes in a 50 ml conical tube. (C) Inverted cell culture flask of buoyant mature adipocytes for de-differentiation into DFAT stem cells.

[0015] FIG. 5 shows a schematic of existing adipose cell isolation methods which suffer from significant drawbacks.

[0016] FIGs. 6A and 6B show isolations of (A) bovine DFAT cells with 0.25% (left), 0.5% (center) and 1.5% (right) poloxamer 188 and (B) porcine DFAT cells with 0.75% (left) and 1.5% (right) poloxamer 188.

[0017] FIG. 7 shows a schematic of one embodiment of the disclosed methods and articles of manufacture.

[0018] FIG. 8 shows Passage 2 bovine dedifferentiated fat cells in culture, isolated using collagenase + poloxamer 188.

[0019] FIG. 9 shows emulsifier-isolated bovine DFAT cell growth over 9 days (Passage 5 to 8). Cells were cultured in media with 2.5% FBS or 0.4 mg / ml rapeseed protein isolate (RPI). Cell doublings were quantified heuristically via cell confluency at the beginning and end of each passage using ilastik, a publicly available open source software plugin for cell counting.DETAILED DESCRIPTION OF THE INVENTION

[0020] Before the present invention is described in further detail, it is to be understood that the invention is not limited to the particular embodiments described. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The scope of the present invention will be limited only by the claims. As used herein, the singular forms "a", "an", and "the" include plural embodiments unless the context clearly dictates otherwise.

[0021] It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpretingthis disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as "comprising" certain elements are also contemplated as "consisting essentially of' and "consisting of' those elements. When two or more ranges for a particular value are recited, this disclosure contemplates all combinations of the upper and lower bounds of those ranges that are not explicitly recited. For example, recitation of a value of between 1 and 10 or between 2 and 9 also contemplates a value of between 1 and 9 or between 2 and 10. Further, as used herein, ranges that are between two particular values should be understood to expressly include those two particular values. For example, “between 0 and 1” means “from 0 to 1” and expressly includes 0 and 1 and anything falling inside these values. Also, as used herein “about” means ±20% of the stated value, and includes more specifically values of ±10%, ±5%, ±2%, ±1%, and ±0.5% of the stated value.Methods of isolating cells

[0022] In one aspect, the present disclosure provides a method of isolating cells, e.g., mature adipocytes, from a tissue comprising incubating the tissue in a solution, the solution comprises an amphiphilic agent and an enzyme. The enzyme may preferably be chosen based on its ability to break down tissue and / or extracellular matrix. Enzymes may include proteases. Other enzymes may not target peptides or proteins, such as hyaluronidase.

[0023] As used herein, the term “isolating” and grammatical variants thereof may refer to a process by which a material is substantially or essentially freed from components that normally accompany it in its native state. For example, isolation of fat cells may include separating the fat cells from the tissue in which it is native. As used herein, the term “mature adipocytes” may refer to terminally differentiated fat cells and may be used interchangeably with “fat” or “fat cells.”

[0024] As used herein, the term “tissue” may refer to a group or groups of cells isolated from an animal.

[0025] As used herein, the term “incubating” and grammatical variants thereof may refer to the maintenance cells or tissue at determined conditions to ensure their growth and / or survival.

[0026] As used herein, the term “solution” may refer to a mixture that is made up of a solute dissolved within a solvent.

[0027] As used herein, the term “amphiphilic agent” may refer to compounds that possess both hydrophilic and lipophilic properties.

[0028] As used herein, the term “enzyme” may refer to proteins capable of breaking down chemical bonds which may be peptide bonds (for example, collagenase which is capable of breaking collagen peptide bonds).

[0029] The inventors found that incorporating an amphiphilic agent such as an emulsifier into an enzyme solution significantly improves the enzyme’s access to the hydrophobic components of tissues which is a critical step for maximizing cell yield in cell isolation protocols. In Examples provided herein, the inventors demonstrate this phenomenon using Pluronic F-68 (which may be referred to as “poloxamer 188”) and collagenase in isolating mature adipocytes from tissue.

[0030] Other suitable pol oxamers may include but are not limited to poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 212, poloxamer 215, poloxamer217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, poloxamer 407, poloxamer105 benzoate, and poloxamer 182 dibenzoate. The poloxamer may be present at or used at about 0.005% (w / v) to about 1.5% (w / v) or any subrange or value therein. The poloxamer may be present at or used at about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, or more.

[0031] While experiments have primarily utilized Poloxamer 188 (Pluronic F-68), other emulsifying agents are expected to work by the same mechanism. The central hypothesis is that the low yield in traditional adipose tissue isolation is due to the inability of the aqueous enzyme solution to efficiently penetrate the hydrophobic fat tissue. An amphiphilic agent, or emulsifier, possesses both hydrophilic (water-attracting) and lipophilic (fat-attracting) properties. This dual nature allows it to act at the interface between the aqueous enzyme solution and the oily tissue, breaking down the lipid barrier. This emulsification process increases the surface area of the fat exposed to the enzymes, allowing them to more effectively access and degrade the extracellularmatrix to liberate the embedded cells. Therefore, any agent with these amphiphilic properties, such as the listed polysorbates (e.g., Tween), phospholipids, lecithins, and others, would be expected to produce a similar increase in cell yield by facilitating the interaction between the digestive enzymes and the hydrophobic tissue components.

[0032] In some aspects, the enzyme is selected from the group consisting of collagenase, trypsin, dispase, hyaluronidase, elastase, papain, and DNAse, and the enzyme may, preferably, be collagenase.

[0033] Although the working examples focus on the use of collagenase, the method is applicable to other enzymes used for tissue dissociation. The function of the enzyme in this process is to degrade the proteins of the extracellular matrix (ECM) that hold the tissue together, freeing the cells for collection and downstream use in cell culture. Collagenase is particularly effective for adipose tissue because collagen is a primary structural component of its ECM. However, other proteases that target ECM components are also commonly used in primary cell isolation and would be expected to benefit from the improved access provided by the emulsifier. For instance, Dispase, which cleaves fibronectin and type IV collagen, and Trypsin, a less specific but potent protease, are viable alternatives for disrupting cell-matrix and cell-cell adhesions. The enzymes may be used singly, or in any combination.

[0034] In some aspects, the enzyme is present in the solution at a concentration of 0.025% to 1%. In some aspects, the enzyme is present in the solution at a concentration of 0.05% to 0.6%. A skilled practitioner may appreciate the inherent variability of enzymatic activity between different enzymes and between batches within the same enzyme. Therefore, the concentration of the enzyme may vary depending on the enzyme used and the specific batch of the enzyme used. For example, one collagenase may comprise 150 units per mg wherein one unit releases one micromole of L- leucine equivalents from collagen in 5 hours at 37°C, pH 7.5. Therefore, 0.1% of this exemplary collagenase would be 1 mg / ml, equivalent to about 150 units / ml of collagenase.

[0035] In some aspects, the amphiphilic agent is an emulsifier, and, in some aspects, the emulsifier is selected from the group consisting of poloxamers, polysorbates, phospholipids such as lecithins (e.g., phosphotidylcholine), mono and diglycerides (including derivatives such as esters, succinylated and ethoxylated glycerides), saponins, sodium dodecyl sulfate, sodium stearoyl lactylate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate), glycerol derivatives (e.g., glyceryl monostearate), propylene glycol derivatives (e.g., PG esters), modifiedfatty acids (e.g., esterification with sucrose AKA sucrose esters), octylphenol ethoxylates, emulsan, lauryl fructose, PEG derivatives other than poloxamer, rhamnolipids, sophorolipids, mannosylerythritol lipids, trehalose lipids, and alkyl polyglucosides and may preferably be poloxamer-188 (which may be referred to herein as “Pluronic F-68”) or may preferably be polyoxy ethyleneorbitan 20 (which may be referred to herein as “Tween”). As used herein, the term “emulsifier” may refer to an agent or substance that allows for the emulsification (or mixing) of two liquids that are typically unmixable. In some aspects, the amphiphilic agent is present in the solution at a concentration of about 0.005% (that is 0.005% (w / v); for example 0.05 g / L) to about 20%. In some aspects, the amphiphilic agent is present in the solution at a concentration of about 0.5% to about 1%. In some aspects, the amphiphilic agent is present in the solution at a concentration of at least 1%. The amphiphilic agent may be present at or used at about 0.005% (w / v) to about 1.5% (w / v) or any subrange or value therein. The amphiphilic agent may be present at or used at about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, or more.

[0036] The inventors demonstrated this phenomenon occurred with an incubation as little as one hour. Therefore, in some aspects, the tissue is incubated in the solution for at least thirty minutes, and, in some aspects, the tissue is incubated in the solution for at least one hour. The tissue may be incubated with the amphiphilic agent for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes, or more. The tissue may be incubated for about 1 minute to about 60 minutes, or any subrange or value therein.

[0037] In some aspects, the tissue is incubated in the solution at room temperature, whereas, in some aspects, the tissue is incubated in the solution at about 37°C. In some aspects, the method further comprises a filtering step following incubation, and, in some aspects, the filtering step comprises filtering the solution through a 750 micron filter and / or a 300 micron filter. As used herein, the term “filtering step” and grammatical variants thereof may refer to processes by which impurities or unwanted materials are removed from a solution. As used herein, the term “following” and grammatical variants thereof may refer to a time period immediately after or an extended period of time after (for example, a filtering step following incubation may occur at the end of the one hour of incubation or the filtering step may occur several minutes, hours, or days after the incubation step). In some aspects, the method further comprises centrifuging the solution, and, in some aspects, the solution is centrifuged at about 100 x g to about 500 x g, or any subrange or value therein. As used herein, the term “centrifuging” and grammatical variants thereof may refer to a mechanical process which involves the use of the centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed.

[0038] In some aspects, the tissue is from an animal selected from the group consisting of bovine, porcine, galline, piscine, hominine, caridean, and homarine. The tissue may be bovine tissue, e.g., isolated from bovid raised for meat production. The tissue may be human tissue, e.g., human adipose tissue, which may be obtained from a surgical specimen or a biopsy, for example.

[0039] In another aspect, the present disclosure provides a method of culturing mature adipocytes comprising culturing mature adipocytes isolated by any one of the methods of isolating mature adipocytes from a tissue disclosed herein.

[0040] As used herein, the term “culturing” and grammatical variants thereof may refer to the process of growing cells in vitro and may include optimization of conditions for which any particular cell type needs to grow. Fat cells have a predisposition to float in aqueous culture conditions. Therefore, in some aspects, the mature adipocytes are cultured via a ceiling culture technique.

[0041] Ceiling culture is a method that uses the buoyant property of adipocytes by allowing them to adhere to the top inner surface of a culture flask (which is often inverted — that is, wherein the adherent side of the flask is above the nonadherent side of the flask, or “upside down”) which isfilled completely with medium. Culturing mature adipocytes as disclosed herein may dedifferentiate the mature adipocytes into dedifferentiated fat (DFAT) cells. Therefore, the methods of culturing mature adipocytes disclosed herein may be utilized as methods of dedifferentiating mature adipocytes. As used herein, the term “dedifferentiating” and grammatical variants thereof may refer to a transient process by which cells become less specialized and return to an earlier cell state within the same lineage.

[0042] Surface markers for DFAT cells are not well defined and may differ from species to species. However, DFAT cells are generally characterized by the expression of mesenchymal stem cell markers (e.g., platelet derived growth factor receptor (PDGFR)) and the concurrent loss of markers for mature adipocytes, e.g., one or more of peroxisome proliferator-activated receptor gamma (PPARg), fatty acid binding protein (FABP), or fatty acid synthase (FASN). Generally, DFAT cells may be classified by their cellular origin and functional characteristics: they are derived from mature, lipid-filled adipocytes which, through a process like ceiling culture, revert to a less specialized, proliferative, stem-cell-like state.Kits and other articles of manufacture

[0043] In another aspects, the present disclosure provides a kit comprising an amphiphilic agent and an enzyme. In some aspects, the enzyme is selected from the group consisting of collagenase, trypsin, and dispase, and may preferably be collagenase. In some aspects, the amphiphilic agent is an emulsifier. In some aspects, the emulsifier is selected from the group consisting of Pluronic F- 68, Tween, polysorbate, poloxamers, polysorbates, phospholipids such as lecithins (e.g., phosphotidylcholine), mono and diglycerides (including derivatives such as esters, succinylated and ethoxylated glycerides), saponins, sodium dodecyl sulfate, sodium stearoyl lactylate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate), glycerol derivatives (e.g., glyceryl monostearate), propylene glycol derivatives (e.g., PG esters), modified fatty acids (e.g., esterification with sucrose AKA sucrose esters), octylphenol ethoxylates, emulsan, lauryl fructose, PEG derivatives other than poloxamer, rhamnolipids, sophorolipids, mannosylerythritol lipids, trehalose lipids, and alkyl polyglucosides, and may preferably be Pluronic F-68. In some aspects, the kit further comprises an antibiotic. As used herein, the term “antibiotic” may refer to an agent which inhibits growth of at least one microorganism. In some aspects, the kit further comprises a 750 micron filter and / or a 300 micron filter.

[0044] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0045] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.EXAMPLESExample 1: Incorporating emulsifiers into collagenase-based mature adipocyte isolation increases yield

[0046] There is often a close-to-zero yield of mature adipocytes during cell isolation from animal adipose tissues. This presents a significant bottleneck for research and applications requiring high- quality mature adipocytes, with unsuccessful isolations translating into significant amounts of wasted resources and time. Each dedifferentiated fat cell (DFAT) isolation attempt to obtain mature adipocytes can require over a week of work.

[0047] Traditionally, adipose tissue is mechanically broken down to increase its surface area in an attempt to enhance the efficacy of collagenase in degrading the extracellular matrix (ECM) to liberate individual cells from the tissue matrix. While effective to a degree, these methods frequently result in suboptimal yields when isolating from adipose tissue, due to the difficulty of the aqueous enzyme solution reaching the interior of the hydrophobic / oily / lipophilic fat tissue. In our previous experience, our method of overcoming this limitation involved performing repeated isolations until an attempt (by chance) yielded sufficient cells (individual isolation attempts have been found to vary greatly in cell yield).

[0048] We found that incorporating an emulsifier, such as Pluronic F-68, into the collagenase solution significantly improves the collagenase’s access to the hydrophobic components of fattissues which is a critical step for maximizing cell yield, especially for mature adipocytes. This methodology particularly addresses challenges faced during dedifferentiated fat (DFAT) cell isolation protocols, highlighting a substantial improvement in mature adipocyte yield upon the addition of 0.5-1% Pluronic F-68, compared to negligible yields without it (FIG. 1). Further experimentation observed improved mature adipocyte yield upon the addition of 0.25%, 0.75%, or 1.5% Pluronic F-68 (FIG. 2). This suggests that a method of isolating mature adipocytes from animal (e.g., bovine) adipose tissues through the utilization of emulsifiers and amphiphilic agents, (e.g., Pluronic F-68), during the cell isolation process is superior to that without emulsifiers and / or amphiphilic agents.

[0049] METHODS

[0050] Preparation of Materials and Media

[0051] Autoclave all spatulas, scissors, tweezers, and bottles for culture media prior to use. Mix and prepare culture media of choice. Additionally, prepare 100 ml of DPBS containing 10X Antibiotic / Antimycotic (Gibco™ Antibiotic-Antimycotic (100X) (Thermofisher 15240062)), (lOOOU / mL penicillin, 1000 pg / mL streptomycin, 2.5 pg / mL amphotericin B final concentration).

[0052] Collagenase + Emulsifier Preparation

[0053] On the day of isolation, prepare collagenase + emulsifier solutions. For a 0.1% collagenase, 1% Poloxamer 188 solution, make 25 ml by combining 2.5 ml anti-anti, 21.25 ml DMEM / F12, 25 mg of collagenase (e.g., Worthington CLSAFAS), and 1.25 ml of 20% Poloxamer 188. The Poloxamer 188 solution is created by dissolving Poloxamer 188 powder in water, followed by 0.22 pm sterile filtration.

[0054] Tissue Processing and Digestion

[0055] Transport bovine adipose tissue to the lab at room temperature. Lightly spray the tissue exterior with 70% ethanol and place it in a large petri dish in a biosafety cabinet. Pour DPBS 10X Anti / Anti over the tissue. Cut 4-5 g of adipose tissue, avoiding connective fascia. Weigh the amount of tissue collected by placing in a pre-weighed 50 ml conical tube and transferring from biosafety cabinet to a weighing scale. Mince the tissue with scissors and / or a scalpel into a pastelike consistency. Transfer minced tissue sample to the collagenase + emulsifier solution, using thesolution to rinse the petri dish. Incubate the tube horizontally at 37°C for 1.5 hours in a shaking incubator at 200 rpm.

[0056] Filtration and Isolation of Mature Adipocytes

[0057] After digestion, filter the tissue solution through 750 um and then 300 um cell strainers, using a serological pipette to scrape the mesh and assist liquid passage. Centrifuge the filtered solution at 400 x g for 5 minutes. During centrifugation, prepare a 15 ml conical tube containing 10 ml of culture media. Collect the floating mature adipocytes from the top the centrifuged tube and transfer into the 15 ml tube. Centrifuge the collected adipocytes again at 400 x g for 5 minutes. If a substantial cell pellet remains after the second centrifugation, perform an additional wash. The fat-like layer on top of the centrifuged culture media contains mature adipocytes.

[0058] DISCUSSION

[0059] The addition of an emulsifier or other amphiphilic agent to enhance enzyme access to hydrophobic fat tissues significantly improves the isolation yield of mature adipocytes. This approach can drastically reduce the failure rate of adipocyte isolation due to lack of yield down to ~0%. This would prove invaluable in at least academic and commercial research laboratories focused on adipose tissue biology, obesity, diabetes, and other metabolic diseases. Additionally, the development of cell therapy and regenerative medicine solutions, as well as cultured meat products, could significantly benefit from this technology.Example 2: Dedifferentiating isolated mature adipocytes

[0060] Stem cell therapy is the most promising treatment option for regenerative medicine. Therapeutic effect of different stem cells has been verified in various disease model. Dedifferentiated fat (DFAT) cells, derived from mature adipocytes, are induced pluripotent stem cells. Compared with ASCs and other stem cells, the DFAT cells have unique advantageous characteristics in their abundant sources, high homogeneity, easily harvest and low immunogenicity. The DFAT cells have shown great potential in tissue engineering and regenerative medicine for the treatment of clinical problems such as cardiac and kidney diseases, autoimmune disease, soft and hard tissue defect (Liang, et al, 2023). Further, per Yuen Jr, et al, (2023), cultivated meat (also cultured, cell-cultured, in vitro meat) uses tissue engineering to produce meat for food (Datar and Betti, 2010; Post, 2012; Post and Hocquette, 2017). Canonically,this research has focused on recreating the muscle component of meat (Post, 2012; Benjaminson et al., 2002; Simsa et al., 2019; Ben-Arye et al., 2020; Furuhashi et al., 2021; Verbruggen et al., 2018). However, fat is key to the taste and texture of meat (He et al., 2020; Moritz et al., 2015; Frank et al., 2016; Frank et al., 2017). For example, peak evaluation scores were achieved with beef samples containing -36% crude fat (lida et al., 2015). Hundreds of volatile compounds are released when meat is cooked, with a majority originating from lipids (Frank et al., 2017; Ba et al., 2012; Mottram et al., 1982). Fat is also responsible for the species-specific flavor of meat, making it important for reproducing the flavors of specific animals (Ramalingam et al., 2019). In addition to cultivated meat, in vitro-grown fat could also be used to enhance existing plant-based meats, as complex species-specific flavors of animals are difficult to recapitulate de novo (Fish et al., 2020; van Vliet et al., 2021; Bohrer, 2019).

[0061] However, the process of dedifferentiating mature adipocytes into DFAT cells proves difficult when considering the difficulty in isolating mature adipocytes during cell isolation from animal adipose tissues, presenting a significant bottleneck for research and applications requiring high-quality mature adipocytes, with unsuccessful isolations translating into significant amounts of wasted resources and time. However, by first ensuring the successful isolation of mature adipocytes using improved methods described at least in Example 1, the bottleneck can be bypassed.

[0062] METHODS

[0063] Pre-Plating of Mature Adipocytes

[0064] Transfer the lipid / adipocyte layer from the twice-centrifuged cell suspension (obtained from a previous isolation protocol such as that described in Example 1) into a T12.5 tissue culture flask. Incubate the flask at 37°C for 2 days to allow attachment of contaminating stromal vascular (SV) cells. After 2 days, proceed to the next step.

[0065] Initiation of Ceiling Culture

[0066] On day 2 , fill a new T12.5 flask to 75% capacity with culture media. Transfer the lipid / mature adipocyte fraction from the pre-plated flask into the new T12.5 flask. Subsequently, fill the flask completely with culture media, ensuring no air bubbles remain at the top surface. Gently flip the flasks upside down, allowing the liquid to wash the flask bottom and sides,collecting the mature adipocytes. Transfer the filled T12.5 flasks to a 37°C incubator and maintain them in the inverted position, using another flask or petri dish for leveling if necessary.

[0067] Monitoring and Further Elimination of Contaminating Cells

[0068] On day 4, examine the 100% filled flask under a microscope to identify lipid-laden, attached cells. Document the locations of any non-lipid holding, contaminating cells. If contaminating cells are present, proceed with cell surgery in a biosafety cabinet. Using a pipette tip or equivalent instrument, gently scrape areas containing contaminating cells to detach them. Rinse the flasks 3 times with DPBS or culture media. Verify under the microscope that all contaminating cells have been removed; if not, repeat the scraping and rinsing process. Refill the T12.5 flasks completely and continue the ceiling culture until day 7.

[0069] DFAT Cell Maintenance and Passaging

[0070] On day 7, which marks 5 days of ceiling culture, flip the flasks back to their regular upright position. Refresh the T12.5 flask with 1.5 ml of new media. The cells present should be DFAT cells. Monitor the cells and passage them when they reach 80-100% confluency or if dense colonies start to form. Use standard cell culture techniques for passaging and maintaining the DFAT cells.

[0071] DISCUSSION

[0072] The addition of an emulsifier or other amphiphilic agent to enhance enzyme access to hydrophobic fat tissues significantly improves the isolation yield of mature adipocytes which, in turn, significantly eases the burden of the practitioner to provide input at step 1 of the above methods. This would prove invaluable in at least academic and commercial research laboratories focused on adipose tissue biology, obesity, diabetes, and other metabolic diseases. Additionally, the development of cell therapy and regenerative medicine solutions, as well as cultured meat products, could significantly benefit from this technology.

[0073] Additionally, one may consider mature adipocytes’ propensity to float in aqueous culture. Therefore, one may consider employing ceiling culture techniques when culturing isolated mature adipocytes using the methods disclosed herein (Sugihara et al., 1986).

[0074] REFERENCES

[0075] Ba HV, Hwang I, Jeong D, Touseef A (2012) Principle of meat aroma flavors and future prospect. Book. IntechOpen.

[0076] Ben-Arye T, Shandalov Y, Ben-Shaul S, Landau S, Zagury Y, lanovici I, Lavon N, Levenberg S (2020) Textured soy protein scaffolds enable the generation of three-dimensional bovine skeletal muscle tissue for cell-based meat. Nature Food 1 :210-220.

[0077] Benjaminson MA, Gilchriest JA, Lorenz M (2002) In vitro edible muscle protein production system (MPPS): stage 1, fish Acta Astronautica 51:879-889.

[0078] Bohrer BM (2019) An investigation of the formulation and nutritional composition of modern meat analogue products. Food Science and Human Wellness 8:320-329.

[0079] Datar I and Betti M (2010) Possibilities for an in vitro meat production system. Innovative Food Science & Emerging Technologies 11 : 13-22.

[0080] Fish KD, Rubio NR, Stout AJ, Yuen JSK, Kaplan DL (2020) Prospects and challenges for cell-cultured fat as a novel food ingredient. Trends in Food Science & Technology 98:53-67.

[0081] Frank D, Joo ST, Warner R (2016) Consumer acceptability of intramuscular fat. Korean Journal for Food Science of Animal Resources 36:699-708.

[0082] Frank D, Kaczmarska K, Paterson J, Piyasiri U, Warner R (2017) Effect of marbling on volatile generation, oral breakdown and in mouth flavor release of grilled beef. Meat Science 133:61-68.

[0083] Furuhashi M, Morimoto Y, Shima A, Nakamura F, Ishikawa H, Takeuchi S (2021) Formation of contractile 3D bovine muscle tissue for construction of millimetre-thick cultured steak. NPJ Science of Food 5:6.

[0084] He J, Evans NM, Liu H, Shao S (2020) A review of research on plant-based meat alternatives: Driving forces, history, manufacturing, and consumer attitudes. Comprehensive Reviews in Food Science and Food Safety 19:2639-2656.

[0085] lida F, Saitou K, Kawamura T, Yamaguchi S, Nishimura T (2015) Effect of fat content on sensory characteristics of Marbled beef from Japanese black steers. Animal Science Journal = Nihon Chikusan Gakkaiho 86:707-715

[0086] Liang, Z., He, Y., Tang, H. el al. Dedifferentiated fat cells: current applications and future directions in regenerative medicine. Stem Cell Res Ther 14, 207 (2023).

[0087] Moritz MSM, Verbruggen SEL, Post MJ (2015) Alternatives for large-scale production of cultured beef: a review Journal of Integrative Agriculture 14:208-216.

[0088] Mottram DS, Edwards RA, Macfie JHH (1982) A comparison of the flavour volatiles from cooked beef and pork meat systems. Journal of the Science of Food and Agriculture 33:934-944.

[0089] Post MJ (2012) Cultured meat from stem cells: Challenges and prospects. Meat Science 92:297-301.

[0090] Post MJ and Hocquette JF (2017) Chapter 16 - new sources of animal proteins: Cultured meat. Book. In: Purslow PP, editors. New Aspects of Meat Quality. Woodhead Publishing, pp. 425-441.

[0091] Ramalingam V, Song Z, Hwang I (2019) The potential role of secondary metabolites in modulating the flavor and taste of the meat. Food Research International 122: 174-182.

[0092] Simsa R, Yuen J, Stout A, Rubio N, Fogelstrand P, Kaplan DL (2019) Extracellular heme proteins influence bovine myosatellite cell proliferation and the color of cell-based meat. Foods 8:521.

[0093] Sugihara H, Yonemitsu N, Miyabara S, Yun K. Primary cultures of unilocular fat cells: characteristics of growth in vitro and changes in differentiation properties. Differentiation. 1986;31(l):42-9

[0094] van Vliet S, Bain JR, Muehlbauer MJ, Provenza FD, Kronberg SL, Pieper CF, Huffman KM (2021) A metabolomics comparison of plant-based meat and grass-fed meat indicates large nutritional differences despite comparable nutrition facts panels. Scientific Reports 11 : 13828.

[0095] Verbruggen S, Luining D, van Essen A, Post MJ (2018) Bovine myoblast cell production in a microcarriers-based system. Cytotechnology 70:503-512.

[0096] Yuen, John Se Kit Jr, et al (2023) Aggregating in vitro-grown adipocytes to produce macroscale cell-cultured fat tissue with tunable lipid compositions for food applications eLife 12:e82120.Example 3: A kit for improved mature adipocyte isolation

[0097] Incorporating an emulsifier such as Pluronic F-68 into an incubating solution for isolation of mature adipocytes from tissue (which may comprise collagenase) significantly improves the mature adipocyte cell yield. Therefore, a kit comprising components needed to isolate mature adipocytes in said improved fashion would be beneficial.

[0098] Said kits may comprise at least one enzyme. The enzyme will depend on the isolationtarget cell type. In the case of mature adipocytes, the enzyme may preferably be collagenase, but other enzymes such as trypsin and / or dispase may be preferred. Addit6ionally, said kit may comprise at least one amphiphilic agent, and that amphiphilic agent may be an emulsifier. Numerous emulsifiers may be of interest including but not limited to Pluronic F-68, Polyoxyethlenesorbitan (Tween), poloxamers, polysorbates, phospholipids such as lecithins (e.g., phosphotidylcholine), mono and diglycerides (including derivatives such as esters, succinylated and ethoxylated glycerides), saponins, sodium dodecyl sulfate, sodium stearoyl lactylate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate), glycerol derivatives (e.g., glyceryl monostearate), propylene glycol derivatives (e.g., PG esters), modified fatty acids (e.g., esterification with sucrose AKA sucrose esters), octylphenol ethoxylates, emulsan, lauryl fructose, PEG derivatives other than poloxamer, rhamnolipids, sophorolipids, mannosylerythritol lipids, trehalose lipids, and alkyl polyglucosides.

[0099] Other components necessary to isolate mature adipocytes may be present in the kit. Such other components may include but is not limited to antibiotics, buffered saline (such as phosphate buffered saline, for example), tubes (for centrifugation and otherwise), tools for mechanically cutting subject tissue, cell strainers, filters of various sizes (including but not limited to 750 micron and 300 micron filters), flasks (including but not limited to T12.5 flasks), ethanol, and standard cell culture media suitable for use in culturing adipose cells.Example 4: The Use of Emulsifiers and Amphiphilic Agents to Vastly Increase Yield During DFAT Cell Isolations

[0100] The inventors tested three different concentrations of pluronic F-68: 0.25%, 0.75%, 1.5%. The inventors found that at 1.5% performance of the method suffered. At 1.5% the cells were too few to count, while the inventors isolated a starting DFAT population of 94,200 cells from the 0.75% pluronic-containing isolation. The inventors are able to isolate about 5 colonies of DFAT cells in ceiling culture, per 3 g of initial starting adipose tissue.

[0101] While the primary hypothesized mechanism is improved enzyme access by emulsification, Poloxamer 188 is also known for its shear-protective properties in bioreactors and cell handling. It's plausible that this protective effect on potentially fragile mature adipocytes during mechanical agitation also contributes to the increased viable cell yield.

[0102] Incorporating an emulsifier during mature fat cell isolation facilitates collagenase entry into adipose tissue, converting an unreliable process into a reproducible method with high cell yield. Current methods for primary cell isolation primarily rely on mechanical disruption of adipose tissue and the degradation of extracellular matrix (ECM) using enzymes such as collagenase. While effective to a degree, these methods frequently result in low-to-zero yields when isolating from adipose tissue, especially when attempting to isolate mature adipocytes to obtain dedifferentiated fat (DFAT) stem cells. We hypothesized that this is due to the difficulty of aqueous enzyme solutions reaching the interior of hydrophobic fat tissue (FIG. 5).

[0103] To address this challenge, we developed a protocol incorporating an amphiphilic agent (e.g., poloxamer 188, also referred to as Pluronic F-68) into the collagenase solution during our primary cell isolation of mature adipocytes (FIG. 7). The addition of 0.5-1% poloxamer 188 likely functions by sequestering lipids into micelles, effectively removing the hydrophobic barrier that typically impedes enzyme access. This allows enzymes to more readily access and degrade ECM components, freeing the cells within.

[0104] Dedifferentiated fat cells produced from adipose cells isolated by the disclosed methods are vigorous and have the potential for continued growth and expansion, e.g., for at least 100 days and about 80 cell doublings (FIG. 3). Therefore, the disclosed methods represent an improvement to current technologies for isolating mature adipose cells.

Claims

CLAIMSWe claim:

1. A method of isolating mature adipocytes from a tissue, the method comprising incubating the tissue in a solution, wherein the solution comprises an amphiphilic agent and an enzyme.

2. The method of claim 1, wherein the amphiphilic agent is an emulsifier.

3. The method of claim 2, wherein the emulsifier is selected from the group consisting of poloxamers, polysorbates, phospholipids, lecithins, phosphotidylcholine, monoglycerides, diglycerides, esters, succinylated glycerides, ethoxylated glycerides, saponins, sodium dodecyl sulfate, sodium stearoyl lactylate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate), glycerol derivatives, glyceryl monostearate, propylene glycol derivatives, propylene glycol esters, modified fatty acids, octylphenol ethoxylates, emulsan, lauryl fructose, PEG derivatives other than poloxamer, rhamnolipids, sophorolipids, mannosylerythritol lipids, trehalose lipids, alkyl polyglucosides, poloxamer-188 (Pluronic F-68), and poly oxy ethyleneorbitan 20.

4. The method of claim 1, wherein the amphiphilic agent is Pluronic F-68.

5. The method of claim 4, wherein the amphiphilic agent is present in the solution at a concentration of 0.005% to 20%.

6. The method of claim 4, wherein the amphiphilic agent is present in the solution at a concentration of 0.1% to 1%.

7. The method of claim 1, wherein the amphiphilic agent is present in the solution at a concentration of at least 1%.

8. The method of any one of the preceding claims, wherein the tissue is incubated in the solution for at least about thirty minutes to about 120 minutes.

9. The method of claim 8, wherein the tissue is incubated in the solution for at least about 60 minutes to about 120 minutes.

10. The method of claim 1, wherein the tissue is incubated in the solution at room temperature or at 37 degrees C.

11. The method of claim 4, wherein the tissue is comprises adipose tissue.

12. The method of claim 1, wherein the enzyme is selected from the group consisting of collagenase, trypsin, and dispase.

13. The method of claim 2, wherein the enzyme is collagenase.

14. The method of any one of the preceding claims, wherein the enzyme is present in the solution at a concentration of about 0.025% to about 1%.

15. The method of claim 1, further comprising a filtering step following incubation.

16. The method of claim 15, wherein the filtering step comprises filtering the solution through a 750 micron filter and / or a 300 micron filter.

17. The method of any one of the preceding claims, further comprising centrifuging the solution.

18. The method of claim 17, wherein the solution is centrifuged at about 100 x g to about 500 x g.

19. The method of any one of the preceding claims, wherein the tissue is from an animal selected from the group consisting of bovine, porcine, galline, piscine, hominine, caridean, and homarine.

20. A method of isolating mature adipocytes from a tissue, the method comprising incubating the tissue in a solution, wherein the solution comprises a poloxamer and collagenase, wherein the poloxamer is present in the solution at a concentration of about 1%, and wherein collagenase is present in the solution at a concentration of about 0.1%.

21. A method of culturing mature adipocytes, the method comprising culturing mature adipocytes isolated by the method of any one of the preceding claims.

22. The method of claim 21, wherein the mature adipocytes are cultured via a ceiling culture technique.

23. The method of claim 21, wherein the mature adipocytes are cultured for at least 4 days.

24. The method of claim 21, wherein the mature adipocytes dedifferentiate in the culture.

25. A kit comprising an amphiphilic agent and an enzyme.

26. The kit of claim 25, wherein the enzyme is selected from the group consisting of collagenase, trypsin, and dispase.

27. The kit of claim 26, wherein the enzyme is collagenase.

28. The kit of claim 25, wherein the amphiphilic agent is an emulsifier.

29. The kit of claim 28, wherein the emulsifier is selected from the group consisting of poloxamers, polysorbates, phospholipids, lecithins, phosphotidylcholine, monoglycerides, diglycerides, esters, succinylated glycerides, ethoxylated glycerides, saponins, sodium dodecyl sulfate, sodium stearoyl lactylate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate), glycerol derivatives, glyceryl monostearate, propylene glycol derivatives, propylene glycol esters, modified fatty acids, octylphenol ethoxylates, emulsan, lauryl fructose, PEG derivatives other than poloxamer, rhamnolipids, sophorolipids, mannosylerythritol lipids, trehalose lipids, alkyl polyglucosides, poloxamer-188 (Pluronic F-68), and poly oxy ethyleneorbitan 20.

30. The kit of claim 29, wherein the emulsifier is Pluronic F-68.

31. The kit of any one of claims 25-30, further comprising an antibiotic.

32. The kit of any one of claims 25-31, further comprising a 750 micron filter and / or a300 micron filter.

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