Compositions and methods for stable trypan blue solutions

Abstract

Compositions, methods, and kits for stabilizing trypan blue solutions are provided. In one example, the method includes cooling the trypan blue solution and filtering the cooled solution. In another example, the method includes mixing a water-soluble polymer with the trypan blue solution. The method may also include adding one or more components, such as an aqueous buffer, an osmotic agent, an acid, a base, a buffer, a cell culture medium, water, or a combination thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application was filed as a PCT international patent application on December 3, 2021, and claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 120,957, entitled “TRYPAN BLUE SOLUTIONS,” filed on December 3, 2020, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0003] The present disclosure generally relates to solutions comprising trypan blue, kits and methods for preparing trypan blue solutions, methods for stabilizing trypan blue solutions, and methods of using trypan blue solutions.

[0004] Trypan blue is an azo dye with a wide range of applications from textiles to biosciences. Trypan blue is commonly used to determine cell viability in dye exclusion tests. When trypan blue is used in dye exclusion tests, cells with intact cell membranes exclude the trypan blue dye and are not stained. This unstained state is considered to be an indicator of cell viability. In contrast, cells with damaged cell membranes allow dye uptake and are stained blue. This stained state is considered to be an indicator of cell non-viability. The ratio of unstained cells to total cells can be used to calculate the percentage of viable cells in a given sample.

[0005] The distinction between stained and unstained cells is typically achieved by visual inspection and counting using an optical microscope and a manual cell counting device (e.g., a hemocytometer). Additionally, analytical instruments are available that automatically count and differentiate stained and unstained cells using imaging devices and image processing algorithms.

[0006] Because counting and differentiation between stained and unstained cells is based on visual inspection or imaging processing, it is important that the dye solution used in the dye exclusion assay remain substantially free of particles and other debris that could interfere with cell identification.

[0007] Trypan blue in phosphate-buffered saline (PBS) is a solution of trypan blue dissolved in PBS and preserved with the aid of sodium azide.

[0008] Dye aggregation describes the phenomenon of self-association between dissolved dye molecules. Significant self-association can lead to precipitation of the dye from solution as insoluble aggregates containing many dye molecules. This self-association is mediated by a variety of attractive intermolecular forces, including hydrogen bonding, electrostatic interactions, van der Waals forces, and pi-stacking.

[0009] The presence of other chemicals in the dye buffer affects the interactions between dye molecules. These effects can increase or decrease the likelihood of dye aggregation. Sodium chloride is added to the dye buffer to ensure osmotic balance; in the absence of this dissolved salt, the resulting hypotonic buffer promotes cell penetration and is unsuitable for dye exclusion assays of cell viability.

[0010] Trypan blue dye usually contains organic impurities. Red impurities appear in trypan blue, which is poorly soluble in PBS and forms filamentous or fibrous red dye aggregates, such as Figure 1 The formation of this red impurity during the synthesis of trypan blue and its structural analogs has been previously described in the scientific literature (see Organic Syntheses 1936, 16, 12; DOI 10.15227 / orgsyn.016.0012). When trypan blue and the red impurity are dissolved separately in solution, they are visually distinct.

[0011] The cosmetic appearance of dye solutions is often marred by the presence of aggregates of impurities that can be confused with biological contaminants or debris and otherwise affect the perceived quality of the dye solution product and can be incorrectly interpreted.

[0012] If the trypan blue contains a large amount of dye aggregates, further filtration or heating may be required to remove or redissolve the dye aggregates. Filtering or heating may be impractical, and these methods cannot prevent the recurrence of dye aggregation or precipitation at storage temperatures, which are typically at or near ambient laboratory temperatures. Therefore, a stable trypan blue solution with reduced or no aggregates is needed. Summary of the Invention

[0013] In one aspect, the present invention provides a trypan blue solution. In one example, the trypan blue solution comprises: trypan blue, an osmotic agent, and an aqueous buffer.

[0014] In some embodiments, the trypan blue solution can have a trypan blue concentration of about 0.01% to about 2% based on the total weight (in w / w%) or total volume (in w / v%) of the trypan blue solution. The buffer can be PBS buffer preserved with sodium azide.

[0015] In some embodiments, the osmotic agent is not a sodium salt.In some embodiments, the osmotic agent is selected from the group consisting of: lithium chloride, D-glucose, L-glycine, or a combination thereof.

[0016] In some embodiments, the trypan blue solution further comprises an acid or a base. The acid may comprise phosphoric acid. The base may comprise an alkaline hydroxide.

[0017] In some embodiments, the trypan blue solution further comprises a water-soluble polymer. The water-soluble polymer may comprise an anionic polymer or a nonionic polymer or both thereof. In some embodiments, the water-soluble polymer comprises a polymer selected from the group consisting of poly(acrylic acid) or a salt thereof, poly(styrene sulfonic acid) or a salt thereof, poly(vinyl pyrrolidone), or any combination thereof. In some embodiments, the water-soluble polymer is a poly(acrylic acid) or an alkali metal salt thereof having a mean molecular weight of about 4,000 to about 6,000 g / mol. In some embodiments, the water-soluble polymer is a poly(4-styrene sulfonic acid) or an alkali metal salt thereof having a mean molecular weight of about 50,000 to about 80,000 g / mol. In some embodiments, the water-soluble polymer is a poly(vinyl pyrrolidone) having a mean molecular weight of about 30,000 to about 50,000 g / mol.

[0018] In some embodiments, the concentration of the water-soluble polymer of the trypan blue solution is about 0.05% to about 4% based on the total weight (w / w%) or total volume (w / v%) of the trypan blue solution. In some embodiments, the concentration of the repeating unit of the water-soluble polymer of the trypan blue solution is about 5 mmol / L to about 500 mmol / L. In some embodiments, the molar ratio of the repeating unit of the water-soluble polymer of the trypan blue solution to the trypan blue is about 1 to about 55.

[0019] In some embodiments, the pH of the trypan blue solution is about 7.0 to about 7.5. In some embodiments, the osmolarity of the trypan blue solution is about 270 to about 310 mOsm kg -1 .

[0020] The trypan blue solution is free or substantially free of precipitated impurities after storage at an ambient temperature of about 15°C to about 30°C for at least about 10 minutes.

[0021] In another example, the trypan blue solution comprises: trypan blue and a water-soluble polymer as described herein. According to the present disclosure, in some embodiments, the trypan blue solution further comprises at least one of: an aqueous buffer, an osmotic agent, an acid, a base, a cell culture medium, or any combination thereof.

[0022] In another aspect, the present disclosure provides a method for at least one of preparing a trypan blue solution, stabilizing a trypan blue solution, purifying a trypan blue solution, using a trypan blue solution, or any combination thereof.

[0023] In one example, the method includes providing a trypan blue solution; and adding a water-soluble polymer described herein to the trypan blue solution. In some embodiments, the method further includes adjusting the trypan blue concentration to a range of about 0.01% to about 2% based on the total weight (in w / w%) or total volume (in w / v%) of the trypan blue solution. In some embodiments, the method further includes adjusting the molar ratio of the repeating units of the water-soluble polymer to the trypan blue to about 1 to about 55.

[0024] In some embodiments, the method further comprises filtering the trypan blue solution before or after adding the water-soluble polymer.In some embodiments, the filtration is performed using at least one filter medium having an average pore size of about 0.2 μm.

[0025] In some embodiments, the method includes cooling the trypan blue solution prior to the filtering step. In some embodiments, the cooling step is performed before, after, or both before and after the addition of the water-soluble polymer. In some embodiments, the trypan blue solution is cooled to a temperature of about 0° C. to about 14° C. In some embodiments, the trypan blue solution is cooled for at least about 1 hour.

[0026] In some embodiments, the method comprises mixing trypan blue with at least one component selected from the group consisting of an osmotic agent, an acid, a base, a pH adjuster, a buffer solution, a cell culture medium, water, or any combination thereof.

[0027] In some embodiments, the method comprises adjusting the pH of the trypan blue solution to a range of about 6.5 to about 8. In some embodiments, the method comprises adjusting the pH of the trypan blue solution to a range of about 6 to about 7 before or during the cooling step. In some embodiments, the method comprises adjusting the pH of the trypan blue solution to a range of about 7 to about 7.5 after the filtering step. In some embodiments, the method comprises adjusting the osmolarity of the trypan blue solution to a range of about 270 to about 310 mOsm kg -1 In some embodiments, the method comprises repeating the cooling and / or filtering steps. In some embodiments, the method comprises packaging the filtered trypan blue solution.

[0028] In another example, the method includes cooling the trypan blue solution; and filtering the cooled solution. In some embodiments, the method further includes mixing the trypan blue solution with one or more ingredients selected from the group consisting of an osmotic agent, an acid, a base, a pH adjuster, a cell culture medium, water, or any combination thereof.

[0029] In some embodiments, the stable trypan blue solution has a trypan blue concentration of about 0.01% to about 2% based on the total weight (in w / w %) or total volume (in w / v %) of the trypan blue solution.

[0030] In some embodiments, the osmotic agent is not a sodium salt.In some embodiments, the osmotic agent is selected from the group consisting of: lithium chloride, D-glucose, L-glycine, or a combination thereof.

[0031] In some embodiments, prior to cooling, the concentration of trypan blue in the solution is near, at, or above the solubility limit. In some embodiments, the trypan blue solution is prepared by mixing trypan blue with an aqueous buffer at ambient or near ambient temperature and pressure prior to cooling.

[0032] In some embodiments, the method further comprises mixing a water-soluble polymer described herein with a solution of trypan blue before cooling or after filtering. In some embodiments, the method further comprises: adjusting the pH of the trypan blue solution, adjusting the osmolarity of the trypan blue solution, or both. In some embodiments, the method further comprises: packaging and / or storing the filtered trypan blue solution. In some embodiments, the filtered trypan blue solution is stored at a temperature of about 0°C to about 25°C. In some embodiments, the filtered trypan blue solution is free of or substantially free of precipitated impurities after being stored at an ambient temperature of about 15°C to about 30°C for at least about 10 minutes.

[0033] In yet another example, a method includes: preparing a trypan blue solution according to the present disclosure; combining the trypan blue solution with a cell culture medium. In some embodiments, the cell culture medium contains cells to be stained with the trypan blue solution. In some embodiments, the method further includes contacting the combined solution with a cell sample.

[0034] In another aspect, the present disclosure provides a kit for performing the methods described herein. In one example, the kit comprises: a trypan blue solution; and at least one filter medium according to the present disclosure. In some embodiments, the kit further comprises a water-soluble polymer stored in a separate container.

[0035] In another example, the kit comprises a trypan blue solution stored in a first container; and a water-soluble polymer stored in a second container.

[0036] According to the present disclosure, in some embodiments, the kits described herein further comprise at least one of: an aqueous buffer, an acid, a base, a pH adjuster, water, an osmotic agent, a cell culture medium, or any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Shown is a microscopic image of a trypan blue solution in which red dye aggregates are present; scale bar is 100 μm.

[0038] Figure 2Shown are microscopic images of nominally 0.4% trypan blue (0.15 mL) in NaCl (untreated trypan blue solution) in the presence of different cell culture media or a control (0.20 mL). Images A to F show trypan blue in DI water; trypan blue in PBS buffer; trypan blue in cell culture medium 1; trypan blue in cell culture medium 3; trypan blue in cell culture medium 2; and trypan blue in cell culture medium 4, respectively. DI = deionized water. PBS = isotonic phosphate buffered saline (pH 7.4). Medium 1 = Gibco TM CD FortiCHO TM Culture medium. Culture medium 2 = Gibco TM CD DG44 medium. Medium 3 = Gibco TM LV-MAX TM Production medium. Medium 4 = Gibco TM FreeStyle TM CHO expression medium. Images of the DI and PBS controls depict the normal degree of precipitation with nominally 0.4% trypan blue in NaCl. Images C, D, E, and F for media 1, 2, 3, and 4, respectively, depict the precipitate formed due to exposure to cell culture medium.

[0039] Figure 3 Shown are microscopic images of three Trypan blue samples in PBS stored at 4°C for 14 hours before and after 0.2 μm filtration. Image A-1 is a microscopic image of sample A before filtration; images A-1 and A-2 show sample A before and after filtration, respectively; images B-1 and B-2 show sample B before and after filtration, respectively; and images C-1 and C-2 show sample C before and after filtration, respectively. Debris consists of aggregates of red dye that appear blue due to lighting conditions. All three Trypan blue samples in PBS were from commercial sources and were not further processed. Sample A has an expiration date of October 23, 2020; sample B has an expiration date of October 10, 2021; and sample C has an expiration date of March 10, 2021.

[0040] Figure 4 Shown are microscopic images of three trypan blue samples (samples A, B, and C) in PBS stored under different conditions: (1) unfiltered dye solution stored at 4°C, (2) filtered dye solution stored at 22°C for five days; (3) filtered dye solution stored at 4°C for five days; (4) filtered dye solution stored at 4°C for five days and then at 22°C for three hours; (5) unfiltered dye solution stored at 4°C for five days and then at 22°C for three hours.

[0041] Figure 5Shown are microscopic images of trypan blue dye solutions after storage for 13 weeks at either 22° C. or 4° C. Images A and F (labeled “Control”) show commercial trypan blue in PBS; images B and G (labeled “NaCl”) show laboratory-prepared trypan blue in PBS; images C and H (labeled “LiCl”) show laboratory-prepared lithium chloride preparations; images D and I (labeled “D-Glucose”) show laboratory-prepared glucose preparations; and images E and J (labeled “L-Glycine”) show laboratory-prepared glycine preparations.

[0042] FIG6 shows the UV-visible absorption spectra of trypan blue solutions after storage at (a) ambient temperature of 22° C. or (b) refrigeration at 4° C.

[0043] Figure 7 Microscopic images of mixtures of (1) nominal 0.4% trypan blue in NaCl (0.20 mL) and PBS (0.20 mL), (2) nominal 0.4% trypan blue in NaCl (0.20 mL) and 0.5 mg mL in PBS (0.20 mL) are shown. -1 Kanamycin, and (3) nominal 0.4% trypan blue in NaCl (0.20 mL) and 0.5 mg mL in PBS (0.20 mL) -1 Spermidine.

[0044] Figure 8 Shown are microscopic images of mixtures of trypan blue solutions and cell culture media according to Table 6 and Example 4. The images show the mixing of 0.20 mL of nominally 0.4% trypan blue in NaCl (with or without polymer additives) with 0.20 mL of Gibco TM CD FortiCHO TM Results of the combination of culture medium or PBS. PC = Positive control = in the presence of NaCl and Gibco TM CDFortiCHO TM Nominal 0.4% Trypan Blue in culture medium. NC = Negative Control = Nominal 0.4% Trypan Blue in NaCl and PBS.

[0045] Figure 9 Shown are UV-Vis absorption spectra of nominal 0.4% Trypan Blue in NaCl according to Example 4 and nominal 0.4% Trypan Blue in NaCl containing 0.05% w / v Na-PAA. DETAILED DESCRIPTION

[0046] Although the concepts of the present disclosure are shown and described in detail in the drawings and description herein, the results of the drawings and description should be considered illustrative in nature and not restrictive; it is understood that only certain exemplary embodiments have been shown and described, and all changes and modifications that come within the spirit of the present disclosure are intended to be protected.

[0047] Unless defined otherwise, scientific and technical terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates.

[0048] Those skilled in the relevant art will appreciate that other suitable modifications and adaptations of the compositions and methods described herein will be readily apparent from the description of the disclosure contained herein, in view of the information known to those skilled in the art, and may be made without departing from the scope of the disclosure or any embodiment thereof. Having now described the disclosure in detail, the disclosure will be more clearly understood by reference to the following examples, which are included herein for illustrative purposes only and are not intended to limit the disclosure.

[0049] Definitions and explanations of selected terms

[0050] As used herein, "weight percent," "weight %," "w / w %," "percent by weight," "% by weight," and variations thereof refer to the concentration of a substance calculated as the weight of the substance divided by the total weight of the composition and multiplied by 100. In some cases, "w / v %' refers to the concentration of the substance calculated as the weight of the substance divided by the total weight of the composition and multiplied by 100. It should be understood that for aqueous compositions having a density (total weight divided by total volume) close to 1 g / mL, the concentration of a substance in the composition calculated as "W / w %" or "w / v %" can have essentially the same value. Therefore, unless otherwise indicated, "percent," "%," and the like as used herein are intended to be synonymous with "weight percent," "weight %," "w / w %'," "w / v %," and the like.

[0051] As used herein, "g" stands for gram; "L" stands for liter; "mg" stands for "milligram (10 -3 grams); "mL" or "cc" represents milliliters (10 -3 One "μL" is equal to one microliter (10 -6 Liter). The unit "g / 100g," "g / 100mL," or "g / L" is a unit of concentration or amount of a component in a composition. One "mg / L" is equal to one ppm (parts per million). "Da" refers to Dalton, a unit of molecular weight; one Dalton is equal to 1 g / mol. Temperatures used herein are in degrees Celsius (°C).

[0052] The term "about" is used in conjunction with a numerical value to include the normal variation in measurements expected by those skilled in the art and is understood to have the same meaning as "approximately" and to cover the usual range of error, such as ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated value. The term "about" also encompasses amounts that differ due to different equilibrium conditions of the composition resulting from a particular initial composition. Whether or not modified by the term "about," the claims include equivalents to the stated quantities.

[0053] It should be noted that, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a / kind" and "the" include plural referents. Thus, for example, reference to a composition comprising a "compound" includes two or more compounds that are the same or different from each other. It should also be noted that, unless the content clearly indicates otherwise, the term "or" generally includes "and / or" in the sense in which it is employed. "And / or" as used herein refers to and encompasses any and all possible combinations of one or more of the relevant listed items, as well as the lack of a combination when interpreted in an alternative manner ("or").

[0054] For the sake of brevity and conciseness, any range of values ​​set forth in this specification contemplates all values ​​within that range and is to be construed as support for claims reciting any sub-range having endpoints that are actual values ​​within the particular range in question. As a hypothetical illustrative example, the disclosure of a range from 1 to 5 in this specification should be considered support for a claim to any of the following ranges: 1 to 5; 1 to 4; 1 to 3; 1 to 2; 2 to 5; 2 to 4; 2 to 3; 3 to 5; 3 to 4; and 4 to 5.

[0055] The term "substantially" is used herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also used herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0056] The term "substantially free of" may refer to any component that the composition of the present disclosure does not have or hardly has. When referring to "substantially free of", it is intended that the component is not intentionally added to the composition of the present disclosure. The term "substantially free of" may refer to any component that the composition of the present disclosure does not have or hardly has. The use of the term "substantially free of" a certain component allows the inclusion of trace amounts of this component in the composition of the present disclosure because they are present in another component. However, it is believed that when a composition is referred to as "substantially free of" a certain component, only trace amounts or deminimus amounts of this component will be allowed to exist. In addition, if a composition is referred to as "substantially free of" a certain component, if this component is present in trace amounts or deminimus, it is understood that it will not affect the effectiveness of the composition. It should be understood that if a certain component is not explicitly included in this article or is not described in this article as possibly including this component, the present disclosure composition may be substantially free of this component. Similarly, explicitly including a certain component allows it to be explicitly excluded, thereby allowing the composition to be substantially free of the clearly stated component.

[0057] As used herein, the terms "comprises / includes" and variations thereof specify the presence of stated features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or combinations thereof.

[0058] As used herein, the transition phrase "consisting essentially of means that the scope of a claim shall encompass the specific materials or steps recited in the claim, as well as those that do not materially affect the basic and novel characteristics of the claimed disclosure. Therefore, when used in the claims of the present disclosure, the term "consisting essentially of is not intended to be interpreted as equivalent to "comprising."

[0059] The term "Trypan Blue" as used herein refers to the compound CAS#72-57-1 or its conjugate base. The molecular formula of Trypan Blue is C 34 H 28 N6O 14 S4, or the water-soluble tetrasodium salt form of C 34 H 24 N6O 14 S4Na4, or a mixture thereof. The chemical structure of neutral trypan blue is:

[0060]

[0061] Trypan blue is a water-soluble azo dye, typically sold as a sodium salt. The structure of the red impurity may be related to trypan blue and may contain similar functional groups.

[0062] As used herein, the term "polymer" firstly comprises a collection of macromolecules that are chemically uniform but differ in degree of polymerization, molar mass and chain length, which are prepared by polymerization reactions (chain growth polymerization, ring-opening polymerization, polyaddition, polycondensation, etc.). The term secondly also comprises derivatives of such a collection of macromolecules from polymerization reactions, i.e. compounds obtained by reaction (e.g. addition or substitution) of functional groups on a given macromolecule, and which may be chemically homogeneous or chemically heterogeneous. As used herein, "polymer" encompasses oligomers, homopolymers, copolymers, random copolymers, block copolymers, branched polymers, grafted polymers, dendrimers or other possible macromolecular structures.

[0063] As used herein, the term "water-soluble polymer" encompasses polymeric substances that dissolve, disperse, or swell in water and thereby alter the physical properties of the aqueous solution. The water-soluble polymer may comprise a polymer containing anionic monomeric units or repeating units, or nonionic monomeric units or repeating units, or both anionic and nonionic monomeric units.

[0064] As used herein, "anionic polymers" or "polyanionic compounds" are a subclass of water-soluble polymers having at least one anionic functional group covalently attached to the polymer backbone. The anionic functional group may be a carboxyl group, a sulfo group, a phosphate group, or a salt thereof, or any combination thereof. Non-limiting examples of anionic polymers used herein include polymaleic acid; polyacrylic acid; a copolymer of acrylic acid and 2-hydroxy-3-allyloxypropanesulfonic acid; a copolymer of acrylic acid and 2-acrylamide-2-methylpropanesulfonic acid; a copolymer of acrylic acid and isoprenesulfonic acid; a copolymer of acrylic acid and 2-hydroxyethyl methacrylate; a copolymer of acrylic acid, 2-hydroxyethyl methacrylate and isopropylenesulfonic acid; a copolymer of maleic acid and pentene; a copolymer of maleic acid and isobutylene, an alkali metal salt of the aforementioned anionic polymers and an alkaline earth metal salt of the aforementioned anionic polymers; poly(vinyl phosphoric acid) or a salt thereof; a salt of poly(vinyl sulfonic acid) or a salt thereof; a poly(2- acrylamide-2-methylpropanesulfonic acid) or its salt; poly(4-styrenesulfonic acid) (PSS); poly(sodium 4-styrenesulfonate) (SPS); poly(sodium styrenesulfonate) (PSSS); acrylate / acrylamide copolymer; acrylate homopolymer; acrylate / methacrylate copolymer; methacrylate / acrylamide copolymer; methacrylate / styrenesulfonate / ester copolymer; acrylate / styrenesulfonate / ester copolymer; methacrylate / vinylsulfonate / ester copolymer; and 2-acrylamido-2-methylpropanesulfonic acid sodium salt homopolymer; sulfomethylated polyacrylamide; alginic acid or its salt; or any combination thereof.

[0065] As used herein, "nonionic polymer" refers to a subclass of water-soluble polymers having at least one nonionic functional group covalently attached to the polymer backbone. Non-limiting examples of nonionic polymers include polymers having: poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol) and copolymers, poly(N-vinyl pyrrolidone) and copolymers, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxypropylmethylcellulose, carboxymethylcellulose, guar gum, hydroxyethylguar gum, hydroxypropylguar gum, gelatin, albumin, hydroxypropylmethylguar gum, carboxymethylguar gum, carboxymethylchitosan, locust bean gum, carrageenan, xanthan gum, gellan gum, pullulan, alginates, chondroitin sulfate, dextran, dextran sulfate, Dextran, aloe vera gel, scleroglucan, schizophyllan, gum arabic, tamarind gum, poly(methyl vinyl ether), ethylene oxide-propylene oxide-ethylene oxide block copolymer, hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, dextran, poly(vinyl methyl ether), polyacrylamides (class), poly(N,N-dimethylacrylamide), poly(N-vinyl acetamide), poly(N-vinyl formamide), poly(2-hydroxyethyl methacrylate), poly(glycerol methacrylate), poly(2-ethyl-2-

[0066] The water-soluble polymers of the present invention may have both nonionic and ionic functional groups attached to the polymer backbone.

[0066] The water-soluble polymer used herein may also include alginic acid or alginates. Alginic acid is a linear heteropolysaccharide comprising β-D-mannuronic acid and α-L-guluronic acid units. Alginic acid may comprise homopolymeric sequences of mannuronic acid, homopolymeric sequences of guluronic acid, and mixed sequences of mannuronic acid and guluronic acid units. The alginates used in the method of the present invention may include alkali metal salts, such as sodium and potassium salts, as well as ammonium and alkanolamine salts.

[0067] As used herein, "ambient temperature" refers to a temperature of about 15 to about 30°C.

[0068] For purposes of this disclosure, chemical elements are identified according to Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Edition, 1986-87, inside cover.

[0069] Overview of technical solutions

[0070] As mentioned above, trypan blue solutions may contain undesirable impurities. For example, commercial products of trypan blue in buffered solution as received often contain precipitates of the dye. These aggregates naturally form, propagate, and precipitate from the solution over time when the dye solution is cooled or stored at relatively low temperatures. Figure 2 As shown, extensive dye precipitation can also be triggered by the combination of trypan blue solution and cell culture medium. Since cells are cultured and processed in suspension in cell culture medium, the combination of trypan blue solution and cell culture medium is a routine and often unavoidable step when performing dye exclusion experiments.

[0071] When dye precipitation is triggered by combination with cell culture medium, the appearance of the solution is negatively impacted. Aggregates or precipitates can be confused with biological contaminants or debris. Furthermore, image processing algorithms used by automated cell viability analyzers, such as those used by automated cell viability analyzers, can occasionally misinterpret precipitates as dead cells, leading to inaccurate results.

[0072] The present disclosure provides several methods that effectively improve trypan blue solution stability and / or minimize dye precipitation over a long period of time.The first method is normally to prepare or reformulate the trypan blue solution that (reformulating) sodium and potassium content reduce.In the present disclosure, have determined that ion such as sodium and potassium cation can contribute to the precipitation of trypan blue and form common large dye aggregates in trypan blue dye solution.Replacing sodium chloride with other types of penetrants can stop this common precipitation pattern.

[0073] The second method generally involves processing or purifying the trypan blue solution by cooling the solution and filtering the cooled solution subsequently. It is found that cooling the solution can effectively separate unwanted impurities, otherwise these impurities can cause and promote dye aggregation. Can effectively remove unwanted impurities from the trypan blue solution by subsequent filtration. It is found that purified trypan blue solution has improved stability and the shelf life of extension when stored for a long period of time at a wide range of temperatures, and has little or no precipitation.

[0074] The third method generally relates to compositions and methods for stabilizing trypan blue solutions by adding or using polymeric additives. In particular, it has been found that the polymeric additives used in the present disclosure can effectively prevent or inhibit precipitation caused by polycationic compounds that may be present in the cell culture medium. Without wishing to be bound by any particular theory, it is believed that the function of the polymeric additives is to (1) capture cationic species in the cell culture medium and / or (2) capture and stabilize trypan blue in the cell culture medium. The polymeric additives used herein can form polyelectrolyte complexes with positively charged cell culture medium components. The formation of such polymeric complexes inhibits or prevents the interaction between trypan blue and the positively charged cell culture medium components. As a result, no dye precipitation is observed or precipitation occurs more slowly. In addition, the polymeric additives used herein bind to trypan blue through nonionic, intermolecular interactions to form soluble dye-polymer adducts. The soluble dye-polymer adducts inhibit or prevent the interaction between trypan blue and the positively charged cell culture medium components. As a result, no dye precipitation is observed or precipitation occurs more slowly.

[0075] Note that the three general methods described above are compatible with each other and can be combined in any manner to synergistically improve the stability of the trypan blue solution.

[0076] Composition

[0077] In one aspect, the present disclosure relates to a composition. In some instances, the composition is an aqueous solution of trypan blue. The composition may also include one or more ingredients, including but not limited to an osmotic agent, an aqueous buffer, an acid, a base, a pH adjuster, a polymer additive, a cell culture medium, a preservative, water, or any combination thereof.

[0078] In some embodiments, the trypan blue weight content or concentration of the trypan blue solution according to the present disclosure is about 0.01% to about 2%, or about 0.05% to about 1%, or about 0.1% to about 0.5%, or about 0.2% to about 0.5%, based on the total weight (w / w%) or total volume (w / v%) of the trypan blue solution. In some embodiments, the concentration of trypan blue is at least about 0.05%, at least about 0.1%, at least about 0.15%, at least about 0.2%, at least about 0.25%, at least about 0.3%, at least about 0.35%, at least about 0.4%, at least about 0.45%, or at least about 0.5%, based on the total weight (w / w%) or total volume (w / v%) of the trypan blue solution.

[0079] In some embodiments, the trypan blue solution comprises an osmotic agent. The amount of osmotic agent and / or the ratio of osmotic agent to trypan blue can be balanced to produce a physiologically isotonic solution comprising approximately 300 mOsm / L dissolved solids.

[0080] In some embodiments, the osmotic agent of the trypan blue solution is non-toxic to cells for the duration of the dye staining period. The osmotic agent can have a sufficiently low molecular weight such that an osmotic volume of about 300 mOsm / L of the trypan blue solution can be achieved without adding excess solids. In some embodiments, the molecular weight of the osmotic agent is less than about 1,000 g / mol, or less than about 500 g / mol, or less than about 250 g / mol.

[0081] In some embodiments, the osmotic agent comprises at least one of the following: an amino acid or a derivative thereof, such as a stereoisomer, a dipeptide, a tripeptide, and a polypeptide derived from an amino acid and its stereoisomer, a natural monosaccharide and a synthetic monosaccharide such as aldohexoses and ketohexoses and pentoses, a diol, a polyol such as a sugar alcohol, a lithium salt, a low molecular weight polymer such as polyethylene glycol, Good's buffer, a low molecular weight physiological osmotic agent (e.g., taurine, sarcosine, betaine, urea, or trimethylamine N-oxide (TMAO)), or any combination thereof. In some embodiments, the osmotic agent of the trypan blue solution comprises lithium chloride, D-glucose, or L-glycine, or any combination thereof.

[0082] In some embodiments, the trypan blue solution comprises an acid or base. The acid or base can be used to adjust the pH of the solution and buffer. The acid or base can be organic or inorganic. The acid can be, for example, a carboxylic acid such as acetic acid or lactic acid, hydrochloric acid, phosphoric acid, etc. The base can be, for example, lithium hydroxide or potassium hydroxide, sodium bicarbonate or potassium bicarbonate, sodium carbonate or potassium carbonate, amine base, ammonia, etc.

[0083] In some embodiments, the trypan blue solution comprises a pH adjuster, such as a buffer. An example of a buffer is phosphate buffered saline (PBS) buffer preserved with sodium azide.

[0084] In some embodiments, the pH of the trypan blue solution is from about 6.0 to about 9.0, or from about 6.5 to about 8.0, or from about 7.0 to about 7.5. In some embodiments, the pH of the trypan blue solution is at least about 6.5, at least about 6.6, at least about 6.7, at least about 6.8, at least about 6.9, at least about 7.0, at least about 7.1, at least about 7.2, at least about 7.3, at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9, at least about 8.0.

[0085] In some embodiments, the trypan blue solution further comprises one or more preservatives. Non-limiting examples of preservatives include sodium azide, 2-phenoxyethanol, formaldehyde, formaldehyde formers (e.g., DMDM ​​hydantoin, imidazolidinyl urea, diazolidinyl urea), sodium hydroxymethylglycinate, 2-bromo-2-nitropropane-1,3-diol or 5-bromo-5-nitro-1,3-diol. Alkanes, isothiazolinones (e.g., methylisothiazolinone, chloromethylisothiazolinone, benzisothiazolinone), benzoic acid and alkali metal salts thereof, sorbic acid and alkali metal salts thereof, p-hydroxybenzoates, or combinations thereof.

[0086] In some embodiments, the osmotic agent of the trypan blue solution of the present invention does not contain sodium chloride. In certain embodiments, the present disclosure contains no or substantially no sodium chloride. Alternatively, sodium chloride is not used as a raw material in the preparation and / or reformulation and / or processing of the trypan blue solution of the present invention.

[0087] In some embodiments, the sodium concentration of the trypan blue solution of the present invention is about 20 mmol / L or less, or about 16 mmol / L or less, or about 12 mmol / L or less, or about 8 mmol / L or less, or about 5 mmol / L, or about 2 mmol / L or less, or about 1.5 mmol / L or less, or about 1 mmol / L or less, or about 0.5 mmol / L or less, or about 0.1 mmol / L or less, or about 0.01 mmol / L or less, or about 0.001 mmol / L or less, or about 0.0001 mmol / L or less. In some embodiments, the trypan blue solution of the present invention contains no or substantially no sodium and its ionic forms.

[0088] In some embodiments, the potassium concentration of the trypan blue solution of the present invention is about 20 mmol / L or less, or about 10 mmol / L or less, or about 5 mmol / L or less, or about 2 mmol / L or less, or about 1 mmol / L, or about 0.5 mmol / L or less, or about 0.1 mmol / L or less, or about 0.01 mmol / L or less, or about 0.001 mmol / L or less, or about 0.0001 mmol / L or less. In some embodiments, the trypan blue solution of the present invention contains no or substantially no potassium and its ionic forms.

[0089] In some embodiments, the lithium concentration of the trypan blue solution of the present invention is at least about 0.5 mmol / L, or at least about 1 mmol / L, or at least about 1.5 mmol / L, or at least about 2 mmol / L, or at least about 5 mmol / L, or at least about 10 mmol / L, or at least about 14 mmol / L.

[0090] In some embodiments, the osmolarity of the trypan blue solution of the present invention is from about 250 to about 350 mOsm kg -1 , or about 270 to about 330 mOsm kg -1 , or about 280 to about 320 mOsm kg -1, or about 290 to about 310 mOsm kg -1 .

[0091] In some embodiments, the trypan blue solution of the present invention further comprises a polymer. The polymer is preferably a water-soluble polymer, such as an anionic polymer, a nonionic polymer, or any combination thereof.

[0092] In some embodiments, the trypan blue solution comprises an anionic polymer selected from the group consisting of poly(acrylic acid) or a salt thereof, poly(4-styrenesulfonic acid) or a salt thereof, poly(vinylpyrrolidone), or any combination thereof.

[0093] In some embodiments, the water-soluble polymer has an average molecular weight of from about 500 g / mol to about 200,000 g / mol, or from about 1,000 g / mol to about 150,000 g / mol, or from about 2,000 g / mol to about 100,000 g / mol, or from about 3,000 g / mol to about 70,000 g / mol, or from about 4,000 g / mol to about 40,000 g / mol, or from about 5,000 g / mol to about 20,000 g / mol, or from about 7,000 g / mol to about 15,000 g / mol, or from about 7,000 g / mol to about 10,000 g / mol.

[0094] In some embodiments, the concentration of the water-soluble polymer in the Trypan blue solution of the present invention is from about 0.01% to about 10%, or from about 0.02% to about 8%, or from about 0.03% to about 6%, or from about 0.05% to about 4%, or from about 0.1% to about 3%, or from about 0.2% to about 2%, or from about 0.5% to about 1%, based on the total weight (w / w%) or total volume (w / v%) of the Trypan blue solution.

[0095] In some embodiments, the concentration of the repeating units of the water-soluble polymer of the trypan blue solution of the present invention, calculated based on the ratio of the total number of moles of the repeating units of the water-soluble polymer to the total weight (w) or total volume (v) of the trypan blue solution, is from about 1 mmol / L to about 1,000 mol / L, or from about 5 mmol / L to about 500 mmol / L, or from about 10 mmol / L to about 100 mmol / L.

[0096] In some embodiments, the trypan blue solution of the present invention has a molar ratio of repeating units of the water-soluble polymer to trypan blue of at least about 1, or at least about 2, or at least about 3, or at least about 5, or at least about 10, or at least about 20, or at least about 30, or at least about 50, or at least about 100, or at least about 200. In some embodiments, the molar ratio of repeating units of the water-soluble polymer to trypan blue is from about 1 to about 100, or from about 2 to about 75, or from about 3 to about 55, or from about 5 to about 26.

[0097] In certain embodiments, trypan blue solution of the present invention comprises poly (acrylic acid) alkali metal salt.The molar percentage of the alkali metal salt of poly (acrylic acid) alkali metal salt can be about 0 to about 100%, or about 10% to about 90%, or about 20% to about 80%, or about 30% to about 70%, or about 40% to about 60%.The molecular weight of poly (acrylic acid) alkali metal salt can be about 500g / mol to about 20,000g / mol, or about 2,000g / mol to about 10,000g / mol, or about 4,000 to about 6,000g / mol.In some embodiments, the alkali metal of poly (acrylic acid) alkali metal salt comprises sodium, potassium or lithium, or its arbitrary combination.In some embodiments, alkali metal comprises lithium, and does not contain or is substantially free of sodium and potassium. The concentration of the poly(acrylic acid) alkali metal salt may be from about 0.01 w / v% to about 5 w / v%, or from about 0.05 w / v% to about 2 w / v%, or from about 0.1 w / v% to about 1 w / v%. The concentration of the repeating unit of the poly(acrylic acid) alkali metal salt may be from about 1 mmol / L to about 500 mmol / L, or from about 5 mmol / L to about 200 mmol / L, or from about 10 mmol / L to about 120 mmol / L. In some embodiments, the molar ratio of the repeating unit of the poly(acrylic acid) alkali metal salt to trypan blue is from about 1 to about 100, or from about 5 to about 75, or from about 25 to about 55.

[0098] In certain embodiments, the trypan blue solution of the present invention comprises poly (4-styrene sulfonic acid) alkali metal salt.Similar to poly (acrylic acid) alkali metal salt, the molar percentage of the alkali metal salt of poly (4-styrene sulfonic acid) alkali metal salt can be about 0 to about 100%, or about 10% to about 90%, or about 20% to about 80%, or about 30% to about 70%, or about 40% to about 60%.The molecular weight of poly (4-styrene sulfonic acid) alkali metal salt can be about 1,000g / mol to about 200,000g / mol, or about 10,000g / mol to about 100,000g / mol, or about 50,000g / mol to about 80,000g / mol.In some embodiments, the alkali metal of poly (4-styrene sulfonic acid) alkali metal salt comprises sodium, potassium or lithium, or its any combination.In some embodiments, alkali metal comprises lithium, and does not contain or is substantially free of both sodium and potassium. The concentration of the poly(4-styrenesulfonic acid) alkali metal salt may be from about 0.02 w / v% to about 10 w / v%, or from about 0.1 w / v% to about 5 w / v%, or from about 0.5 w / v% to about 2.5 w / v%. The concentration of the repeating unit of the poly(4-styrenesulfonic acid) alkali metal salt may be from about 1 mmol / L to about 500 mmol / L, or from about 5 mmol / L to about 200 mmol / L, or from about 10 mmol / L to about 120 mmol / L. In some embodiments, the molar ratio of the repeating unit of the poly(4-styrenesulfonic acid) alkali metal salt to trypan blue is from about 1 to about 100, or from about 5 to about 75, or from about 25 to about 55.

[0099] In certain embodiments, the trypan blue solution of the present invention comprises poly(vinyl pyrrolidone). The molecular weight of the poly(vinyl pyrrolidone) can be from about 1,000 g / mol to about 200,000 g / mol, or from about 10,000 g / mol to about 100,000 g / mol, or from about 30,000 g / mol to about 50,000 g / mol. The concentration of the poly(vinyl pyrrolidone) can be from about 0.01 w / v% to about 10 w / v%, or from about 0.05 w / v% to about 5 w / v%, or from about 0.2 w / v% to about 2 w / v%. The repeating unit concentration of the poly(vinyl pyrrolidone) can be from about 1 mmol / L to about 500 mmol / L, or from about 5 mmol / L to about 200 mmol / L, or from about 10 mmol / L to about 120 mmol / L. In some embodiments, the molar ratio of the repeating units of poly(vinyl pyrrolidone) to trypan blue is from about 1 to about 100, or from about 5 to about 75, or from about 25 to about 55.

[0100] In some embodiments, the trypan blue solution of the present invention further comprises cell culture medium. Cell culture medium can be obtained from commercial products, such as Gibco TM CD FortiCHO TMCulture medium. Gibco TM CD DG44 medium, Gibco TM LV-MAX TM Production medium, Gibco TM FreeStyle TM Expression medium.

[0101] In a specific example, the trypan blue solution of the present invention comprises trypan blue, a buffer, an osmotic agent and a water-soluble polymer. In some embodiments, the osmotic agent is sodium chloride. In other embodiments, the trypan blue solution does not contain or is substantially free of a sodium salt as a component of the trypan blue solution. In some embodiments, the osmotic agent is selected from lithium chloride, D-glucose or L-glycine or any combination thereof. In some embodiments, the water-soluble polymer is selected from poly (acrylic acid) or a salt thereof, poly (4-styrene sulfonic acid) or a salt thereof, poly (vinyl pyrrolidone) or any combination thereof.

[0102] In some embodiments, the prepared trypan blue solution of the present invention is stable and does not contain or is substantially free of precipitated impurities after being stored for a period of time at a certain temperature. The temperature can be from about 4°C to about 50°C, or from about 8°C to about 43°C, or from about 12°C to about 37°C, or from about 16°C to about 30°C, or from about 20°C to about 23°C. The time period can be at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 2 weeks, or at least about 3 weeks, or at least about 4 weeks, or at least about 1 month, or at least about 2 months, or at least about 3 months, etc.

[0103] Methods and kits

[0104] In some aspects, the present disclosure relates to methods for at least one of preparing a stable trypan blue solution, treating a trypan blue solution, stabilizing a trypan blue solution, purifying a trypan blue solution, increasing the stability of a trypan blue solution, and staining cells with a trypan blue solution.

[0105] In some embodiments, the method for preparing a trypan blue solution comprises mixing trypan blue with at least one component selected from the group consisting of an osmotic agent, a buffer solution, an acid, a base, a pH regulator, a preservative, a water-soluble polymer, a cell culture medium, water, or any combination thereof. The components and various aspects of the trypan blue solution have been described above and will not be repeated. The components can be added in any order. The mixing can be stirring, shaking, or continuous flow mixing or turbulent mixing. In some embodiments, the components can be in any physical form, such as a solid, a powder, a concentrate, or a stock solution. A skilled person will calculate the required amount of each component to obtain a trypan blue solution having a desired concentration and composition.

[0106] Generally speaking, a method for preparing, treating, stabilizing, or purifying a trypan blue solution comprises at least one of the following processing operations (or steps): preparing or providing a trypan blue solution; mixing the trypan blue solution with a water-soluble polymer; mixing the trypan blue solution with one or more ingredients; cooling the trypan blue solution; filtering the cooled solution; adjusting the pH of the trypan blue solution; adjusting the osmolarity of the trypan blue solution; adjusting the molar ratio of the water-soluble polymer to the trypan blue; and repeating at least one of the above operations. These operations can be performed in any combination or in any order. These operations can be performed simultaneously, sequentially, or in other coordinated ways.

[0107] The trypan blue solution before treatment / stabilization / purification can be prepared or provided before cooling. The trypan blue solution before treatment can be any trypan blue solution described above. The trypan blue solution before treatment can be obtained from a commercial source, or freshly prepared, or aged / stored for a period of time. The trypan blue solution before treatment may be unstable or contain visible impurities, or aggregates or precipitates.

[0108] The trypan blue solution before treatment / stabilization / purification can be a buffered solution of trypan blue, wherein the content or concentration of trypan blue is about 0.01% to about 2% of the gross weight (in w / w%) or total volume (in w / v%) of the trypan blue solution, or about 0.05% to about 1%, or about 0.1% to about 0.5%, or about 0.2% to about 0.5%. In some embodiments, before treatment, trypan blue can be mixed with an aqueous buffer at an environment or near-environmental temperature and pressure. Trypan blue can also be mixed with an aqueous buffer at a temperature higher than ambient temperature.

[0109] In some embodiments, prior to any processing step, the trypan blue solution further comprises at least one of: an osmotic agent, an acid or base, a water-soluble polymer, a cell culture medium, or any combination thereof.

[0110] In one example, a method for preparing or treating or stabilizing or purifying a trypan blue solution comprises mixing a trypan blue solution with a water-soluble polymer according to the present disclosure. In some embodiments, the method further comprises adjusting the molar ratio of repeating units of the water-soluble polymer to trypan blue to at least about 1, or at least about 2, or at least about 3, or at least about 5, or at least about 10, or at least about 20, or at least about 30, or at least about 50, or at least about 100, or at least about 200. In some embodiments, the molar ratio of repeating units of the water-soluble polymer to trypan blue is from about 1 to about 100, or from about 2 to about 75, or from about 3 to about 55, or from about 5 to about 26. The water-soluble polymer can be from a source of stock solution as described above.

[0111] In some embodiments, the method further comprises filtering the trypan blue solution before or after adding the water-soluble polymer. The filtering step can be performed using a filter medium (e.g., a syringe filter or a canister filter common in the art). The average pore size of the filter medium can be from about 0.05 μm to about 1 μm or from about 0.1 μm to about 0.5 μm. Common filters, such as Pall Acrodisc 0.2 μm or 0.45 μm nylon syringe filters can be used for the filtering step. In some embodiments, multiple rounds of filtration can be performed.

[0112] In some embodiments, the method further comprises: cooling the trypan blue solution before the filtering step. The cooling step can be performed before, after, or before and after the addition of the water-soluble polymer. In some embodiments, the trypan blue solution is cooled to a certain temperature for a period of time. The temperature is about 14°C or lower, about 13°C or lower, about 12°C or lower, about 11°C or lower, about 10°C or lower, about 9°C or lower, about 8°C or lower, about 7°C or lower, about 6°C or lower, about 5°C or lower, about 4°C or lower, about 3°C ​​or lower, about 2°C or lower, about 1°C or lower, or about 0°C. In some embodiments, the temperature is in the range of about 0°C to about 14°C, or about 3°C ​​to about 10°C, or about 4°C to about 6°C. The time for cooling the trypan blue solution can be at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 7 days.

[0113] In some embodiments, the method further comprises mixing the trypan blue with at least one component selected from the group consisting of an osmotic agent, an acid, a base, a pH adjuster, a buffer solution, a cell culture medium, water, or any combination thereof according to the present disclosure.

[0114] In some embodiments, the method also includes adjusting the pH of the trypan blue solution. The adjustment of pH can be carried out by adding a pH adjusting agent, a buffer, an acid or a base to the trypan blue solution before, during or after the filtering step. In some embodiments, the pH of the trypan blue solution is first adjusted to a first pH to promote the precipitation of impurities. The first pH can be relatively acidic, for example, from about 6 to about 7. After filtering, the pH of the filtered (or remaining) trypan blue solution is adjusted to a relatively alkaline, for example, from about 7 to about 8, at which pH, ​​trypan blue is more stable in solution.

[0115] In some embodiments, the method further comprises adjusting the osmolarity of the trypan blue solution. Adjusting the osmolarity can be performed by adding an osmotic agent to the trypan blue solution before, during, or after the filtration step.

[0116] In some embodiments, the method further comprises packaging the trypan blue solution in packaging after filtering. The packaging can be carried out at ambient temperature. The packaging can be a bottle, a rigid or flexible plastic container, a glass container, a metal container, or a metal drum. After packaging, the trypan blue solution can be stored at a temperature of about 0°C to about 50°C, or about 10°C to about 45°C, or about 20°C to about 40°C, or about 23°C to about 37°C.

[0117] The following embodiments also describe exemplary methods. In one embodiment, the method comprises mixing a water-soluble polymer with a trypan blue solution without a cooling or filtering step. In another embodiment, the method comprises: mixing a water-soluble polymer with a trypan blue solution; and filtering the trypan blue solution. In yet another embodiment, the method comprises: mixing a water-soluble polymer with a trypan blue solution; cooling the trypan blue solution; and filtering the trypan blue solution. In another embodiment, the method comprises: providing or preparing a trypan blue solution; cooling the trypan blue solution; filtering the trypan blue solution; and adding a water-soluble polymer to the cooled solution.

[0118] In another example, a method for preparing or treating or stabilizing or purifying a trypan blue solution includes: cooling the trypan blue solution; and filtering the cooled solution.

[0119] In some embodiments, the method further comprises providing or preparing a trypan blue solution prior to cooling. In some embodiments, according to the present disclosure, providing the trypan blue solution comprises preparing the trypan blue solution by mixing trypan blue with at least one component selected from the group consisting of an osmotic agent, an acid, a base, a buffer solution, a pH adjuster, a water-soluble polymer, a cell culture medium, water, or any combination thereof. In some embodiments, the method further comprises adding a component to the trypan blue solution before, during, or after the cooling step. In some embodiments, the method further comprises adding a component to the trypan blue solution before or after the filtering step.

[0120] In some embodiments, the method further comprises: repeating the cooling and / or filtering step. For example, another filtering step can be performed before cooling the trypan blue solution.

[0121] In some embodiments, the trypan blue solution before cooling is a buffered solution of trypan blue, wherein the content or concentration of trypan blue is about 0.01% to about 2%, or about 0.05% to about 1%, or about 0.1% to about 0.5%, or about 0.2% to about 0.5% based on the gross weight (with w / w%) or total volume (with w / v%) of the trypan blue solution. In some embodiments, trypan blue can be mixed with an aqueous buffer at an ambient or near ambient temperature and pressure before cooling. Trypan blue can also be mixed with an aqueous buffer at a temperature higher than ambient temperature.

[0122] In some embodiments, the trypan blue solution prior to cooling further comprises at least one of: an osmotic agent, an acid or a base, a water-soluble polymer, a cell culture medium, or any combination thereof.

[0123] In some embodiments, the trypan blue solution is cooled to a temperature for a period of time. The temperature is about 14°C or lower, about 13°C or lower, about 12°C or lower, about 11°C or lower, about 10°C or lower, about 9°C or lower, about 8°C or lower, about 7°C or lower, about 6°C or lower, about 5°C or lower, about 4°C or lower, about 3°C ​​or lower, about 2°C or lower, about 1°C or lower, or about 0°C. In some embodiments, the temperature is in the range of about 0°C to about 14°C, or about 3°C ​​to about 10°C, or about 4°C to about 6°C.

[0124] In some embodiments, the method also includes adjusting the pH of the trypan blue solution. The pH can be adjusted by adding a pH adjusting agent, a buffer, an acid or a base to the trypan blue solution before, during or after the filtering step. In some embodiments, the pH of the trypan blue solution is first adjusted to a first pH value to promote the precipitation of impurities. The first pH can be relatively acidic, for example, from about 6 to about 7. After filtering, the pH of the filtered (or remaining) trypan blue solution is adjusted to a second pH value. The second pH value can be relatively alkaline, for example, from about 7 to about 8, at which pH value, trypan blue is more stable in solution.

[0125] In some embodiments, the method further comprises adjusting the osmolarity of the trypan blue solution. The osmolarity can be adjusted by adding an osmotic agent to the trypan blue solution before, during, or after the filtration step.

[0126] In some embodiments, the method further comprises adding a water-soluble polymer to the trypan blue solution, as described above. In other embodiments, no water-soluble polymer is used in the methods of the present invention.

[0127] The methods described herein can reduce solid impurities in treated / stabilized trypan blue solutions. Solid impurities can be reduced by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100%.

[0128] The present method can make the treated trypan blue solution stable at a temperature and a period of time sufficient for cell staining tests, and free of or substantially free of aggregates and precipitates. In some embodiments, the trypan blue solution treated by the present method is stable for at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 2 weeks, or at least about 3 weeks, or at least about 4 weeks, or at least about 1 month, or at least about 2 months, or at least about 3 months, etc. The temperature range in which the treated trypan blue solution can remain stable is about 0 to about 50°C, or about 10°C to about 45°C, or about 20°C to about 40°C, or about 23°C to about 37°C.

[0129] In some aspects, the present disclosure relates to methods for staining cells. In one example, the method comprises: providing a trypan blue solution as described herein; mixing the trypan blue solution with a cell culture medium; and contacting the combined solution with a cell sample to be stained. The cell culture medium may contain cells to be stained with the trypan blue solution. In some embodiments, the present disclosure relates to providing a trypan blue solution comprising treating, stabilizing, or purifying the trypan blue solution using the methods described herein or any of the procedures thereof.

[0130] In some aspects, the present disclosure relates to a kit comprising components useful for performing any of the methods disclosed herein, such as for preparing or treating or stabilizing or purifying a trypan blue solution. The kit may include a plurality of components disclosed herein, a prepared trypan blue solution, a filter medium disclosed herein, instructions, or a combination thereof. The plurality of components may be stored in different containers.

[0131] The description disclosed herein can be present in this test kit in various forms, wherein one or more can be present in the test kit. A form in which these instructions can exist is as printed information on a suitable medium or substrate (e.g., one or more sheets of paper on which information is printed), in the packaging of the test kit, in a package insert, etc. Another means is a computer-readable medium, such as an electronic memory, a proximity integrated circuit card (PICC), a floppy disk, a magnetic tape or a CD, etc., on which information has been recorded. Another possible mode is a website address, which can be used to access the information of the removed site via the Internet. Any convenient method can be present in the test kit.

[0132] In one example, a kit according to the above description comprises a trypan blue buffer solution stored in a container; and a filter medium. In another example, a kit according to the above description comprises a trypan blue buffer solution stored in a container; and a water-soluble polymer stored in a separate container. The water-soluble polymer can be in solid or powder form, or as a stock solution of a defined concentration.

[0133] In some embodiments, the kit comprises a trypan blue solution stored in a container; a water-soluble polymer stored in a separate container; and a filter medium. In some embodiments, the kit described above may further comprise one or more components selected from the group consisting of an osmotic agent, an acid, a base, a buffer, a pH adjuster, a cell culture medium, or any combination thereof. The components are stored in separate containers.

[0134] In some embodiments, the kit further comprises instructions providing guidance to the user for performing the methods according to the present disclosure.

[0135] All publications, patents, and patent applications mentioned in the specification are indicative of the levels of those skilled in the art to which the present disclosure pertains.

[0136] Example

[0137] The following non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of some representative embodiments presently contemplated. These examples should not be construed as limiting any embodiments described in this specification, including those relating to methods for preparing, treating, stabilizing, or purifying trypan blue solutions, or methods for staining cells.

[0138] Example 1 - Treatment of Trypan Blue Solution

[0139] A study was conducted to determine the effect of cold filtration on trypan blue in PBS. Three different commercial samples of trypan blue solutions were studied (sample A, sample B, sample C). All samples contained approximately 0.2% w / v trypan blue. The samples were examined microscopically as received without further processing and were found to contain aggregates of red impurities. The sample solutions were then stored at 4°C for 14 hours to promote additional aggregation of the red dye. A portion of each cold sample solution was filtered through a Pall Acrodisc 0.2μm nylon syringe filter and imaged microscopically. For comparison, a portion of each cold sample solution was not filtered and imaged microscopically under the same conditions. The microscopic images are summarized in Figure 3 As can be seen, little or no dye aggregation was observed for all filtered trypan blue solutions (from samples A, B, and C). In contrast, significant dye aggregates were found in all unfiltered trypan blue solutions.

[0140] The sample solutions were each divided into two parts and stored at 4°C or 22°C for five days. After storage, the sample solutions were imaged by microscopy and the results were summarized in Figure 4 As can be seen, dye aggregates did not reappear in all three trypan blue solutions (from samples A, B and C) that were filtered and stored at 22°C for five days. In contrast, a large number of dye aggregates were found in the filtered solution stored at 4°C. However, it was found that warming the cold filtrate to ambient temperature at 22°C for 3 hours resulted in the dissolution of most of the dye aggregates. In contrast, warming the unfiltered trypan blue solution under the same conditions did not result in a large amount of dissolution of the dye aggregates.

[0141] The filtered and unfiltered solutions were stored at ambient temperature (22° C.) for an additional two days. After this storage period, the filtered solution was found to be essentially free of dye aggregates by microscopic examination. The unfiltered solution showed dye aggregates typical of trypan blue in PBS.

[0142] If the Trypan Blue solution is not cooled and filtered, dye aggregates readily form and persist (at either 4°C or 22°C), reflecting the normal behavior of untreated Trypan Blue solutions. After cooling and subsequently filtering the Trypan Blue solution, aggregates reappear after storage at the filtration temperature of 4°C, but do not persist at 22°C.

[0143] These unexpected results support that treating the trypan blue solution by cooling and filtering effectively removes most of the insoluble dye aggregates from the trypan blue solution and improves the stability of the trypan blue solution.

[0144] Example 2 - Reformulation of Trypan Blue Solution

[0145] In preliminary experiments, it was found that adding 250 mM sodium chloride to a 0.2% w / v aqueous solution of trypan blue caused rapid precipitation of the dye. After 16 hours of storage at 4°C in the presence of 150 mM sodium chloride, 0.2% w / v trypan blue precipitated. Red aggregates of the trypan blue impurity were the predominant form of precipitation. In contrast, trypan blue dissolved in deionized water at 0.2% w / v did not form red dye aggregates after two weeks at 4°C.

[0146] At a concentration of 150 mM, the alkali metal salts potassium chloride (KCl) and cesium chloride (CsCl) produced dye aggregation. Sodium azide (NaN3), sodium dihydrogen phosphate (NaH2PO4), and sodium sulfite (Na2SO3) also promoted dye aggregation at a sodium concentration of 150 mM, indicating that this effect is largely dependent on the cation. In contrast, lithium chloride did not promote dye aggregation at concentrations up to 600 mM.

[0147] Exemplary reconstituted formulations (Table 1) were prepared based on lithium chloride, D-glucose, and L-glycine. Each exemplary reconstituted formulation consisted of 0.2% w / v trypan blue dissolved in sodium azide-preserved sodium phosphate buffer. The amount of lithium chloride, D-glucose, or L-glycine added was sufficient to produce a theoretical osmolarity of approximately 300 mOsm / kg.

[0148] Commercial trypan blue in PBS formulation was used as a control. A similar sodium phosphate-based trypan blue solution containing sodium chloride was reconstituted as a comparative reconstituted formulation. The prepared reconstituted formulations and controls were each filtered (0.2 μm), subjected to three freeze-thaw cycles (2 hours, -20°C), and heated at 50°C for five days. After this temperature cycle, the osmolarity of each prepared solution was measured (Table 2).

[0149] Table 1. Composition of exemplary reformulated formulations according to Example 2 and controls.

[0150]

[0151]

[0152]

[0153] *Trypan blue added during preparation meets spectrophotometric specifications.

[0154] **Add phosphoric acid and sodium hydroxide as needed to produce a final pH of 7.1 to 7.5.

[0155] Table 2. Osmolarity of trypan blue solution according to Table 1.

[0156] preparation <![CDATA[Osmolality (mOsm kg -1 )]]> comparison 273±2 Comparative Reformulated Formulation 1 (Sodium Chloride) 277±1 Exemplary Reformulated Formulation 1 (Lithium Chloride) 277±2 Exemplary Reconstituted Formulation 2 (D-Glucose) 303±2 Exemplary Reformulated Formulation 3 (L-Glycine) 286±2

[0157] The reported values ​​are the mean ± SD of n = 3 replicate measurements.

[0158] The prepared and treated solutions were then divided into two parts and stored in separate bottles. These bottles were stored at an ambient laboratory temperature of 22°C or in a refrigerator at 4°C. The microscopic appearance, pH, and UV-visible absorbance of each solution were recorded at two-week intervals for six weeks. After a total of 13 weeks, the final microscopic appearance, pH, and UV-visible absorbance of the solutions were recorded.

[0159] like Figure 5 (Images C, D, E, H, I, and J) and Table 3 show that little or no dye aggregation was observed in exemplary reformulated formulations 1 to 3 after 13 weeks of storage at 4°C or 22°C. In contrast, the control ( Figure 5 Images A and F) and a comparative reformulated formulation containing sodium chloride ( Figure 5 Images B and G) show significant dye aggregation. In the control and comparative reconstituted formulation solutions, aggregation occurred within one week of storage at 22°C or 4°C. In contrast, exemplary reconstituted formulation solutions containing lithium chloride, D-glucose, or L-glycine prevented dye aggregation even when refrigerated at 4°C (a temperature that was found to exacerbate aggregation of untreated trypan blue solutions). This result suggests that replacing sodium chloride in the trypan blue buffer formulation can reduce or eliminate the formation of dye aggregates.

[0160] Table 3. Tests of Trypan Blue Solution at 22°C or 4°C over 13 weeks

[0161]

[0162]

[0163] After 13 weeks of storage at either 22°C or 4°C, the pH of each reconstituted formulation remained relatively stable.

[0164] As shown in Figure 6, the absorbance at 600nm of all exemplary reformulated formulations increased by about 12% compared to the control, which clearly shows that the soluble trypan blue in the exemplary reformulated formulations increased compared to the control. These comparative results further support that the treatment of the trypan blue solution effectively stabilizes trypan blue and maximizes the soluble trypan blue in the solution. Without wishing to be bound by any particular theory, it is believed that the trypan blue solution treated by the method of the present invention can remove impurities that would otherwise provide nuclei to initiate and promote aggregation in the trypan blue solution.

[0165] In addition, the absorbance at 600 nm of the solution remained stable after storage at 22°C or 4°C for 13 weeks, further supporting that the replacement of sodium chloride has no substantial effect on the spectroscopic properties of trypan blue.

[0166] Cell viability performance of exemplary reformulated Formulation 1 (containing lithium chloride) and control (containing sodium chloride) was performed using a Vi-CELL BLU instrument (Beckman Coulter) with Chinese hamster ovary (CHO) cells, and the results are compared and summarized in Table 4.

[0167] Table 4. Comparative results from cell viability testing of exemplary reformulated Formulation 1 and controls.

[0168] parameter comparison lithium chloride difference(%) P-value Cell counting 15543±649 15921±1050 2.4 .424 living cells 14741±621 15196±1019 3.1 .327 <![CDATA[Total (×10 6 ) cells / mL]]> 5.72±0.30 5.92±0.39 3.5 .295 <![CDATA[Live (×10 6 ) cells / mL]]> 5.48±0.23 5.65±0.38 3.1 .329 Viability (%) 94.8±0.3 95.4±0.2 0.6 .005 Average diameter (μm) 16.03±0.11 16.02±0.12 -0.1 .868 Average diameter of living organisms (μm) 16.23±0.11 16.18±0.11 -0.3 .423 Average roundness 0.91±0.01 0.92±0.01 0.7 067 Average roundness of the live 0.92±0.00 0.92±0.00 0.0 n / a Average cells per image 155±6 159±11 2.3 .443 Average background intensity 133±2 134±1 0.6 .295 LED power 17.09±0.44 19.31±0.38 13.0 ＜.001

[0169] Values ​​reported are means and 95% confidence intervals of n = 5 replicate measurements.

[0170] Statistical analysis was performed using a two-tailed Welch's unequal variance t-test at a 95% confidence level (α=0.05).

[0171] The results of the comparison do not support significant differences in behavior between Exemplary Reformulated Formulation 1 and the control as shown in Table 4. The largest difference in the single parameter was observed for LED power, which increased by 13% to account for the greater absorbance of Exemplary Reformulated Formulation 1 containing lithium chloride.

[0172] Example 3 - Dye Precipitation in Cell Culture Medium Containing Trypan Blue

[0173] Trypan blue (also known as Direct Blue 14) is a water-soluble azo dye derived from o-tolidine and is usually sold as sodium tetrasulfonate. The chemical structure of trypan blue contains four sulfonic acid groups (-SO3H), which are mainly deprotonated at slightly acidic to alkaline pH to form a negatively charged sulfonate group (-SO3 - ). Therefore, the trypan blue molecule has a net negative charge in the pH range relevant to cell culture applications.

[0174] Cell culture medium is a chemically complex mixture that generally comprises salt, pH buffer, amino acid, vitamin, protein and sugar. Antibiotic, supplement and other additives may also be included in the preparation of cell culture medium. Positively charged polyamino acids such as spermine and spermidine or aminoglycoside antibiotics such as streptomycin, geneticin (G418), kanamycin, gentamicin and neomycin may also be components of specific cell culture medium preparations. Some antibiotic may be added to cell culture medium as a selective agent to select genetically modified cells with antibiotic resistance.

[0175] Positively charged cell culture medium components can interact with trypan blue to cause dye precipitation. This can be achieved through the formation of polyelectrolyte complexes. Polyelectrolyte complexes are formed by the association of positively charged chemicals with negatively charged counterparts. The cationic (positively charged) and anionic (negatively charged) components of the complex are bound together by electrostatic interactions. The polyelectrolyte complex may be less soluble or completely insoluble than either parent substance.

[0176] It is known that some kinds of positively charged cell culture medium additives form insoluble polyelectrolyte complexes with trypan blue or chemical analogs of trypan blue.For example, Evans blue is a positional isomer of trypan blue, which forms an insoluble blue precipitate (Anal.Sci.2017, 33, 499-504) with neomycin (a polycationic aminoglycoside).The precipitate is insoluble in the time of pH 8.5, but dissolves in the time of pH 10.4.It is reported that the pKa value of neomycin is 5.7 and 7.6 to 8.8, which shows that the compound exists mainly in the form of positively charged cations in the time of pH 8.5, and exists mainly in the form of neutral non-cations in the time of pH 10.4.When pH is 10.4, neomycin is not positively charged, and therefore the polyelectrolyte complex containing Evans blue is unstable and the precipitate dissolves.

[0177] In this study, trypan blue was mixed with various cell culture media containing polycationic compounds. Nominal 0.4% trypan blue in NaCl was mixed with PBS (negative control), kanamycin (a positively charged aminoglycoside), and spermidine (a positively charged polyamine). Figure 7 As shown, both kanamycin and spermidine triggered immediate precipitation of trypan blue. This effect can reasonably be extended to other polycationic compounds, especially those containing amine functional groups.

[0178] Example 4 - Effect of polymer additives on the stabilization of trypan blue solution

[0179] In this study, the ability of polymer additives to inhibit the trypan blue precipitation caused by cell culture medium was studied. Various polymers were dissolved in a nominal 0.4% trypan blue solution in NaCl (Table 5). In this study, a total of 24 examples were prepared to represent six different polymers under four different concentrations. The composition of the 24 examples is summarized in Table 6. Polymer is composed of a repeating unit chain derived from a monomer. Each repeating unit comprises one or more chemical functional groups that can be combined with a cationic substance or dye. The mass of each polymer additive was calculated to produce an equimolar repeating unit concentration in the dye solution. Because the molecular weight of the repeating unit is different, this results in different weight volume percentages (% w / v) concentrations of each polymer.

[0180] Table 5. Polymer additives used in Example 4.

[0181]

[0182] Table 6. Exemplary Trypan Blue Solutions Containing Polymer Additives According to Example 4

[0183]

[0184]

[0185] For Na-PAA, polymer concentrations were arbitrarily chosen to represent four different levels of 1.00, 0.50, 0.10, and 0.05% w / v. Na-PAA was chosen to define the concentration range because it is the anionic polymer with the lowest repeat unit mass. This concentration range limits the amount of polymer dissolved in the dye solution. At all concentrations, the repeat unit concentration (mol / L) exceeded the nominal trypan blue concentration (where the ratio was greater than 1). The concentration of cationic species in proprietary cell culture medium formulations is unknown and may vary by product and manufacturer.

[0186] Poly(acrylic acid), poly(styrene sulfonic acid), and alginic acid were polymers used to test the hypothesis that anionic polymers would inhibit or prevent dye precipitation triggered by cell culture media. These polymers were used in the form of water-soluble anionic sodium salts (poly(acrylic acid sodium salt) (Na-PAA), poly(sodium 4-styrene sulfonate) (Na-PSS), and sodium alginate (Na-Alg)). In aqueous solution, the polymers carry anionic carboxylate or sulfonate groups that are counteracted by sodium cations.

[0187] Poly(vinyl alcohol) (PVA) and poly(vinylpyrrolidone) (PVP) are nonionic polymers that are known to bind or may bind to trypan blue-like azo dyes. Polyethylene glycol (PEG) is a nonionic polymer that is less likely to bind or interact with trypan blue.

[0188] Each of the 24 example solutions according to Table 6 was mixed with Gibco TM CD FortiCHO TM Each sample mixed with cell culture medium was qualitatively assessed for dye precipitation by microscopy, and the microscopy results are summarized in Figure 8 The positive control consisted of nominally 0.4% trypan blue in NaCl (no polymer additive) mixed with cell culture medium. As expected, this combination resulted in significant cell culture medium-triggered precipitation immediately after mixing.

[0189] The negative control consisted of a nominal 0.4% trypan blue solution in NaCl (no polymer additives) mixed with PBS. This combination did not result in trypan blue precipitation. However, upon receipt of the product, dye precipitation was often already present in the nominal 0.4% trypan blue in NaCl. Therefore, varying levels of dye precipitation were present in some images of the negative control.

[0190] like Figure 8 As shown, all four examples (A-1 to A-4) containing 0.05% to 1.00% w / v Na-PAA significantly prevented the formation of dye precipitates when the trypan blue solution was mixed with the cell culture medium. Similarly, the four examples (B-1 to B-4) containing 0.11% to 2.19% w / v Na-PSS prevented the formation of new dye precipitates relative to the positive control. This protective effect is temporary, and prolonged exposure to the cell culture medium (>10 minutes) will gradually lead to the appearance of trypan blue precipitation at low polymer concentrations. However, the dye exclusion test occurs in a relatively short period of time. Therefore, in this study, permanent protection is not required or targeted.

[0191] In contrast, Examples C-1 to C-4, which contained 0.12% to 2.41% w / v Na-Alg, did not prevent dye precipitation, but did so to a lesser extent relative to the positive control. This result is attributed to the fact that Na-Alg is poorly soluble in the nominal 0.4% trypan blue in NaCl. The gel-like, insoluble portion of Na-Alg was stained by trypan blue and appeared to promote dye precipitation. The extent of apparent polymer staining or dye precipitation worsened with increasing Na-Alg concentration. Trypan blue is one of the known dyes for cotton textiles and is primarily composed of the polysaccharide cellulose. Na-Alg is also a polysaccharide, which can lead to an affinity for trypan blue. These results suggest that polymer additives with relatively high solubility and that are not prone to strong interactions with trypan blue are preferred when used in stable trypan blue solutions.

[0192] Examples D-1 to D-4, which included PEG at concentrations ranging from 0.02% to 0.47% w / v, showed no inhibition or prevention of dye precipitation. This result may indicate that PEG has a low affinity for cationic polyamines and trypan blue. PEG and other neutral polymers that do not provide hydrogen bonds are less likely to interact with charged organic compounds. In contrast, Examples E-1 to E-4, which included PVA, were found to inhibit or prevent dye precipitation at concentrations ranging from 0.03% to 0.56% w / v. Although PVA is nonionic and does not form polyelectrolyte complexes, it is both a hydrogen bond donor and acceptor. These properties may allow it to bind to trypan blue or cationic polyamines in cell culture media.

[0193] Examples F-1 to F-4 containing PVP were found to prevent dye precipitation at concentrations of 0.59% and 1.18% w / v, while moderate inhibition occurred at concentrations of 0.06% and 0.12% w / v. This performance can be attributed to the concentration-dependent binding of the N-alkylpyrrolidone repeating units of PVP to trypan blue (Bull. Chem. Soc. Jpn. 1989, 62, 295-303). This binding can be facilitated by charge separation in the N-alkylpyrrolidone units due to amide-imide tautomerism. This mode of inhibition is less desirable than binding of cationic cell culture medium components because it can reduce the availability of the dye or cause changes in its absorption properties.

[0194] Due to the particularly favorable behavior of Na-PAA at low concentrations, further tests were performed on nominally 0.4% trypan blue in NaCl containing 0.05% w / v Na-PAA. Figure 9 As shown, the addition of 0.05% w / v Na-PAA did not change the absorbance properties of trypan blue. Because dye exclusion assays rely on optical recognition of stained cells in dye-rich medium, it is important that the color and relative opacity of the dye is not altered by polymer additives.

[0195] The Vi-CELLBLU cell viability analyzer was used to perform dye exclusion tests using a nominal 0.4% trypan blue solution in NaCl with and without 0.05% Na-PAA. Chinese hamster ovary (CHO) cells were exposed to a trypan blue solution containing 0.05% Na-PAA and a control lacking any polymer additives. The cell viability results are summarized in Table 7.

[0196] Table 7. Cell viability results according to Example 4.

[0197]

[0198] As shown in Table 7, the percent viability of the cells was not adversely affected by the presence of 0.05% w / v Na-PAA. The hypothesis that the mean results for trypan blue and trypan blue containing 0.05% w / v Na-PAA were equivalent was tested using a Welch t-test with unequal variances (α = 0.05). The hypothesis was not rejected for total cell concentration, viable cell concentration, and percent viability, supporting that 0.05% Na-PAA did not affect cell viability. The hypothesis was rejected for mean diameter (p < 0.001), which could indicate an effect on the osmotic balance of the solution. Any soluble additive would be expected to cause a change in osmotic pressure, and this change could be corrected by minor formulation adjustments.

[0199] Numbered clauses

[0200] The following numbered clauses further define example aspects and features of the compositions, methods, and techniques of the present disclosure:

[0201] 1. Trypan blue solution, comprising: trypan blue, osmotic agent and aqueous buffer.

[0202] 2. The trypan blue solution of clause 1, wherein the trypan blue solution has a trypan blue concentration of about 0.01% to about 2% based on the total weight (in w / w %) or total volume (in w / v %) of the trypan blue solution.

[0203] 3. The trypan blue solution of any one of clauses 1 to 2, wherein the osmotic agent is not a sodium salt.

[0204] 4. The trypan blue solution of any one of clauses 1 to 3, wherein the osmotic agent is selected from lithium chloride, D-glucose, L-glycine, or a combination thereof.

[0205] 5. The trypan blue solution of any one of items 1 to 4, further comprising an acid or a base.

[0206] 6. The trypan blue solution of clause 5, wherein the acid comprises phosphoric acid.

[0207] 7. The trypan blue solution of clause 5, wherein the base comprises an alkaline hydroxide.

[0208] 8. The trypan blue solution of any one of clauses 1 to 7, wherein the buffer is PBS buffer preserved with sodium azide.

[0209] 9. The trypan blue solution of any one of items 1 to 8, further comprising a water-soluble polymer.

[0210] 10. The trypan blue solution of clause 9, wherein the water-soluble polymer is selected from the group consisting of poly(acrylic acid) or a salt thereof, poly(styrene sulfonic acid) or a salt thereof, poly(vinyl pyrrolidone), or any combination thereof.

[0211] 11. The trypan blue solution of any one of clauses 9 to 10, wherein the concentration of the water-soluble polymer in the trypan blue solution is from about 0.05% to about 4% based on the total weight (w / w %) or total volume (w / v %) of the trypan blue solution.

[0212] 12. The trypan blue solution of any one of clauses 9 to 11, wherein the trypan blue solution has a repeat unit concentration of the water-soluble polymer of about 5 mmol / L to about 500 mmol / L.

[0213] 13. The trypan blue solution of any one of clauses 9 to 12, wherein the molar ratio of repeating units of the water-soluble polymer of the trypan blue solution to trypan blue is from about 1 to about 55.

[0214] 14. The trypan blue solution of any one of clauses 9 to 13, wherein the water-soluble polymer is poly(acrylic acid) or an alkali metal salt thereof having an average molecular weight of about 4,000 to about 6,000 g / mol.

[0215] 15. The trypan blue solution of any one of clauses 9 to 13, wherein the water-soluble polymer is poly(4-styrenesulfonic acid) or an alkali metal salt thereof having an average molecular weight of about 50,000 to about 80,000 g / mol.

[0216] 16. The trypan blue solution of any one of clauses 9 to 13, wherein the water-soluble polymer is poly(vinyl pyrrolidone) having an average molecular weight of about 30,000 to about 50,000 g / mol.

[0217] 17. The trypan blue solution of any one of clauses 1 to 16, further comprising cell culture medium.

[0218] 18. The trypan blue solution of any one of clauses 1 to 17, wherein the pH of the trypan blue solution is from about 7.0 to about 7.5.

[0219] 19. The trypan blue solution of any one of clauses 1 to 18, wherein the trypan blue solution has an osmolarity of about 270 to about 310 mOsm kg -1 .

[0220] 20. The trypan blue solution of any one of clauses 1 to 19, wherein the trypan blue solution is free or substantially free of precipitated impurities after storage at ambient temperature for at least about 10 minutes.

[0221] 21. A method for preparing a stable trypan blue solution, the method comprising: providing a trypan blue solution; and adding a water-soluble polymer to the trypan blue solution.

[0222] 22. The method of clause 21, wherein the water-soluble polymer is selected from poly(acrylic acid) or a salt thereof, poly(styrene sulfonic acid) or a salt thereof, poly(vinyl pyrrolidone), or any combination thereof.

[0223] 23. The method of any one of clauses 21 to 22, further comprising adjusting the trypan blue concentration to a range of about 0.01% to about 2% based on the total weight (in w / w %) or total volume (in w / v %) of the trypan blue solution.

[0224] 24. The method of any one of clauses 21 to 23, further comprising adjusting the molar ratio of repeating units of the water-soluble polymer to trypan blue from about 1 to about 55.

[0225] 25. The method of any one of clauses 21 to 24, further comprising filtering the trypan blue solution before or after adding the water-soluble polymer.

[0226] 26. The method of Clause 25, wherein the filtering is performed using at least one filter medium having an average pore size of about 0.2 μm.

[0227] 27. The method of any one of clauses 25 to 26, further comprising cooling the trypan blue solution prior to the filtering step.

[0228] 28. The method of clause 27, wherein the cooling step is performed before, after, or before and after the addition of the water-soluble polymer.

[0229] 29. The method of any one of clauses 27 to 28, wherein the trypan blue solution is cooled to a temperature of about 0°C to about 14°C.

[0230] 30. The method of any one of clauses 27 to 29, wherein the trypan blue solution is cooled for at least about 1 hour.

[0231] 31. The method of any one of clauses 21 to 30, further comprising mixing the trypan blue with at least one component selected from the group consisting of an osmotic agent, an acid, a base, a pH adjuster, a buffer solution, a cell culture medium, water, or any combination thereof.

[0232] 32. The method of any one of clauses 21 to 31, further comprising adjusting the pH of the trypan blue solution to a range of about 6.5 to about 8.

[0233] 33. The method of any one of clauses 27 to 32, further comprising adjusting the pH of the trypan blue solution to about 6 to about 7 before or during the cooling step.

[0234] 34. The method of clause 33, further comprising adjusting the pH of the trypan blue solution to about 7 to about 7.5 after the filtering step.

[0235] 35. The method of any one of clauses 21 to 34, further comprising adjusting the osmolarity of the trypan blue solution to about 270 to about 310 mOsm kg -1 range.

[0236] 36. The method of any one of clauses 21 to 35, further comprising repeating the cooling and / or filtering steps.

[0237] 37. The method of any one of clauses 21 to 36, further comprising packaging the filtered trypan blue solution.

[0238] 38. A method for preparing a stable trypan blue solution, the method comprising: cooling the trypan blue solution; and filtering the cooled solution.

[0239] 39. The method of clause 38, further comprising mixing the trypan blue solution with one or more components selected from the group consisting of an osmotic agent, an acid, a base, a pH adjuster, a cell culture medium, water, or any combination thereof.

[0240] 40. The method of any one of clauses 38 to 39, wherein the stable trypan blue solution has a trypan blue concentration of about 0.01% to about 2% based on the total weight (in w / w %) or total volume (in w / v %) of the trypan blue solution.

[0241] 41. The method of any one of clauses 39 to 40 wherein the osmotic agent is not a sodium salt.

[0242] 42. The method of any one of clauses 39 to 41 wherein the osmotic agent is selected from lithium chloride, D-glucose, L-glycine, or a combination thereof.

[0243] 43. The method of any one of clauses 39 to 42 wherein the acid comprises phosphoric acid.

[0244] 44. The process of any one of clauses 39 to 42, wherein the base comprises an alkaline hydroxide.

[0245] 45. The method of any one of clauses 38 to 44, wherein the buffer is sodium azide preserved PBS buffer.

[0246] 46. ​​The method of any one of clauses 38 to 45, wherein prior to cooling, the concentration of trypan blue in solution is near, at or exceeds the solubility limit.

[0247] 47. The method of any one of clauses 38 to 46, wherein the trypan blue solution is prepared by mixing trypan blue with an aqueous buffer at ambient or near ambient temperature and pressure prior to cooling.

[0248] 48. The method of any one of clauses 38 to 47, further comprising mixing the water-soluble polymer with the trypan blue solution before cooling or after filtering.

[0249] 49. The method of any one of clauses 38 to 48, further comprising adjusting the pH of the trypan blue solution.

[0250] 50. The method of Clause 49, further comprising adjusting the pH of the trypan blue solution to about 6 to about 7 before or during the cooling step.

[0251] 51. The method of clause 50, further comprising adjusting the pH of the trypan blue solution to about 7 to about 7.5 after the filtering step.

[0252] 52. The method of any one of clauses 38 to 51, further comprising adjusting the osmolarity of the trypan blue solution to about 270 to about 310 mOsm kg -1 range.

[0253] 53. The method of any one of clauses 38 to 52, further comprising repeating the cooling and / or filtering steps.

[0254] 54. The method of any one of clauses 38 to 53, further comprising packaging the filtered trypan blue solution.

[0255] 55. The method of any one of clauses 38 to 54, further comprising storing the filtered trypan blue solution.

[0256] 56. The method of clause 55, wherein the filtered trypan blue solution is stored at a temperature of about 0°C to about 25°C

[0257] 57. The method of any one of clauses 38 to 56, wherein the filtered trypan blue solution is free or substantially free of precipitated impurities after storage at ambient temperature for at least about 10 minutes.

[0258] 58. A method for cell staining, the method comprising: preparing a stable trypan blue solution according to any one of clauses 21 to 57; combining the stable trypan blue solution with a cell culture medium; and contacting the combined solution with a cell sample.

[0259] 59. A kit for performing the method according to any one of clauses 21 to 58, the kit comprising: a trypan blue solution; and at least one filter medium.

[0260] 60. The kit of Clause 59, further comprising a water-soluble polymer stored in a separate container.

[0261] 61. A kit for performing the method of any one of clauses 21 to 58, the kit comprising: a trypan blue solution stored in a first container; and a water-soluble polymer stored in a second container.

[0262] 62. The kit of clause 61, further comprising a filter medium.

[0263] According to the present disclosure, all compositions and methods disclosed and claimed herein can be manufactured and implemented without excessive experimentation. Although the compositions and methods of the present disclosure have been described according to some of the aforementioned embodiments, it will be apparent to those skilled in the art that, without departing from the true concept, spirit and scope of the present disclosure, modifications, changes, modifications and variations can be applied to the steps or sequence of steps of compositions, compositions, trypan blue solutions, methods and methods described herein. More specifically, it will be apparent that certain reagents, additives and compositions similar to those described herein can replace the reagents, additives and compositions described herein while achieving identical or similar results. It will be apparent to those skilled in the art that all such similar substitutions and modifications are considered to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

Claims

1. A method for preparing a stable trypan blue solution, comprising: (a) Cooling a solution of trypan blue in phosphate-buffered saline (PBS) preserved with sodium azide to a temperature of less than 15°C; (b) storing the solution from step (a) at a temperature of less than 15° C. for at least 14 hours; as well as (c) filtering the cooled solution from step (b).

2. The method of claim 1, further comprising filtering the trypan blue solution at ambient temperature and pressure prior to step (a).

3. The method of claim 1, wherein the trypan blue solution is cooled to a temperature of less than 5°C.

4. The method of claim 1, wherein the trypan blue solution is cooled to a temperature of 0°C to 14°C.

5. The method of claim 1, wherein the concentration of trypan blue in the solution is near, at, or above the solubility limit.

6. The method of claim 1, wherein the trypan blue solution is prepared by mixing trypan blue with PBS at ambient or near ambient temperature and pressure prior to cooling.

7. The method of claim 1 , further comprising adding a water-soluble polymer to the trypan blue solution before cooling and / or after filtering, wherein the water-soluble polymer is selected from the group consisting of poly(acrylic acid) or a salt thereof, poly(styrene sulfonic acid) or a salt thereof, poly(vinyl pyrrolidone), or any combination thereof. 8 . The method of claim 7 , wherein the trypan blue solution has a molar ratio of the repeating unit of the water-soluble polymer to trypan blue of 1 to 55.

9. The method of claim 1, further comprising storing the filtered trypan blue solution at a temperature of 0°C to 50°C.

10. The method of claim 1, wherein a water-soluble polymer is added to the trypan blue solution before the cooling step (a), during the filtering step (c), or after the filtering step (c).

11. The method of claim 10, wherein the water-soluble polymer is selected from the group consisting of poly(acrylic acid) or a salt thereof, poly(styrene sulfonic acid) or a salt thereof, poly(vinyl pyrrolidone), or any combination thereof.

12. The method of claim 10, further comprising filtering the trypan blue solution before or after adding the water-soluble polymer.

13. The method of claim 1, further comprising mixing the solution at a temperature of less than 15°C for at least 1 hour after the cooling step (a) and before the filtering step (c).

14. The method of claim 1, further comprising mixing the solution at a temperature of less than 15°C for at least 3 hours after the cooling step (a) and before the filtering step (c).

15. The method of claim 1, further comprising mixing the solution at a temperature of less than 15°C for at least 6 hours after the cooling step (a) and before the filtering step (c).

16. The method of claim 1, further comprising mixing the solution at a temperature of less than 15°C for at least 12 hours after the cooling step (a) and before the filtering step (c).

17. The method of claim 1, wherein steps (a) and (b) are performed at a temperature of less than 5°C.

18. The method of claim 1, wherein steps (a) and (b) are performed at a temperature of 0°C to 14°C.

19. The method of claim 1, further comprising mixing the solution at a temperature of less than 15°C for at least 1 hour after the cooling step (a) and before the filtering step (c).

20. The method of any one of claims 1 to 9 and 13 to 19, wherein the filtered trypan blue solution is free or substantially free of precipitated impurities after storage at a temperature of 0°C to 50°C for at least 10 minutes.

Images