Quality control method for serum or serum substitute
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Filing Date
- 2026-05-14
- Publication Date
- 2026-06-08
Abstract
Description
Quality control methods for serum or serum substitutes
[0001] The present invention relates to a method for controlling the quality of serum or serum substitutes, and in particular to a method for evaluating differences in quality between storage conditions or lots of serum or serum substitutes.
[0002] Serum is an essential supplement for cell culture. It is a highly complex solution containing millions of proteins, including growth factors and cytokines, as well as a wide variety of organic and inorganic substances, including lipids, carbohydrates, vitamins, electrolytes, trace elements, and other undefined components. Fetal bovine serum (FBS) is commonly used in cell culture, and its quality is affected by processes such as collection, processing, safety testing, and removal of contaminants, as well as differences in the infrastructure, regulations, and management of the country of origin and the manufacturer (Non-Patent Document 1).
[0003] Serum requires strict quality control because it significantly influences cell proliferation, phenotypes, and differentiation. However, due to its complex composition, the components in serum that affect cell culture and how they affect it are not fully understood. Furthermore, because serum is derived from animals, its cell culture support performance typically varies significantly between lots. Despite this, manufacturers only provide limited information, such as the content of a few protein components and the results of endotoxin and virus tests and growth tests for specific cell lines. As a result, end users must actually culture the desired cells to verify the serum's quality (so-called lot-to-lot verification), which requires a great deal of time and effort. Furthermore, lot checks depend on the condition of the cells and the skill of the experimenter, making reliable and consistent quality control often difficult.
[0004] Serum substitutes with defined compositions have been developed to ensure consistent quality and eliminate pathogen contamination. However, most serum substitutes also contain animal-derived components, and there are lot-to-lot variations, and their quality is prone to deterioration depending on storage conditions. Furthermore, the composition of many commercially available serum substitutes is not disclosed, making it difficult for end users to verify their quality, just as it is with serum.
[0005] P. Hawkes et al., "Fetal Bovine Serum - Country of Origin, Geographic Relevance, and Labeling", BioProcess. J., 2019, Vol. 18
[0006] The present invention aims to solve the problems of the prior art and to provide an easy, rapid, and consistent quality control technology for serum and serum substitutes, regardless of the state of cells or the skill of the experimenter.
[0007] The present inventors have successfully used a cross-reactive sensing method to distinguish between samples containing a wide variety of proteins and samples containing microflora (WO 2018 / 088510, WO 2020 / 262413). Based on the above method, the present inventors have established a method for determining serum quality with high accuracy.
[0008] That is, according to one embodiment, the present invention provides a method for controlling the quality of serum or serum substitute, comprising: (1) dissolving probes in a plurality of solvents having different ionic strengths and / or pHs, wherein the probes comprise: (a) a cationic polymer having at least five primary amino groups per molecule and a weight-average molecular weight of 1,000 to 500,000; and (b) an environmentally responsive fluorophore, the fluorophore being covalently bonded to some of the primary amino groups in the cationic polymer; (2) adding an analytical sample containing serum or a serum substitute to the plurality of probe solutions prepared in step (1); (3) measuring the fluorescence intensities of the plurality of probe solutions to which the analytical sample has been added in step (2); and (4) comparing the fluorescence intensity pattern obtained in step (3) with the fluorescence intensity pattern obtained for a reference sample.
[0009] The environmentally responsive fluorophore is preferably selected from the group consisting of a fluorophore having a naphthalenesulfonic acid skeleton, a fluorophore having a benzofurazan skeleton, a fluorophore having a xanthene skeleton, a fluorophore having a pyrene skeleton, and an aggregation-induced emission fluorophore.
[0010] The cationic polymer is preferably a linear or branched polyamino acid, polyallylamine, polyamidoamine or polyalkyleneimine.
[0011] It is preferable that the environmentally responsive fluorescent group be covalently bonded to 1 to 50% of the primary amino groups in the cationic polymer.
[0012] It is preferable that at least a portion of the primary amino groups of the cationic polymer to which the environmentally responsive fluorophore is not covalently bonded be modified with an acyl group or an amino acid.
[0013] The measurement of the fluorescence intensity in (3) above is preferably carried out at a plurality of excitation wavelengths and fluorescence wavelengths.
[0014] The step (4) makes it possible to evaluate differences in quality between origins, manufacturers, or lots of the serum or serum substitute.
[0015] Alternatively, step (4) can be used to evaluate the difference in quality of the serum or serum substitute due to storage conditions.
[0016] Preferably, the quality is the ability to support the survival, proliferation, transformation or differentiation of cultured cells.
[0017] According to the method of the present invention, the quality of serum or serum substitute can be determined with high accuracy, ease, and consistency by simply obtaining a fluorescence intensity pattern that reflects the sum of nonspecific interactions between all biological components and the probes using only one or a few types of probes, without requiring comprehensive analysis of the components in serum or serum substitute or actual cell culture. Therefore, according to the method of the present invention, it is possible to perform quality control of serum or serum substitute, for example, by simply comparing the fluorescence intensity pattern with a serum or serum substitute of guaranteed quality as a reference sample.
[0018] Figure 1A shows the change in fluorescence spectrum when human serum was added to 300 nM probe 1 (-None) / 20 mM MOPS (pH 7.0). Figure 1B shows the change in fluorescence intensity when various concentrations of human serum were added to 300 nM probe 1 (-None), probe 2 (-Phe), probe 4 (-Suc), or probe 5 (-Pht) / 20 mM MOPS (pH 7.0). Figure 2 shows the change in fluorescence intensity (I-I) obtained from serum (16 animal species) x 2 solvent conditions x 6 probes x 2 wavelength sets x 10 measurements. ０ ) is a graph showing the change in fluorescence intensity (I-I) obtained by measuring fetal bovine serum albumin (4 producing countries x 2 lots) x 2 solvent conditions x 6 probes x 2 wavelength sets x 10 measurements. FIG. 3 shows a heat map representation of the data in FIG. 2 and a dendrogram obtained by unsupervised hierarchical cluster analysis of the data in FIG. 2. FIG. 4 shows a plot of the results up to the second principal component obtained by analyzing the data in FIG. 2 by unsupervised principal component analysis. FIG. 5 shows a plot of the results up to the second discriminant score obtained by analyzing the data in FIG. 2 by supervised linear discriminant analysis. FIG. 6 shows the change in fluorescence intensity (I-I) obtained by measuring fetal bovine serum albumin (4 producing countries x 2 lots) x 2 solvent conditions x 6 probes x 2 wavelength sets x 10 measurements. ０) is a heat map. Figure 7 is a diagram plotting the first and second discriminant scores (left) and the first and third discriminant scores (right) obtained by analyzing the data of Figure 6 by linear discriminant analysis. Figure 8 is a diagram plotting the results up to the second discriminant score obtained by analyzing the data of Figure 6 by linear discriminant analysis without distinguishing between lots. Figure 9 is a diagram plotting the results up to the second discriminant score obtained by integrating the data of Figures 2 and 6 and analyzing it by linear discriminant analysis. Figure 10 is a diagram plotting the change in fluorescence intensity (I-I) obtained by measuring fetal bovine serum (untreated or 7 storage / treatment conditions) x 2 solvent conditions x 6 probes x 2 wavelength sets x 10 measurements. ０ ) is a heat map. Figure 11 is a plot of the second discriminant scores obtained by linear discriminant analysis of the data in Figure 10 . Figure 12 is a graph showing the increase in cell number in human dermal fibroblasts (NHDF) cultured with fetal bovine serum (untreated or under seven storage / treatment conditions). Figure 13 is a graph showing the change in fluorescence intensity upon addition of various concentrations of CDM1 to 300 nM of probe 1 (-None) or probe 5 (-Pht) in 20 mM MOPS (pH 7.0). Figure 14 is a microscopic image showing the results of culturing HCN4-EGFP_409B2 cells with CDM1 containing N2 and B27, untreated or treated at various temperatures (top: bright-field image; bottom: fluorescent image). Figure 15 is a graph showing the incidence of GFP-positive (HCN4-positive) cells in cultures of HCN4-EGFP_409B2 cells using CDM1 containing N2 and B27, either untreated or treated at various temperatures. Figure 16 shows the change in fluorescence intensity (I-I) obtained by measuring CDM1 (untreated or with four treatment conditions containing N2 / B27) × 2 solvent conditions × 6 probes × 2 wavelength sets × 10 measurements. ０) is a heat map. Figure 17 is a diagram plotting the results up to the second principal component obtained by analyzing the data in Figure 16 by principal component analysis. Figure 18 is a diagram plotting the results up to the second discriminant score obtained by analyzing the data in Figure 16 labeled with differentiation induction performance (Good, Acceptable, or Unacceptable) by linear discriminant analysis. Figure 19 is a diagram showing changes in fluorescence intensity when various concentrations of KSR are added to 300 nM Probe 1 (-None) or Probe 5 (-Pht) in 20 mM MOPS (pH 7.0). Figure 20 is an image of 409B2 cell cultures using KSR with different storage periods (top: stained image of undifferentiated cell colonies, bottom: bright-field microscopy image of cells). Figure 21 is a graph showing changes in colony and cell counts (relative values) in the cultures shown in Figure 20. FIG. 22 shows the change in fluorescence intensity (I-I) obtained by KSR (5 storage periods) x 2 solvent conditions x 6 probes x 2 wavelength sets x 10 measurements. ０ ) is a heat map. Figure 23 is a diagram plotting the second discriminant scores obtained by analyzing the data in Figure 22 by linear discriminant analysis. Figure 24 is a diagram showing the structural formulas of the probes (probes 1 to 6: -None, -Phe, -Nle, -Suc, -Pht, -Pyr) used in the examples. The values in parentheses indicate the ClogP values of the sites where functional groups were introduced. Figure 25 is a diagram showing changes in fluorescence intensity when various concentrations of yeast extract were added to 300 nM probe 1 (-None) or probe 5 (-Pht) / 20 mM MOPS (pH 7.0). Figure 26 shows the change in fluorescence intensity (I-I) obtained by yeast extract (2 manufacturers x 4 autoclave times) x 2 solvent conditions x 6 probes x 2 wavelength sets x 6 measurements. ０ 27 is a diagram in which the data in FIG. 26 is analyzed by linear discriminant analysis, and the obtained second discriminant scores are plotted.
[0019] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in this specification.
[0020] According to a first embodiment, the present invention provides a method for controlling the quality of serum or a serum substitute, comprising: (1) dissolving probes in a plurality of solvents having different ionic strengths and / or pHs, wherein the probes comprise: (a) a cationic polymer having at least five primary amino groups per molecule and a weight-average molecular weight of 1,000 to 500,000; and (b) an environmentally responsive fluorophore, the fluorophore being covalently bonded to some of the primary amino groups in the cationic polymer; (2) adding an analytical sample containing serum or a serum substitute to the plurality of probe solutions prepared in step (1); (3) measuring the fluorescence intensities of the plurality of probe solutions to which the analytical sample has been added in step (2); and (4) comparing the fluorescence intensity pattern obtained in step (3) with the fluorescence intensity pattern obtained for a reference sample.
[0021] First, the probe used in the method of this embodiment will be described. The probe used in the method of this embodiment contains (a) a cationic polymer having at least five primary amino groups per molecule and a weight-average molecular weight of 1,000 to 500,000, and (b) an environmentally responsive fluorophore, with the fluorophore covalently bonded to some of the primary amino groups in the cationic polymer.
[0022] The cationic polymer (a) of the probe in this embodiment may be any polymer as long as it has a weight-average molecular weight of 1,000 to 500,000 and at least five primary amino groups per polymer molecule. Here, "polymer" refers to a compound formed by polymerizing two or more monomers, which may be the same or different, and therefore may be a homopolymer or a copolymer. The degree of polymerization of the polymer is not particularly limited, and therefore "polymer" also includes oligomers formed by polymerizing several (e.g., 3 to 20) monomers.
[0023] The weight average molecular weight of the cationic polymer of the probe in this embodiment is 1,000 to 500,000, preferably 1,500 to 200,000, and particularly preferably 2,000 to 100,000.
[0024] The cationic polymer of the probe in this embodiment has at least 5, preferably 7 or more, and particularly preferably 10 or more primary amino groups in one molecule of the polymer.
[0025] In this embodiment, the cationic polymer that can be used for the probe is preferably a linear or branched polyamino acid, polyallylamine, polyamidoamine, or polyalkyleneimine. Furthermore, these cationic polymers may be copolymerized with polyethylene glycol.
[0026] In this embodiment, the polyamino acid that can be used for the probe may be a polymer of the same type of amino acid residues, or may be a polymer of different types of amino acid residues. Furthermore, the amino acid residues that make up the polyamino acid may be either L- or D-isomers. Examples of polyamino acids include polylysine, polyornithine, random copolymers of lysine and phenylalanine, and random copolymers of lysine and tyrosine. A preferred polyamino acid in this embodiment is polylysine or polyornithine.
[0027] In this embodiment, examples of polyalkyleneimine that can be used for the probe include polyethyleneimine, polypropyleneimine, polybutyleneimine, etc. A preferred polyalkyleneimine in this embodiment is polyethyleneimine.
[0028] The environmentally responsive fluorophore (b) of the probe in this embodiment may be any fluorophore whose fluorescence properties change depending on the environment around the fluorescent molecule. Examples of such fluorophores include, but are not limited to, fluorophores whose fluorescence properties change depending on the polarity around the fluorescent molecule, fluorophores whose fluorescence properties change depending on the pH around the fluorescent molecule, and fluorophores whose fluorescence properties change depending on the degree of crowding around the fluorescent molecule.
[0029] Examples of fluorophores whose fluorescent properties change depending on the polarity around the fluorescent molecule include fluorophores having a naphthalenesulfonic acid skeleton, such as 5-dimethylaminonaphthalene-1-sulfonyl (dansyl), 1-anilinonaphthalene-8-sulfonic acid (ANS), N-methyl-2-anilinonaphthalene-6-sulfonic acid (MANS), and 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS); fluorophores having a benzofurazan skeleton, such as 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD), 7-nitro-2,1,3-benzoxadiazole (NBD), 4-(aminosulfonyl)-2,1,3-benzoxadiazole (ABD), and ammonium 2,1,3-benzoxadiazole-4-sulfonate (SBD); and fluorescent derivatives thereof.
[0030] Examples of fluorophores whose fluorescence properties change depending on the pH around the fluorescent molecule include fluorophores having a xanthene skeleton, such as fluorescein, fluorescein isothiocyanate (FITC), 5(6)-carboxyfluorescein (5(6)-FAM), 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and seminaphthalodafluorescein (SNARF); fluorophores having a pyrene skeleton, such as 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HTPS); and fluorescent derivatives thereof.
[0031] Examples of fluorophores whose fluorescence properties change depending on the degree of crowding around the fluorescent molecule include aggregation-induced emission (AIE) fluorophores such as tetraphenylethylene (TPE), 10,10',11,11'-tetrahydro-5,5'-bisbenzo[a,d][7]annulenylidene (THBA), and 1,1,2,3,4,5-hexaphenylsilole (HPS), as well as fluorescent derivatives thereof.
[0032] A preferred environmentally responsive fluorophore in this embodiment may be dansyl, NBD, DBD or TPE or a fluorescent derivative thereof.
[0033] The probe in this embodiment comprises a cationic polymer (a) having an environmentally responsive fluorophore (b) covalently bonded to a portion of the primary amino groups in the cationic polymer (a). Here, "a portion" preferably refers to 1 to 50%, and particularly preferably 5 to 20%, of the primary amino groups in the cationic polymer molecule.
[0034] The probe in this embodiment can be synthesized by a conventionally known chemical synthesis method, or the chemical synthesis method described in the examples of WO 2018 / 088510 or WO 2020 / 262413, or a chemical synthesis method equivalent thereto. Specifically, for example, the probe can be prepared by labeling the amino group of the cationic polymer (a) with an environmentally responsive fluorophore (b) activated by an active ester group such as an N-hydroxysuccinimide (NHS) ester group, a pentafluorophenyl (PFP) ester group, or an O-acylisourea group, an isothiocyanate group, or a halogenated alkyl group.
[0035] In the probe of this embodiment, at least a part of the primary amino groups to which an environmentally responsive fluorophore has not been introduced may be modified with a modifying group. In other words, the primary amino groups to which an environmentally responsive fluorophore has not been introduced (-NH ２In at least a portion of the probes, a hydrogen atom (H) may be substituted with a monovalent modifying group (X) (-NHX). The modifying group may have either a negative or positive charge and may preferably be selected from the group consisting of a guanidino group, an acyl group, and an amino acid. Examples of amino acids include leucine, valine, isoleucine, tyrosine, tryptophan, phenylalanine, serine, asparagine, glutamine, or derivatives thereof. The probe in this embodiment may be modified with one or more modifying groups selected from the above.
[0036] Modification of an amino group with a modifying group can be carried out by conventionally known chemical synthesis methods. Modification of an amino group with a guanidino group can be carried out by guanidinylating the amino group using, for example, 1H-pyrazole-1-carboxamidine hydrochloride. Modification of an amino group with an acyl group can be carried out by, for example, a nucleophilic acyl substitution reaction using a carboxylic acid anhydride such as acetic anhydride, phthalic anhydride, or naphthalenedicarboxylic acid anhydride. Modification of an amino group with an amino acid can be carried out by, for example, dehydration condensation of the carboxyl group of the amino acid with the amino group.
[0037] The probe in this embodiment can interact nonspecifically with any biomolecule. The "biomolecule" targeted by the probe in this embodiment may be any type of compound present in a living organism and may have any molecular weight. Examples of such biomolecules include, but are not limited to, proteins, peptides, amino acids, nucleic acids, nucleotides, lipids, polysaccharides, monosaccharides, vitamins, hormones, etc. Furthermore, the biomolecules targeted by the probe in this embodiment may include not only known biomolecules but also unknown biomolecules.
[0038] The probe in this embodiment preferably interacts primarily with proteins and their peptide fragments, and can non-specifically interact with any protein and its peptide fragments, regardless of the type of protein or the type of post-translational modification.
[0039] In the method of this embodiment, the probe is dissolved in a plurality of solvents with different ionic strengths and / or pHs. The solvent for dissolving the probe may be an aqueous solvent containing any buffer and / or salt. Examples of buffers include MES, MOPS, EPPS, HEPES, Tris, phosphoric acid, acetic acid, citric acid, boric acid, and glycine. Examples of salts include NaCl, KCl, and MgCl. ２ , Na ２ SO ４ , K. ２ SO ４ , MgSO ４ , NaI, NaSCN, etc. In this embodiment, the pH of the solvent is preferably 4.0 to 10.0, and particularly preferably 5.0 to 7.0. In this embodiment, the ionic strength of the solvent is preferably 10 to 500 mM. In this embodiment, the concentration of the probe is preferably 0.1 to 100 μg / mL.
[0040] In the method of this embodiment, solvents with, for example, two or more different ionic strengths and / or pH conditions can be used, preferably three or more, and particularly preferably six or more. Furthermore, in the method of this embodiment, one type of probe may be used, but it is more preferable to use multiple types of probes, for example, two or more, preferably three or more, and particularly preferably six or more. For example, by using two types of solvents and three types of probes, six types of probe solutions can be prepared, thereby obtaining six-dimensional data for one analytical sample. For example, by using five types of solvents and three types of probes, fifteen types of probe solutions can be prepared, thereby obtaining fifteen-dimensional data for one analytical sample. In this way, by increasing the number of solvents and probes, more multidimensional data can be obtained.
[0041] An assay sample containing serum or a serum substitute is then added to the multiple probe solutions.
[0042] The "serum" in this embodiment may be derived from any animal, but is preferably derived from a mammal such as a cow, horse, sheep, goat, pig, rabbit, rat, mouse, or human. The age of the animal from which the serum is derived is not particularly limited, and the animal may be of any age, including fetus, newborn, and adult. The country / region of origin of the serum is also not particularly limited. Serum derived from a wide variety of animals produced in various countries / regions is commercially available, and the method of this embodiment can analyze any of them.
[0043] "Serum replacement" is a general term for serum-free supplements used in cell culture in place of serum. Serum replacements generally contain several to several dozen factors selected from growth factors, cytokines, hormones, etc. Various serum replacements are commercially available, such as N2 supplement (Cell Cult. Neurosci. 1985; pp. 3-43) and its improved products, B27 supplement (J. Neurosci. Res. 1993; 35(5): 567-76) and its improved products, G5 supplement (Int. J. Dev. Neurosci. 1984; 2(6): 575-84) and its improved products, Gibco™ KnockOut™ Serum Replacement (KSR) (Thermo Fisher Scientific), StemSure™ Serum Replacement (SSR) (Fujifilm Wako Pure Chemical Industries), XF212 XerumFree (TNC BIO Examples of serum substitutes include, but are not limited to, human platelet lysate, plant hydrolysate, corn extract, yeast extract, soy extract, etc. Although the specific compositions of many commercially available serum substitutes are not published, the method of the present embodiment can analyze any of them.
[0044] The analysis sample in this embodiment may consist solely of serum or a serum substitute, or may be a medium containing serum or a serum substitute. The final concentration of serum or serum substitute added to the probe solution may be, for example, 0.001 vol% to 99.9 vol%, and preferably 0.01 vol% to 10 vol%. If the concentration of serum or serum substitute in the analysis sample is unknown, the sample may be serially diluted as appropriate and added to the probe solution.
[0045] In this step, the probe interacts non-specifically with any biomolecules contained in the serum or serum substitute.
[0046] Next, the fluorescence intensities of the multiple probe solutions to which the analytical sample has been added are measured. In the method of this embodiment, fluorescence intensities can be measured at an excitation wavelength of 300 to 500 nm and a fluorescence wavelength of 400 to 700 nm. Furthermore, in the method of this embodiment, it is preferable to measure the fluorescence intensities for each measurement target at multiple excitation wavelength / fluorescence wavelength sets (e.g., excitation wavelength (nm) / fluorescence wavelength (nm): 340 / 520, 330 / 480, 345 / 505, 360 / 530, etc.). For example, fluorescence intensities can be measured using two, three, or four sets of excitation wavelength / fluorescence wavelength.
[0047] The fluorescence intensity of the probe solution varies depending on the type, amount, and state of the biomolecules contained in the serum or serum substitute, as well as the conditions of the solvent the probe is dissolved in. Therefore, this step allows us to obtain a fluorescence intensity pattern (fluorescence fingerprint) specific to the sample.
[0048] The fluorescence intensity pattern obtained for the analytical sample is then compared with that obtained for the reference sample. The fluorescence intensity pattern for the reference sample may be obtained by measuring the fluorescence intensity in parallel with the analytical sample, or may be a predetermined fluorescence intensity pattern prepared in advance. The comparison of the fluorescence intensity patterns is preferably performed by multivariate analysis such as principal component analysis, linear discriminant analysis, or hierarchical cluster analysis to reduce the number of dimensions and compress the differences between the fluorescent fingerprints into two or three dimensions for comparison.
[0049] The method of this embodiment can determine the quality of serum or serum substitutes based solely on differences in fluorescence intensity patterns. Here, the "quality" of serum or serum substitutes refers to their ability to support cells when added to a culture medium and used in culture, specifically, their ability to support characteristics such as survival, proliferation, transformation, or differentiation of cultured cells. These characteristics can be determined by various activities such as adhesion, motility, division, metabolism, and secretion of growth factors and cytokines. Therefore, the ability to support the characteristics of cultured cells can refer to their ability to support, maintain, or control cellular activities such as adhesion, motility, division, metabolism, and secretion of growth factors and cytokines. Typically, the quality of serum or serum substitutes varies significantly depending on the place of origin, manufacturer, lot, etc., but this does not provide sufficient information for determining quality. Furthermore, the quality of serum or serum substitutes deteriorates depending on the storage temperature and duration, but it is extremely difficult and impractical to individually identify which components, to what extent, and how changes cause quality deterioration. In contrast, the method of this embodiment obtains a fluorescence intensity pattern (fluorescence fingerprint) that reflects the sum of nonspecific interactions between various biomolecules in serum and the probe, and compares it with the fluorescence intensity pattern for a reference sample, thereby making it possible to detect differences in the overall quality of serum or serum substitutes.
[0050] Therefore, in certain embodiments, for example, a fluorescent fingerprint obtained for a serum or serum substitute currently being used for culture (reference sample) is compared with a fluorescent fingerprint obtained for a serum or serum substitute (analytical sample) from a different origin / manufacturer A, B, or C. As a result, if the fluorescent fingerprint obtained for the serum or serum substitute from origin / manufacturer A is probabilistically closest to the distribution of the fluorescent fingerprint obtained for the reference sample, it can be estimated or identified that the serum or serum substitute from origin / manufacturer A can be used in place of the serum or serum substitute currently being used.
[0051] In another specific embodiment, for example, a fluorescent fingerprint obtained for a serum or serum substitute currently being used for culture (reference sample) is compared with a fluorescent fingerprint obtained for a serum or serum substitute (analytical sample) from the same place of origin / manufacturer but from different lots A, B, or C. As a result, if the fluorescent fingerprint obtained for the serum or serum substitute from lot A is probabilistically closest to the distribution of the fluorescent fingerprint obtained for the reference sample, it can be estimated or identified that the serum or serum substitute from lot A can be used in place of the serum or serum substitute currently being used.
[0052] In another specific embodiment, for example, a fluorescent fingerprint obtained for a serum or serum substitute (reference sample) immediately after purchase is compared with a fluorescent fingerprint obtained for a serum or serum substitute (analytical sample) from the same place / manufacturer / lot but stored under different conditions A, B, or C (e.g., different storage temperatures and / or periods). As a result, if the fluorescent fingerprint obtained for the serum or serum substitute stored under condition A is probabilistically closest to the distribution of the fluorescent fingerprint obtained for the reference sample, it can be estimated or determined that the quality of the serum or serum substitute stored under condition A has not deteriorated.
[0053] The method of this embodiment allows the quality of serum or serum substitute to be evaluated without the need to actually culture cells and test their proliferation or differentiation ability. Therefore, the method of this embodiment enables easy, rapid, and consistent quality control of serum or serum substitute that is not affected by the state of the cells or the skill of the experimenter.
[0054] The present invention will be further described below with reference to examples, which should not be construed as limiting the scope of the present invention.
[0055] <1. Materials and Reagents> (1-1) Probe In this example, the nonspecific fluorescent probe synthesized in the examples of WO 2020 / 262413 was used. Probe 1: A probe obtained by introducing tetraphenylethylene (TPE) into polyethylene glycol-block-poly-L-lysine trifluoroacetate (PEG-b-PLL, Mw: 17200) [number of ethylene glycol repeating units: 104, number of L-lysine trifluoroacetate repeating units: 52] (hereinafter referred to as "-None") Probe 2: A probe obtained by dehydration condensation reaction of the amino group in -None (probe 1) with phenylalanine (hereinafter referred to as "-Phe") Probe 3: A probe obtained by dehydration condensation reaction of the amino group in -None (probe 1) with norleucine (hereinafter referred to as "-Nle") Probe 4: A probe obtained by nucleophilic acyl substitution reaction of the amino group in -None (probe 1) with succinic anhydride (hereinafter referred to as "-Suc") Probe 5: -None (probe 1) by nucleophilic acyl substitution reaction with phthalic anhydride (hereinafter referred to as "-Pht") Probe 6: -None (probe 1) by nucleophilic acyl substitution reaction with 2,3-pyrazinedicarboxylic anhydride (hereinafter referred to as "-Pyr") Probe 7: -None (probe 1) by nucleophilic acyl substitution reaction with phthalic anhydride (hereinafter referred to as "-Pyr")
[0056] The structural formulas of probes 1 to 6 (-None, -Phe, -Nle, -Suc, -Pht, -Pyr) are shown in FIG.
[0057] (1-2) Serum The serum used in this example is shown in Table 1.
[0058] Table 1. serum
[0059] (1-3) Serum Substitutes The serum substitutes used in this example are shown in Table 2.
[0060] Table 2. serum substitute
[0061] (1-4) Media and Reagents The media and reagents used in this example are shown in Table 3.
[0062] Table 3. Media and reagents
[0063] <2. Changes in Probe Fluorescence Induced by Human Serum> Probes 1, 2, 4, and 5 (-None, -Phe, -Suc, -Pht) were dissolved in 20 mM MOPS (pH 7.0) to prepare four types of probe solutions (333 nM). Each probe solution was added to a 96-well microplate (Corning, 3650) at 180 μL / well using an automated pipetting device (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity at an excitation wavelength of 330 nm (fluorescence wavelength: 480 nm) and the fluorescence spectrum (fluorescence wavelength: 372-700 nm) were measured using a microplate reader (Cytation5, BioTek). Then, using an automatic pipetting device, 20 μL / well of a solution of human serum (serum number 13) at various concentrations in 20 mM MOPS (pH 7.0) was added, and the plate was incubated at 35°C for 10 minutes. The fluorescence intensity and fluorescence spectrum were measured under the same conditions (final concentrations: 300 nM probe, 0 to 0.5 vol% human serum, 20 mM MOPS (pH 7.0)).
[0064] The change in the fluorescence spectrum of probe 1 (-None) upon addition of 0 to 0.075 vol% human serum is shown in Figure 1. The fluorescence intensity of probe 1 (-None) increased in a serum concentration-dependent manner, and the fluorescence intensity at 460 nm of probe 1 (-None) in 0.075 vol% human serum increased approximately 63-fold compared to the fluorescence intensity in 0 vol% human serum.
[0065] Figure 2 shows the changes in 480 nm fluorescence intensity of probes 1, 2, 4, and 5 (-None, -Phe, -Suc, -Pht). The fluorescence intensity of probe 1 (-None) increased in a serum concentration-dependent manner when the serum addition amount was 0.075 vol% or less, but began to decrease when the serum addition amount exceeded 0.075 vol%. The fluorescence intensities of probe 4 (-Suc) and probe 5 (-Pht) increased monotonically in a serum concentration-dependent manner. The fluorescence intensity of probe 2 (-Phe) hardly changed even when serum was added. These results suggest that nonspecific fluorescent probes exhibit diverse responses to serum.
[0066] <3. Discrimination of sera from different animal species> Probes 1 to 6 (-None, -Phe, -Nle, -Suc, -Pht, -Pyr) were dissolved in the following two buffer solutions: 22.2 mM MOPS (pH 7.0) or 22.2 mM acetic acid (pH 5.0) to prepare 12 probe solutions (333 nM). Each probe solution was added to a 96-well half-well microplate (Corning, 3993) at 180 μL / well using an automatic pipetting device (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity (I ) was measured using a microplate reader (Cytation5, BioTek) at the following two sets of excitation wavelength (nm) / emission wavelength (nm). ０ ) were measured: (Ch1) 330 / 480, (Ch2) 360 / 530. Then, using an automated dispenser, serum solutions from various animals (serum numbers 1-16, each 1 vol% serum / pure water) were added at 12 μL / well, and after incubation at 35°C for 10 minutes, the fluorescence intensity (I) was measured under the same conditions (final concentrations: 300 nM probe, 0.1 vol% serum, 20 mM buffer). Each measurement (16 sera x 2 solvent conditions x 6 probes x 2 wavelength sets) was performed 10 times.
[0067] The results are shown in Figure 2. The change in fluorescence intensity (I-I) before and after the addition of serum ０ ) was normalized using the following formula: z = (x - μ) / σ (z is the normalized value, x is the raw data of the change in fluorescence intensity, μ is the population mean, and σ is the population standard deviation). In the figure, error bars indicate standard deviation. The fluorescence intensity of each probe changed differently depending on the type of serum and solvent conditions, and unique fluorescence patterns were obtained for each serum from different animal species.
[0068] The above data were analyzed by unsupervised hierarchical clustering analysis, and the resulting tree is shown in Figure 3. Sera from all animal species were clustered. These results demonstrate the feasibility of using nonspecific fluorescent probes to identify the animal species from which serum originates.
[0069] The data were analyzed using unsupervised principal component analysis, and the results, plotted up to the second principal component, are shown in Figure 4. Metaclusters corresponding to Bovidae and Rodentia (shown by dotted lines) were found, and Laurasia and Eucoptera were distinguished (shown by dashed lines). These results suggest that the fluorescence patterns reflect information about the animal's phylogeny.
[0070] The data were analyzed by supervised linear discriminant analysis, and the results obtained up to the second discriminant score are plotted in Figure 5. The clusters of serum derived from each animal were distributed without overlapping with each other. Furthermore, when these results were analyzed by the jackknife method and the holdout method, it was confirmed that the serum derived from each animal could be distinguished with 100% accuracy in both cases.
[0071] 4. Discrimination of Fetal Bovine Serum from Different Origins and Lots. Fetal bovine sera (serum numbers 18-21) (two lots each) from the United States, Australia, Ireland, and Chile were analyzed using the same procedures and conditions as in Section 3 above. Figure 6 shows a heat map of the measurement results (four origins, two lots, two solvent conditions, six probes, and two wavelength sets). The fluorescence intensity of each probe varied depending on the type of fetal bovine serum and the solvent conditions, resulting in unique fluorescence patterns for each fetal bovine serum from different origins and lots. The fluorescence patterns were also analyzed by linear discriminant analysis. The plots of the first and second discriminant scores, as well as the first and third discriminant scores, are shown in Figure 7. The clusters for each fetal bovine serum were distributed without overlapping. Furthermore, when these results were analyzed by the jackknife and holdout methods, it was confirmed that each fetal bovine serum could be distinguished with 96% and 88% accuracy, respectively. The fluorescence patterns were also analyzed by linear discriminant analysis without distinguishing between lots. The plots of the obtained second discriminant scores are shown in Figure 8. The clusters for each country of origin were distributed without overlapping with each other. Furthermore, when the results were analyzed using the jackknife and holdout methods, it was confirmed that each fetal bovine serum could be identified with 96% and 84% accuracy, respectively.
[0072] Furthermore, the data obtained here (serum numbers 18-21 x 2 lots x 2 solvent conditions x 6 probes x 2 wavelength sets) was integrated with the data obtained in 3 above (serum numbers 1-16 x 2 solvent conditions x 6 probes x 2 wavelength sets), analyzed by linear discriminant analysis, and the results up to the second discriminant score were plotted, as shown in Figure 9. The formation of a cluster for bovine sera revealed that properties common to bovine sera are reflected in their fluorescence patterns, even if the origins and lots differ.
[0073] 5. Identification of fetal bovine serum stored under different conditions Fetal bovine serum (serum number 18) was dispensed into microtubes and stored or treated under the following conditions in a sealed, light-shielded state: (1) 4°C for 2 weeks, (2) 4°C for 4 weeks, (3) 37°C for 1 week, (4) 37°C for 2 weeks, (5) 37°C for 4 weeks, (6) 56°C for 30 minutes, or (7) 56°C for 60 minutes. Analysis was then performed using the same procedures and conditions as in 3 above. Immediately after thawing, fetal bovine serum (serum number 18) was used as a control (untreated).
[0074] A heat map of the measurement results is shown in Figure 10. Unique fluorescence patterns were obtained for each fetal bovine serum stored under different conditions. The fluorescence patterns were analyzed using linear discriminant analysis, and the results up to the second discriminant score were plotted. Figure 11 shows the results. All sera stored at 4°C overlapped with the control (untreated), whereas sera stored at 37°C or treated at 56°C were not overlapping. When the third and fourth discriminant scores were plotted, the control (untreated), sera stored at 4°C for 2 weeks, and sera stored at 4°C for 4 weeks clustered without overlapping (data not shown). Furthermore, analysis of these results using the jackknife and holdout methods confirmed that the serum storage and treatment conditions could be distinguished with 98% and 97% accuracy, respectively.
[0075] Subsequently, human dermal fibroblasts (NHDF) (PromoCell GmbH) were cultured using the sera stored or treated under the above conditions (1) to (7) and the control (untreated) serum, and the ability of each serum to support cell proliferation was compared. NHDF (4 × 10 ４The cells / mL were added to a 48-well plate (AGC Techno Glass) at 200 μL / well, and each serum was added at 22.2 μL / well. ２ After culturing for 48 hours under the conditions, the number of viable cells was counted using Cell Counting Kit-8 (Dojindo Laboratories).
[0076] The results are shown in Figure 12. The increase in absorbance at 450 nm (ΔAbs ４５０ The larger the ΔAbs value, the higher the number of viable cells, and the higher the cell proliferation and viability. ４５０ The mean ± standard error (n = 6) is shown. One-way ANOVA and Tukey's post-hoc test showed significant differences compared to untreated serum for serum stored at 37°C for 1 week (p = 0.0362), 37°C for 2 weeks (p = 0.0083), 37°C for 4 weeks (p ≦ 0.0001), and 56°C for 30 minutes (p = 0.0028) (* in the figure). However, no difference was observed between untreated serum and serum stored at 4°C.
[0077] These results indicate that changes in the performance of fetal bovine serum on cell proliferation due to differences in storage conditions are reflected in the fluorescence pattern, and suggest that it may be possible to detect changes in the performance of serum on cell proliferation using a nonspecific fluorescent probe.
[0078] 6. Changes in Probe Fluorescence Due to Serum Substitutes for Differentiation Induction Probes 1 and 5 (-None, -Pht) were dissolved in 20 mM MOPS (pH 7.0) to prepare two types of probe solutions (400 nM). Each probe solution was added to a 384-well microplate (Corning, 3575) at 45 μL / well using an automated pipetting device (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity at an excitation wavelength of 330 nm (fluorescence wavelength 480 nm) was measured using a microplate reader (Cytation 5, BioTek). Cardiac differentiation induction medium 1 (DM / F-12 supplemented with 1% N-2 supplement, 2% B-27 supplement, 1% NEAA, 1% GlutaMAX, 1% PS, and 0.1 mM 2-ME) (hereinafter referred to as "CDM1") was prepared, and various concentrations of CDM1 / 20 mM MOPS (pH 7.0) solutions were added at 15 μL / well using an automated pipetting device. After incubation at 35°C for 10 minutes, fluorescence intensity was measured under the same conditions (final concentrations: 300 nM probe, 0-2 vol% CDM1, 20 mM MOPS (pH 7.0)).
[0079] The results are shown in Figure 13. The Y axis represents the change in fluorescence intensity before and after the addition of CDM1 (I-I ０ ) are shown as the mean ± standard error (n=3). The fluorescence intensity of both probe 1 (-None) and probe 5 (-Pht) increased in a CDM1 concentration-dependent manner, and the response saturated at approximately 1 vol%.
[0080] 7. Identification of serum substitutes for differentiation induction treated under different conditions N-2 supplement (hereinafter simply referred to as "N2") and B-27 supplement (hereinafter simply referred to as "B27") were each dispensed into microtubes and treated in a sealed, light-shielded state under the following conditions: 45°C, 55°C, 65°C, or 75°C for 30 minutes. CDM1 was prepared using untreated N2 and B27 or after each treatment. HCN4-EGFP_409B2 cells (Regen. Ther. 2022;21:239-249) (409B2 cells (human iPS cells) transfected with a BAC vector that knocked in EGFP at the HCN4 locus) were suspended in mouse embryonic fibroblast-conditioned medium (MEF-CM) and plated on Geltrex (Thermo Fisher Scientific, A1413302). The following day, the medium was replaced with CDM1 supplemented with 100 ng / mL Noggin and 3.3 μM CHIR99021, and then replaced with fresh CDM1 until day 3. On day 4, the medium was replaced with CDM1 supplemented with 3.3 μM CHIR99021 and 5 μM IWP-2. On day 5, the cells were dispersed and collected by trypsinization, and plated at 1 x 10 cells per well on a PrimeSurface™ 96U low-attachment plate using cardiac differentiation induction medium 2 (RPMI 1640 supplemented with 1% pyruvic acid, 1% GlutaMAX, 1% ITS-G supplement, 2 mM L-ascorbic acid, 10 mM nicotinamide, 0.2 μM dexamethasone, 0.5% FBS, 1% PS, 10 ng / mL bFGF, and 10 ng / mL BMP4) (hereinafter referred to as "CDM2"). ４ The cells were seeded at a density of 1000 cells / well. The medium was then replaced with fresh CDM2 every 2-3 days. On day 19 or 20, GFP fluorescence was observed using a fluorescence microscope (Olympus, IX73). 2 mg / mL collagenase II (Worthington, CLS2) was then added to the cells and incubated overnight at room temperature. The cells were dispersed and collected, and GFP-positive (i.e., HCN4-positive) cells were counted using a FACSAria™ III cell sorter (BD biosciences).
[0081] The results of the fluorescence microscopy observation are shown in Figure 14, and the quantification results of GFP-positive cells are shown in Figure 15. The number of GFP-positive cells decreased as the heat treatment temperature of N2 / B27 increased. These results confirmed that the differentiation-inducing ability of N2 and B27 deteriorated due to heat treatment.
[0082] Probes 1 to 6 (-None, -Phe, -Nle, -Suc, -Pht, -Pyr) were dissolved in the following two buffer solutions: 24 mM MOPS (pH 7.0) or 24 mM acetic acid (pH 5.0) to prepare 12 probe solutions (360 nM). Each probe solution was added to a 384-well microplate (Corning, 3575) at 50 μL / well using an automatic pipetting device (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity (I ) was measured using a microplate reader (Cytation5, BioTek) at the following two sets of excitation wavelength (nm) / emission wavelength (nm). ０ ) were measured: (Ch1) 330 / 480, (Ch2) 360 / 530. Then, 10 μL / well of CDM1 solution (3 vol% CDM1 / pure water) containing untreated or heat-treated N2 / B27 was added using an automated pipetting device. After incubation at 35°C for 10 minutes, the fluorescence intensity was measured under the same conditions (final concentrations: 300 nM probe, 0.5 vol% CDM1, 20 mM buffer).
[0083] A heat map of the measurement results is shown in Figure 16. A unique fluorescence pattern was obtained for each CDM1 containing N2 / B27 heated at different temperatures. The fluorescence patterns were analyzed by linear discriminant analysis, and the results, up to the second discriminant score, are plotted in Figure 17. Treatment at 55°C and 65°C significantly shifted the clusters. Meanwhile, little change was observed between untreated and 45°C treatment, and between 65°C and 75°C treatment. CDM1 containing N2 / B27 treated at 45°C, which showed little change in both differentiation-inducing ability and fluorescence pattern, was labeled "Good." CDM1 containing N2 / B27 treated at 55°C, which showed a significant change in fluorescence pattern but little change in differentiation-inducing ability, was labeled "Acceptable." CDM1 containing N2 / B27 treated at 65°C and 75°C, which almost completely lost its differentiation-inducing ability, was labeled "Unacceptable." The results were analyzed by linear discriminant analysis, and the results, up to the second discriminant score, are plotted in Figure 18. The clusters of each ability were distributed without overlapping with each other. Furthermore, when these results were analyzed by the jackknife method and the holdout method, it was confirmed that the differentiation induction ability of CDM1 could be identified with 100% accuracy in both cases.
[0084] These results indicate that even slight deterioration in the quality of CDM1, which could not be detected based on differentiation-induced culture, is reflected in the fluorescence pattern, and that the quality of N2 and B27 can be discriminated with high accuracy using nonspecific fluorescent probes.
[0085] 8. Changes in Probe Fluorescence Due to Serum Substitutes for Cell Proliferation. Probes 1 and 5 (-None, -Pht) were dissolved in 20 mM MOPS (pH 7.0) to prepare two probe solutions (400 nM). Each probe solution was added to a 384-well microplate (Corning, 3575) at 45 μL / well using an automated pipetting device (Andrew+, Andrew Alliance). After 10 minutes of incubation at 35°C, the fluorescence intensity at an excitation wavelength of 330 nm (fluorescence wavelength: 480 nm) was measured using a microplate reader (Cytation 5, BioTek). Various concentrations of KSR / 20 mM MOPS (pH 7.0) solutions were added at 15 μL / well using the automated pipetting device. After incubation at 35° C. for 10 minutes, the fluorescence intensity was measured under the same conditions (final concentrations: 300 nM probe, 0 to 0.1 vol% KSR, 20 mM MOPS (pH 7.0)).
[0086] The results are shown in Figure 19. The Y axis represents the change in fluorescence intensity before and after the addition of KSR (I-I ０ ) are shown as the mean ± standard error (n=3). The fluorescence intensity of both probe 1 (-None) and probe 5 (-Pht) increased in a KSR concentration-dependent manner, and the response saturated at approximately 0.1 vol%.
[0087] 9. Identification of Serum Substitutes for Cell Growth with Different Storage Periods KSR stored at −80°C and within the manufacturer-specified expiration date (In-date 1, In-date 2, In-date 3) and KSR that had expired (Expired 1 (approximately 4 years since the expiration date) and Expired 2 (approximately 12 years since the expiration date)) were used to prepare DM / F-12 supplemented with 20% KSR, 1% NEAA, 1% GlutaMAX, 1% PS, and 0.1 mM 2-ME (hereinafter referred to as "iPS cell growth medium"). 409B2 cells (human iPS cells) were suspended in iPS cell growth medium supplemented with 5 ng / mL bFGF and 10 μM Y27632, and plated at 1 × 10 cells / well onto a 6-well plate (Corning, 3506) coated with Matrigel (Corning, 356231). ４The cells were seeded at a density of 1 x 10 cells / well. Thereafter, the medium was replaced with fresh iPS cell growth medium supplemented with 5 ng / mL bFGF every three days and cultured. On the 9th day after seeding, undifferentiated cell colonies were stained using an AP Staining Kit (SBI, AP100B-1), and the number of stained colonies was counted. To count the cell number, 1 x 10 cells were used. ５ 409B2 cells were seeded at a density of 100 cells / well and cultured with fresh iPS cell growth medium supplemented with 5 ng / mL bFGF every day. Microscopic images were taken four days after seeding, and the cells were then dispersed by trypsinization, collected, and counted using a hemocytometer.
[0088] The results are shown in Figures 20 and 21. In Figure 20, the upper panel shows a stained image of the colonies, and the lower panel shows a bright-field microscope image of the cells. In Figure 21, the left panel shows the change in the number of stained colonies, and the right panel shows the change in cell number (normalized to 1 as the number of colonies in culture using In-date 1). Statistical analysis showed that proliferation was significantly suppressed in culture using Expired 1 KSR (mean ± standard deviation, n = 4, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, one-way ANOVA and Tukey post-hoc test).
[0089] Twelve probe solutions (360 nM) were prepared by dissolving probes 1 to 6 (-None, -Phe, -Nle, -Suc, -Pht, and -Pyr) in two buffer solutions: 24 mM MOPS (pH 7.0) or 24 mM acetic acid (pH 5.0). Each probe solution was added to a 384-well microplate (Corning, 3575) at 50 μL / well using an automated pipetting device (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, fluorescence intensity was measured using a microplate reader (Cytation5, BioTek) at two excitation / emission wavelengths (nm): (Ch1) 330 / 480 and (Ch2) 360 / 530. Then, 10 μL / well of KSR solution (0.02 vol% KSR / pure water) was added using an automatic dispenser. After incubation at 35°C for 10 minutes, the fluorescence intensity was measured under the same conditions (final concentrations: 300 nM probe, 0.02 vol% KSR, 20 mM buffer).
[0090] A heat map of the measurement results is shown in Figure 22. Unique fluorescence patterns were obtained for each KSR with different storage periods. The fluorescence patterns, consisting of [-None, pH 7.0 and pH 5.0, Ch1 and Ch2], [-Nle, pH 7.0, Ch1 and Ch2], [-Phe, pH 7.0, Ch1], [-Phe, pH 5.0, Ch1 and Ch2], [-Suc, pH 7.0 and pH 5.0, Ch1], [-Pht, pH 5.0, Ch2], and [-Pyr, pH 7.0 and pH 5.0, Ch1 and Ch2], were analyzed by linear discriminant analysis. The results, plotted up to the second discriminant score, are shown in Figure 23. Clusters of KSR with different storage periods were distributed without overlapping. Furthermore, when these results were analyzed by the jackknife and holdout methods, it was confirmed that each KSR could be distinguished with 97% and 93% accuracy, respectively.
[0091] These results indicate that deterioration in the quality of KSR, which could not be detected based on growth culture, is also reflected in the fluorescence pattern, demonstrating that the quality of KSR can be determined with high accuracy using a nonspecific fluorescent probe.
[0092] 10. Identification of serum substitutes for cell growth treated under different conditions Yeast extract powders from different manufacturers (Becton Dickinson and Oxoid) (hereinafter referred to as "YE-B" and "YE-O," respectively) were dissolved in purified water at a concentration of 1%. 10 mL of each solution was added to a 15 mL centrifuge tube and autoclaved at 121°C for 10 to 30 minutes. Probes 1 and 5 (-None, -Pht) were dissolved in 20 mM MOPS (pH 7.0) to prepare two probe solutions (1800 nM). Untreated or autoclaved yeast extract solution for 30 minutes was diluted to various concentrations with 20 mM MOPS (pH 7.0) and added to a 384-well microplate (Corning, 3575) at 50 μL / well using an automated pipetting device (Assist Plus, Integra Biosciences). After 10 minutes of incubation at 35 °C, the fluorescence intensity at an excitation wavelength of 330 nm (fluorescence wavelength 480 nm) was measured using a microplate reader (Synergy H1, Agilent Technologies). 10 μL / well of 1800 nM probe solution / 20 mM MOPS (pH 7.0) was added using the automated pipetting device. After incubation at 35° C. for 10 minutes, the fluorescence intensity was measured under the same conditions (final concentrations: 300 nM probe, 0 to 0.3% yeast extract, 20 mM MOPS (pH 7.0)).
[0093] The results for YE-B (simply referred to as "YE" in the figure) are shown in Figure 25. The Y axis represents the change in fluorescence intensity (II) after addition of the probe. ０ The mean ± standard error (n = 3) of the values of the probes is shown. The fluorescence intensity of both probe 1 (-None) and probe 5 (-Pht) increased depending on the concentration of the yeast extract solution, and the response saturated at approximately 0.10% for probe 1 and approximately 0.30% for probe 5.
[0094] Twelve probe solutions (1800 nM) were prepared by dissolving probes 1 to 6 (-None, -Phe, -Nle, -Suc, -Pht, and -Pyr) in purified water. A 0.12% solution of each yeast extract in either 24 mM MOPS (pH 7.0) or 24 mM acetic acid (pH 5.0) was added to a 384-well microplate (Corning, 3575) at 50 μL / well using an automated pipetting device (Assist Plus, Integra Biosciences). After incubation at 35°C for 10 minutes, fluorescence intensity was measured using a microplate reader (Synergy H1, Agilent Technologies) at two sets of excitation wavelength (nm) / emission wavelength (nm): (Ch1) 330 / 480, (Ch2) 360 / 530. Then, 10 μL / well of probe solution (1800 nM probe / pure water) was added using an automated pipetting device. After incubation at 35°C for 10 minutes, fluorescence intensity was measured under the same conditions (final concentrations: 300 nM probe, 0.1% yeast extract, 20 mM buffer).
[0095] A heat map of the measurement results is shown in Figure 26. A unique fluorescence pattern was obtained for each yeast extract solution from a different manufacturer and autoclaving time. The fluorescence patterns were analyzed by linear discriminant analysis, and the results up to the second discriminant score were plotted. The results are shown in Figure 27. The clusters of yeast extract solutions from different manufacturers and autoclaving times were distributed without overlapping. Furthermore, when these results were analyzed by the jackknife method, it was confirmed that each yeast extract solution could be distinguished with 96% accuracy.
[0096] These results indicate that differences in yeast extract quality between manufacturers and changes in yeast extract quality due to differences in autoclaving time are reflected in the fluorescence patterns, demonstrating that the quality of yeast extract can be determined with high accuracy using a nonspecific fluorescent probe.
Claims
1. (1) A step of dissolving the probe in several solvents with different ionic strengths and / or pH, wherein the probe is (a) A cationic polymer having at least five primary amino groups in one molecule and a weight-average molecular weight of 1,000 to 500,000, (b) Environmentally responsive fluorophores and The material contains, and the fluorescent group is covalently bonded to a portion of the primary amino group in the cationic polymer. (2) Adding an analytical sample containing serum or a serum substitute to a plurality of probe solutions prepared in step (1), (3) A step of measuring the fluorescence intensity of multiple probe solutions to which the analytical sample was added in step (2), (4) A step of comparing the fluorescence intensity pattern obtained in step (3) with the fluorescence intensity pattern obtained for a reference sample, thereby evaluating differences in quality between the origin, manufacturer, or lot of the serum or serum substitute, or evaluating differences in quality due to the storage conditions of the serum or serum substitute. A method for evaluating differences in the quality of serum or serum substitutes, including [specific components / conditions].
2. The method according to claim 1, wherein the environmentally responsive fluorophore is selected from the group consisting of a fluorophore having a naphthalene sulfonic acid skeleton, a fluorophore having a benzofurazan skeleton, a fluorophore having a xanthene skeleton, a fluorophore having a pyrene skeleton, and an aggregation-induced luminescence fluorophore.
3. The method according to claim 1, wherein the cationic polymer is a linear or branched polyamino acid, polyallylamine, polyamidoamine, or polyalkyleneimine.
4. The method according to claim 1, wherein 1 to 50% of the primary amino groups in the cationic polymer are covalently bonded to the environmentally responsive fluorophores.
5. The method according to claim 1, wherein at least a portion of the primary amino groups of the cationic polymer that are not covalently bonded to the environmentally responsive fluorophore are modified with an acyl group or an amino acid.
6. The method according to claim 1, wherein the measurement of fluorescence intensity in step (3) is performed for a plurality of excitation wavelengths and fluorescence wavelengths.
7. The method according to claim 1, wherein the quality is the ability to support the survival, proliferation, trait, or differentiation of cultured cells.