RNA-free animal serum

Abstract

To provide an animal serum, mainly FBS, that does not contain RNA or contains a minimum amount of RNA, enabling research in cell cultures without risk of contamination or interference by the RNA of the serum.SOLUTION: The present invention relates to RNA-free mammal serums which can be useful for cell culture or for producing pharmaco-biological products due to the fact that they maintain their supplementation features. Another embodiment of the present invention relates to a method for removing RNA from mammal serum through the application of sequential serum heating, alkalization, and neutralization steps.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of cell biology, and more particularly to the field of cell culture supplements. Generally speaking, the present invention relates to serum, preferably fetal bovine serum, having reduced ribonucleic acid (RNA) or minimal RNA content below the limit of detection, and a method useful for removing RNA from serum. This RNA-reduced serum is useful for RNA expression analysis from cell cultures without the interference of RNA normally present in animal serum.

[0002] Cell culture is one of the primary tools for in vitro basic and biomedical research. It involves maintaining cells from a variety of animal species, from insects to mammals, in controlled environments, including temperature, humidity, and carbon dioxide levels. To keep cells alive and continually dividing, cell cultures are maintained in liquid media containing the appropriate amount of salts to allow the cells to perform their most important metabolic functions (Swain P. Basic Techniques and Limitations in Establishing Cell Culture: a Mini Review. Adv Anim Vet Sci (2014) doi:10.14737 / journal.aavs / 2014 / 2.4s.1.10; Arora M. Cell Culture Media: A Review. Mater Methods (2013) doi:10.13070 / mm.en.3.175).

[0003] A key component of in vitro cell culture is serum. Serum contains many of the growth factors, lipids, and proteins necessary for cells to continually divide. As such, serum is added as a nutritional supplement to synthetic liquid media used to maintain cell cultures. Serum used for this purpose can be derived from human, equine, or bovine sources, with fetal bovine serum (FBS) being the most widely used worldwide (Arora M. Cell Culture Media: A Review. Mater Methods (2013) doi:10.13070 / mm.en.3.175.).

[0004] In addition to the components mentioned above, FBS is rich in nucleic acids, the most abundant of which are various biologically functional forms of RNA, such as transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), Piwi-associated small RNA (pi-RNA), and nucleolar RNA (snoRNA) (Chen X, et al. Cell Res. 2008 Oct;18(10):997-1006). Among the various types of RNA, some are contained within extracellular vesicles (EVs) and some are contained outside of them (Keerthikumar S. et al. J Mol Biol. 2016 Feb 22;428(4):688-692).

[0005] EVs are tiny lipid bilayer structures with diameters ranging from 50 to 400 nm that contain proteins, RNAs with various biological functions, and several small molecules. EVs are composed of subgroups that vary in diameter and molecular weight, with exosomes, which are 50 to 1200 nm in diameter, being the most widely studied (Colombo M. et al. Annu Rev Cell Dev Biol. 2014;30:255-89). An important feature of EVs is that they have been shown to be able to transfer their contents to recipient cells both in vitro and in vivo. EVs have been proposed as a cell-cell communication system, potentially having significant implications for metabolism and the development of diseases such as cancer and diabetes (Muralidharan-Chari V. et al. J Cell Sci. 2010 May 15;123(Pt 10):1603-11 and Bobrie A. et al. Traffic. 2011 Dec;12(12):1659-68).

[0006] Studies using cultured cells have demonstrated that FBS-derived EVs are capable of transferring their RNA content to human and murine cell lines, highlighting the potential for bovine RNA to be transferred to cell cultures when FBS is used. This finding highlights the high likelihood that scientific literature reporting on the detection and analysis of miRNA expression since the discovery of miRNAs to date contains a mixture of bovine RNA and RNA from the species from which the cell lines in question were derived. The extent of interference with both bovine RNA detection and its possible function in cell culture remains unclear. The primary reason for this is that over 70% of bovine miRNAs are identical to miRNAs from other mammals, including human miRNAs (Wei Z. et al. Sci Rep. 2016 Aug 9;6:31175).

[0007] In this regard, Wei et al. state that FBS contains various types of RNA, including both protein-coding and regulatory RNAs, such as messenger RNA, microRNA (miRNA), ribosomal RNA, and small nuclear RNA, and that up to 70% of these may remain in the serum even after ultracentrifugation.

[0008] Wei et al. point out that RNA-free serum is preferable for such studies because FBS-derived RNA is co-isolated with cell culture-derived RNA, which can interfere with or mislead subsequent RNA analysis (Tosar JP. J Extracell Vesicles. 2017 Jan 12;6(1):1272832).

[0009] To reduce potential interference and misinterpretation due to the presence of endogenous EVs in serum used for cell culture, several methods have been developed to remove EVs from serum.

[0010] In this regard, US Patent No. 9,005,888 describes a method for isolating EVs produced by animal cells and producing plasma or serum with a reduced EV content, demonstrating that some of the miRNAs originally present in the plasma or serum are no longer detectable by quantitative PCR (qPCR) after applying the method. However, the method described in US Patent No. 9,005,888 has several drawbacks, such as the use of a precipitation solution containing polyethylene glycol (PEG), the residues of which remain in the treated serum and may be considered contaminants.

[0011] For example, it has been reported that PEG induces heterokaryon formation, i.e., cell fusion to generate multinucleated cells (Davidson RL, Gerald PS. Somatic Cell Genet. 1976 Mar;2(2):165-76.), and therefore, PEG remaining in FBS may alter the biological function of cells and potentially alter the results of molecular biology experiments.

[0012] Furthermore, it cannot be denied that PEG remaining in serum may affect RNA extraction using conventional methods such as Trizol, and the effect of little or no RNA detection described in Patent US9,005,888 is thought to be due to an artificial structure caused by PEG, not due to removal of RNA.

[0013] Of note, as Wei et al. noted, most of the RNA in serum is contained outside EVs, not inside them, so using PEG-treated serum significantly reduces the certainty that cell cultures are free of RNA contamination. Furthermore, although PEG precipitation can precipitate highly concentrated proteins from serum, it is unclear whether it can completely remove exogenous circulating RNA and associated protein complexes outside of EV inclusions (Tosar et al.).

[0014] It should also be noted that the method described in Patent US9,005,888 requires procedures and operations to ensure the sterility of serum, and that the use of FBS in sterile cell culture may require additional steps to restore sterility to serum once it has been treated with PEG. This increases the time and cost required to produce EV-free serum. Another important point is that the PEG method cannot completely remove EVs present in serum; one of the methods described involves removing EVs up to 10 per ml. 4 We define EV-reduced serum as a vesicle concentration of 0.1 or less.

[0015] Kornilov et al. (Kornilov R. et al. J Extracell Vesicles. 2018 Jan 21;7(1):1422674) described a method for removing EVs from FBS by ultrafiltration. While this method offers a methodologically simple option for EV removal on a laboratory scale, it requires centrifugation and the use of expensive materials (e.g., ultrafiltration materials and equipment), making it less practical and less applicable on an industrial scale. Furthermore, although this method can reduce the amount of EVs, it cannot remove RNA outside of EVs, and therefore, a large amount of FBS RNA remains in serum treated with this method.

[0016] In addition to the methods described above for treating FBS to reduce EV and RNA content, commercially available FBS substitutes for cell culture are available. These serum substitutes are synthetic preparations that contain some of the natural components of serum, allowing some cell lines to grow optimally under controlled culture conditions (Barnes D, Sato G. Anal Biochem. 1980 Mar 1;102(2):255-70). However, synthetic serum substitutes are often significantly more expensive than FBS and are designed for culturing specific cell types. Therefore, they are generally considered effective as FBS substitutes only for specific cell lines and culture conditions, such as stem cells (Ohnuma K. et al. J Neurosci Methods. 2006 Mar 15;151(2):250-61).

[0017] To study the biological effects of exosomes in cell culture and to prevent miRNAs from serum from contaminating the culture and interfering with experimental conclusions, miRNA-reduced FBS products are already commercially available (Paszkiet B. et al. Development of an improved process for the depletion of exosomes from fetal bovine serum. Thermo Fisher Scientific Inc. 2016). However, these products contain large amounts of other types of RNA, and their effects on cell culture have not been investigated. Summary of the Invention [Problem to be solved by the invention]

[0018] Ideally, to avoid RNA contamination within cell culture, the serum used to supplement the medium should be RNA-free or substantially reduced, EV-free or substantially reduced, and devoid of additional chemical compounds, such as PEG, but this serum must retain other serum components essential for cell culture.

[0019] Given the current state of the art, there is a clear need for FBS-based animal sera that are RNA-free or contain minimal RNA, enabling cell culture studies without the risk of contamination or interference from the serum's own RNA. Similarly, there is a clear need for a simple, low-cost technique for removing RNA from animal serum used for cell culture that does not compromise serum sterility and is industrially scalable without exponentially increasing the cost of the product. [Means for solving the problem]

[0020] The present invention relates to animal-derived serum that is free or substantially free of RNA, which is free or substantially free of extracellular vesicles (EVs) while retaining other components of serum essential for cell culture, making it useful for conducting cell culture-based studies without the risk of contamination with EVs or RNA inherent in serum.

[0021] Various types of serum derived from animals, such as bovine, horse, human, mouse, rat, and goat serum, and fetal serum of each of these, are encompassed within the scope of the present invention.

[0022] Furthermore, this application relates to a method useful for removing or substantially reducing the amount of RNA present in animal serum, primarily fetal bovine serum (FBS), by sequentially applying heating, cooling, alkalinization, and neutralization steps. This method degrades endogenous EVs present in animal serum, thereby denaturing and degrading free RNA in the serum, thereby removing or significantly reducing the amount of RNA present in the serum. Degradation of EVs negates their inherent function. [Brief explanation of the drawings]

[0023] [Figure 1] Bar graph summarizing the percentage and abundance of RNA classes in various commercially available FBS products. [Figure 2] Graph showing the concentration of RNA recovered after RNA removal using different process combinations applied to FBS samples. [Figure 3] A histogram showing the amount and size of particles contained in FBS [Figure 4] A bar graph showing the RNA concentration of various commercially available FBS products before and after application of the method of the present invention. [Figure 5] Graph showing cell proliferation in cell cultures using RNA-free or substantially RNA-free FBS as a complement according to the present invention. [Figure 6] Morphology of cells cultured under different supplementation conditions [Figure 7]Bar graph comparing cell viability when cultured under different supplementation conditions [Figure 8] Ratio of proliferation rates of cell lines supplemented with RNA-free FBS to those supplemented with untreated serum as a control. [Figure 9] Ratio of cell viability between lines supplemented with RNA-free FBS and untreated serum (control) [Figure 10] Graph showing the abundance of various miRNAs in untreated serum (control) and the same serum after treatment with the method of the present invention. [Figure 11] Graph showing the abundance of other classes of RNA in untreated serum (control) and the same serum after treatment with the method of the present invention. [Figure 12] Graph showing the abundance of various miRNAs in untreated serum (control) and the same serum after treatment with the method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] Figure 1 is a bar graph summarizing the proportion and abundance of RNA classes in various commercially available FBS products. Each bar represents a different presentation of a different commercially available FBS product. 1) Regular FBS A, 2) Regular FBS B, 3) characterized FBS, 4) graded FBS, 5) Certified FBS, 6) triple filtered FBS, 7) FBS reduced in exosomes. While the class content and RNA abundance differ from commercially available presentations, and some do not contain miRNAs, RNA is present in all classes. Black bars represent miRNAs, dark gray bars represent non-annotated RNAs (sequences present in the Bos taurus genome but that could not be associated with specific functional RNA types), light gray bars represent tRNAs, and white bars represent other RNAs not present in the Bos taurus genome that may be of non-bovine origin.

[0025] Figure 2 shows the concentration of RNA recovered after RNA removal using different process combinations applied to FBS samples. The processes used and their order are listed in Table 1. RNA was removed using three different conditions (bars 11, 13, and 18). IND indicates no detectable RNA concentration. The inactivation combination of heating, cooling, alkalinization, and neutralization is one combination that can remove or significantly reduce RNA in FBS. Other combinations that reduce the amount of RNA include an additional ultracentrifugation step.

[0026] Figure 3 shows histograms of the amount and size of particles present in FBS. The gray histogram corresponds to the size and amount of particles present in the FBS sample treated with the method of the present invention (combination #11 according to Table 1), while the black histogram corresponds to the untreated control sample. Typical EV-sized particles (50-400 nm in diameter), including exosomes, were reduced in size compared to untreated serum (control), with the majority reaching a diameter of approximately 30 nm after heating, cooling, alkalinization, and neutralization.

[0027] Figure 4 is a bar graph showing the RNA concentration of various commercially available FBS products before and after application of the method of the present invention: 1) regular FBS A, 2) regular FBS B, 3) characterized FBS, 4) graded FBS, 5) certified FBS, 6) triple filtered FBS, and 7) FBS reduced in exosomes. Regardless of the commercial product and its initial RNA concentration, it was confirmed that applying the RNA removal method described in this application resulted in an RNA concentration that was undetectable (IND).

[0028] A graph showing cell proliferation in cell cultures using the RNA-free or substantially RNA-free FBS described in the present invention as a complement is shown in Figure 5. HEK293 cells were cultured under different supplementation conditions. Open circles represent untreated (control) FBS, filled circles represent RNA-free or substantially RNA-free FBS of the present invention (obtained in combination #11 in Table 1), and gray triangles represent synthetic serum replacement. Cells cultured in RNA-free or substantially RNA-free FBS showed growth similar to that of cells cultured in untreated FBS (control) and superior to that of synthetic replacement, demonstrating that the RNA-free or substantially RNA-free FBS obtained by applying the methods of the present invention maintains the properties necessary to effectively promote cell proliferation.

[0029] Figure 6 shows the morphology of cells cultured under different supplementation conditions: untreated FBS (control), RNA-free or substantially RNA-free FBS (obtained in Combination #11 in Table 1) according to the present invention, and synthetic serum supplement. The RNA-free or substantially RNA-free FBS according to the present invention did not adversely affect cell morphology compared to the control or synthetic serum, and it was confirmed that the properties necessary for cell growth were maintained.

[0030] Figure 7 is a bar graph comparing cell viability when cultured under different supplementation conditions. Untreated FBS (control) is represented by white bars, RNA-free or substantially RNA-free FBS of the present invention is represented by black bars, and synthetic serum substitutes are represented by gray bars. It was confirmed that there was no significant difference in cumulative viability on day 6 between cells supplemented with control FBS, RNA-free FBS, and synthetic substitutes, confirming that RNA-free serum maintains its supplementary properties even after application of the method of the present invention.

[0031] Figure 8 shows the ratio of proliferation rates of cell lines supplemented with RNA-free FBS to those supplemented with untreated serum as a control. 1) HEK 293, 2) HeLa, 3) CHO, 4) MCF7, and 5) MEF cells were incubated with untreated FBS (control), RNA-free, or essentially RNA-free FBS, and the ratio of proliferation in both supplemented conditions was determined. None of the cell lines cultured with RNA-free FBS showed any difference in cumulative proliferation at day 6 compared to those supplemented with untreated FBS.

[0032] Figure 9 shows the cell viability ratios for lines supplemented with RNA-free FBS versus untreated serum (control). 1) HEK293, 2) HeLa, 3) CHO, 4) MFC7, and 5) MEF cells were incubated with untreated FBS supplemented with RNA-free or RNA-substantially free FBS, and the viability ratios for both supplemented conditions were calculated. None of the cell lines cultured with RNA-free FBS showed any difference in cumulative viability at day 6 compared to their respective control FBS.

[0033] Figure 10 is a graph showing the abundance of various miRNAs in untreated serum (control) and the same serum after treatment with the method of the present invention. 1) Bta-miR-143, 2) Bta-miR-181a, 3) Bta-miR-192, 4) Bta-miR-380-3p, 5) Hsa-miR-25-3p The miRNAs were detected by quantitative reverse transcription PCR (qRT-PCR). Open bars represent untreated FBS, and closed bars represent FBS treated with the method of the present invention. In all cases, the method of the present invention reduced the abundance of miRNAs to levels below the detection limit (approximately 38 Ct), demonstrating the effectiveness of the method for removing miRNAs from FBS.

[0034] Figure 11 is a graph showing the abundance of other classes of RNA in untreated serum (control) and the same serum after treatment with the method of the present invention. 1) U47 and 2) unannotated RNA #49627 were detected by qRT-PCR. White bars indicate untreated FBS (control), and black bars indicate FBS treated with the method of the present invention. This demonstrates that the method of the present invention is effective in removing various classes of RNA.

[0035] Figure 12 is a graph showing the abundance of various miRNAs in untreated serum (control) and the same serum after treatment with the method of the present invention. 1) hsa-miR-486, 2) hsa-miR-423-5p, 3) hsa-miR-10b were detected by qRT-PCR. The open bars represent untreated FBS (control), and the closed bars represent FBS treated with the method of the present invention. As can be seen, these miRNAs are present in low abundance in the serum samples used, but can be removed to undetectable levels by the method of the present invention.

[0036] The cultivation of cell lines and primary tissues is one of the primary tools for biomedical research. Cell culture is typically performed by culturing cells in a liquid medium containing supplements to keep them alive or in a constant state of proliferation. Fetal bovine serum (FBS) is a key component of culture media because it contains a complex mixture of small molecules, including proteins, nucleic acids, hormones, growth factors, lipids, vitamins, and minerals, all of which are important for cell growth.

[0037] FBS contains a mixture of various classes of RNA, among which miRNAs are highly conserved, with miRNAs with identical sequences and sizes even found in evolutionarily distant species. Due to their high degree of conservation and specificity, miRNAs are considered important regulators of gene expression (Bartel DP Metazoan MicroRNAs. Cell (2018) 173:20-51).

[0038] Recently, it has been reported that when serum is used in cell culture, RNA present in FBS interferes with the detection of intracellular miRNAs (Wei Z. et al. Sci Rep. 2016 Aug 9;6:31175). Because the majority of studies characterizing RNA gene expression in cell cultures use supplemental FBS, the results may be altered by the mixing of miRNAs derived from cultured cells and miRNAs derived from FBS. Further exacerbating this situation is the fact that it is impossible to distinguish the species of origin of a significant number of miRNAs. It is possible that miRNAs and other RNAs contained in supplemental serum may exert biological functions on cultured cells by altering gene expression and physiological conditions, thereby affecting the results of the experiments performed.

[0039] One possible source of bovine miRNAs being transferred to cultured cells is extracellular vesicles (EVs) contained in FBS. However, because miRNAs and other types of RNA exist in equal proportions both inside and outside EVs, bovine RNA contamination can also originate from media other than EVs. Therefore, removing EVs from serum does not completely remove contaminating RNA.

[0040] Finally, RNA in FBS is highly stable both inside and outside EVs and must therefore be protected by RNA-binding proteins, as FBS naturally contains numerous ribonucleases (RNAases) that would degrade RNA if not effectively protected (Chen X, et al. Cell Res. 2008 Oct;18(10):997-1006).

[0041] Several methods for reducing or removing EVs from FBS, including ultrafiltration, ultracentrifugation, and precipitation with chemical agents, have been described. However, these methods focus on removing EVs and the biological materials they contain. However, none of these methods or FBS preparations are RNA-free or substantially RNA-free, as they cannot reduce or remove free-form RNA in serum, which can account for up to 60% of the RNA in mammalian serum. An alternative is the use of synthetic supplements, but these are often much more expensive than FBS and are not effective for the growth of all cell types.

[0042] Therefore, there is a need for an RNA-free or substantially RNA-reduced mammalian serum that allows for in vitro cell culture without potentially contaminating or altering the results of molecular biology experiments while maintaining its properties and functionality as a supplement. In this sense, it would also be desirable to have a method for producing RNA-free or substantially RNA-reduced serum without altering its functionality as a supplement or leaving behind chemical residues that could interfere with cell culture and molecular biology experiments using it.

[0043] The present invention overcomes the shortcomings of the prior art by providing RNA-free or substantially RNA-reduced serum and a method for efficiently removing RNA contained in mammalian serum, particularly FBS. This method can efficiently remove RNA present in mammalian serum, regardless of whether it is contained in EVs, thereby producing RNA-free or substantially reduced serum from different mammalian species.

[0044] Another technical advantage is the simplicity of the method, which does not require centrifugation, ultracentrifugation, or the use of filters, thereby reducing application and industrial scale-up costs. Furthermore, this method can be performed without the need to transfer serum to a different container, making it easier to maintain the required sterility during use. The serum produced by the method described herein is free of or substantially reduced in EVs and total RNA, achieving a higher RNA removal rate than other sera described in the prior art.

[0045] The present invention is based on the unexpected finding that sequential treatment of mammalian serum with heating, cooling, alkalinization, and neutralization substantially removes or reduces RNA naturally present in serum. While these processes are commonly used and the effects of each process on various biomolecules present in serum are known, the specific sequence of these four processes combined to achieve this effectiveness in removing RNA has not previously been described. As described in this invention, applying these processes in this specific sequence can increase the efficiency of removing RNA present in mammalian serum.

[0046] The present invention is based on applying the processes of heating, cooling, alkalizing, and neutralization in a specific sequence that is contrary to the current state of the art, and a person skilled in the art with average knowledge in this field would not be expected to reach the same conclusion. Applying these processes separately or in a different sequence to serum would not produce the same results, and therefore the method is not apparent from the state of the art.

[0047] RNA-free or substantially reduced serum can be used as a supplement for all types of in vitro cell culture (HeLa, HEK293, CHO, MFC7, MEF, etc.) and any molecular biology experiment aimed at investigating the metabolic or physiological state of the cells under study. Similarly, RNA-free or substantially reduced serum is useful as a complement to all studies related to gene expression analysis, more specifically, the analysis of the expression of various classes of RNA, such as miRNA, tRNA, lncRNA, mRNA, snRNA, and piRNA. Similarly, RNA-free or substantially reduced serum can be used as a supplement for culturing cells useful for the production of therapeutics, such as hormones, recombinant proteins, antibodies, clotting factors, and vaccines (Ashish Verma, Anchal Singh Academic Press, 4 Nov. 2013).

[0048] As used herein, the terms "RNA-free serum" and "substantially reduced RNA serum" refer to serum from animals, preferably mammals, that has undergone an RNA removal process and whose endogenous RNA content is at a level that is undetectable by specific RNA sequences detected by conventional, highly sensitive methods useful for measuring nucleic acid concentrations in aqueous samples, such as spectrophotometry and spectrofluorometry, or by methods that detect the presence and thus relative abundance of specific RNA sequences. A nearly or undetectable RNA sequence is typically interpreted as an "absent" or "undetectable" sequence. In the case of qRT-PCR, where the amplification cycle (Ct) is an indicator of the abundance of a sequence of interest, an abundance signal with a Ct greater than 38 cycles is considered to indicate the absence or undetectability of the sequence of interest. In sequence-specific detection techniques such as next-generation sequencing (RNA-seq, small RNA-seq), the number of reads is traditionally considered to indicate the abundance of the sequence of interest, with a sequence with a zero read count indicating either no RNA or a value below the detection limit of the technique.

[0049] In developing this invention, we first analyzed the composition and relative abundance of various RNA species present in serum (Figure 1). To this end, samples from different commercially available FBS products (1) regular FBS A, 2) regular FBS B, 3) characterized FBS, 4) qualified FBS, 5) certified FBS, 6) triple-filtered FBS, and 7) exosome-reduced FBS) were analyzed using RNA sequencing (Illumina). Bioinformatics analysis revealed that FBS contains different classes of RNA in varying amounts. Among the most abundant RNA classes are miRNAs, tRNAs, and unannotated RNAs (RNAs sequenced in the Bos taurus genome but unable to be associated with a specific RNA type). It is important to note that while the proportions of different RNA species vary among commercial products, with some containing lower amounts of miRNAs than others, all products contain some RNA.

[0050] To determine the optimal conditions for removing RNA from serum, we tested various processes and their combinations, including heating, cooling, alkalinization, addition of enzymes (ribonuclease), and ultracentrifugation. The results of testing different process combinations are shown in Table 1. After each process, RNA was extracted with TRIzol reagent (Thermo) and quantified using a Qubit spectrofluorometer (Thermo).

[0051] (Table 1) TIFF2025131694000001.tif200141

[0052] Figure 2 shows the RNA removal efficiency of different processes and their combinations in FBS samples. Only three specific process combinations remove RNA to undetectable levels (IND), below the spectrofluorometric detection limit of 250 pg / μL. One combination (Figure 2, bar #11) involves heating and cooling followed by alkalinization and neutralization. Another effective combination (Figure 2, bar #13) involves heating and cooling followed by ultracentrifugation. The third effective combination (Figure 2, bar #18) involves the sequential steps of ultracentrifugation, heating and cooling, alkalinization, and neutralization.

[0053] Although other processes and combinations show some degree of effectiveness in removing RNA from serum, none of them are useful for completely removing RNA or reducing it to below the detection limit. To remove RNA from serum, it is important to note that not only do four processes—heating, cooling, alkalinization, and neutralization—need to be applied in combination, but the order in which these processes are applied is particularly important. This is because the effectiveness of removing RNA from serum is not the same whether the three processes are combined or applied in a different order (Figure 2, bar graph #12). In conclusion, applying these processes separately or in a different order to serum will not remove the RNA contained therein.

[0054] Because some of the RNA present in serum is contained in EVs, to verify the effect of the method of the present invention on EVs present in FBS, we performed nanoparticle quantification by nanoparticle tracking analysis (NTA) using a NanoSight instrument (Malvern) (Figure 3).

[0055] The size of particles naturally present in FBS ranges from 50 to 400 nm in diameter, with two peaks in particle concentration near 100 nm and 180 nm (Figure 3, Control, black contour histogram). After applying the methods described herein, a significant decrease in the size of particles present in FBS was observed, with the largest peak in particle concentration near 30 nm in diameter (Figure 3, #11, gray histogram).

[0056] These results demonstrate that application of the RNA depletion method degrades EVs in serum, including exosomes. The peak observed around 30 nm (1.5 × 108 particles) correlates with the sum of the particle amounts of the two main peaks in the control sample, indicating that the natural state of EVs is degraded, resulting in the absence or significant reduction of EV content in serum. EV decomposition results in more efficient removal of total RNA in animal serum, likely by releasing the RNA present within EVs for further degradation.

[0057] This result is thought to be due to the fact that EVs are denatured by heating and cooling, releasing the RNA they contain, and then the RNA is alkaline hydrolyzed by treatment with a strong base, and the RNA degradation is further promoted by adding a strong acid, resulting in improved efficiency of RNA removal from serum. The RNA remaining in the serum after this treatment is non-functional fragments of degraded RNA molecules and is therefore thought to have no effect on cultured cells.

[0058] To validate the effectiveness of our method for removing RNA from serum, we quantified RNA from seven different commercially available FBS products before and after treatment with the method described here. RNA was extracted with TRIzol Reagent (Thermo) and quantified using a Qubit spectrofluorometer (Thermo).

[0059] The RNA removal method described in this application is effective for all commercially available FBS products tested: 1) regular FBS A, 2) regular FBS B, 3) characterized FBS, 4) qualified FBS, 5) certified FBS, 6) triple-filtered FBS, and 7) exosome-reduced FBS. Figure 4 shows that the method is able to remove RNA from different commercial products to levels below the detection limit, despite having different RNA content classes as previously determined (Figure 1), indicating that the method is effective regardless of the concentration or type of RNA present in the serum and is therefore applicable to all types of animal serum.

[0060] Next, we evaluated whether the RNA-free or substantially reduced FBS described in this invention would alter its ability to complement cell culture after applying the RNA removal method. To this end, we performed growth curves of human cells (HEK293 cells) supplemented with RNA-free serum and compared the proliferation, morphology, and viability over a 6-day period to those obtained from cells cultured in medium supplemented with untreated serum and synthetic serum replacement (a synthetic formulation containing components necessary for growth of several cell types in culture, but which, by its synthetic nature, does not contain RNA or EVs).

[0061] Figure 5 shows a comparison of cell proliferation in three supplemented conditions. HEK293 cells were cultured under standard conditions until they reached 60% confluence in culture medium. The culture medium was then changed to medium supplemented with one of the variants (control FBS, RNA-free FBS, or synthetic serum supplement), and cell density was quantified (days 0–6). Results showed no difference in cell proliferation between culture medium supplemented with RNA-free serum (black circles) and untreated FBS (control, open circles). The proliferation obtained with RNA-free serum was superior to that of cells cultured in medium supplemented with synthetic serum replacement (gray triangles).

[0062] Figure 6 shows the morphology of cells cultured under the three supplemented conditions. RNA-free FBS was found to have no effect on cell morphology compared to the control or synthetic serum, demonstrating that it maintains the properties necessary to promote cell proliferation without secondary effects on cell physiology.

[0063] Cell viability was determined by quantifying HEK293 cells using a trypan blue exclusion assay to assess membrane integrity. Figure 7 shows the cumulative cell viability on day 6 for cells cultured under three supplemented conditions (control FBS, white bars; RNA-free FBS, black bars; synthetic surrogate, gray bars). The cumulative cell viability on day 6 did not change depending on the supplement used, indicating that the RNA-free FBS described in this invention does not adversely affect cell viability and can be used safely.

[0064] These viability and proliferation assays were repeated with other cell lines (Figures 8 and 9, respectively). Figure 8 shows the ratios of proliferation rates for cell lines cultured in media supplemented with RNA-free FBS to those cultured in media supplemented with untreated serum (control). 1) HEK293, 2) HeLa, 3) CHO, 4) MFC7, and 5) MEF cells were cultured in media supplemented with untreated FBS and free or substantially free FBS RNA, and the proliferation ratios for both supplemented conditions were determined. As with HEK293 cells, the proliferation ratios for all tested cell lines were close to 1, indicating no difference in the supplementation capacity between RNA-free FBS and control FBS.

[0065] Figure 9 shows the cumulative viability of the same cell line under the two culture conditions on day 6, and shows that the viability ratio for both supplemented conditions is close to 1. This result shows that there is no significant difference in cell viability when serum treated by the method of the present invention is used compared to the control serum.

[0066] To confirm the effectiveness of this method for removing RNA from serum, we measured several miRNAs before and after application of this method. To this end, we extracted RNA from a commercially available FBS product, which is rich in miRNAs, as described previously (Figure 1). Subsequently, we performed bioinformatics analysis of the results of extensive sequencing to identify the relative abundance of the most representative miRNAs in serum using qRT-PCR.

[0067] The probes used to determine each miRNA are listed in Table 2. As can be seen in Figure 10, all evaluated miRNAs, including 1) Bta-miR-143, 2) Bta-miR-181a, 3) Bta-miR-192, 4) Bta-miR-380-3p, and 5) Hsa-miR-25-3p, were detected in the control serum (open bars), but no miRNAs below 38 Ct (dotted line) were detected in the serum treated with the method of the present invention (black bars), indicating that the most abundant miRNAs in FBS were eliminated or reduced below the detection limit by applying the method of the present invention.

[0068] Some commercially available FBS products contain low levels of miRNAs but high levels of other RNAs (Fig. 1, bars 4 and 7). Computational analysis of the various RNA classes in one of these commercial products revealed that it was enriched in two RNA sequences: an RNA classified as U47 and an RNA classified as "unannotated" (here, #49627).

[0069] To confirm that other RNA types were excluded, we performed qRT-PCR using specific probes to assess the abundance of these two RNAs. The probes used to measure U47 are listed in Table 2.

[0070] (Table 2) For the unannotated RNA sequence #49627 (TIFF2025131694000002.tif121150), SYBR-green fluorescence was detected using the primer oligonucleotides listed in Table 3 as previously reported.

[0071] (Table 3) TIFF2025131694000003.tif54141

[0072] Figure 11 shows the abundance of 1) U47 and 2) #49627 in untreated commercial serum (open bars) and the same serum after treatment with the method of the present invention (black bars). Both RNAs are present at levels below 20 Ct in untreated serum, and application of the method of the present invention reduces the levels to below the detection limit of 38 Ct, eliminating both types of RNA.

[0073] An important aspect of commercially available products that appear to be miRNA-free is that they contain detectable amounts of miRNAs, including Bta-miR-143, Bta-miR-181a, Bta-miR-192, Bta-miR-380-3p, and Hsa-miR-25-3p, despite containing undetectable levels of other miRNAs. Figure 12 shows qRT-PCR analysis of one of these products for 1) Hsa-miR-486, 2) Hsa-miR-423-5p, and 3) Hsa-miR-10b before treatment (open bars) and after application of the method of the present invention (black bars). In untreated FBS, these miRNAs were detected at around 34 Ct. Application of the method of the present invention confirmed that these miRNAs were reduced to below the detection limit of 38 Ct.

[0074] In accordance with the teachings set forth herein, the invention described herein relates to RNA-free or substantially RNA-reduced mammalian serum and methods for obtaining such RNA-free or substantially RNA-reduced serum, which maintains its ability to support cell culture and is free of chemical residues that may adversely affect cell culture.

[0075] To practice the methods of the present invention, any methodology described in the prior art useful for controlled heating of serum samples can be employed, preferably in sterile vessels or containers, or industrial containers, including, but not limited to, bathing and immersion in temperature-controlled water, incubation in a temperature-controlled cabinet (oven), or use of other controlled heating devices.

[0076] The serum heating step can be performed at a temperature of 52° C. to 63° C., preferably 55° C. to 37° C. The heating time is 25 to 50 minutes, preferably 35 minutes.

[0077] The cooling step may be gradual without temperature manipulation by heating and then cooling to room temperature, or may be accelerated by the use of cooling means, such as immersion in water or liquid below room temperature, or other controlled cooling devices, with the final cooling temperature being between 8°C and 25°C, preferably 16°C.

[0078] Various alkaline or basic compounds or salts that release hydroxide ions (OH-) can be used, in solution or in anhydrous form, including, but not limited to, potassium hydroxide (KOH), magnesium hydroxide (Mg(OH)2), calcium hydroxide (Ca(OH)2), and sodium hydroxide (NaOH). One skilled in the art can routinely standardize the optimal temperatures and times for the heating and cooling steps required in this method.

[0079] The alkali concentration may vary between 10 and 12 N, preferably 12 N. A person skilled in the art can routinely standardize the optimum concentration of the alkalinization process required in the present method.

[0080] The serum should be alkalinized to between pH 10 and 12, preferably pH 12.

[0081] The exposure time of the serum to alkali varies depending on the concentration and volume of RNA present, but should be maintained for at least 3 minutes, but may vary between 3 and 20 minutes, preferably 5 to 10 minutes.

[0082] A variety of compounds or salts with acidic chemical properties can be used for the neutralization process, including, but not limited to, phosphoric acid (H3PO4), nitric acid (HNO3), acetic acid (CH3COOH), hydrochloric acid (HCl), and other compounds with similar properties.

[0083] The acid concentration may vary between 0.1 and 2 N, preferably a concentration of 1 N may be used. A person skilled in the art can routinely standardize the optimum concentration for the neutralization treatment required in this method.

[0084] The serum must be acidified to a physiological pH of 7.2 to 7.5, preferably 7.4.

[0085] In a preferred embodiment of the present invention, serum is heated to a temperature of 55°C to 57°C for 30 to 45 minutes, gradually cooled to room temperature of 20°C, alkalized to a pH of 12 using anhydrous NaOH (powder or granules) over 10 minutes, and then the pH of the alkalized serum is lowered using 1N HCl until it is neutralized to a physiological pH of 7.4.

[0086] If the DNA concentration is very high, for example above 45 or 50 ng / mL, the method of the invention can be applied repeatedly in succession to ensure complete removal of RNA.

[0087] In one embodiment of the present invention, the methods described herein may incorporate an additional ultracentrifugation step at between 80,000 x g and 100,000 x g. If the ultracentrifugation process compromises the sterility of the serum, additional sterilization procedures should be performed using techniques known in the art, such as filtration through 0.2 micron pore membranes.

[0088] Any type of animal serum can be used to practice the present invention, including, but not limited to, bovine, equine, human, mouse, rat, and goat serum, as well as respective fetal serum.

[0089] The present invention is further illustrated by the following examples, which are not to be construed as limitations on the scope of the claims in any way, but rather are presented to provide a better understanding of the practice of the invention and are understood to represent only some of the embodiments of the invention.

[0090] Example 1 FBS RNA removal. Starting with a regular commercial FBS product, the concentration of total RNA was first determined by extraction with Trizol product according to the manufacturer's recommendations, resulting in a concentration of 35 ng / mL. The FBS was heated at 56°C for 35 minutes and then slowly cooled to room temperature at 20°C. The serum was then alkalinized to pH 12 with 12N anhydrous NaOH and held for 15 minutes, after which the pH of the alkalinized serum was lowered to physiological pH 7.4 with 1N HCl. After applying this method, the total RNA concentration was measured using the above-mentioned Trizol extraction method, and the concentration obtained was below the detection limit by spectrofluorometry.

[0091] Example 2 Removal of RNA from FBS by combining the method of the invention with ultracentrifugation. Starting with a standard commercial FBS product found to have an RNA concentration of 40 ng / mL, the serum was ultracentrifuged at 100,000 x g for 7 hours at 4°C. At the end of the ultracentrifugation, the sample supernatant was transferred to a new sterile container without disturbing the resulting button. To maintain sterility, the serum supernatant was passed through a 0.2 micron pore size sterile filter. The FBS supernatant was then heated at 56°C for 35 minutes, then slowly cooled to room temperature at 20°C. The serum was alkalinized to pH 12 using anhydrous NaOH and held for 15 minutes. The pH of the alkalinized serum was then lowered to physiological pH 7.4 using 1N HCl. After applying this method, the total RNA concentration was measured using the above-mentioned Trizol extraction method, and the concentration obtained was below the detection limit by spectrofluorometry.

[0092] Example 3 Results of different replicates of RNA removal from FBS using the method of the present invention It is expected that the RNA content of different types of commercially available serum will vary. For serum samples with high RNA content, the method of the present invention can be repeated to give additive results of its RNA removal efficiency. Serum with an RNA content of 45 ng / mL or more was heated at 56°C for 35 minutes, then slowly cooled to room temperature at 20°C. The serum was alkalinized to pH 12 with anhydrous NaOH and held for 15 minutes. The pH of the alkalinized serum was then lowered to physiological pH 7.4 with 1N HCl. The heating, cooling, alkalizing, and neutralization processes were then repeated under the same conditions. After two successive applications of this method, the total RNA concentration was measured using the Trizol extraction method described above, and concentrations below the detection limit were obtained by spectrofluorometry.

Claims

1. Serum derived from an animal, characterized by being free or substantially free of RNA.

2. 2. The serum of claim 1, wherein the serum is bovine, horse, human, mouse, rat, goat, or a variant of each of the fetal serums.

3. 3. The serum according to claim 1, wherein the serum is fetal bovine serum.

4. 1. A method useful for removing RNA from mammalian serum, comprising: a) heating the serum to a temperature between 52°C and 63°C; b) cooling the serum to a temperature between 8°C and 25°C; c) alkalizing the serum to a pH between 10 and 12; d) Neutralizing the serum to physiological pH wherein these steps are performed sequentially.

5. 5. The method of claim 4, wherein the serum is bovine, horse, human, mouse, rat, goat, or a variant of each of the fetal serums.

6. 6. The method of claim 4, further comprising the step of heating the serum to a temperature between 55°C and 57°C.

7. A method according to any one of claims 4 to 6, characterized in that the temperature of step a) is maintained for an additional 30 to 45 minutes.

8. 8. A method according to any one of claims 4 to 7, characterized in that the whey (serum) is alkalized to a pH of 12.

9. Sodium hydroxide (NaOH), potassium hydroxide (KOH), magnesium hydroxide (Mg(OH) ２ ), calcium hydroxide (Ca(OH) ２ 9. The method according to claim 4, further comprising alkalizing the whey with a compound having basic chemical properties selected from the group consisting of:

10. 10. The method according to any one of claims 4 to 9, wherein the alkalinization is maintained for a period of 3 to 20 minutes.

11. 11. The method according to any one of claims 4 to 10, wherein the alkalinization is maintained for an additional 5 to 10 minutes.

12. The serum was neutralized using phosphate (H ３ P.O. ４ ), nitric acid (HNO 3 ), acetic acid (CH ３ 12. The process according to any one of claims 4 to 11, further carried out with a compound of acidic chemical nature selected from the group consisting of: HCl (HCl), COOH (COOH), and hydrochloric acid (HCl).

13. 13. The method of any one of claims 4 to 12, further characterized in that the neutralization of the serum is carried out until a pH between 7.2 and 7.5 is reached.

14. 14. A method according to any one of claims 4 to 13, characterized in that the whey is neutralised to a pH of 7.

4.

15. 15. The method according to any one of claims 4 to 14, further comprising an ultracentrifugation step as the first or last step.

16. 1. A method useful for removing RNA from mammalian serum, comprising: a) heating the serum to a temperature between 52°C and 63°C; b) cooling the serum to a temperature between 8°C and 25°C; c) ultracentrifuging the serum at between 80,000 x g and 100,000 x g for at least 5 to 7 hours; wherein the steps are performed in this sequential order.

17. 17. The method of claim 16, wherein the serum is bovine, horse, human, mouse, rat, goat, or a variant of each of the fetal serums.

18. 18. The method of claim 16 or 17, wherein the serum is heated to a temperature between 52°C and 63°C.

19. 19. The method according to any one of claims 16 to 18, wherein the temperature of step a) is maintained for 30 to 45 minutes.

20. 20. The method according to any one of claims 16 to 19, characterized in that the ultracentrifugation is carried out at 100,000 x g for 7 hours.

21. The method according to any one of claims 16 to 20, wherein the ultracentrifugation is carried out for 5 to 10 hours.

22. 19. The method of any one of claims 16 to 18, further comprising the step of sterilizing the serum.

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