RNA-free animal serum

Abstract

The present invention relates to mammalian serum free of RNA, which can be used for cell culture or for the production of pharmaceutical biological products, since this fact preserves their supplementary properties. Another embodiment of the present invention relates to a method for removing RNA from mammalian serum, the method comprising applying successive steps of serum heating, alkalinization and neutralization.

Description

Technical Field

[0001] The present invention relates to the field of cell biology, particularly to the field of cell culture supplements. Generally speaking, the present invention relates to a serum, preferably fetal bovine serum, whose ribonucleic acid (RNA) is reduced or has a minimum RNA content below the detection limit. The present invention also relates to a method for removing RNA from serum. This RNA-reduced serum can be used to analyze the RNA expression of cell cultures without interfering with the analysis of RNA normally present in animal sera. Background Art

[0002] Cell culture is one of the main tools for basic and biomedical in vitro research, including maintaining cells from very different types of animals (from insects to mammals) under controlled conditions of temperature, humidity, and carbon dioxide percentage. To keep cells viable and continuously dividing, cell cultures are maintained in a liquid medium containing an appropriate amount of salts to enable the cells to perform their most basic 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 and Arora M. Cell Culture Media: A Review. Mater Methods (2013) doi:10.13070 / mm.en.3.175).

[0003] An important component of in vitro cell culture is serum, as it contains high levels of growth factors, lipids, proteins, and other necessary factors for keeping cells continuously dividing. In this way, serum is added as a nutritional supplement to the synthetic liquid medium that maintains cell culture. The serum used for this purpose can be sourced from humans, horses, or cattle, and the most widely used worldwide is fetal bovine serum (SFB) (Arora M. Cell Culture Media: A Review. Mater Methods (2013) doi:10.13070 / mm.in.3.175.).

[0004] In addition to the above components, SFB also contains high levels of nucleic acids, the most abundant of which is RNA in various biologically functional forms, such as transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), Piwi-interacting RNA (pi-RNA), small nucleolar RNA (snoRNA), etc. (Chen X, et al. Cell Res. 2008 Oct; 18(10):997-1006). Some different types of RNA are contained in extracellular vesicles (EVs), while other types of RNA are included in extracellular vesicles (Keerthikumar S. et al. J Mol Biol. 2016 Feb 22; 428(4):688-692).

[0005] EVs are microstructures with a diameter of 50 - 400 nm, composed of a lipid bilayer, containing proteins, various types of biologically functional RNA, and some small molecules. EVs are composed of its subsets, and the diameters and molecular contents that make up them are different from each other. Among them, EVs with a diameter of 50 - 120 nm, exosomes, are the most widely studied (Colombo M. et al. Annu Rev Cell Dev Biol. 2014; 30:255-89). An important aspect of EVs is that they have been shown to be able to transfer their contents to recipient cells in vitro and in vivo; therefore, they are considered a cell-to-cell communication system and may be of great significance in 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 on cultured cells have demonstrated that the EVs of SFB can transfer their RNA contents to human and murine cell lines; emphasizing the possible transfer of bovine RNA in cell cultures when using SFB. This finding reveals that since miRNA was discovered to date, the scientific literature on the detection and analysis of miRNA expression is likely to be a mixture of bovine RNA and RNA from the species cell lines in the study at hand. To date, the extent of interference with the detection of bovine RNA and its possible functions in cell cultures is not clear. The main reason is that more than 70% of cow miRNAs are the same as those of other mammals, including humans (Wei Z. et al. Sci Rep. 2016 Aug 9; 6:31175).

[0007] In this sense, Wei et al. described that SFB contains various types of RNA, including protein-coding RNA and regulatory RNA, including messenger RNA, small molecule RNA (miRNA), ribosomal RNA, and small nuclear RNA. Even after applying ultracentrifugation, up to 70% of the RNA can remain in the serum during the process.

[0008] Wei et al. pointed out that the RNA unique to SFB was isolated together with the RNA from cell cultures, which might cause interference or misunderstanding in subsequent RNA analysis. This is why serum without RNA is needed for such studies (Tosar JP. J Extracell Vesicles. 2017 Jan 12;6(1):1272832).

[0009] To reduce possible interference or misunderstanding, several methods have been developed to eliminate VE in serum due to the presence of endogenous VE in the serum used for cell culture.

[0010] In this sense, US Patent 9,005,888 described a method for isolating VE produced by animal cells and a method for producing plasma or serum with reduced VE content, indicating that after applying the method, some of the initially present miRNAs could not be detected by quantitative PCR (qPCR). However, the method described in US Patent 9,005,888 has several drawbacks, including the use of a precipitation solution containing polyethylene glycol (PEG), and its residues remain in the treated serum and can thus be regarded as contaminant elements.

[0011] For example, it has been described that PEG can induce the formation of heterokaryons, that is, cell fusion to obtain multinucleated cells (Davidson RL, Gerald PS. Somatic Cell Genet. 1976 Mar;2(2):165 - 76.), so the PEG remaining in SFB can change the biological functions of cells, thus producing altered results in molecular biology experiments.

[0012] In addition, it cannot be excluded that the remaining PEG in the serum will affect the extraction of RNA by conventional methods (such as Trizol), and the little or undetectable RNA described in US Patent 9,005,888 is affected by artifacts caused by PEG rather than the RNA itself being removed.

[0013] It is worth noting that, as described by Wei et al., most of the serum RNA is not contained within the VE but rather outside of it. This greatly limits the certainty of having no RNA contamination in cell cultures when using PEG-treated serum. Similarly, although high concentrations of proteins can also be precipitated from serum using the PEG precipitation method, it is unclear whether the circulating RNA-associated protein complexes, which are exogenous to the VE content (Tosar et al.), can be completely removed.

[0014] It is also important to note that the steps and serum manipulations required by the method described in U.S. Patent 9,005,888 jeopardize its aseptic conditions, which may force the need to include an additional step to restore the serum to aseptic status after PEG treatment; this is for the use of SFB in aseptic cell culture, which will increase the time and cost of producing serum without VE. Another important aspect is that the PEG method cannot completely remove the VE present in the serum, as one of the described embodiments defines serum with reduced VE having a concentration of no more than 104 vesicles per milliliter.

[0015] On another part, Kornilov et al. (Kornilov R. et al. J Extracell Vesicles. 2018 Jan 21;7(1):1422674) described a method for removing VE from SFB by ultrafiltration. Although providing a methodologically simpler laboratory-scale alternative for eliminating VE, this method still requires centrifugation and the use of expensive materials (such as ultrafiltration materials and equipment), which reduces the practicality of the method and its application at the industrial level. In addition, this method reduces the number of VE but does not remove the external RNA, so the serum treated by this method still contains a large amount of RNA from the SFB.

[0016] In addition to the above methods for treating SFB and reducing the VE content and its RNA content, there are also commercial alternatives to SFB for cell culture. Such serum alternatives are synthetic preparations containing some of the natural components of serum, such that some cell lines are able to grow optimally under controlled culture conditions (Barnes D, Sato G. Anal Biochem. 1980 Mar 1;102(2)255 - 70). However, synthetic serum substitutes tend to be much more expensive than SFB and are generally designed for culturing specific types of cells. Thus, generally speaking, they only represent effective SFB substitutes for culturing cell lines and / or specific culture conditions, such as for stem cells (Ohnuma K. et al. J Neurosci Methods. 2006 Mar 15;151(2):250 - 61).

[0017] The need to study the biological effects of exosomes in cell culture and prevent serum miRNA contamination of the culture has hampered the conclusions of experiments. There are already SFB products with reduced miRNA on the market (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 contain large amounts of other types of RNA, and the effects of these on the culture have not yet been studied.

[0018] Ideally, to avoid RNA contamination in cell cultures, the serum used to supplement the culture medium should be RNA-free or should substantially reduce the RNA it contains, and should be VE-free or should substantially reduce the VE it contains, but it is also desirable that it be free of any other compounds, for example, such as PEG. However, such serum should retain the other serum components necessary for cell culture.

[0019] Based on the current state of the art, the need for animal sera (primarily SFB) that are RNA-free or have a minimal RNA content is obvious, as this will allow them to be used in cell cultures without the risk of contaminating or interfering with serum RNA. Equally obvious is the need for a method to remove RNA from animal-derived sera used in cell culture, a method that uses simple and low-cost techniques, does not put the serum at risk of sterility, and can be scaled up for industrial applications without substantially increasing the cost of the serum. Summary of the Invention

[0020] The present invention relates to animal sera that are RNA-free or substantially RNA-free. The sera are free or substantially free of extracellular vesicles (VE), but they retain the other components of the serum that are crucial for cell culture, thus facilitating cell culture-based research without the risk of contamination by VE or RNA in the serum.

[0021] In embodiments of the present invention, different types of animal sera are included, such as sera from cows, horses, mice, rats, goats, and humans, as well as sera from fetal cows, fetal horses, fetal mice, fetal rats, or fetal goats.

[0022] The present application also relates to a method for removing or substantially reducing the amount of RNA present in animal sera, particularly fetal bovine serum (FBS), by successively applying heating, cooling, alkalinization, and neutralization processes. The method decomposes endogenous VE present in the animal sera, thereby denaturing and degrading the free RNA in the sera, such that the RNA present in the sera is removed or substantially reduced, and the decomposition of VE thus counteracts its natural function. Description of the Drawings

[0023] Figure 1 A bar graph is shown that summarizes the ratios of the categories and abundances of RNA in different commercial FBS products. Each bar graph shows a different FBS set. 1) Conventional FBS A; 2) Conventional FBS B; 3) Characterized FBS; 4) Qualified FBS; 5) Certified FBS; 6) Triple-filtered FBS; 7) Exosome-reduced FBS. It is noted that the content and quantity of different categories of RNA vary in terms of packaging, and although some do not contain miRNA, all contain RNA. The black bars represent miRNA; the dark gray bars represent unannotated RNA (sequences found in the Bos taurus genome but not associated with a specific functional RNA type); the light gray bars represent tRNA; the white bars represent other RNAs not found in the Bos taurus genome, and their origin may not be bovine.

[0024] Figure 2 A graph is shown that depicts the concentration of RNA recovered after applying different procedures to FBS samples to achieve RNA removal. The procedures used and their order are described in Table 1. RNA was removed under three different conditions (lines 11, 13, and 18), and IND indicates that the RNA concentration could not be detected. The combination of inactivation by heating, cooling, alkalinization, and neutralization in a specific order is one of the combinations that results in the removal or significant reduction of RNA in FBS, and other combinations for reducing the amount of RNA involve additional ultracentrifugation steps.

[0025] Figure 3 A histogram is shown that describes the number and size of the particles contained in FBS. The gray histogram corresponds to the size and number of the particles present in the FBS sample treated using the method of the present invention (combination #11 according to Table 1), and the black outlined histogram corresponds to the untreated control sample. VE particles (including exosomes) with representative sizes (diameter 50 to 400 nm) become smaller in size compared to the untreated serum (control group), and after heating, cooling, alkalinization, and neutralization, the vast majority have a size of approximately 30 nm in diameter.

[0026] Figure 4The bar graph shown displays the RNA concentration of several commercial SFB products before and after applying the method of the present invention. 1) Conventional SFB A; 2) Conventional SFB B; 3) Characterized SFB; 4) Qualified SFB; 5) Certified SFB; 6) Triple-filtered SFB; 7) Exosome-reduced SFB. It should be noted that regardless of the commercial product and its initial RNA concentration, after applying the RNA removal method described in the present application, the RNA concentration is non-detectable (IND).

[0027] Figure 5 is a graph showing the cell proliferation of cell cultures, in which the RNA-free or substantially RNA-free SFB described in the present invention is used as a complement. HEK 293 cells were grown under different supplementation conditions: white circles represent untreated SFB (control group), black circles represent the RNA-free or substantially RNA-free SFB of the present invention (obtained by combination #11 in Table 1), and grey triangles represent synthetic serum replacement. Cells cultured with the RNA-free or substantially RNA-free SFB showed similar proliferation to cells cultured with untreated SFB (control group) and higher proliferation than the synthetic replacement, indicating that the obtained RNA-free or substantially RNA-free SFB after applying the method of the present invention retains the properties required to effectively promote cell proliferation.

[0028] Figure 6 Shows the cell culture morphology using different supplementation conditions. Untreated SFB (control group), the RNA-free or substantially RNA-free SFB described in the present invention (obtained by combination #11 described in Table 1), and synthetic serum replacement were used. It should be noted that the RNA-free or substantially RNA-free SFB of the present invention has no negative impact on the cell morphology of the control group and no negative impact on the synthetic serum, demonstrating that it retains the properties required for cell proliferation.

[0029] Figure 7 Shows a bar graph in which the cell viability of cultures under different supplementation conditions is compared. The white bars are for untreated SFB (control group); the black bars are for the RNA-free or substantially RNA-free SFB of the present invention, and the grey bars are for synthetic serum replacement. It is worth noting that on day 6, there was no significant difference in the cumulative viability between cells supplemented with the control group SFB and cells with RNA-free SFB and synthetic replacement, confirming that the RNA-free serum still retains its supplementation properties after using the method of the present invention.

[0030] Figure 8Shows the proliferation ratios of cell lines supplemented with RNA-free SFB and cell lines using untreated serum as a control: 1) HEK 293, 2) HeLa, 3) CHO, 4) MCF7, and 5) MEF cells, the proliferation rates obtained under two supplementation conditions, using untreated SFB as a supplement (control group) and cells cultured with RNA-free or substantially RNA-free SFB. Compared with using untreated SFB as a supplement, on day 6, none of the cell lines cultured with RNA-free SFB showed a cumulative proliferation difference.

[0031] Figure 9 Shows the ratio of cell viability between cells supplemented with RNA-free SFB and cells using untreated serum (control group). 1) HEK 293, 2) HeLa, 3) CHO, 4) MCF7, and 5) MEF cells, cultured with untreated SFB as a supplement and with RNA-free or substantially RNA-free SFB, the survival rates obtained under two supplementation conditions. The cell lines cultured with RNA-free SFB showed no cumulative viability difference on day 6 compared to their respective SFB control groups.

[0032] Figure 10 Shows a graph of the amounts of several miRNAs in untreated serum (control group) and the same serum treated with the method of the present invention. 1) Bta-miR-143; 2) Bta-miR-181a; 3) Bta-miR-192; 4) Bta-miR-380-3p, and 5) Hsa-miR-25-3p miRNAs were detected by quantitative reverse transcription PCR (qRT-PCR). The white bars represent untreated SFB, and the black bars represent SFB treated with the method of the present invention. In all cases, the method of the present invention reduced the amount of miRNA to a level below the detection limit (about 38 Ct), showing the effectiveness of the method of the present invention in removing miRNAs from SFB.

[0033] Figure 11 Shows a graph of the amounts of other types of RNA in untreated serum (control group) and the same serum treated with the method of the present invention. 1) U47 and 2) unannotated RNA#49627 were detected for RNA by qRT-PCR. The white bars represent untreated SFB (control group), and the black bars represent SFB treated with the method of the present invention, clearly showing that the method of the present invention can effectively remove several types of RNA.

[0034] Figure 12A graph showing the amounts of several miRNAs in untreated serum (control group) and the same serum treated by the method of the present invention. 1) hsa-miR-486; 2) hsa-miR-423-5p; 3) hsa-miR-10b. The miRNAs were detected by qRT-PCR. The white bars represent untreated SFB (control group), and the black bars represent SFB treated by the method of the present invention. It can be seen that the contents of these miRNAs in the serum samples used are very low. However, the method of the present invention can remove them to undetectable levels. Detailed implementation

[0035] The culture of cell lines and primary tissues is one of the main tools in biomedical research. Generally, cell culture is carried out by culturing cells in a liquid medium containing additives, which can keep the cells alive or continuously proliferate. Fetal bovine serum (SFB) can be used as a supplement to the medium because it consists of a complex mixture of proteins, nucleic acids, hormones, growth factors, lipids, and other small molecules (such as vitamins and minerals important for cell growth).

[0036] SFB contains a mixture of different classes of RNA, including highly conserved miRNAs. Therefore, there are miRNAs with exactly the same sequence and size between species with a relatively large evolutionary distance. Due to their high conservation and specificity, miRNAs are considered important regulators of gene expression (Bartel D.P. Metazoan MicroRNAs. Cell (2018) 173: 20-51).

[0037] Recently, it has been reported that when serum is used as a supplement in cell cultures, the RNA present in SFB interferes with the detection of intracellular miRNAs (Wei Z. et al. Sci Rep. 2016 Aug 9; 6: 31175). This finding is of great significance in molecular biology because the vast majority of studies characterizing RNA gene expression in cell culture use SFB as a complement, and the results obtained may be altered by the combined effects of the miRNAs of the cultured cells themselves and the miRNAs from SFB. This situation is even more serious when considering that it is impossible to distinguish the species from which they come among a large number of miRNAs. It is also not excluded that the miRNAs or other RNAs in the serum used as a supplement may exert biological functions on the cultured cells by altering gene expression or their physiological state, and thus affect the subsequent experimental results.

[0038] One of the possible sources of bovine miRNAs transferred into cell cultures is the extracellular vesicles (EVs) contained in SFB. However, contamination by bovine RNA also comes from the extracellular medium, since the amounts of miRNAs and other types of RNA are present in equal proportions inside and outside the EVs. Thus, removing EVs from the serum is not sufficient to completely remove the contaminating RNA.

[0039] Finally, it should be noted that the RNA in SFB is very stable and should be protected by RNA-binding proteins both inside and outside the VEs. If the RNA is not effectively protected, the large amount of RNases in SFB will naturally degrade the RNA (Chen X, et al. Cell Res. 2008 Oct; 18(10): 997-1006).

[0040] In the prior art, several methods for reducing or removing EVs from SFB have been described, including ultrafiltration, ultracentrifugation, and precipitation using chemical reagents. However, the focus of these methods is on eliminating the EVs and the biological materials they contain, but they cannot reduce or remove the RNA present in the serum in free form, which can account for 60% of the RNA contained in mammalian serum. Thus, none of these methods or SFB preparations are RNA-free or substantially RNA-free. A possible alternative is to use synthetic supplements, but these are generally much more expensive than using SFB and are not effective for all types of cell growth.

[0041] This is why there is a need for mammalian serum that is RNA-free or substantially reduced in RNA, which allows for cell culture in vitro without causing contamination or potentially altering the results of molecular biology experiments, while maintaining its characteristics and function as a supplement. In this sense, there is also a need for a method that allows for the production of serum that is RNA-free or substantially reduced in RNA, without altering its function as a supplement and without leaving chemical reagent residues that could interfere with cell culture or molecular biology experiments.

[0042] The present invention overcomes the drawbacks of the prior art by providing serum that is RNA-free or substantially reduced in RNA, as well as an effective method for removing the RNA contained in mammalian serum (especially SFB). This method can effectively eliminate the RNA present in mammalian serum, whether it is inside or outside the EVs, and thus can produce serum that is RNA-free or substantially reduced in RNA from different species of mammals.

[0043] Another technical advantage of the method is that it is a simple method that does not require centrifugation or ultracentrifugation, nor the use of filters, thus reducing the application cost and the industrial scale required. In addition, the method can be carried out without the need to transfer the serum to different containers, which helps to maintain the required aseptic conditions. The serum produced by the method described in the present application, which does not contain VE and RNA, or in which the total amount of VE and RNA is significantly reduced, achieves a higher RNA removal rate compared to other sera described in the prior art.

[0044] The present invention is further based on the unexpected fact that applying the processes of heating, cooling, alkalization, and neutralization to mammalian serum in a specific order results in a significant elimination or reduction of the naturally occurring RNA in the serum. Although these processes are commonly used and the effect of each of them on the different biomolecules present in the serum is known, combining these four processes with this specific order of execution and having this effective method for removing RNA has not been described so far. As described in the present invention, this specific order of application of these methods improves the efficiency of RNA removal from mammalian serum.

[0045] Since the present invention is based on applying the processes of heating, cooling, alkalization, and neutralization in a specific order, which is contrary to the intuition of the existing known art, it is not expected that a person with ordinary knowledge would arrive at a similar conclusion. Applying these processes individually or in a different order in the serum does not produce the same result, and thus this method is non-obvious compared to the existing known art.

[0046] Serum that does not contain RNA or substantially reduced RNA can be used as a supplement in any type of in vitro cell culture (such as HeLa, HEK293, CHO, MCF7, MEF, etc.) and in any molecular biology experiment seeking to study the cell metabolism or physiological state. In addition, in all studies related to gene expression analysis, especially in studies related to the expression analysis of several RNA species (such as miRNA, tRNA, lncRNA, mRNA, snRNA, and piRNA, etc.), serum that does not contain RNA or substantially reduced RNA can be used as a complement. Similarly, serum that does not contain RNA or substantially reduced RNA can be used as a supplement in cell cultures that can be used for the production of therapeutic agents, such as hormones, recombinant proteins, antibodies, clotting factors, and vaccines, to name just a few. (Ashish Verma, Anchal Singh Academic Press, Nov 4, 2013)

[0047] As described in the specification of the present application, the terms "RNA-free serum" and "serum with substantially reduced RNA" refer to animal sera, preferably of mammalian origin, which have undergone an RNA removal process and the content of whose endogenous RNA cannot be detected by conventional highly sensitive methods for measuring the nucleic acid concentration of aqueous samples, such as spectrophotometry and fluorescence spectrophotometry, or by methods for detecting the presence and relative amounts of specific RNA sequences, and the rare or non-detection of RNA sequences is generally interpreted as "absent" or "undetectable" sequences, as in the case of qRT-PCR, where the amplification cycle (Ct) represents the quantity of the sequence of interest, considering that a signal quantity with a Ct greater than 38 cycles indicates the absence or undetectability of the sought sequence. In specific sequence detection techniques such as next-generation sequencing (RNA-seq, small RNA-seq), it is generally assumed that the number of reads represents the quantity of the sequence of interest, and thus a sequence with a read count of 0 indicates the absence of RNA or its content is below the detection limit of the technique.

[0048] For the development of the present invention, first, the composition and relative amounts of the different types of RNA present in the sera were analyzed ( Figure 1 ). To this end, samples of different commercial SFB products: 1) conventional SFB A; 2) conventional SFB B; 3) characterized SFB; 4) qualified SFB; 5) certified SFB; 6) triple-filtered SFB and 7) SFB with reduced exosomes analyzed by the RNAseq method (Illumina). The results of the bioinformatics analysis showed that the SFBs contain different quantities of different types of RNA. Among the most abundant RNA categories, we found miRNA, tRNA, and unannotated RNA (whose sequences are located in the Bos taurus genome but cannot be associated with a specific type of RNA). It should be noted that the proportions of the different types of RNA vary among the different commercial products, some of which show a lower miRNA proportion compared to other products, but all show some type of RNA.

[0049] To determine the most suitable conditions for the process of removing the RNA present in the sera, different processes and their combinations were tested, including: heating, cooling, alkalization, addition of enzymes (ribonucleases), and ultracentrifugation. The tested combinations of the different processes are described in Table 1. After applying these processes, RNA was extracted using TRIzol (Thermo) reagent, and the extracted RNA was quantified using a Qubit-type fluorescence spectrophotometer (Thermo).

[0050] Table 1

[0051]

[0052]

[0053] Figure 2 shows the RNA removal efficiency of different processes and their combinations in the SFB sample. Only specific combinations of three different processes result in RNA removal to non-detectable levels (IND), which are below the detection limit of 250 pg / μL by fluorospectrophotometry. One of the combinations ( Figure 2 , Article 11) includes heating and cooling, followed by alkalization, and then neutralization. Another effective combination ( Figure 2 , Article 13) includes heating and cooling, followed by ultracentrifugation. The third effective combination ( Figure 2 , Article 18) is a combination of ultracentrifugation steps, followed by heating and cooling, then alkalization, and then neutralization, in this order.

[0054] Other methods and combinations show a certain degree of effectiveness in removing RNA from serum, but none of them can be used for complete removal or below the detection limit. It should be emphasized that the removal of RNA from serum is not only carried out by applying the four described processes: heating, cooling, alkalization, and neutralization, but also the order of application of these processes is particularly important because the combination of these three processes but in different orders ( Figure 2 , Article 12) has different effects on the removal of RNA from serum; this is surprising and non-obvious in the prior art. In summary, applying these processes alone or in different orders in serum does not remove the RNA contained therein.

[0055] Since part of the RNA present in serum is contained in VE, the NanoSight (Malvern) instrument ( Figure 3 ) is used to quantify the nanoparticles by nanoparticle tracking analysis (NTA) to verify the effect of the method of the present invention on the VE present in SFB.

[0056] The particle sizes naturally contained in SFB are between 50 nm and 400 nm in diameter, and the two particle concentration peaks are close to 100 nm and 180 nm ( Figure 3 , control group, black outline histogram). After using the method described in this application, the particle sizes present in SFB are significantly reduced, and the diameter of the largest particle concentration peak is close to 30 nm ( Figure 3 , #11, gray histogram).

[0057] These results indicate that the application of the RNA removal method decomposes VE present in serum, including exosomes, as the number of peaks seen near 30 nm (1.5 x 108 particles) correlates to the number of two main peaks of the control group sample, indicating the decomposition of the natural state of VE, removing or significantly reducing the content of VE in serum. The decomposition of VE makes the removal of total RNA present in animal serum more effective and may release the RNA present in VE for subsequent degradation.

[0058] This result can be explained by the fact that the heating and cooling processes denature VE and release the RNA they contain, subsequent treatment with strong base produces alkaline hydrolysis of RNA, and the addition of strong acid promotes greater RNA degradation, thus increasing the efficiency of RNA removal from serum. The RNA remaining in serum after this treatment is likely composed of non-functional fragments, which are products of RNA molecule degradation and thus will no longer have an impact on cells in culture.

[0059] To confirm the efficiency of the method for removing RNA from serum, the RNA of seven different commercial SFB products was quantified before and after treatment with the method described in this application. RNA was extracted using TRIzol (Thermo) reagent and quantified by Qubit-type fluorospectrophotometry (Thermo).

[0060] The RNA removal method described in this application is effective for all tested commercial SFB products: 1) conventional SFB A; 2) conventional SFB B; 3) characterized SFB; 4) qualified SFB; 5) certified SFB; 6) triple-filtered SFB and 7) exosome-reduced SFB. Figure 4 It shows that, although containing different classes of RNA, as previously determined ( Figure 1 ), the method is capable of removing the RNA of different commercial products to levels below the detection limit, indicating that the method is effective regardless of the RNA concentration and its presence in serum, and thus the method is applicable to any type of animal serum.

[0061] Subsequently, the supplementation ability of the SFB described in this application that is RNA-free or substantially reduced in RNA was evaluated to determine if it changed after the application of the RNA removal method. To this end, the growth curves of human cells (HEK 293 cells) supplemented with RNA-free serum were plotted, and their proliferation, morphology, and viability were measured over 6 days and compared to cells cultured in medium supplemented with untreated serum, as well as to cells cultured using a synthetic serum substitute, a synthetic formulation that contains the components necessary for the growth of certain types of cell lines in culture and, due to its synthetic nature, is RNA-free or VE-free.

[0062] Figure 5 Shows the comparison of cell proliferation under three supplementary conditions. HEK 293 cells were grown in medium under normal conditions until they reached 60% confluence. After that, the medium was changed, and medium supplemented with any variant (control SFB, SFB without RNA, and synthetic serum substitute) was added, and the cell density (days 0 - 6) was quantified. The results showed that there was no difference in cell proliferation in cultures supplemented with serum without RNA (black circles) compared to untreated SFB (control group, hollow circles). The proliferation obtained using serum without RNA was higher than that of cells grown in medium supplemented with synthetic serum substitute (grey triangles).

[0063] Figure 6 Shows the cell morphology of cells cultured under three supplementary conditions. Notably, compared to the control group, SFB without RNA did not affect the cell morphology, nor did it affect the synthetic serum, indicating that it maintained the properties necessary to promote cell proliferation without any side effects on cell physiology.

[0064] Cell viability was determined by quantifying HEK 293 cells using the trypan blue exclusion assay, which assesses the integrity of the membrane. Figure 7 Shows the cumulative cell viability on day 6 of cells cultured under three supplementary conditions (white bars: control SFB; black bars: SFB without RNA; grey bars: synthetic substitute). The results showed that the cumulative cell viability on day 6 did not vary between the different supplements used, indicating that the SFB without RNA described in the present invention had no negative impact on cell viability and was thus safe to use.

[0065] These viability and proliferation assays were repeated in other cell lines (respectively Figure 8 and Figure 9 ). Figure 8 Shows the proliferation ratio between cell lines grown in medium supplemented with SFB without RNA and cell lines grown in medium supplemented with serum but untreated (control group). Cultured cells: 1) HEK 293, 2) HeLa, 3) CHO, 4) MCF7, and 5) MEF were grown in medium supplemented with untreated SFB and medium supplemented with SFB without RNA or substantially without RNA, and the proliferation rate profiles under the two supplements were obtained. As with HEK 293 cells, for all tested cell lines, the proliferation ratio between the two supplementary conditions was close to 1, indicating no difference in the supplementary ability between SFB without RNA and control SFB.

[0066] Figure 9Shows the cumulative viability of the same cell line on day 6 under two culture conditions, noting that the viability ratio between the two supplementation conditions is close to 1. This result indicates that there is no significant difference in cell viability when using the serum treated by the method of the present invention compared to the serum of the control group.

[0067] To verify the effectiveness of the method for removing RNA from serum, some miRNA assays were performed before and after applying the method. For this purpose, RNA was extracted from a commercial SFB product with a rich miRNA content as described previously ( Figure 1 ), and subsequently, according to bioinformatics analysis of the massive sequencing results, the relative amounts of the most representative miRNAs in the serum were determined.

[0068] Probes were used to assay each miRNA and are described in Table 2. As Figure 10 shown, all miRNAs evaluated: 1) Bta-miR-143; 2) Bta-miR-181a; 3) Bta-miR-192; 4) Bta-miR-380-3p and 5) Hsa-miR-25-3p were found in the control group serum (white bars), while miRNAs below 38 Ct (dashed line) were not detected in the serum treated with the method of the present invention (black bars), indicating that when the method of the present invention is applied, a large amount of miRNAs in SFB are removed or reduced below the detection limit.

[0069] Some commercial SFB products have a low miRNA content but contain a large amount of other RNAs ( Figure 1 , articles 4 and 7). Computational analysis of several classes of RNAs contained in one of these commercial products showed that two RNA sequences were abundant: RNA classified as U47 and another RNA classified as "unannotated" (designated here as #49627).

[0070] To confirm the elimination of other types of RNAs, qRT-PCR was performed using specific probes to evaluate the amounts of these two RNAs. The probe used to assay U47 is shown in Table 2.

[0071] Table 2

[0072] miRNA assay identifier bta-mi-R-143 ID:006735_mat bta-miR-181a ID:005861_mat bta-miR-192 ID:006776_mat bta-miR-380-3p ID:006377_mat hsa-miR-10b ID:002218 hsa-miR-25-3p ID:000403 hsa-miR-423-5p ID:00234 hsa-miR-486 ID:001278 U47 ID:001223

[0073] For the unannotated RNA sequence #49627, SYBR-green type fluorescence was used with the previously reported technique and the primer oligonucleotides described in Table 3.

[0074] Table 3

[0075]

[0076] Figure 11Shows the amounts of 1) U47 and 2) #49627 in commercial (white strips) untreated serum and the same serum (black strips) treated using the method of the present invention. The levels of both RNAs were found to be below 20 Ct in the untreated serum, and the application of the method of the present invention removed both types of RNAs, reducing their levels below the detection limit of 38 Ct.

[0077] An important aspect of those commercial products that seemingly do not contain miRNAs is that although certain miRNAs seemingly have undetectable levels, such as Bta-miR-143, Bta-miR-181a, Bta-miR-192, Bta-miR-380-3p, and Hsa-miR-25-3p, they do contain some miRNAs that are still detectable. Figure 12 Shows the qRT-PCR analysis for determining: 1) Hsa-miR-486; 2) Hsa-miR-423-5p; and 3) Hsa-miR-10b, before treatment (white strips) and after application of the method of the present invention (black strips) in one of these commercial products. Notably, in the untreated SFB, these miRNAs were detected around 34 Ct, and when using the method of the present invention, these miRNAs were reduced below the detection limit of 38 Ct.

[0078] According to the description in the present application, the invention described herein relates to mammalian serum that is RNA-free or substantially reduced in RNA and a method for obtaining such RNA-free or substantially reduced RNA serum. The serum obtained by this method retains its ability to supplement cell cultures and is free of chemical residues that may be harmful to cell cultures.

[0079] To implement the method of the present invention, any method for controlled heating of serum samples described in the prior art can be used, preferably in a sterile container or vessel, or in an industrial container, including but not limited to bathing in a bath and immersing in temperature-controlled water, culturing in a temperature-controlled cabinet (oven), or using any other controlled heating device.

[0080] Temperatures between 52 °C and 63 °C, preferably between 55 °C and 57 °C can be used for the serum heating process. The heating time can be between 25 and 60 minutes, preferably 35 minutes.

[0081] The cooling process can be carried out stepwise after heating, without controlling the temperature by cooling it to room temperature, or by using cooling means to accelerate the process, such as immersing in water or liquid at room temperature or below room temperature, or using any other controlled cooling device. The range of the final cooling temperature can be between 8 °C and 25 °C, preferably 16 °C.

[0082] For the alkalization process, several alkaline or basic compounds or salts, either in solution or anhydrous, which release hydroxyl ions (OH-), can be used, including but not limited to: potassium hydroxide (KOH), magnesium hydroxide (Mg(OH)₂), calcium hydroxide (Ca(OH)₂), sodium hydroxide (NaOH), and other substances of similar nature. A person of ordinary skill in the art can routinely standardize the optimal temperature and time for the heating and cooling processes required in this method.

[0083] The alkali concentration can be between 10 - 12N, preferably 12N. A person of ordinary skill in the art can routinely standardize the optimal concentration for the alkalization process required in this method.

[0084] The serum should be alkalized to a pH value between 10 and 12, preferably a pH of 12.

[0085] The time the serum is exposed to the alkali can vary depending on the RNA concentration and volume present, but it must be maintained for at least 3 minutes, can be between 3 and 20 minutes, and preferably between 5 and 10 minutes.

[0086] For the neutralization process, several compounds or salts with acidic chemical properties can be used, including but not limited to: phosphoric acid (H₃PO₄), nitric acid (HNO₃), acetic acid (CH₃COOH), hydrochloric acid (HCl), and other compounds of similar nature.

[0087] The acid concentration can be in the range between 0.1 and 2N, preferably 1N can be used as the concentration. A person of ordinary skill in the art can routinely standardize the optimal concentration for the neutralization process required in this method.

[0088] The serum must be acidified until a physiological pH value between 7.2 and 7.5 is obtained, preferably a pH value of 7.4.

[0089] In a preferred embodiment of the present invention, the serum is heated at a temperature between 55°C and 57°C for 30 to 45 minutes; it is gradually cooled until it reaches room temperature of 20°C, the serum is alkalized using anhydrous NaOH (powder or granules) until it reaches pH 12 for 10 minutes, and then the pH of the alkalized serum is reduced using 1N concentration of HCl until neutralization is achieved at a physiological pH value of 7.4.

[0090] If the DNA concentration is very high, for example, higher than 45 or 50 ng / mL, the method of the present invention can be continuously and repeatedly applied to ensure the removal of RNA.

[0091] In one embodiment of the present invention, the method described in the present application may incorporate an additional ultracentrifugation step (between 80,000 xg and 100,000 xg). If ultracentrifugation is performed and the process jeopardizes the sterility of the serum, an additional sterilization step must be performed using any technique known in the prior art, such as filtration through a 0.2 micron pore membrane.

[0092] Any type of animal serum can be used to practice the present invention, including but not limited to serum from cows, horses, mice, rats, goats, and humans, as well as serum from bovine fetuses, equine fetuses, murine fetuses, rat fetuses, or caprine fetuses.

[0093] The present invention is further illustrated by the following examples, which do not limit the scope of the claims. On the contrary, these examples are presented to better understand the practice of the present invention and it is to be understood that they only represent some embodiments of the present invention.

[0094] Description of the implementation mode of the present invention

[0095] Example 1

[0096] Removing RNA from SFB

[0097] Starting from a conventional commercial SFB product, the total RNA concentration was initially determined by extraction using Trizol product according to the manufacturer's recommendations and found to be 35 ng / mL.

[0098] The SFB was heated at 56 °C for 35 minutes and slowly cooled to room temperature until it reached 20 °C. Subsequently, the serum was alkalinized using 12N anhydrous NaOH until a pH of 12 was reached and maintained in this way for 15 minutes, and then the pH of the alkalinized serum was lowered using 1N HCl until a physiological pH of 7.4 was reached.

[0099] After applying this method, the total RNA concentration was determined by the above Trizol extraction method, and a concentration below the detection limit was found by fluorescence spectrophotometry.

[0100] Example 2

[0101] Combining the method of the present invention and ultracentrifugation to remove RNA from SFB

[0102] Starting from a conventional commercial SFB product with an RNA concentration of 40 ng / mL, the serum was ultracentrifuged at 100,000 xg for 7 hours at 4 °C. At the end of ultracentrifugation, the sample supernatant was transferred to a new sterile container without disturbing the bottom fraction. To maintain sterile conditions, the serum supernatant was passed through a sterile filter with a pore size of 0.2 microns.

[0103] Subsequently, the SFB supernatant was heated at 56 °C for 35 minutes and slowly cooled to room temperature until it reached 20 °C; subsequently, the serum was alkalized with anhydrous NaOH until the pH reached 12 and maintained at this state for 15 minutes, and then the pH of the alkalized serum was reduced with 1N HCl until the physiological pH of 7.4 was reached.

[0104] After applying this method, the total RNA concentration was determined by the above Trizol extraction method, and the concentration below the detection limit was found by fluorescence spectrophotometry.

[0105] Example 3

[0106] The method of the present invention was used to remove RNA from SFB in different iterations

[0107] Different serum sets were expected to contain higher amounts of RNA than other sera. In the case of serum samples with high RNA content, the method of the present invention can be repeated to produce an accumulative result of its efficiency in RNA removal.

[0108] Starting from serum with an RNA content quantification of more than 45 ng / mL, it was heated at 56 °C for 35 minutes and slowly cooled to room temperature until it reached 20 °C; subsequently, the serum was alkalized with anhydrous NaOH until the pH reached 12 and maintained for 15 minutes, and then the pH value of the alkalized serum was reduced with 1N HCl until the physiological pH value of 7.4 was reached.

[0109] Subsequently, the processes of heating, cooling, alkalization, and neutralization were repeated under the same conditions.

[0110] After applying the method continuously twice, the total RNA concentration was determined by the above Trizol extraction method, and the concentration below the detection limit was found by fluorescence spectrophotometry.

Claims

1. A method for removing RNA from mammalian serum, the method comprising: (a) heating the serum to a temperature of 52 °C to 63 °C; (b) cooling the serum to a temperature of 8 °C to 25 °C; (c) alkalizing the serum to a pH value of 10 to 12; (d) neutralizing the serum to physiological pH, wherein these steps are carried out in this order.

2. The method according to claim 1, wherein, The serum is from cattle, horses, mice, rats, goats, or humans.

3. The method according to claim 2, wherein, The serum is from bovine fetuses, equine fetuses, murine fetuses, rat fetuses, or goat fetuses.

4. The method according to claim 1, wherein The serum is heated to a temperature of 55 °C to 57 °C.

5. The method according to claim 1, wherein The temperature is maintained for 30 minutes to 45 minutes.

6. The method according to claim 1, wherein, The alkalization of the serum is carried out until a pH of 12 is reached.

7. The method according to claim 1, wherein The alkalization of the serum is carried out using a compound having basic chemical properties selected from the following: sodium hydroxide (NaOH), potassium hydroxide (KOH), magnesium hydroxide (Mg(OH)2), calcium hydroxide (Ca(OH)2).

8. The method according to claim 1, wherein, The alkalization is maintained for 3 minutes to 20 minutes.

9. The method according to claim 1, wherein, The alkalization is maintained for 5 minutes to 10 minutes.

10. The method according to claim 1, wherein the neutralization of the serum is carried out using a compound having acidic chemical properties selected from the following: phosphoric acid (H3PO4), nitric acid (HNO3), acetic acid (CH3COOH), hydrochloric acid (HCl).

11. The method according to claim 1, wherein, The neutralization of the serum is carried out until a pH of 7.2 to 7.5 is reached.

12. The method according to claim 1, wherein The neutralization of the serum is carried out until a pH of 7.4 is reached.

13. The method according to claim 1, the method further comprising an initial or final ultracentrifugation step.

14. A method for removing RNA from mammalian serum, the method comprising: (a) heating the serum to a temperature of 52 °C to 63 °C; (b) cooling the serum to a temperature of 8 °C to 25 °C; (c) ultracentrifuging the serum at 80,000 xg to 100,000 xg for at least 5 hours to 7 hours, wherein these steps are carried out in this order.

15. The method according to claim 14, wherein, The serum is from cattle, horses, mice, rats, goats, or humans.

16. The method according to claim 15, wherein, The serum is from bovine fetuses, equine fetuses, murine fetuses, rat fetuses, or goat fetuses.

17. The method according to claim 14, wherein, The serum is heated to a temperature of 55 °C to 57 °C.

18. The method according to claim 14, wherein, The temperature is maintained for 30 minutes to 45 minutes.

19. The method according to claim 14, wherein The ultracentrifugation is carried out at 100,000 xg for 7 hours.

20. The method according to claim 14, wherein, The ultracentrifugation is carried out for 5 hours to 10 hours.

21. The method according to claim 14, further comprising an additional serum sterilization step.

22. An animal serum free of RNA, the animal serum being prepared by the method according to any one of claims 1 - 21.

23. The RNA-free animal serum according to claim 22, wherein, The serum is from cattle, horses, mice, rats, goats, or humans.

24. The RNA-free animal serum according to claim 23, wherein, The serum is from bovine fetuses, equine fetuses, murine fetuses, rat fetuses, or goat fetuses.

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