Reduction of albumin in a cell culture medium.
Patent Information
- Application Number
- NL2038950
- Authority / Receiving Office
- NL · NL
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-10-29
- Publication Date
- 2026-06-04
- Estimated Expiration
- 2044-10-28
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Abstract
Description
1 / 22 DESCRIPTION TITLE: Reduction of albumin in a cell culture medium. TECHNICAL FIELD The invention is in the general field of biotechnology, specifically in the sub-field of media for cell cultures. BACKGROUND OF THE INVENTION It is known that in cell cultures of many animal, or more broadly eukaryote cell types that albumin (blood-derived or from recombinant origin) may be used as a component of the culture media to promote, or enhance, the proliferation of the cells. The use of blood-derived and / or recombinant albumin as a cell culture supplement is not ideal or undesirable considering a number of aspects. Blood-derived albumin poses ethical issues considering the fact that blood has to be drawn (repeatedly) from animals or animals have to be slaughtered to obtain the blood from which albumin can be purified. Human albumin can allo be used, which is similarly obtained from withdrawn blood. As a result of the source, there is a limited supply for blood-derived albumins and increasing demands for animal blood- derived albumins would increase animal suffering and / or slaughter. Considering the ability of albumin to bind a wide range of molecules (e.g. hormons, lipids, metals, ions, hydrophilic and hydrophobic substances), there is substantial batch-to-batch variation in biological effect of blood-derived albumins as the albumin's 'cargo', i.e. the molecules bound to albumin, will be different depending on factors like, among others, the specific animal, time of year and time of day. The 'cargo' can add to albumin's biological effects in cell culture, as exemplified for albumin-associated lipids (e.g. Keenan et al., Separation of growth-stimulating activity of BSA fraction V from the bulk of albumin using Heparin Sepharose Chromatography, 1995 Cytotechnology 19, 63-72 and Garcia-Gonzalo and Belmonte, Albumin-Associated Lipids 25 Regulate Human Embryonic Stem Cell Self-Renewal, 2008 PLOS ONE 3(1): e1384), but will vary across batches depending on the aforementioned variables. Likewise, the albumin molecule can dynamically exist in several oxidation states which influence its biological activity by, for example, influencing ligand binding (Oettl and Stauber, Physiological and pathological changes in the redox state of human serum albumin critically influence its binding 2 / 22 properties, 2007 Br. J. Pharmacol. 151, 580-590). Thus, the proportion of albumin oxidation states and associated bioactivity can change across batches. Next to blood-derived albumins, recombinant albumin produced by a heterologous expression system (e.g. plants, bacteria, animal cells or live animals) can be used as a cell culture supplement, which would circumvent the ethical issues associated with withdrawing blood from or slaughtering animals. However, in addition to the supply being limited by current manufacturing capability, the advanced technology required to produce recombinant albumin translates into a high selling price, making it less attractive for activities requiring large scale usage of albumin-supplemented culture media. Despite the fact that recombinant albumin may be produced in an environment that is biologically more controlled compared to being sourced from live animals, recombinant albumin will still exhibit biological variation. For example, the glycation status of recombinant albumin has been described to differ across supplier and batches (Frahm et al., Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa, 2014 PLOS ONE 9(10): e109893) and glycation status determines the proliferation-enhancing effect of albumin Interaction between glycated serum albumin and AGE-receptors depends on (Indurthi et al., structural changes and the glycation rengent, 2012 Arch Biochem Biophys 528(2) 185-196). Moreover, sine albumin is able to bind a range of molecules, conditions of the heterologous expression system will influence the type and amount of molecules bound to the recombinant albumin that can be retained during purification. This variation will lead to batch-to-batch variation in the biological effect of the albumin. Different methods exist to purify both blood-derived and recombinant albumin. These methods can differentially affect the albumin protein and the bound molecules (e.g. lipids being removed during ethanol precipitation), which impacts its biological effect and the variation in 25 biological activity across suppliers, products and batches. Or, in other words, the variation in lipids, oxidation and glycation affect the structure of the albumin protein, and the structure affects the function of the albumin. Many functions have been ascribed to albumin. For example, albumin can act as an antioxidant, in part owing to (the oxidation status of) its free cysteine residue, and can bind 30 several metal ions, hormones, lipids, nitric oxide, and vitamins. Moreover, albumin is a ligand to several receptors, can act as a chaperone (Pomier et al., Interactions of intrinsically disordered proteins with the unconventional chaperone human serum albumin: From 3 / 22 mechanisms of amyloid inhibition to therapeutic opportunities, 2022 Biophysical chemistry, 282, 106743) and a shear protectant, and possesses several enzymatic activities (Belinskaia et al., Serum Albumin in Health and Disease: Esterase, Antioxidant, Transporting and Signaling Properties, 2021 Int. J. Mol. Sci., 22(19), 10318). Despite years of research and many elucidated functions of albumin, it is still unclear which function(s) is / are crucial to promote proliferation for specific cell types. Protein isolates of rapeseed (Stout et al., A Beeft'-R culture medium: Replacing albumin with rapeseed protein isolates, 2023 Biomaterials, 296, 122092) and pea or chickpea (W02021148955A1) have been suggested as an albumin replacement. However, these solutions were only described when applied to relatively short culture durations, specific culture conditions (e.g. adherent or suspension cultures) and specific cell type(s). The inventor has tested rapeseed protein isolate and identified the albumin-replacing potential of rapeseed protein to be lacking in suspension cultures and the albumin-replacing potential of pea and chickpea protein to be insufficient. Additionally, phenotypic changes to cells can be induced by supplementing rapeseed protein to the culture medium (e.g. differentiation potential: Alashi et al., Effects of canola proteins and hydrolysates on adipogenic differentiation of C3H10T / 2 mesenchymal stem cells, 2015 Food Chemistry, 185, 226-232) which was also observed in experiments using rapeseed protein isolate conducted by the inventor. More specifically, the adipogenic potential of cells was found to be severely hampered upon exposure to rapeseed protein. W02022132974A1 describes peptides comprising superoxide dismutase activity and Cu+, Zn+ chelating activity, superoxide scavengers, vitamin E analog(s), and hydrogen peroxide reducing reagents, that are claimed to be able to replace albumin for specific functions. The inventor tested these and was not able to use them in order to replace the 25 proliferation-promoting capabilities of albumin. It is clear that none of the existing solutions to this problem of replacing albumin are fully reproducible and / or applicable to a variety of cell types and / or culture conditions and there is thus a need to ameliorate these problems, as disclosed with the present invention. SUMMARY OF THE INVENTION 30 In a preferred embodiment, the invention is a method to reduce the amount of albumin in a cell culture, characterised by comprising the steps of a) providing a proliferation cell 4 / 22 culture with at least a cell type that benefits from improved proliferation in the presence of albumin; b) adding whey to the cell culture medium in a concentration that provider the same proliferation improvement as albumin; c) allowing the cells to proliferate in the whey containing cell culture. More preferably the invention is a method wherein the whey is a whey protein isolate or whey protein concentrate, also preferably wherein the cell culture is of an animal cell type, more particularly of a mammalian cell type, more particularly of a mesenchymal lineage, more particularly a primary cell, more particularly a fibro-adipogenic progenitor (FAP) or a satellite cell (SC), more particularly of a bovine origin; also preferably wherein whey is added to the cell culture at a concentration that replaces the desired effect of albumin, usually at a relationship of 1:1 in mass (1 g of whey for 1 g of albumin) or more (more than 1 g of whey for 1 g of albumin) or slightly less, depending on the cell type; also preferably wherein for bovine FAPs and SCs the relationship is 1:1; also preferably wherein no albumin is added to the cell culture medium or wherein an amount of albumin not enough to maximise the proliferation potential of the culture is added to the cell culture medium; also preferably wherein the whey, preferably in the form of whey protein isolate or whey protein concentrate is present in the culture medium at a concentration of about 0.01 mg / mL to about 10 mg / ml; also preferably wherein the medium is serum free medium; also preferably wherein the culture medium further comprises a basal medium, preferably a carbon source, and preferably a growth factor. In another preferred embodiment the invention is a medium for a cell culture characterised by comprising a base medium, preferably further comprising a carbon source, preferably further comprising an appropriate growth factor, where the culture is of a cell type or types that would benefit from the presence of albumin, asserted by an improvement in the proliferation outcome being verified experimentally when albumin is present, wherein the 25 actual culture either has no albumin added to it or does not have enough albumin to substantially increase the proliferation performance of the culture, and wherein whey is added at a concentration that replaces the desired effect of albumin; also preferably wherein the medium comprises a basal medium, a carbon source, a growth factor, and whey protein isolate or whey protein concentrate as a substitute for albumin; also preferably wherein the whey or 30 whey protein isolate or whey protein concentrate is present at a concentration of about 0.01 mg / mL to about 10 mg / ml. 5 / 22 In another preferred embodiment the invention is in the use of whey as an albumin replacement in a cell culture. DESCRIPTION OF THE DRAWINGS Fig. 1 — A comparison of the population doublings obtained at 3 days of adherent cell cultivation of bovine primary fibro-adipogenic progenitors in well plates with medium containing different albumins. Manufacturer-to-manufacturer and batch-to-batch performance variations are demonstrated. A1buMAX I is shown to be one of most potent albumins for enhancing proliferation. Fig. 2 - Panel A shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs for different concentrations of A1buMAX I and whey protein isolate or concentrate. Panel B shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs for different concentrations of AlbuMAX I and different whey protein isolate products from different suppliers. The successful replacement of albumin with whey is proven. Fig. 3 - A comparison of the population doublings obtained at 3 days of adherent cell cultivation of bovine primary fibro-adipogenic progenitors in well plates with medium containing one of whey protein isolate or a whey protein hydrolysate up to a maximum concentration of 3 mg / ml. Isolate is shown to outperform hydrolysate. Fig. 4 - Panel A shows the population doublings obtained in 4 days of culture of primary bovine FAPs grown in suspension in low-attachment 24-well plates with AlbuMAX I, whey protein isolate and without albumin or whey. Panel B shows the cell density (shown as eens / mi) obtained in 6 days of culture of bovine FAPs grown in suspension in 100 ml spinner flasks with 3 mg / m1 AlbuMAX I, 3 mg / m1 whey protein isolate with 91.3% protein and a whey protein concentrate with 79.0% protein. Whey is shown to outperform albumin. 25 Fig. 5 - A comparison of the population doublings obtained up to 8 days of cell cultivation of bovine primary fibro-adipogenic progenitors in 3L bioreactors with medium containing one of A1buMAX I, or a whey protein isolate at a concentration of 3 mg / ml. Whey is shown to outperform albumin. 6 / 22 Fig. 6 - A comparison of the number of cells obtained after 3 days of adherent cell cultivation of bovine primary fibro-adipogenic progenitors in well plates with medium with different basal media containing one of A1buMAX I, or a whey protein isolate at a concentration of 3 mg / ml. Whey is shown to replace albumin independently of the base media used. Fig. 7 - A comparison of the population doublings obtained at 3 days of adherent cell cultivation of bovine primary satellite cells in well plates with medium containing one of A1buMAX I, or a whey protein isolate up to a maximum concentration of 3 mg / ml. Whey is shown to replace albumin independently of the cell type used within the same species. Fig. 8 - A comparison of the population doublings obtained at 3 days of cell cultivation of ovine primary fibro-adipogenic progenitors in well plates with medium containing one of A1buMAX I, or a whey protein isolate up to a maximum concentration of 10 mg / ml. Whey is shown to replace albumin independently of the species. In this case, a higher concentration of whey is required to reach the same effect as of albumin, but albumin is still successfully replaced by whey. Fig. 9 - Fat droplets as bright points in confocal microscopy images of differentiated FAP cultures, previously proliferated in spinner flask cultures in the presence of A1buMAX I, whey protein isolate or whey protein concentrate. Fig. 10 - Whey protein isolate and AlbuMAX I performance compared at a high concentration, 10 mg / ml. DETAILED DESCRIPTION OF THE INVENTION The invention described herein aims primarily to solve the problem of reducing the amount of, or eliminating, albumin used in medium when performing cell cultures. 25 Cell cultures consist of cells, usually animal, or more broadly eukaryote, cell types isolated from their original living organism in the form of multicellular organisms, or isolated single cells organisms, subject to artificial conditions that replicate the biological processes that allow them to proliferate or differentiate. These artificial conditions may comprise placing the cells in an appropriate bioreactor, spinner flask, well-plate, Petri dish or any other industrial 7 / 22 or laboratory equipment adequate for the culture of cells. The cells are mostly surrounded by, or immersed in, a cultivation medium appropriate for culturing the specific cell type. This medium can be specifically tailored to promote proliferation, differentiation, or both. Proliferation is understood to be the phase of the lifetime of a cell during which the cell divides, creating new cells, but maintaining its unipotent, pluripotent, multipotent or totipotent abilities, i.e. the ability to differentiate into specific cell types. Differentiation is understood to be the phase of the lifetime of a cell during which it attains a specific phenotype, for example becoming an adipose cell or a muscle cell, and losing its uni, pluri, toti or multipotency. Albumin refers to a family of globular proteins, of which serum albumins are the most common members. -Serum albumins" refer specifically to the albumins found in the blood of vertebrates, specifically in the serum portion. Typical serum albumins are human serum albumin (HSA) or bovine serum albumin (BSA). Serum albumin, i.e. HSA and BSA, are the albumins most commonly used in cell cultures, and are usually referred to as simply "albumin". A cell culture with "albumin" may thus contain one (or more) of several alternative albumins. For the purposes of this specification, when "albumin" is discussed, especially in terms of albumin reduction or elimination in cell culture media, any suitable type of albumin for cell culture media is meant. Albumin may also be of recombinant origin, i.e., manufactured by expression from a genetically modified organism, such as a bacteria. Serum, in the context of this invention, is meant to refer to blood serum, from which albumin can be purified. Serum, in the context of this invention does not mean the full composition of, for example, Fetal Bovine Serum or equivalent serums, that is sometimes added to cell culture media. If this type of serum is meant, it is explicitly referred to as such. In a typical cell culture, of the cell types discussed in this invention, it is customary and extremely well established in the art that a basal medium should be used, such as Minimum 25 Essential Medium (MEM), Eagle Medium, Dulbecco's Modified Eagle Medium (DMEM), Ham's F-12, Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (DMEM F12), or Roswell Park Memorial Institute (RPMI) 1640. A basal medium is formulated to provide baseline conditions for the survivability of the cultureel cells and typically contains amino acids, glucose, and minerals containing calcium, magnesium, potassium, sodium, and / or phosphate, 30 considered essential for the survival of the cells. 8 / 22 Typically, basal media by itself allow the cells to proliferate (i.e. expand) but not necessarily to differentiate. Proliferation (quantified as maximum population doublings for example) can usually be improved from the levels obtained by culturing cells with basal media by providing supplements to a certain medium. These supplements may be hormones, growth factors, vitamins or amino acids with a profile that is different from the one provided in the used basal medium. These supplements may allo be "small molecules", in the pharmacological meaning of the term. Differentiation usually requires a more advanced choice of supplements to drive the cell's phenotype to a desired type. It is known that many cell types respond positively to being cultured in the presence of albumin added to a basal medium, otherwise supplemented or not. This positive response is typically seen by increased maximum population doublings or decreased time between doublings, i.e., faster and / or prolonged growth of the cells in culture. Some examples of proliferation media that contain albumin are described in W02021158103A1 or W02023133441A2. Many others exist in published papers, patents and even commercial formulations. Whey is the part of milk that remains after the formation of curds. Whey is a typical subproduct of the cheese making process. The manufacture of whey is considered to belong to the common general knowledge and is not part of the invention, except where the type of processing whey goes through in some way affects its performance. The inventor proposes that albumin, in a cell culture medium, to be applied in a culture of any type of cells that benefits from the presence of an albumin, for example of animal cells (or eukaryote), more specifically of mammalian cells, more specifically of a mesenchymal lineage, more specifically of a typical farm animal, more specifically of bovine origin, more specifically FAPs (fibro-adipogenic precursors) or SCs (satellite cells), for example by expanding (proliferating) more quickly, can be partially or completely replaced by whey. This is to say, a reduction (reduction interpreted to include elimination, so, reduction to zero) of albumin is achieved by using whey in its stead. The inventor tested several cell types from several species. Fig.1 panel A shows the results of an evaluation of the effect of thirty-four different albumins on the proliferation of a cell culture of primary bovine fibro-adipogenic progenitor (FAP) cells. The methodology included using concentration ranges of 0.01 up to 2.5 mg / ml of 9 / 22 albumin (horizontal axis of Fig. 1) as a supplement to an otherwise identical medium for each trial. The number of population doublings (PDs) in 3 days (vertical axis) was measured and used to evaluate the performance of each albumin. Data is shown as mean + standard error of the mean (SEM) based on 4 replicates. The cultures were performed in collagen-coated 96- well plates, or what is considered a 2D adherent culture, in the normai parlance of the cell cultivation technical area. Cells were seeded with a density of 5.000 cells / cm2 and cultured in DMEWF12 supplemented with 1% v / v PSA (Penicillin-Streptomycin-Amphotericin), 17.5 mM glucose, 2 mM L-alanyl-L-glutamine, 50 µg / mi L-ascorbic acid 2-phosphate, 1 µg / m1 a- linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 µg / mi ethanolamine, 10 µg / m1 insulin, 5.5 1.1g / m1 transferrin, 10 ng / ml FGF2 (fibroblast growth factor 2), 10 ng / ml PDGF-BB (platelet-derived growth factor with subunits BB), and 0.1 ng / ml TGFI31 (Transforming Growth Factor Beta 1). This corresponds to an optimal proliferation medium for this cell type, but the assay could have been run with a simpler medium, the differente being that lower numbers of total cells would have been achieved. The chart is admittedly difficult to read due to the amount of data, but, at 2.5 mg / ml of albumin concentration, three albumins displaying a superior performance are visible. These correspond to A1buMAX I (commercial name for a lipid rich bovine serum albumin manufactured by GIBCO, catalogue number 11020021 as of July 2024), A1buMAX II (commercial name for a lipid rich bovine serum albumin manufactured by GIBCO, catalogue number 11021037 as of July 2024) and CorningTM rhAlbumin Media (supplier catalogue number CorningTM 62450RF). Wishing for the replacement to albumin to display the best possible performance, one of these three, namely AlbuMAX I, was selected as the albumin against which whey is compared. So, in this specification, A1buMAX I should be read as a synonym to "best performing albumin". Figure 1 panel B depicts the averaged performance of A1buMAX I and A1buMAX II based on the data in Figure 1 panel A (shown as circles). The averaged performance of the blood-derived albumins, except the A1buMAX samples (shown as squares), and recombinant albumins (shown as triangles) indicate that, on average, the source of the albumin does not influence its performance and that the average albumin (maximum performance indicated by dotted line) is only 75% as potent as A1buMAX I or A1buMAX II (maximum performance 30 indicated by dashdotted line). The full list of albumins tested is as follows: 10 / 22 Supplier Sample name (catalogue number) Source Aspira Scientific Recombinant HSA (800001) recombinant Bovogen BovoLep (BSAL) blood-derived Biologicals Bovogen blood-derived BovoStar (BSAS) Biologicals BSA Diagnostic grade (BSA-DG) blood-derived Capricorn Capricorn BSA Protease free (BSA-PF-1U) blood-derived Capricorn BSA Standard Grade (BSA-1U, Lot CP22-5091) blood-derived Capricorn BSA Standard Grade (BSA-1U, Lot CP21-4306) blood-derived CorningTM rhAlbumin Media (62450RF) recombinant Coming - Fisher Gibco A1buMAX II (11021037) blood-derived Gibco A1buMAX I (11020021) blood-derived Optibumin® — Recombinant human albumin InVitria recombinant (777HSA047 / 555HSA0074) batch Recombinant human albumin (RHAC-NW20; Laurus Bio recombinant 21 / 023) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 024) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 025) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 027) Recombinant human albumin (RHAC-NW20; batch recombinant Laurus Bio 21 / 028) Recombinant human albumin (RHAC-NW20; batch recombinant Laurus Bio 21 / 029) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 030) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 031) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 033) Recombinant human albumin (RHAC-NW20; batch recombinant Laurus Bio 21 / 034) AlbumiNZTM Bovine Albumin Microbiological Grade MPBio blood-derived (180620) AlbumiNZTM Bovine Albumin Low Free Fatty Acid MPBio blood-derived (199899) Recombinant human serum albumin from Rice Grain Oryzogen recombinant (Oryza Sativa) (HYCOO2M03) 11 / 22 Recombinant human serum albumin from Rice Grain Oryzogen recombinant (Oryza Sativa) (HYCOO2M02) Recombinant human serum albumin from Rice Grain Oryzogen recombinant (Oryza Sativa) (HYC0O2M01; lot C002202103002) Recombinant human serum albumin from Rice Grain Oryzogen recombinant (Oryza Sativa) (HYCOO2M01; lot C002201911007) PanBiotech Panbio Bovine Serum Albumin, fraction V (P06-1391050) Proliant AlbuRich P140 (67675) blood-derived Proliant AlbuRich PRP (67685) blood-derived Sartorius Bio-Pure Human Serum Albumin (HSA) (05- Sartorius blood-derived 720-1B) Human Serum Albumin recombinant from P. pastoris, Validogen recombinant 200 mg / mL Sigma Bovine Serum Albumin (05470) blood-derived Cellastim; Recombinant human albumin expressed in rice Sigma recombinant (A9731) The type of whey used in the trials for this invention was mainly whey protein isolate but whey protein concentrate was also tested to verify any of the forms work. Both of these correspond to the concentrated and dried powder obtained by processing liquid whey recovered directly from cheese production, wherein the isolate typically contains a purer protein fraction (circa 90%) than the concentrate (circa 80%) as a result of additional processing. Whey in this powdered form is the most typically sold. This choice of dried whey, in contrast to the natural whey with high water content, was taken so the replacement of albumin could be compared by simple weight equivalence of the powder. It is shown further ahead in this specification (see Fig. 3 and its discussion on hydrolysates) that the intact proteins are required for whey to function most potently as an albumin replacement, so the (intact) protein content is taken to be the most important factor in adjusting the whey concentration in a culture medium, between different wheys of different protein concentration. The composition of the whey protein isolate typically used is: Typical whey protein isolate composition Grams / 100 grams powder Protein 86-93 Carbohydrates <0.2 - 3.9 12 / 22 Fat <0.2 - 1.1 Fibre 0- 1.6 Salt 0.1 - 0.5 The composition of the whey protein concentrate typically used is: Typical whey protein concentrate composition Grams / 100 grams powder Protein 78-80 Carbohydrates 4-5 Fat 6-8 Fibre 0 - 1 Salt 0.1 - 0.9 The first test performed to verify the performance of whey in stimulating cell proliferation and its albumin-replacing potential was a simple 1:1 weight substitution of 5 A1buMAX I for a commercial whey protein isolate, and also a commercial whey protein concentrate. The result of this test was positive as can be seen in Fig 2, panel A. Fig. 2 panel A shows the population doublings obtained in 3 days of culture of primary bovine FAPs for different concentrations of A1buMAX I and whey protein isolate or concentrate. Whey protein isolate or concentrate can be added to the medium in its final concentration or by diluting a sterile stock solution (e.g. 100-200 mg / ml) in an appropriate solvent (e.g. water or phosphate-buffered saline) that can be prepared using conventional methods generally known to the common practitioner in the technical field (this method of preparing whey for use in medium was used throughout the testing described in this specification). Data is shown as mean + SEM based on 4 replicates. The horizontal dotted line indicates the population doublings obtained when no whey or albumin sample was added. Culture conditions were identical to those described for Fig. 1. A1buMAX I, with data points represented as circles can be seen to be successfully replaced with a whey isolate with 91.3% protein (data points shown as squares), or with a whey protein concentrate with 79.0% protein (data points shown as triangles with an apex pointing up). It is clear that all wheys used do 13 / 22 replace the effect of AlbuMAX I, and by extension, albumin. It is thus part of this invention the claim of the use of whey in a culture media as a replacement for albumin. Fig. 2 panel B shows the population doublings obtained in 3 days of culture of primary bovine FAPs for different concentrations of A1buMAX I and different whey protein isolate products from different suppliers. Data is shown as mean + SEM based on 4 replicates. The horizontal dotted line indicates the population doublings obtained when no whey sample was added. Culture conditions were identical to those described for Fig. 1. It is clear that all different whey protein isolates (WPI) replace the effect of AlbuMAX I, and by extension, albumin, while showing virtually no supplier-to-supplier or product-to-product variation. This is true even for isolates with variable protein content, such as the ones tested that vary from 86.6% to 93.0%. To determine if the (intact) protein content of whey is an important factor in the results, one of the isolates tested in Fig. 2, panel B, was compared with whey hydrolysates. Hydrolysates are composed of cleaved proteins, and it was hypothesised that their performance should be worse than that of an isolate. Fig. 3 shows the population doublings obtained in 3 days of culture of bovine FAPs at different concentrations of a whey protein isolate with 91.3% protein (data points shown as squares) and a whey protein hydrolysate with 79.6% protein (data points shown as diamonds). Data is shown as mean + SEM based on 4 replicates. The horizontal dotted line indicates the population doublings obtained when no whey sample was added. Culture conditions were identical to those described for Fig. 1. It is clear that the maximum performance of the whey protein isolate obtained at 3 mg / ml is not matched by the whey protein hydrolysate, indicating that intact proteins in whey are more potent in promoting population doublings and therefore replacing the albumin effect. It is known, in the cell cultivation field, especially for animal cells, that the results of cell cultures performed in a certain device do not always translate to others. To better explain: a cell culture that is successful in adherent cultures using well plates may or may not be successful in suspension cultures using shake plates, spinner flasks and bioreactors, and a cell culture that is successful in a spinner flask may or may not be successful in a bioreactor. There is thus a hierarchy from well plates to spinner flasks to bioreactors. An unsuccessful experiment lower in this hierarchy will remain unsuccessful further up, so, if for example a whey protein isolate or concentrate cannot replace the effect of albumin in a well plate, it will not replace it 14 / 22 in a spinner or bioreactor. For this reason, whey was tested as an albumin replacement in suspension cultures using shake plates and spinner flasks to verify that the albumin- replacement effect is still present. Fig. 4 panel A shows the population doublings obtained in 4 days of culture of primary bovine FAPs grown in suspension in low-attachment 24-well plates placed on a shake plate (speed: 90 rpm) with AIbuMAX I (horizontal hatched bar), whey protein isolate (diagonal hatched bar) and without albumin or whey (white bar, labelled as 'neg ctrl'). Data is shown as mean + SEM based on 4 replicates. Cells were seeded with a density of 25.000 cells / ml and cultured in DMEM / F12 supplemented with 1% PSA, 17.5 mM glucose, 2 mM L-alanyl-L- glutamine, 50 µg / m1 L-ascorbic acid 2-phosphate, 1 µg / m1 a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 µg / m1 ethanolamine, 10 µg / m1 insulin, 5.5 µg / m1 transferrin, 10 ng / ml FGF2, 10 ng / ml PDGF-BB, and 1 ng / ml TGF[31. It is clear that whey protein isolate' s performance is equipotent to A1buMAX I. Fig. 4 panel B shows the cell density (shown as cells / ml) obtained in 6 days of culture of bovine FAPs grown in suspension in 100 ml spinner flasks (impeller rotation 75 rpm) with 3 mg / ml A1buMAX I (data points shown as circles), 3 mg / ml whey protein isolate with 91.3% protein (data points shown as squares) and a whey protein concentrate with 79.0% protein (data points shown as triangles with an apex pointing up). Data is shown as mean + standard deviation (SD) based on 2 replicates. Cells were seeded with a density of 15.000 eens / mi and cultured in the same medium described for Fig.4 panel A. Medium changes were performed every 24h from day 3 onwards; during medium changes the addition of TG931 was omitted. It is clear that whey protein isolate and whey protein concentrate can promote cell proliferation in suspension at densities higher than lx10^6 eens / mi and that both whey conditions outperform A1buMAX I. Since the replacement effect of albumin by whey protein isolate and concentrate was confirmed for spinner flasks, the potential to replace albumin was tested in a larger volume suspension culture in a bioreactor of 3L. Fig. 5 shows the cell density (shown as cells / ml) obtained in 8 days of culture of bovine FAPs grown in suspension in 3L bioreactors with 3 mg / ml A1buMAX I (data points shown as circles) or 3 mg / ml whey protein isolate with 91.3% protein (data points shown as squares). This experiment replicated culture conditions closer to real-life (commercial) biotechnological 15 / 22 applications outside the laboratory. All bioreactors were operated under the same set parameters of pH, dissolved oxygen, agitation, etc. It can clearly be seen that, as also observed in Fig.4, whey protein isolate actually outperformed A1buMAX I. After proving without doubt that, in both adherent and suspension cultures, whey can replicate the effects of AIbuMAX I and by the transitive property also the effects of other albumins, the next step was to verify if the effects of whey can be isolated from the base media or the cell type used. Fig. 6 shows the number of FAP cells after 3 days of culture in different conditions. Data is shown as mean + SEM based on 4 replicates. All conditions start with 1600 cells seeded in a 96-well plate' s well (5000 cells / cm2) (quantity indicated by the horizontal dotted line), and cultivation proceeds with different basal media and extra compounds. The first set of conditions (labelled with `nothing') corresponds to conditions in which cells were cultured in DMEM / F12 with 17.5 mM glucose and 10 ng / ml FGF2 or RPMI-1640 with 11.1 mM glucose, 2.1 mM L- glutamine and 10 ng / ml FGF2 but without AlbuMAX I or 91.3% whey protein isolate . After three days the number of cells in these conditions is lens than 200 and therefore smaller than the initial 1600 showing that basal media with a carbon source (glucose and / or L-glutamine) and a growth factor (10 ng / ml FGF2) alone, whichever the type, is not enough to keep the seeded cells alive. The second set of conditions labelled as `AlbuMAX 3 mg / ml' relates to the same two basal media complemented with the carbon source and growth factor but this time the conditions are supplemented with 3 mg / ml A1buMAX I. In this case it can be seen that for A1buMAX I addition the final number of cells is almost the same as the initial, only very slightly lower, indicating that AlbuMAX I promotes survival but no growth of cells in these media. For whey-supplemented media the cells show marked growth, growing to about 2700 cells in both basal media (conditions labelled as `whey 3 mg / m1'). The conclusion here is that A1buMAX I, in the presence of basal media and the minimum components required to keep the cells alive does not promote growth, but that whey protein isolate, together with the minimum components required to keep the cells alive actually actively promotes growth, independently of the basal medium used. This proves that the verified effect of whey as a replacement for albumin is not linked to the basal media used, so, there is no special synergy between the whey and basal media that would imply a limitation of the use of whey to certain basal media. Of note, the difference between for example Fig. 2 and Fig. 6, where in Fig. 2 there is a marked cell growth with A1buMAX I and where in Fig. 6 there is only the maintenance of the same 16 / 22 amount of cells is that the medium used in the experiment of Fig. 2, as discussed, was an enriched supplemented medium optimised for growth and in Fig. 6 only the minimum components that are required to keep the cells from dying were used. After proving independence of the whey effect from the basai media used, independence of cell type was checked. Growth for bovine FAPs had been shown already in figures 1 to 6. Fig. 7 shows the population doublings obtained in 3 days of culture of primary bovine satellite cells (SCs) for different concentrations of A1buMAX I (data represented as circles) and whey protein isolate (91.3% protein; data represented as squares). Data is shown as mean + SEM based on 4 replicates. The horizontal dotted line indicates the population doublings obtained when no albumin or whey sample was added. The cultures were performed in laminin- coated 96-well plates, or what is considered a 2D adherent culture, in the normal parlance of the cell cultivation technical area. Cells were seeded with a density of 5.000 cells / cm2 and cultured in DMEM / F12 supplemented with 1% PSA, 17.5 mM glucose, 2 mM L-alanyl-L- glutamine, 50 µg / ml L-ascorbic acid 2-phosphate, 1 lig / m1 a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 µg / m1 ethanolamine, 10 µg / m1 insulin, 5.5 µg / m1 transferrin, 10 ng / ml FGF2, 50 ng / ml HGF, and 20 ng / ml 3,3',5-Triiodo-L-thyronine. It can be seen that for the tested concentration range the whey protein isolate induces the same as or more population doublings than A1buMAX I. Both these SCs in Fig. 7 and FAPs in Figs 1-6 are of bovine origin, so, at least in the bovine species there is sufficient proof to say that whey as a replacement to albumin is cell type independent. Clearly, bovine cells that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey. After proving independence of the whey effect from the cell type used, independence of cell species was checked. Growth for bovine FAPs had been shown already in Figures 1 to 25 6. Fig. 8 shows figures for primary ovine FAPs. Fig. 8 shows the population doublings obtained in 3 days of culture of primary ovine FAPs for different concentrations of A1buMAX I and whey protein isolate. Data is shown as mean + SEM based on 4 replicates. The horizontal dotted line indicates the population doublings obtained when no albumin or whey sample was added. Culture conditions were 30 identical to those described for Fig. 1. The performance of 3 mg / m1 A1buMAX I (with data points represented as circles) can be seen to be successfully replaced with 10 mg / ml of a whey 17 / 22 isolate with 91.3% protein (data points shown as squares). It should be noted that some differences in proliferation performance seem to exist comparing bovine FAPs versus ovine FAPs. Firstly, the shapes of the A1buMAX I and whey concentration-response curves of bovine FAPs (Fig 2) are different from those of ovine FAPs (Fig 8) indicating that different species respond differently to proliferation-promoting culture medium supplements. However, the shapes of the curves for the same species are very similar irrespective whether cells are grown with A1buMAX I or whey protein isolate. Secondly, while bovine FAPs barely grow, but also do not die, in absence of A1buMAX I or whey (dotted line in Fig 2), ovine FAPs do die when cultured without A1buMAX I or whey (dotted line in Fig 8). This difference between species likely is a result of the fact that the used medium is optimised for bovine FAPs but may contain supplements that are not beneficial or hampering the growth of ovine FAPs. Lastly, while whey can replace albumin using equivalent weights for bovine FAPs, the performance of 3 mg / ml A1buMAX I on ovine FAPs is matched by adding a higher concentration of 10 mg / m1 of whey protein isolate. This proves that the effect of whey as an albumin replacement is independent of the species of the cell. It should be noted that whey contains a small amount of Bovine Serum Albumin. Typically, the proportion of BSA in whey is approximately 5% (Foegeding & Luck, MILK PROTEINS 1 Whey Protein Products, 2002 Encyclopedia of Dairy Sciences, 1957-1960). Supplementing whey to culture medium at 2.5 mg / ml would thereby introduce BSA in a range of 0.1 mg / ml (for whey protein concentrate composed of around 80% protein) to 0.1125 mg / ml (for whey protein isolates composed of around 90% protein). BSA enters cow milk via the blood, and therefore the BSA in whey can be regarded as similar to albumin derived from cow blood. The approximately 0.1 mg / ml BSA introduced by adding whey at 2.5 mg / ml to a medium cannot explain the equipotency of whey protein concentrate or whey protein isolate to A1buMAX I, as 0.1 mg / ml of the average blood-derived albumin only reaches about 40% of the potency of the AlbuMAX samples (see Fig 1, panel B). Therefore, the small amount of BSA in whey is not an explanation for the unexpected effect of positive replacement of the properties of albumin in a cell culture for whey. Besides all the technical effects and advantages disclosed so far as regards to whey in the context of this invention, it is also noted that sourcing whey is much more animal-friendly and scalable, being derived from milk and not from blood, meaning that the animal needs not be killed or periodically weakened by blood harvesting during its lifetime. There is also a limit 18 / 22 as to how much blood can be sourced from an animal without killing it, which is much lower currently than the amount of milk that can be obtained from a healthy, well-handled dairy cow. Another advantage in terms of sourcing is that whey is a natural product with a currently well developed processing pipeline and several providers for the general public, unlike albumin which remairs a quasi pharmaceutical ingredient, extremely expensive and is sold at price and availability ranges not unlike those of a scarce commodity. The inventor also tested a whey advertised as a "animal-free whey protein isolate", and the results are the same as for animal based whey protein. The fabrication method of this product (or types of products) is unclear, but, this result seems to confirm that any whey is suitable for the replacement of albumin. The media used to generate the data presented in this specification are serum-free media. Serum-free in this context means free of serums such as FBS (foetal bovine serum) or FHS (foetal horse serum) amongst others. These types of serums are non-chemically defined, usually contain albumin in varying dosages and are used mainly to improve the performance of cell cultures. There is no reason to assume the invention would not work in the presence of media with these types of serum, actually it would simply improve the performance of those media even further. In general, serum-free media are more appropriate at least to the field of cellular agriculture, where a reduction in compounds sourced from unbom butchered animals is desired. It is thus preferable, for the inventor, to use serum-free media with this albumin replacement. An extra advantage of whey when compared with albumin relates to shelf life and stability. Albumin is currently added to media close to the moment of use sine the shelf life of albumin in stock solution is very short; the product manual of A1buMAX states: `Sterile solutions of A1buMAX® Lipid-Rich BSA are stable for up to 30 days when stored at 2°C to 8°C, tightly capped and protected from light.'. The inventor has determined that sterile whey solutions stay equipotent for at least 120 days when stored at 2°C to 8°C, tightly capped and protected from light, by showing equipotent performance to freshly made solutions of A1buMAX and promoting cell proliferation to the same extent as shown in Fig. 2. It is also important to stress that, independent of, in albumin, the current lack of clarity from the scientific establishment of which function(s) is / are crucial to promote proliferation 30 for specific cell types, whey simply works to replace whatever that function(s) is( / are). 19 / 22 Another important detail to stress is that even if all the testing was done with one type of cell per culture, cultures can be co-cultures, i.e. have different cells in the same culture vessel. For example, bovine SCs and FAPs could be cultured together. As a further result, cells originating from the same experiment from which the data of Fig. 4, panel B was obtained were subjected to a differentiation medium, taken from the description in W02023003470, so, a purposefully designed FAP differentiation medium. Fig. 9 is a set of confocal microscopy images of the differentiated cultures that were differentiated upon proliferation for 4 days in presence of A1buMAX I, whey protein isolate or whey protein concentrate (proliferation data in Fig 4, panel B). The bright points correspond to fat droplets and it can be clearly seen that the samples cultured with whey differentiated well, probably better even than the one cultured with AlbuMAX I. Contrary to the inventor' s experience with rapeseed substitutes (as described above in the background section), the replacement of albumin with whey does not impede the differentiation capacity, or stemness, of the proliferated cells. The inventor performed a final check on whether the whey replacement effect of albumin holds true at A1buMAX I concentrations even higher than the ones currently used in cell cultures, and ran a final trial, using the same culture details as those used for Fig. 2 panel A, but with a A1buMAX I or whey protein isolate (91.3% protein content) concentration of 10 mg / ml. Fig. 10 shows the result of this trial. It is clearly seen that whey, in this case isolate, but as proven before concentrate will follow the same trend, replaces the effect of albumin in a cell culture even at higher concentrations than those typically used in cell cultures. There is no reason to assume that this trend of whey indistinguishably following the performance of albumin over increasing concentrations, as can be seen from the progression of the population doublings from Fig. 2 panel A into Fig. 10, will not stay true even beyond the maximum tested 25 of 10 mg / ml. It is submitted that whey will replace the effect of albumin in any given concentration of A1buMAX I, of course with the necessary adjustment for those types of cells, such as ovine FAPs as discussed before, where a larger amount of whey is required. The proof point that whey is a viable substitute for albumin in FAPs and SCs, both of a mesenchymal lineage, in different species, shows that BLG can be seen to work as an albumin 30 replacement for cells of a mesenchymal lineage in general. 20 / 22 Exemplary Embodiments of the Invention There are a myriad of possible embodiments for this invention, since it can be applied in several types of cultures under many different conditions, but in basic terms a preferred embodiment of the invention is as, a medium for, or as an animal cell culture, comprising at least a basal medium, such as Minimum Essential Medium (MEM), Eagle Medium, Dulbecco's Modified Eagle Medium (DMEM), Ham's F-12, Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (DMEM / F12), Roswell Park Memorial Institute (RPMI) 1640, etc; preferably with a carbon source, preferably with an appropriate growth factor, where the culture is of a cell type or types (the culture may be a co-culture mix of different cell types, or even from different species) that would benefit from the presence of albumin, asserted by an improvement in the culture outcome being verified experimentally when albumin is present, wherein the actual culture either has no albumin added to it or does not have enough albumin to substantially increase the performance of the culture, and wherein whey is added at a concentration that replaces the desired effect of albumin, usually at a relationship of 1:1 in mass 15 (1 g of whey for 1 g of albumin) or more (more than 1 g of whey for 1 g of albumin) or slightly less, depending on the cell type. Usually, maximum performance of a culture is desired so the relationship should be the correct one that gives the best possible effect, as determined experimentally for the cell type, species or both. The culture may be run on any of several types of common culture vessels, in adherent or suspension cultures, such as well plates, spinner flasks or bioreactors. A preferred embodiment is also, as explained throughout this specification, the straightforward claim of the use of whey in a culture medium as a replacement for albumin, specifically in cell cultures that benefit from the presence of albumin, such benefit being increased proliferation of the cells in the culture when compared to albumin' s (or equivalent 25 molecules') absence. 21 / 22
Claims
1. A method to reduce the amount of albumin in a cell culture, characterized by the following steps: a. Providing a proliferation cell culture with at least one cell type that benefits from improved proliferation in the presence of albumin; b. The addition of whey to the cell culture medium in a concentration that offers the same proliferation improvement as albumin; c. Allowing the cells to proliferate in the whey-containing cell culture.
2. A method according to claim 1 whereby the whey is a whey protein isolate or whey is protein concentrate.
3. A method according to claims 1 and / or 2 whereby the cell culture of an animal cell type is, more specifically of a mammalian cell type, more specifically of a mesenchymal lineage, more specifically a primary cell, more 15 in particular a fibroadipogenic precursor (FAP) or a satellite cell (SC), more in particular of bovine origin.
4. A method according to conclusion 3 where whey is added to the cell culture in a concentration that replaces the desired effect of albumin, usually in a ratio of 1:1 by mass (1 g whey for 1 g albumin) or more (more than 1 g whey for 1 g albumin) or slightly less, depending on the cell type.
5. A method in accordance with claim 4 whereby for bovine FArs and SCs the ratio is 1:
1.
6. A method according to one of the preceding conclusions in which no albumin is added to the cell culture medium or whereby an amount of albumin 25 which is not sufficient to the proliferation potential of the cell culture maximize, is added to the cell culture medium.
7. A method according to one of the preceding conclusions, whereby the meadow, at preference in the form of whey protein isolate or whey protein concentrate, is present in the culture medium at a concentration of approximately 0.01 mg / ml to approximately 10 30 mg / ml. 22 / 22 8. A method according to one of the preceding conclusions, whereby the medium is serum-free medium.
9. A method according to one of the preceding conclusions, whereby the culture medium furthermore comprises a basic medium, preferably with a carbon source, and with preference a growth factor.
10. A medium for a cell culture, characterized by the inclusion of a basal medium, preferably further comprising a carbon source, preferably further comprising a suitable growth factor, whereby the culture of a cell type or cell types is that would benefit from the presence of albumin, confirmed by a improvement in proliferation outcome that is experimentally verified when albumin is present, whereby the actual culture either has no albumin has added or does not have enough albumin to the proliferation performance of the to substantially increase cultivation, and where whey is added in a concentration that replaces the desired effect of albumin.
11. A medium according to conclusion 10, comprising: a. a basal medium; b. a carbon source; c. a growth factor; and d. whey protein isolate or whey protein concentrate as a replacement for albumin.
12. The cell culture medium of claims 10 and / or 11, whereby the whey or the whey- protein isolate or the whey protein concentrate is present in a concentration of approximately 0.01 mg / ml to approximately 10 mg / ml.
13. Use of whey as an albumin substitute in a cell culture. 1 / 6 Fig. 1 A B 4 o average Albumax 1 and Albumax 11 average blood-derived albumins ❑ il- average recombinant albumins 1 I i 0.01 0.1 1 mg / m1