Reduction of albumin in a cell culture medium

Abstract

The present invention relates to cell culture media, and more specifically, to a method of reducing or replacing albumin in cell culture media. The inventor has demonstrated that whey can effectively replace the role of albumin in promoting cell proliferation in various cell types and culture conditions. This replacement not only offers a cost-effective and scalable alternative to albumin but also addresses ethical concerns associated with the use of certain animal-derived products. The invention has broad applicability in the field of biotechnology, particularly in cell culture-based applications such as cellular agriculture and regenerative medicine.

Description

[0001] DESCRIPTION

[0002] TITLE: Reduction of albumin in a cell culture medium.

[0003] TECHNICAL FIELD

[0004] The invention is in the general field of biotechnology, specifically in the sub-field of media for cell cultures.

[0005] BACKGROUND OF THE INVENTION

[0006] It is known that in cell cultures of many animal, or more broadly eukaryote cell types, 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.

[0007] 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 Human albumin can also be used, which is similarly obtained from withdrawn blood.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. hormones, 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 ofBSA fraction V from the bulk of albumin using Heparin Sepharose Chromatography, 1995 Cytotechnology 19, 63-72 and Garcia-Gonzalo and Belmonte, Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-Renewal, 2008 PLOS ONE 3(1): el384), 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 properties, 2007 Br. J. Pharmacol. 151, 580-590). Thus, the proportion of albumin oxidation states and associated bioactivity can change across batches.

[0008] 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 saliva, 2014 PLOS ONE 9(10): el09893) and glycation status determines the proliferation-enhancing effect of albumin (Indurthi et al., Interaction between glycated serum albumin and AGE-receptors depends on structural changes and the glycation reagent, 2012 Arch Biochem Biophys 528(2) 185-196). Moreover, since 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.

[0009] 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 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.

[0010] 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 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 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.

[0011] Protein isolates of rapeseed (Stout et al., A Beefy-R culture medium: Replacing albumin with rapeseed protein isolates, 2023 Biomaterials, 296, 122092) and pea or chickpea (WO2021148955A1) 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.

[0012] WO2022132974A1 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 proliferation-promoting capabilities of albumin.

[0013] The publication "Bovine Whey: A Substitute forFBS in CH0-K1 Cell Cultures’’’ , by J. Capiaumont et al., published in In Vitro Cellular & Developmental Biology. Animal, Vol. 32, No. 1 , pp.8-12, from January 1996 deals with using bovine whey as a substitute for Fetal Bovine Serum (FBS) in CHO-K1 cell cultures. Albumin is never mentioned and no research into its effects or replacement is shown. In the current invention, a serum free cell culture, supplemented with albumin is the starting point for the subsequent replacement of the albumin by whey. In this paper, the starting point is a cell culture with FBS - it is not demonstrated in any way what the effect of adding just albumin is, and whether that effect could be replicated by whey. The results obtained actually contradict the current invention in that growth of the cells was only achieved when whey was supplemented with 1% FBS (see page 1, column 2, line 10). This means a (blood derived) serum free medium was not achieved in any way. It is also clearly said (page 2, column 2) that “Bovine whey and foetal calf serum (FCS) are very complex mixtures, and the significance of particular components for cell growth or product synthesis is often unknown”. Here, the authors clearly express their lack of knowledge of what individual components of FCS or FBS actually affect, signalling a teaching away from the future development of supplementing serum free cell cultures with albumin (it is not shown if isolated albumin would have had any effect in this particular type of culture), and the current invention of replacing that albumin with whey (which is not a blood derived serum product). We submit that “Bovine Whey: A Substitute for FBS in CHO-K1 Cell Cultures” clearly teaches away from using whey in serum free cultures, by showing positive results only in combination with serum (1% FBS in this case).

[0014] Another noticeable publication is “Bovine whey protein and other biological fluids as alternative to fetal bovine serum in supplementing cell culture media", by Prajakta Paradkar et al. published in the Indian Journal of Experimental Biology, Vol. 57, pgs. 123-130, in February 2019. There, experiments are described where FBS was replaced in varying quantities with whey, in a cell culture of CHO-K1 and Jurkat E6.1 cells.. The relevant results are shown in Fig. 3, with accompanying text on column 1 of page 126, first paragraph. Here, an experiment where the medium is supplemented with a total of 10% of either FBS, FBS plus whey or just whey is shown. Data points are for 10% FBS / 0% whey, 6%FBS / 4% whey, 4% FBS / 6% whey, 2%FBS / 8% whey and 0%FBS / 10% whey. Along the horizontal axis of fig.3 the decrease in culture growth can be seen as the amount of whey is increased, until reaching the absence of FBS and the worst possible results. These results teach away from having a (blood derived) serum free culture in the first place, provide no teachings on using albumin in itself as a compound in a cell culture, and as such say nothing about replacing albumin by whey. Also, the 10% whey results are described as “cell growth was poor [...] as well as not sustained [...] in the complete absence of FBS”, so whey is taught as being improper for use in a cell culture in the absence of FBS (serum free culture), teaching away from the invention.

[0015] W02024007033A1, from Good Meat Inc. published on January 4th 2024, relates to cultivated animal cells adapted for growth in low amounts, and / or the absence of direct growth factors, indirect growth factors, animal serum, and / or animal components. Albumin, either its presence in serum or its addition as an independent compound to a culture is not discussed. A mention of whey appears as a protein for a cell culture scaffold (paragraph 205), or as an added protein in a final product (paragraph 36), not as a cell culture medium component.

[0016] 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.

[0017] SUMMARY OF THE INVENTION

[0018] In a preferred embodiment the invention is a method to improve cell proliferation or improve the survival of cells in a cell culture characterised by the steps of: a. Providing a serum-free medium, comprising a basal medium; b. Providing the cell culture with at least a mammalian cell type that benefits from enhanced proliferation in the presence of albumin; c. Supplementing the serum-free medium with whey in a concentration that provides the same cell proliferation or cell survival improvement as albumin, for a specific mammalian cell type, preferably in a range of whey concentration in the cell culture media of 0.1 to 10 mg / ml. d. Allowing the cells to proliferate in the whey containing cell culture.

[0019] In a further preferred embodiment the invention is a method wherein the whey is a whey protein isolate or whey protein concentrate.

[0020] In a further preferred embodiment the invention is a method characterised by the cell types being of mammalian origin, preferably of mesodermal (exhibiting endothelial, mesenchymal, or epithelial morphology) or ectodermal origin (exhibiting epithelial morphology).

[0021] In a further preferred embodiment the invention is a method 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. In a further preferred embodiment the invention is a method wherein no supplemental albumin is added to the serum-free cell culture medium.

[0022] In a further preferred embodiment, the invention is a method wherein the whey concentration is 0.1 mg / ml to about 10 mg / ml, preferably 1 to 10 mg / ml, preferably 2.5 to 10 mg / ml, preferably 3 to 10 mg / ml, more preferably 2.5 or 3 mg / ml.

[0023] In a further preferred embodiment the invention is a method wherein the culture medium comprises besides a basal medium, preferably a carbon source, and preferably a growth factor.

[0024] In another preferred embodiment, the invention is a serum-free medium for a cell culture comprising a base medium, whey and no supplemental albumin, and wherein whey is added at a concentration that replaces the desired effect of albumin.

[0025] In a further preferred embodiment, the invention is a serum-free medium wherein the concentration of whey is 0.1 mg / mL to about 10 mg / ml, preferably 1 to 10 mg / ml of whey, preferably 2.5 to 10 mg / ml, preferably 3 to 10 mg / ml, more preferably 2.5 or 3 mg / ml.

[0026] In a further preferred embodiment, the invention is a medium further comprising: a carbon source; A growth factor; and Whey protein isolate or whey protein concentrate as a substitute for albumin.

[0027] In another preferred embodiment, the invention is the use of whey as an albumin replacement in a cell culture.

[0028] In a further preferred embodiment, the invention is the use of whey according to claim 11, in a concentration of 0.1 mg / mL to about 10 mg / ml, preferably 1 to 10 mg / ml of whey, preferably 2.5 to 10 mg / ml, preferably 3 to 10 mg / ml, more preferably 2.5 or 3 mg / ml.

[0029] 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 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 provides 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.

[0030] 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 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 whey protein isolate or whey protein concentrate is present at a concentration of about 0.01 mg / mL to about 10 mg / ml.

[0031] In another preferred embodiment the invention is in the use of whey as an albumin replacement in a cell culture.

[0032] DESCRIPTION OF THE DRAWINGS

[0033] 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 serum-free medium containing different albumins. Manufacturer-to-manufacturer and batch-to-batch performance variations are demonstrated. AlbuMAX I is shown to be one of most potent albumins for enhancing proliferation.

[0034] Fig. 2 - Panel A shows the population doublings obtained in 3 days of serum-free adherent culture of primary bovine FAPs for different concentrations of AlbuMAX 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.

[0035] Fig. 3 - A comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of bovine primary fibro-adipogenic progenitors in well plates with medium containing concentrations ranges of one of whey protein isolate or a whey protein hydrolysate.

[0036] Fig. 4 - Panel A shows the population doublings obtained in 4 days of serum-free 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 cells / ml) obtained in 6 days of serum-free culture of bovine FAPs grown in suspension in 100 ml spinner flasks with 3 mg / ml AlbuMAX I, 3 mg / ml whey protein isolate with 91.3% protein and a whey protein concentrate with 79.0% protein.

[0037] Fig. 5 - A comparison of the population doublings obtained up to 8 days of serum-free cell cultivation of bovine primary fibro-adipogenic progenitors in 3L bioreactors with medium containing one of AlbuMAX I, or a whey protein isolate at a concentration of 3 mg / ml.

[0038] Fig. 6 - Panel A shows a comparison of the number of cells obtained after 3 days of serum-free adherent cell cultivation of bovine primary fibro-adipogenic progenitors in well plates with medium with different basal media containing one of AlbuMAX 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. Panel B shows a comparison of the number of cells obtained after 3 days of serum -free adherent cell cultivation of bovine primary fibro-adipogenic progenitors in well plates with medium with different minimal compositions and additional supplementation with one of AlbuMAX I, or a whey protein isolate at a concentration of 3 mg / ml, or nothing. Fig. 7 - Panel A shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of bovine primary satellite cells in well plates with medium containing concentration ranges of AlbuMAX I or a whey protein isolate. Panel B shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of bovine primary dermal fibroblasts in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate. Panel C shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of bovine subcutaneous fat-derived stromal vascular fraction cells in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate. Panel D shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of bovine endothelial cells in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate.

[0039] Fig. 8 - Panel A shows a comparison of the population doublings obtained at 3 days of serum-fee adherent cell cultivation of ovine primary fibro-adipogenic progenitors in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate. Panel B shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of ovine primary dermal fibroblasts in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate. Panel C shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of ovine subcutaneous fat-derived stromal vascular fraction cells in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate. Panel D shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of porcine endothelial cells in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate.

[0040] Fig. 9 - Panel A shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of human bone marrow-derived mesenchymal stem cells in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate. Panel B shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of human embryonic kidney (HEK293T) cells in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate. Panel C shows a comparison of the population doublings obtained at 3 days of serum- free adherent cell cultivation of African green monkey epithelial kidney (Vero) cells in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate. Panel D shows a comparison of the population doublings obtained at 3 days of serum- free adherent cell cultivation of human retinal epithelial (ARPE-19) cells in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate.

[0041] Fig. 10 - Panel A shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of Syrian hamster kidney (BHK-21) cells in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate. Panel B shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of Chinese hamster ovary cells in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate. Panel C shows a comparison of the population doublings obtained at 3 days of serum-free adherent cell cultivation of primary murine fibroblasts in well plates with medium containing concentration ranges of one of AlbuMAX I, or a whey protein isolate.

[0042] Fig. 11 - Fat droplets as bright points in confocal microscopy images of differentiated FAP cultures, previously proliferated in spinner flask cultures in the presence of AlbuMAX I, whey protein isolate or whey protein concentrate.

[0043] Fig. 12 - Panel A shows the population doublings obtained at 3 days of serum-free adherent cell cultivation of bovine primary fibro-adipogenic progenitors (FAPs) in well plates with medium containing different concentrations of a whey protein isolate in presence of different ‘baseline’ concentrations of AlbuMAX I. Panel B shows whey protein isolate and AlbuMAX I performance compared at a high concentration, 10 mg / ml, in bovine primary fibro- adipogenic progenitors (FAPs).

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] 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.

[0046] 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 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.

[0047] A medium may not be well optimized for a specific cell type and in some cases the measured PDs are negative, which means cell death is occurring, and albumin may be observed to reduce this effect (for example shown in Figure 7, panel D). Whey is also able to replicate the function of albumin in this specific case, of preventing cell death, that is, promoting cell survival. This is to say that even if proliferation is not shown to be happening as reflected in PDs being negative, there may in fact be a positive effect of albumin in a culture, and also of the whey used instead of albumin.

[0048] 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.

[0049] 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. Whey is sometimes referred to as a serum, but it should be noted that milk serum is not blood serum, and in cell cultures these two serum types, even if they are homonyms, are not the same thing. Blood serums are the typical serums used in cell culture, for example bovine fetal serum or horse fetal serum. Albumin is a specific protein within the components of a serum, especially a blood serum where it exists in high quantities.. Albumin is not, by any of the previous definitions, a serum, so, neither a blood serum or a milk derived serum,. Blood serum in itself has different bio-activity than albumin in isolation, when used under the same conditions (same cell type, same culture conditions, same remaining medium). Albumin is indeed present in low percentage in milk serum, also known as whey, but its effect is not important, being negligible if used independently of the full whey in those quantities, as described further down.

[0050] 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 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 cultured cells and typically contains amino acids, vitamins, and minerals containing calcium, magnesium, potassium, sodium, and / or phosphate, considered essential for the survival of the cells.

[0051] Typically, basal media by itself allow the cells to proliferate (i.e. expand) or simply survive but not necessarily to differentiate. Proliferation (quantified as 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 also 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.

[0052] Most cell culture media contains Fetal Bovine Serum or equivalent serums, these serums usually containing albumin. It has long been a wish in this technical area to remove Fetal Bovine Serum or equivalent serums from media compositions due to several considerations, namely ethical, due to the origin of these types of serums, batch to batch variations, safety due to contaminants (prions, etc), and economical, due to their high cost. These types of media are called serum-free media. During the last decade, several compositions of serum-free media have been disclosed to the public, and it is noticeable that, besides other supplements designed to replace certain functions of the Fetal Bovine Serum or equivalent serums, albumin still needs to be supplemented, in addition of other components, to these media in noticeable quantities to maintain a level of performance (i.e. cell growth, or other factors) similar to that given in the presence of Fetal Bovine Serum or equivalent serums (see for example Kolkman et al., Development of a Chemically Defined Medium for in vitro Expansion of Primary Bovine Satellite Cells, Vol, 10, August 2022).

[0053] It is thus known that many cell types respond positively to being cultured in the presence of albumin added / supplemented to a basal medium, otherwise supplemented with compounds besides albumin 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, or simply improved survival of the cells. Some examples of proliferation media that contain albumin are described in W02021158103A1 or WO2023133441 A2. Many others exist in published papers, patents and even commercial formulations.

[0054] Whey is the part of milk that remains after the formation of curds, sometimes also called “milk serum” or just “serum”, not the same and not to be confused with “blood serum”, or “serum” in the context of “serum” added to cell cultures. Whey is a typical subproduct of the cheese making process. The manufacture of liquid and or powdered 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.

[0055] 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 mesodermal or ectodermal 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, or in terms of stimulating cell survival or cell proliferation, can be partially or completely replaced by whey. This is to say, a reduction (reduction interpreted to include elimination, so, reduction to zero) of supplemental albumin is achieved by using whey in its stead. The inventor tested several cell types from several species. Fig. l 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 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 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% v / v PSA (Penicillin-Streptomycin-Amphotericin), 17.5 mM glucose, 2 mM L-alanyl-L-glutamine, 50 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml 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 TGFpi (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 difference 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 AlbuMAX I (commercial name for a lipid rich bovine serum albumin manufactured by GIBCO, catalogue number 11020021 as of July 2024), AlbuMAX II (commercial name for a lipid rich bovine serum albumin manufactured by GIBCO, catalogue number 11021037 as of July 2024) and Corning™ rhAlbumin Media (supplier catalogue number Corning™ 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, AlbuMAX I should be read as a synonym to “best performing albumin”.

[0056] Figure 1 panel B depicts the averaged performance of AlbuMAX I and AlbuMAX II based on the data in Figure 1 panel A (shown as circles). The averaged performance of the blood-derived albumins, except the AlbuMAX 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 AlbuMAX I or AlbuMAX II (maximum performance indicated by dashdotted line). The full list of albumins tested is as follows:

[0057] 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 that 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 - this is not to mean that liquid whey would not serve the same function, but that the amounts used would have to be adapted based on the dry matter content of the whey solution. 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, usually specified in weight percentage or equivalent, is taken to be the most important factor in adjusting the whey concentration in a culture medium, between different wheys of different protein concentration.

[0058] The composition of the whey protein isolate typically used is:

[0059] The composition of the whey protein concentrate typically used is:

[0060] 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 AlbuMAX 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.

[0061] Fig. 2 panel A shows the population doublings obtained in 3 days of culture of primary bovine FAPs for different concentrations of AlbuMAX 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. AlbuMAX I (data points shown 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 replace the effect of AlbuMAX I, and by extension, albumin. It is thus part of this invention the use of whey in a culture media as a replacement for, or for the effect of, albumin.

[0062] Fig. 2 panel B shows the population doublings obtained in 3 days of culture of primary bovine FAPs for different concentrations of AlbuMAX 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%.

[0063] 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 a whey hydrolysate. Hydrolysates are composed of cleaved proteins, and it was hypothesised that their performance should be worse than that of an isolate.

[0064] 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.

[0065] 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 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 albuminreplacement effect is still present.

[0066] 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 AlbuMAX I (grey bar), whey protein isolate (hatched grey 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 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, 10 ng / ml FGF2, 10 ng / ml PDGF-BB, and 1 ng / ml TGFpi. It is clear that whey protein isolate’ s performance is equipotent to AlbuMAX I.

[0067] 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 AlbuMAX 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 cells / ml 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 TGFpi was omitted. It is clear that whey protein isolate and whey protein concentrate can promote cell proliferation in suspension at densities higher than lxlOA6 cells / ml and that both whey conditions outperform AlbuMAX I.

[0068] 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 AlbuMAX 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 applications outside the laboratory. All bioreactors were operated under the same set parameters of pH, dissolved oxygen, agitation, et cetera. It can clearly be seen that, as also observed in Fig.4, whey protein isolate actually outperformed AlbuMAX I.

[0069] After proving without doubt that, in both adherent and suspension cultures, whey can replicate the effects of AlbuMAX 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, additional medium supplements or the cell type used.

[0070] Fig. 6 panel A 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 less 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 AlbuMAX I. In this case it can be seen that for AlbuMAX 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 / ml’). The conclusion here is that AlbuMAX 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 AlbuMAX I and where in Fig. 6 there is only the maintenance of the same 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.

[0071] Fig. 6 panel B shows the number of primary bovine 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 medium compositions. The first set of conditions (labelled with ‘DMEM / F12’) corresponds to conditions in which cells were cultured in DMEM / F12. The second set of conditions (labelled with ‘DMEM / F12 +17.5mM glucose) corresponds to conditions in which cells were cultured in DMEM / F12 with 17.5 mM glucose. The third set of conditions (labelled with ‘DMEM / F12 +17.5mM glucose + 170 pM L-ascorbic acid) corresponds to conditions in which cells were cultured in DMEM / F12 with 17.5 mM glucose and 170 pM L-ascorbic acid 2-phosphate sesquimagnesium. In addition to the medium components described for these sets, medium was not supplemented with anything (i.e. negative control) indicated by white bars, supplemented with 3 mg / ml AlbuMAX I (grey bars) or supplemented with 3 mg / ml whey protein isolate (hatched bars). After three days the number of cells in these conditions is smaller than the initial 1600 showing that basal media with a carbon source (i.e. glucose) and an antioxidant (i.e. L-ascorbic acid 2-phosphate sesquimagnesium) alone, whichever the type, is not enough to keep all the seeded cells alive. However, it is clear that in any of the medium formulations whey protein promotes cell survival just as well as or better than albumin when comparing to the surviving cell number in the negative control condition. The absence of net proliferation is a result of using a suboptimal medium lacking crucial mitogenic components. The first set of conditions (i.e. the three left bars) clearly indicate that albumin or whey allow for improved cell survival when supplemented to a basal medium (e.g. DMEM / F12), but the absence of crucial mitogenic factors may result in slow or no proliferation - an expert in the art would be able to define these mitogenic factors if needed, depending on cell types. As is clear from the graph, adding a carbon source (i.e. glucose) and an antioxidant (i.e. L-ascorbic acid 2-phosphate sesquimagnesium) can further improve survival. After proving independence of the whey effect from the basal media and additional medium supplements used, independence of cell type was checked by testing primary bovine satellite cells (SCs), primary bovine dermal fibroblasts, primary bovine endothelial cells, and bovine adipose-derived stromal vascular fraction. Growth for bovine FAPs had been shown already in figures 1 to 6.

[0072] Fig. 7 panel A shows the population doublings obtained in 3 days of culture of primary bovine satellite cells (SCs) for different concentrations of AlbuMAX 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 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt , 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, 10 ng / ml FGF2, 50 ng / ml HGF, and 20 ng / ml 3,3',5- Triiodo-L-thyronine (T3). It can be seen that for the tested concentration range the whey protein isolate induces the same as or more population doublings than AlbuMAX I. Both these SCs in Fig. 7 panel A 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 SCs that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey.

[0073] Fig. 7 panel B shows the population doublings obtained in 3 days of culture of primary bovine dermal fibroblasts for different concentrations of AlbuMAX 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. Culture conditions were identical to those described for Fig. 1. It can be seen that for the tested concentration range the whey protein isolate can induce the same population doublings as AlbuMAX I. Both these dermal fibroblasts in Fig. 8, and FAPs and SCs in Figs 1-7 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 dermal fibroblasts that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey.

[0074] Fig. 7 panel C shows the population doublings obtained in 3 days of culture of primary bovine endothelial cells for different concentrations of AlbuMAX 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 fibronectin-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 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, 5 ng / ml FGF2, 15 ng / ml LR3-IGF1, 5 ng / ml EGF, and 5 ng / ml VEGF. It can be seen that for the tested concentration range the whey protein isolate induces the same population doublings as AlbuMAX I. It should be noted that the population doublings obtained after 3 days of culture are negative which indicates that the number of cells at the end of culture is lower than seeded at the start of culture. Compared to the negative control condition, indicated by the horizontal dotted line, both AlbuMAX I and whey protein isolate improve survival to the same extent. Clearly, bovine endothelial cells that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey. The absence of net positive population doublings is likely a result of using a suboptimal medium lacking crucial mitogenic components. As exemplified in Figure 6, a minimalistic medium composition including an albumin or a whey sample can allow for cell survival, but the absence of crucial mitogenic factors may result in slow or no proliferation - an expert in the art would be able to define these mitogenic factors if needed. The fact that whey acts as a replacement to albumin for the bovine mesenchymal cells in Fig. 2 (FAPs), Fig. 7 panel A (SCs), Fig. 7 panel B (dermal fibroblasts) in addition to the bovine endothelial cells shows that, at least in the bovine species and for cells of mesodermal origin, there is sufficient proof the replacement effect of whey is cell type and cell morphology-type independent.

[0075] Considering the albumin-replacing effect of whey for bovine mesenchymal and endothelial cells, it was tested if albumin can be replaced by whey in co-cultures containing mixtures of aforementioned cells. Therefore, stromal vascular fractions were obtained from bovine adipose tissue and cultured.

[0076] Fig. 7 panel D shows the population doublings obtained in 3 days of culture of adipose- derived bovine stromal vascular fractions for different concentrations of AlbuMAX 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 collagen-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 10.000 cells / cm2 and cultured in DMEM / F12 supplemented with 1% PSA, 17.5 mM glucose, 2 mM L-alanyl-L-glutamine, 50 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, 10 ng / ml FGF2, 10 ng / ml PDGF-BB, 20 ng / ml IL-6, and 10 ng / ml LR3-IGF1. It can be seen that for the tested concentration range the whey protein isolate induces the same as or more population doublings than AlbuMAX I. This shows that, in addition to cell cultures using specific cell types, also in cell cultures comprising mixtures of cell types, whey acts as an albumin replacement in serum-free media.

[0077] After proving independence of the whey effect from the cell type used, independence of cell species was checked for additional species from the same taxonomic order (i.e. Artiodactyla, even-toed hoofed mammals). Growth for bovine mesenchymal and endothelial cells from mesodermal origin has been shown already in Figures 1 to 7. Figure 8 shows results for ovine and porcine cells; specifically primary ovine FAPs, primary ovine dermal fibroblasts, ovine adipose-derived stromal vascular fraction and primary porcine endothelial cells.

[0078] Fig. 8 panel A shows the population doublings obtained in 3 days of culture of primary ovine FAPs for different concentrations of AlbuMAX I (data points represented as circles) and whey protein isolate (data points 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. Culture conditions were identical to those described for Fig. 1. It can be seen that for the tested concentration range the whey protein isolate induces the same population doublings as AlbuMAX I. It should be noted that some differences in proliferation performance seem to exist comparing bovine FAPs versus ovine FAPs. Firstly, the shapes of the AlbuMAX 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 AlbuMAX I or whey protein isolate. Secondly, while bovine FAPs barely grow, but also do not die, in absence of AlbuMAX I or whey (dotted line in Fig 2), ovine FAPs do die when cultured without AlbuMAX 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. All this proves that the effect of whey as an albumin replacement is independent of the species of the cell within the mammalian order of Artiodactyla.

[0079] Fig. 8 panel B shows the population doublings obtained in 3 days of culture of primary ovine dermal fibroblasts for different concentrations of AlbuMAX I (data shown as circles) and a whey protein isolate with 91.3% protein (data shown 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. Culture conditions were identical to those described for Fig. 1, except for the seeding density, which was 3.200 cells / cm2 for ovine dermal fibroblasts. It can be seen that for the tested concentration range the whey protein isolate induces the same population doublings as AlbuMAX I.

[0080] Fig. 8 panel C shows the population doublings obtained in 3 days of culture of adipose- derived ovine stromal vascular fractions for different concentrations of AlbuMAX 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 collagen-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 10.000 cells / cm2 and cultured in DMEM / F12 supplemented with 1% PSA, 17.5 mM glucose, 2 mM L-alanyl-L-glutamine, 50 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, 10 ng / ml FGF2, 10 ng / ml PDGF-BB, 20 ng / ml IL-6, and 10 ng / ml LR3-IGF1. It can be seen that for the tested concentration range the whey protein isolate induces the same population doublings as AlbuMAX I. Fig. 8 panel D shows the population doublings obtained in 3 days of culture of primary porcine endothelial cells for different concentrations of AlbuMAX 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 fibronectin-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 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, 5 ng / ml FGF2, 15 ng / ml LR3-IGF1, 5 ng / ml EGF, and 5 ng / ml VEGF. It can be seen that for the tested concentration range the whey protein isolate induces the same population doublings as AlbuMAX I. It should be noted that the population doublings obtained after 3 days of culture are negative which indicates that the number of cells at the end of culture is lower than seeded at the start of culture. Compared to the negative control condition, indicated by the horizontal dotted line, both AlbuMAX I and whey protein isolate improve survival to the same extent. Clearly, porcine endothelial cells that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey. The absence of net positive population doublings is likely a result of using a suboptimal medium lacking crucial mitogenic components as also described for Figure 7 panel C.

[0081] Figure 7, panels A to D and figure 8, panels A to D, prove the effect of whey to be independent from the cell type and species of taxonomic families (i.e. Suidae and Bovidae) within the order (i.e. Artiodactyla, even-toed hoofed mammals). Additional proof for the cell type and cell species independency of whey as an albumin replacement was checked using cells from other taxonomic orders (i.e. Primates) from the same taxonomic class (i.e. Mammalia) as the Artiodactyla cells shown in Figures 1 to 8. More specifically, human bone marrow-derived mesenchymal stem cells, human kidney epithelial cells, African green monkey kidney epithelial cells, and human retinal epithelial cells were tested.

[0082] Fig. 9 panel A shows the population doublings obtained in 3 days of culture of bone marrow-derived human mesenchymal stem cells (BM-MSCs) for different concentrations of AlbuMAX 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 gelatine-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 10.000 cells / cm2 and cultured in DMEM / F12 supplemented with 1% PSA, 17.5 mM glucose, 2 mM L-alanyl-L-glutamine, 50 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, 10 ng / ml PDGF-BB and 10 ng / ml FGF2. It can be seen that for the tested concentration range the whey protein isolate induces the same as or more population doublings than AlbuMAX I. Clearly, human cells and cells exhibiting a mesenchymal phenotype from mesodermal lineage that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey.

[0083] Fig. 9 panel B shows the population doublings obtained in 3 days of culture of human embryonic kidney cells (HEK293T) for different concentrations of AlbuMAX 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 collagen-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 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, and 100 ng / ml LR3-IGF1. It can be seen that for the tested concentration range the whey protein isolate induces the same population doublings as AlbuMAX I. Clearly, human cells and cells from mesodermal lineage exhibiting an epithelial phenotype that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey.

[0084] Fig. 9 panel C shows the population doublings obtained in 3 days of culture of African green monkey epithelial kidney cells (Vero) for different concentrations of AlbuMAX 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 uncoated 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 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, and 5 ng / ml EGF. It can be seen that for the tested concentration range the whey protein isolate induces the same as or more population doublings than AlbuMAX I. Clearly, primate cells and cells from mesodermal lineage exhibiting an epithelial phenotype that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey.

[0085] Fig. 9 panel D shows the population doublings obtained in 3 days of culture of human retinal pigment epithelial cells (ARPE-19) for different concentrations of AlbuMAX 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 uncoated 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 10.000 cells / cm2 and cultured in DMEM / F12 supplemented with 1% PSA, 17.5 mM glucose, 2 mM L-alanyl-L-glutamine, 81 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, 8 ng / ml EGF, 10 ng / ml FGF2, 50 ng / ml all-trans retinoic acid, 7 ng / ml 3,3',5-Triiodo-L-thyronine (T3). It can be seen that for the tested concentration range the whey protein isolate induces the same as or more population doublings than AlbuMAX I. Clearly, human cells from ectodermal lineage that exhibit an epithelial phenotype benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey.

[0086] After establishing whey as an albumin replacement in mesenchymal and epithelial cells from mesodermal and ectodermal (human retinal epithelial cells) origin of different species from taxonomical orders (Primates and Artiodactyla), the applicability was tested further in cells from additional mammalian species that are evolutionary further apart (i.e. Rodentia). Figure 10, panels A, B, and C display results for Syrian hamster (Mesocricetus auratus) kidney cells (BHK-21), Chinese hamster (Cricetulus griseus) ovary cells (CHO-K1) and primary murine fibroblasts. Fig. 10 panel A, shows the population doublings obtained in 3 days of culture of Syrian hamster (Mesocricetus auratus) kidney cells (BHK-21) for different concentrations of AlbuMAX 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 fibronectin-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 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, and 100 ng / ml FGF2. It can be seen that for the tested concentration range the whey protein isolate induces the same as or more population doublings than AlbuMAX I. Clearly, in addition to cells from ungulates and primates, also cells derived from rodents, e.g. Syrian hamster, benefit from the presence of albumin in the same way, or very similarly, to the presence of whey.

[0087] Fig. 10 panel B shows the population doublings obtained in 3 days of culture of Chinese hamster ovary cells (CHO-K1) for different concentrations of AlbuMAX 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 fibronectin-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 8.125 cells / cm2 and cultured in DMEM / F12 supplemented with 1% PSA, 17.5 mM glucose, 2 mM L-alanyl-L-glutamine, 50 pg / ml L-ascorbic acid 2-phosphate sesquimagnesium salt, 1 pg / ml a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 pg / ml ethanolamine, 10 pg / ml insulin, 5.5 pg / ml transferrin, and 50 ng / ml LR3-IGF1. It can be seen that for the tested concentration range the whey protein isolate induces the same as or more population doublings than AlbuMAX I. Clearly, rodent cells that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey.

[0088] Fig. 10 panel C shows the population doublings obtained in 3 days of culture of primary murine fibroblasts for different concentrations of AlbuMAX 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. Culture conditions were identical to those described for Fig. 1. It can be seen that for the tested concentration range the whey protein isolate induces the same population doublings as AlbuMAX I. Clearly, rodent cells that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey. It should be noted that the population doublings obtained for most conditions after 3 days of culture are negative which indicates that the number of cells at the end of culture is lower than seeded at the start of culture. Compared to the negative control condition, indicated by the horizontal dotted line, both AlbuMAX I and whey protein isolate improve survival to the same extent. Clearly, primary murine fibroblasts that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of whey. The absence of net positive population doublings is likely a result of using a suboptimal medium lacking crucial mitogenic components as also described for Figure 7 panel C.

[0089] Figures 1 to 10 clearly indicate that whey can act as a replacement for albumin in serum- free media to culture mammalian cells from mesodermal (exhibiting endothelial, mesenchymal, or epithelial morphology) or ectodermal origin (exhibiting epithelial morphology)

[0090] The mammalian cells used in the assays described within this specification can be obtained by third parties and are not in any way unavailable to the public. Either commercially, or with very low effort, not requiring inventive skills of any kind, equivalent cells can be obtained. The described cells were obtained as follows:

[0091] • Bovine / ovine fibro-adipogenic progenitor (FAP) cells o Obtained by isolation from muscle tissue as described by Messmer et al., Singlecell analysis of bovine muscle-derived cell types for cultured meat production, Front. Nutr., 13 September 2023.

[0092] • Bovine / ovine / murine dermal fibroblasts o Obtained by isolation from skin tissue as described by Nejaddehbashi et al. Isolating human dermal fibroblasts using serial explant culture, Stem Cell Investigation, Vol 6, August 2019. Bovine satellite cells (SC) o Obtained by isolation from muscle tissue as described by Messmer et al., Singlecell analysis of bovine muscle-derived cell types for cultured meat production, Front. Nutr., 13 September 2023.

[0093] • Bovine / ovine stromal vascular fraction (SVF) o Obtained by isolation from subcutaneous fat as described by Mitic et al., A simplified and defined serum-free medium for cultivating fat across species, i Science, Volume 26, Issue 1, 20 January 2023.

[0094] • Bovine / porcine endothelial cells o Obtained by isolation from muscle tissue as described by Messmer et al., Singlecell analysis of bovine muscle-derived cell types for cultured meat production, Front. Nutr., 13 September 2023.

[0095] • Obtained commercially via American Type Culture Collection o HEK293T (CRL-3216), BHK-21 (CCL-10), CHO-K1 (CCL-61), ARPE-19 (CRL-2302), Vero (CCL-81), bone marrow-derived human mesenchymal stem cells (PCS-500-012)

[0096] The protocol described in Messmer et al. never includes (blood) serum. This means the cells used in this invention obtained according to these protocols, so, all cells except the ones obtained commercially from American Type Culture Collection, dermal fibroblasts and vascular stromal fractions were never exposed to (blood) serum at any point in their culture.

[0097] It should be noted that whey contains a small amount of Bovine Serum Albumin (BSA). Typically, the proportion of BSA in whey is approximately 5% (Foegeding & Luck, MILK PROTEINS | 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 AlbuMAX 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.

[0098] 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 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 remains 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.

[0099] 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 horse serum amongst others. These types of serums 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 unborn butchered animals is desired. It is thus preferable, for the inventor, to use serum-free media with this albumin replacement. Most serum-free media compositions disclosed to the public include supplemental albumin. Supplemental albumin is considered the quantity of albumin required to exert a noticeable effect in whatever metric is being used in an evaluation, such as cell survival or cell proliferation rate in the cell culture. The typical quantity of supplemented albumin is 0.1-10 mg / ml with optimal concentrations being dependent on the albumin product which corresponds to the variability seen in Figure 1, panel A. For AlbuMAX I, shown to induce a maximum number of population doublings in Figure 1, panel A, the manufacturer has stated that supplementation “at 0.5-2.5 mg / mL has been proven effective in growth promotion of anchorage dependent and independent cell lines” (https: / / documents.thermofisher.eom / TFS-Assets / LSG / manuals / 3117.pdf) which corresponds to effective concentrations found in Figures 1 to 10 for a variety of cell types. As explained before, whey consists of around 5% bovine serum albumin in the protein fraction. For example, medium supplementation of 2.5 mg / ml whey protein isolate (91.3% protein) would introduce 2.5*0.913*0.05=0.11 mg / ml bovine serum albumin. Since albumin shows a concentrationdependent effect (e.g. Figure 1 panel A), the introduction of small amounts of bovine serum albumin by whey supplementation does not explain the albumin-replacing effect of whey protein. From Figure 1 panel A it can be seen that for any albumin the performance of 0.11 mg / ml is lower than 2.5 mg / ml indicating that the albumin-replacing effect of whey is not a result of introducing equal albumin concentrations into the medium.

[0100] The media used to generate the data presented in this specification are in general also “albumin free”, except when partial replacement is discussed. Albumin free media means media where albumin is either completely absent or has not been supplemented or added as an ingredient in a quantity in which it is normally used in cell culture, such as for cell survival or proliferation purposes. For example, as explained above a medium with some quantity of whey added to it does introduce albumin but not in a concentration that explains the albuminreplacing effect of whey, and as such the media is considered albumin-free by the present definition. In another point of view, the media of the present inventions can be said not to be supplemented with albumin when the amount of albumin incidentally present in the supplemented whey does not reach the usual amount supplemented to a serum free media of the prior art for a desired effect. In other words, the medium does not contain supplemented albumin in an amount effective to provide the functions and / or results desired.

[0101] To recap, the media used in this invention is serum-free and free of supplemented albumin.

[0102] 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 since the shelf life of albumin in stock solution is very short; the product manual of AlbuMAX states: ‘Sterile solutions of AlbuMAX® 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 360 days when stored at 2°C to 8°C, tightly capped and protected from light, by showing equipotent performance to freshly made solutions of AlbuMAX and promoting cell proliferation to the same extent as shown in Fig. 2.

[0103] It is also important to stress that the current lack of clarity from the scientific establishment of which function(s) of albumin is / are crucial to promote proliferation or survival for specific cell types, whey simply works to replace whatever that(those) function(s) is( / are).

[0104] Another important detail to stress is that even if most of 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 as shown for Figure 7 panel D and Figure 8 panel C which feature cultures of stromal vascular fractions of bovine and ovine origin, respectively, and which comprises different cell types (e.g. mesenchymal stem cells, fibroblasts and endothelial cells).

[0105] 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. 11 is a set of confocal microscopy images of the differentiated cultures that were differentiated upon proliferation for 4 days in presence of AlbuMAX 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 sternness, of the proliferated cells.

[0106] Based on the similarity in curve shapes for albumin and whey in the aforementioned figures, the survival and / or proliferative response to either albumin or whey may be based on a similar mechanism. As the observations in Figures 1 to 10 indicated that the full effect of albumin can be replaced by whey, it was investigated whether partial substitution of albumin by whey would add up to the effect that would be elicited by just albumin.

[0107] Figure 12 panel A shows the population doublings obtained in 3 days of culture of primary bovine FAPs for different concentrations of whey protein isolate (91.3% protein) on top of different concentrations of AlbuMAX I. Presence of AlbuMAX I in the concentration of 3 mg / ml is represented as circles, 0.75 mg / ml is represented as squares, 0.1875 mg / ml is represented as triangles with apex pointing up, 0.0469 mg / ml is represented as triangles with apex pointing down, 0.0117 mg / ml is represented as hexagons, and 0 mg / ml is represented as diamonds. Data is shown as mean + SEM based on 4 replicates. The horizontal dotted lines indicate the population doublings obtained when no whey sample was added on top of the AlbuMAX I concentration (0-0.75 mg / ml). The horizontal dashed line indicates the population doublings obtained when no whey sample was added on top of 3 mg / ml AlbuMAX I. Culture conditions were identical to those described for Fig. 1. It can be clearly seen that adding whey protein in concentrations up to 3 mg / ml on top of 0 to 0.75 mg / ml AlbuMAX I can promote population doublings up to the level of 3 mg / ml AlbuMAX I (dashed horizontal line). This shows that the effect elicited by a concentration of albumin can be matched by fully or partially replacing albumin with whey.

[0108] Albumin is typically supplemented at concentrations ranging from 0.1 to 10 mg / ml and AlbuMAX I is suggested to be used at 0.5-2.5 mg / ml according to the manufacturer. This corresponds to the plateauing effect on population doublings obtained in Figure 1 panel A and Figure 12 panel A for the highest tested concentrations of albumin and / or whey,

[0109] The inventor performed a final check on whether the whey replacement effect of albumin holds true at AlbuMAX I concentrations at the highest end of the albumin concentration range typically 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 AlbuMAX I or whey protein isolate (91.3% protein content) concentration of 10 mg / ml. Figure 12 panel B shows the result of this trial. It is clearly seen that whey, in this case whey protein isolate, but as proven before concentrate will follow the same trend (Figure 2, panel A), replaces the effect of albumin in a cell culture even at higher concentrations. 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. 12 panel B, will not stay true even beyond the maximum tested of 10 mg / ml. It is submitted that whey will replace the effect of albumin in any given concentration of AlbuMAX 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 mammalian cells from mesodermal (exhibiting endothelial, mesenchymal, or epithelial morphology) or ectodermal origin (exhibiting epithelial morphology), in different species, shows that whey can be seen to work as an albumin replacement for mammalian cells at least of the origins and exhibiting the morphology (or phenotypes) disclosed in this specification. We submit, considering the preponderance of evidence towards and none against, that there is no reason to believe that whey does not work as an albumin replacement for cultures of any mammalian originated cell types.

[0110] It is also important to stress once more that all the experimental data was obtained with the best performing albumin in the market. It is clear that, when replacing inferior albumins, whey can be used in smaller concentrations for the same effect (proliferation, survival), also an even better effect can be achieved when using whey, since it performs at par with the best albumin. This also means that, at any point when replacing albumin with whey, the same performance would be achieved with less whey for a lesser performing albumin, meaning that the ideal replacement point is always a range, with an upper limit at the upper limit of performance of the best albumin, and increasingly lower as the quality of the used albumin decreases.

[0111] Exemplary Embodiments of the Invention

[0112] 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 (e.g. cell survival and / or cell proliferation) being verified experimentally when albumin is present, wherein the actual culture either has no supplemental 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 (1 g of whey for 1 g of albumin) or more (more than 1 g of whey for 1 g of albumin) 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.

[0113] The preferred dosage of whey in the cell culture media based on the results obtained in all the trials is between 0.1 and 10 mg / ml, simply because above 10 mg / ml no better effect should be detectable, which is not to say more whey couldn’t be added; preferably 1 to 10 mg / ml of whey, preferably 2.5 to 10 mg / ml, more preferably 3 to 10 mg / ml, more preferably 2.5 or 3 mg / ml.

[0114] 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 or survival of the cells in the culture when compared to albumin’s (or equivalent molecules’) absence.

[0115] Further preferred embodiments may be taken from the specification as a whole, and this section should not be considered as limiting - there is a preponderance of data leading to the conclusion that whey is beneficial for cell survival and / or proliferation, in general, so in many dosages and combined with many other media components.

Claims

38 / 39Claims2. A method to improve cell proliferation or improve the survival of cells in a cell culture characterised by the steps of: a. Providing a serum-free medium, comprising a basal medium; b. Providing the cell culture with at least a mammalian cell type that benefits from enhanced proliferation in the presence of albumin; c. Supplementing the serum-free medium with whey in a concentration that provides the same cell proliferation or cell survival improvement as albumin, for a specific mammalian cell type, preferably in a range of whey concentration in the cell culture media of 0.1 to 10 mg / ml. d. Allowing the cells to proliferate in the whey containing cell culture.

3. A method according to claim 1 wherein the whey is a whey protein isolate or whey protein concentrate.

4. A method according to any of the preceding claims characterised by the cell types being of mammalian origin, preferably of mesodermal (exhibiting endothelial, mesenchymal, or epithelial morphology) or ectodermal origin (exhibiting epithelial morphology).

5. A method according to any of the preceding claims 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.

6. A method according to any of the preceding claims wherein no supplemental albumin is added to the serum-free cell culture medium.

7. A method according to any of the preceding claims, wherein the whey concentration is 0.1 mg / ml to about 10 mg / ml, preferably 1 to 10 mg / ml,39 / 39 preferably 2.5 to 10 mg / ml, preferably 3 to 10 mg / ml, more preferably 2.5 or 3 mg / ml.

8. A method according to any of the preceding claims, wherein the culture medium comprises besides a basal medium, preferably a carbon source, and preferably a growth factor.

9. A serum-free medium for a cell culture characterised by comprising a base medium, whey and no supplemental albumin, and wherein whey is added at a concentration that replaces the desired effect of albumin.

10. A serum-free medium according to claim 8, wherein the concentration of whey is 0.1 mg / mL to about 10 mg / ml, preferably 1 to 10 mg / ml of whey, preferably 2.5 to 10 mg / ml, preferably 3 to 10 mg / ml, more preferably 2.5 or 3 mg / ml.

11. A medium according to claims 8 or 9, further comprising: a. A carbon source; b. A growth factor; and c. Whey protein isolate or whey protein concentrate as a substitute for albumin.

12. Use of whey as an albumin replacement in a cell culture.

13. Use of whey according to claim 11, in a concentration of 0.1 mg / mL to about 10 mg / ml, preferably 1 to 10 mg / ml of whey, preferably 2.5 to 10 mg / ml, preferably 3 to 10 mg / ml, more preferably 2.5 or 3 mg / ml.

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