Reduction of albumin in a cell culture medium.

Abstract

1 / 1 The present invention discloses a method for reducing or eliminating the use of albumin in cell culture media by replacing it with P-lactoglobulin ((3LG), optionally supplemented with sunfiower lecithin. The invention demonstrates that PLG can effectively replace albumin's proliferation-enhancing effects in various cell types, including fibro-adipogenic progenitors and satellite cells, cultured in both adherent and suspension conditions. This replacement offers a cost-effective, ethically sound, and readily available alternative to traditional albumin supplementation, with potential applications in large-scale biotechnological applications.

Description

1 / 25 DESCRIPTION TITLE: Reduction of albumin in a cel) culture medium. TECHNICAL FIELD The invention is in the general field of biotechnology, specifically in the sub-field of media for cel) cultures. BACKGROUND OF THE INVENTION It is known that in cel) cultures of many animal, or more broadly eukaryote cel) types that albumin (blood-derived or from recombinant origin) may be used as a component of the culture media to promote, or enhance, the proliferation of the cells. The use of blood-derived and / or recombinant albumin as a cel) 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 Human albumin can also be used, which is similarly obtained from withdrawn blood.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, 15 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 20 different depending on factors like, among others, the specific animal, time of year and time of day. The 'cargo' can add to albumin's biological effects in cell culture, as exemplified for albumin-associated lipids (e.g. Keenan et al., Separation of growth-stimulating activity of BSA fraction V from the bulk of albumin using Heparin Sepharose Chromatography, 1995 Cytotechnology 19, 63-72 and Garcia-Gonzalo and Belmonte, Albumin-Associated Lipids 25 Regulate Human Embryonic Stem Cell Self-Renewal, 2008 PLOS ONE 3(1): e1384), but will vary across batches depending on the aforementioned variables. Likewise, the albumin molecule can dynamically exist in several oxidation states which influence its biological activity by, for example, influencing ligand binding (Oettl and Stauber, Physiological and pathological changes in the redox state of human serum albumin critically influence its binding 2 / 25 properties, 2007 Br. J. Pharmacol. 151, 580-590). Thus, the proportion of albumin oxidation states and associated bioactivity can change across batches. Next to blood-derived albumins, recombinant albumin produced by a heterologous expression system (e.g. plants, bacteria, animal cells or live animals) can be used as a cell culture supplement, which would circumvent the ethical issues associated with withdrawing blood from or slaughtering animals. However, in addition to the supply being limited by current manufacturing capability, the advanced technology required to produce recombinant albumin translates into a high selling price, making it less attractive for activities requiring large scale usage of albumin-supplemented culture media. Despite the fact that recombinant albumin may be produced in an environment that is biologically more controlled compared to being sourced from live animals, recombinant albumin will still exhibit biological variation. For example, the glycation status of recombinant albumin has been described to differ across supplier and batches (Frahm et al., Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa, 2014 PLOS ONE 9(10): e109893) and glycation status determines the proliferation-enhancing effect of albumin (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. 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. Many functions have been ascribed to albumin. For example, albumin can act as an antioxidant, in part owing to (the oxidation status of) its free cysteine residue, and can bind 30 several metal ions, hormones, lipids, nitric oxide, and vitamins. Moreover, albumin is a ligand to several receptors, can act as a chaperone (Pomier et al., Interactions of intrinsically disordered proteins with the unconventional chaperone human serum albumin: From 3 / 25 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. A Beeft-R culture medium: Replacing albumin Protein isolates of rapeseed (Stout et al., with rapeseed protein isolates, 2023 Biomaterials, 296, 122092) and pea or chickpea (W02021148955A1) have been suggested as an albumin replacement. However, these solutions were only described when applied to relatively short culture durations, specific culture conditions (e.g. adherent or suspension cultures) and specific cell type(s). The inventor has tested rapeseed protein isolate and identified the albumin-replacing potential of rapeseed protein to be lacking in suspension cultures and the albumin-replacing potential of pea and chickpea protein to be insufficient. Additionally, phenotypic changes to cells can be induced by supplementing rapeseed protein to the culture medium (e.g. differentiation potential: Alashi et al., Effects of canola proteins and hydrolysates on adipogenic differentiation of C3H10T / 2 mesenchymal stem cells, 2015 Food Chemistry, 185, 226-232) which was also observed in experiments using rapeseed protein isolate conducted by the inventor. More specifically, the adipogenic potential of cells was found to be severely hampered upon exposure to rapeseed 20 protein. W02022132974A1 describes peptides comprising superoxide dismutase activity and Cu+, Zn+ chelating activity, superoxide scavengers, vitamin E analog(s), and hydrogen peroxide reducing reagents, that are claimed to be able to replace albumin for specific functions. The inventor tested these and was not able to use them in order to replace the 25 proliferation-promoting capabilities of albumin. It is clear that none of the existing solutions to this problem of replacing albumin are fully reproducible and / or applicable to a variety of cell types and / or culture conditions and there is thus a need to ameliorate these problems, as disclosed with the present invention. SUMMARY OF THE INVENTION 30 4 / 25 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 enhanced proliferation in the presence of albumin; b. Adding P-lactoglobulin to the cell culture medium in a concentration that provides the same or approximately the same proliferation improvement as albumin; c. Allowing the cells to proliferate in the 3-lactoglobulin containing culture medium; more preferably 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; more preferably a method wherein p- lactoglobulin is added to the cell culture at a concentration that replaces the desired effect of albumin, usually at a preferable relationship of 1:1 in mass (1 g of (3-lactoglobulin for 1 g of albumin) or optionally more (more than 1 g of (3-lactoglobulin for 1 g of albumin) or optionally less, depending on the cell type; more preferably a method wherein P-lactoglobulin is supplemented with sunflower lecithin, to improve the effectiveness of replacing albumin for p- lactoglobulin or surpass the effect of albumin; more preferably a method wherein the sunflower lecithin is added at a concentration of ideally 1.8% (w / w); more preferably a method wherein 20 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; more preferably a method wherein the f3-lactoglobulin is present in the culture medium at a concentration of about 0.3 µM to about 150 µM, more preferably a method wherein the medium is a serum free medium; more preferably a method wherein the P-lactoglobulin is of bovine 25 origin, preferably the genetic variant A; more preferably a method wherein the 13-lactoglobulin is supplemented with (lyso)phospholipids, preferably those containing a linoleic acid, preferably with a choline as phospholipid polar head, and preferably with a palmitic or stearic acid in addition to the linoleic acid in case of a phospholipid. In another preferred embodiment the invention is a medium for a cell culture 30 characterised by comprising a base medium, where the culture is of a cell type or types that 5 / 25 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 P-lactoglobulin is added at a concentration that replaces the desired effect of albumin, or wherein p-lactoglobulin supplemented with sunflower lecithin are added at a concentration that replaces the desired effect of albumin; more preferably a cell culture medium wherein the P-lactoglobulin is present at a concentration of about 0.3 µM to about 150 µM; more preferably a cell culture medium, wherein the f3-lactoglobulin is of bovine origin, preferably the genetic variant A; more preferably a cell culture medium wherein the 13-lactoglobulin is supplemented with (lyso)phospholipids, preferably those containing a linoleic acid, preferably with a choline as phospholipid polar head, and preferably with a palmitic or stearic acid in addition to the linoleic acid in case of a phospholipid. In another preferred embodiment the invention is the use of P-lactoglobulin as an albumin replacement in a cell culture. In another preferred embodiment the invention is a medium for a cell culture containing an albumin or a replacement for albumin, supplemented with (lyso)phospholipids, preferably those containing a linoleic acid, preferably with a choline as phospholipid polar head, and preferably with a palmitic or stearic acid in addition to the linoleic acid in case of a 20 phospholipid; more preferably a medium wherein the source of the (lyso)phospholipids is sunflower lecithin. DESCRIPTION OF THE DRAWINGS Fig. 1 — A comparison of the population doublings obtained at 3 days of cell cultivation of bovine primary fibro adipogenic progenitors (FAPs) in well plates with medium containing 25 different albumins. Manufacturer-to-manufacturer and batch-to-batch performance variations are demonstrated. Fig. 2 - Panel A shows the population doublings obtained in 3 days of culture of primary bovine FAPs for different concentrations of A1buMAX I and P-Lactoglobulin ((3LG). Panel B shows the normalized population doublings obtained in 3 days of culture of primary bovine FAPs for different concentrations of PLG and different lecithins. The successful replacement 30 of albumin with I3LG or PLG supplemented with lecithin is proven. Panel C shows the 6 / 25 normalized population doublings obtained in 3 days of culture of primary bovine FAPs for different concentrations of sunflower lecithin added to 1.67 mg / ml PLG. Fig. 3 - Population doublings obtained in 3 days of adherent culture of primary bovine FAPs for different concentrations of A1buMAX I (data points indicated as circles), pLG (data points indicated as squares) and PLG + 1.8% w / w sunflower lecithin (data points indicated as triangles). Fig. 4 - Panel A shows the population doublings obtained in 4 days of culture of primary bovine FAPs grown in suspension in well plates with A1buMAX I (grey bar), without albumin or PLG (white bar, labelled as 'neg ctrf ), 150 µM PLG (single hatched bar) or 150 µM PLG + 1.8% w / w sunflower lecithin (double hatched bar). Panel B shows 20x magnification microscopic images of the suspension cultures of the different conditions after 4 days of culture. Fig. 5 - Population doublings obtained in 3 days of adherent culture of primary bovine FAPs grown with 37.5 µM A1buMAX I (grey bars), no albumin or PLG (white bars) or 150 15 µM PLG with 1.8% w / w sunflower lecithin (hatched bars) in different base media. Fig. 6 - Population doublings obtained in 3 days of adherent culture of primary bovine satellite cells (SCs) for different concentrations of A1buMAX I (data represented as circles), PLG (data represented as squares) and PLG with 1.8% w / w sunflower lecithin (data represented as triangles). 20 Fig. 7 - Population doublings obtained in 3 days of culture of primary murine FAPs for different concentrations of A1buMAX I (data represented as circles), I3LG (data represented as squares) and 150 µM PLG with the addition of a concentration range of sunflower lecithin (data represented as triangles). Fig. 8 - Population doublings obtained in 3 days of adherent culture of primary bovine 25 FAPs for 150 [..iM PLG comprising an undefmed mix of different variants (data point indicated as square) and different concentrations of PLG variant A (data points indicated as triangles) and PLG variant B (data points indicated as diamonds). Fig. 9 - Lipidomics analysis results of A1buMAX I, PLG and I3LG supplemented with sunflower lecithin. The mean peak areas of the lipid species are plotted. 7 / 25 Fig. 10 - Lipidomics results for (lyso)phospholipid species present in sunflower lecithin and rapeseed lecithin. DETAILED DESCRIPTION OF THE INVENTION 5 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. 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 10 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, 15 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. 20 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". 25 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. 8 / 25 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, Foetal Bovine Serum or equivalent serums, that is sometimes added to cell culture media. If this type of serum is meant, it is explicitly referred to as such. In a typical cell culture, of the cell types discussed in this invention, it is customary and extremely well established in the art that a basal medium should be used, such as Minimum 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, glucose, and minerals containing calcium, magnesium, potassium, sodium, and / or phosphate, considered essential for the survival of the cells. Typically, basal media by itself allow the cells to proliferate (i.e. expand) but not necessarily to differentiate. Proliferation (quantified as maximum population doublings for example) can usually be improved from the levels obtained by culturing cells with basal media by providing supplements to a certain medium. These supplements may be hormones, growth factors, vitamins or amino acids with a profile that is different from the one provided in the used basal medium. These supplements may 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. It is known that many cell types respond positively to being cultured in the presence of albumin added to a basal medium, otherwise supplemented or not. This positive response is typically seen by increased maximum population doublings or decreased time between doublings, i.e., faster and / or prolonged growth of the cells in culture. Some examples of 25 proliferation media that contain albumin are described in W02021158103A1 or W02023133441A2. Many others exist in published papers, patents and even commercial formulations. P-Lactoglobulin ((3LG) is the major whey protein of cow and sheep's milk and many other ruminant species and other mammals, but not all. It is notably absent from human and 30 rodent milk. In several species 13LG exists as several genetic variants (Godovac-Zimmermann et al. Genetic Variants of Bovine 13-Lactoglobulin. A Novel Wild-type fl-Lactoglobidin W and 9 / 25 its Primary Sequence. 1990, Biol. Chem. Hoppe-Seyler, 371(1), 255-260) with for bovine species the most prevalent ones being variants A and B, which only differ by two amino acids. The presence of each of these two variants affects the properties of the milk due to the A and B variant PLG molecules not having the same physico-chemical properties. Bovine PLG genetic variant A is most prevalent in the world cow population. PLG is a member of the lipocalin family of proteins and features the ability to bind hydrophobic and amphiphilic molecules. PLG also possesses a free cysteine and can exert antioxidative effects. Like albumin, despite years of research and a thorough study of the molecular structure, the physiological role of f3LG is not fully understood. 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 mammal cells, more specifically of a mesenchymal lineage, more specifically of a typical farm animal, more specifically of bovine origin, more specifically FAPs (fibro-adipogenic precursors) or SCs (satellite cells), for example by expanding (proliferating) more quickly, can be partially or completely replaced by P-Lactoglobulin ((3LG). The inventor tested several cell types from several species. In this specification, bovine PLG was used in all trials. This is a result of the fact that bovine PLG is the most common species type in the market, and other types are actually difficult to obtain. It is expected that 13LG from other species will have similar effects, especially ones with higher homology to and similar functionality as bovine PLG. As such, 20 PLG in this specification may be read as "bovine J3LG" or "PLG from other species with similar performance to bovine PLG". Fig.1 panel A shows the results of an evaluation of the effect of thirty-four different albumins on the proliferation of a cell culture of primary bovine fibro-adipogenic progenitor 25 (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- 30 well plates, or what is considered a 2D culture, in the normal parlance of the cell cultivation technical area. Cells were seeded with a density of 5.000 cells / cm' - and cultured in DMEM / F12 supplemented with 1% v / v PSA (Penicillin-Streptomycin-Amphotericin), 17.5 mM glucose, 2 10 / 25 mM L-alanyl-L-glutamine, 50 pg / m1L-ascorbic acid 2-phosphate, 1 µg / m1 a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 lig / m1 ethanolamine, 10 µg / m1 insulin, 5.5 µg / mi 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 TGF(31 (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 CorningTM rhAlbumin Media (supplier catalogue number CorningTM 62450RF). Wishing for the replacement to albumin to display the best possible performance, one of these three, namely A1buMAX I, was selected as the albumin against which fl-Lactoglobulin ((3LG) is compared. So, in this specification, A1buMAX I should be read as a synonym to "best performing albumin". Figure 1 panel B depicts the averaged performance of AlbuMAX I and A1buMAX II based on the data in Figure 1 panel A (shown as circles). The averaged performance of the blood-derived albumins, except the A1buMAX samples, (shown as squares) and recombinant albumins (shown as triangles) indicate that, on average, the source of the albumin does not influence its performance and that the average albumin (maximum performance indicated by I or AlbuMAX II (maximum performance of dotted line) is only 75% as potent as A1buMAX each indicated by the dashed and the dotted line). The full list of albumins tested is as follows: Supplier Sample name (catalogue number) Source Aspira Scientific Recombinant HSA (800001) recombinant Bovogen BovoLep (BSAL) blood-derived Biologicals Bovogen BovoStar (BSAS) blood-derived Biologicals Capricorn BSA Diagnostic grade (BSA-DG) blood-derived Capricorn BSA Protease free (BSA-PF-1U) blood-derived Capricorn BSA Standard Grade (BSA-1U, Lot CP22-5091) blood-derived 11 / 25 BSA Standard Grade (BSA-1U, Lot CP21-4306) blood-derived Capricorn CorningTM rhAlbumin Media (62450RF) recombinant Coming - Fisher Gibco AlbuMAX II (11021037) blood-derived Gibco AlbuMAX I (11020021) blood-derived f4 Optibumin — Recombinant human albumin InVitria recombinant (777HSA047 / 555HSA0074) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 023) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 024) Recombinant human albumin (RHAC-NW20; batch recombinant Laurus Bio 21 / 025) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 027) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 028) Recombinant human albumin (RHAC-NW20; batch recombinant Laurus Bio 21 / 029) Recombinant human albumin (RHAC-N'W20; batch Laurus Bio recombinant 21 / 030) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 031) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 033) Recombinant human albumin (RHAC-NW20; batch Laurus Bio recombinant 21 / 034) AlbumiNZTM Bovine Albumin Microbiological Grade MPBio blood-derived (180620) AlbumiNZTM Bovine Albumin Low Free Fatty Acid MPBio blood-derived (199899) Recombinant human serum albumin from Rice Grain recombinant Oryzogen (Oryza Sativa) (HYCOO2M03) Recombinant human serum albumin from Rice Grain Oryzogen recombinant (Oryza Sativa) (HYCOO2M02) Recombinant human serum albumin from Rice Grain Oryzogen recombinant (Oryza Sativa) (HYCOO2M01; lot C002202103002) Recombinant human serum albumin from Rice Grain Oryzogen recombinant (Oryza Sativa) (HYCOO2M01; lot C002201911007) PanBiotech Panbio Bovine Serum Albumin, fraction V (P06-1391050) Proliant AlbuRich P140 (67675) blood-derived Proliant AlbuRich PRP (67685) blood-derived 12 / 25 Sartorius Bio-Pure Human Serum Albumin (HSA) (05- Sartorius blood-derived 720-1B) Human Serum Albumin recombinant from P. pastoris, Validogen recombinant 200 mg / mL Sigma Bovine Serum Albumin (05470) blood-derived Cellastim; Recombinant human albumin expressed in rite Sigma recombinant (A9731) Fig. 2 panel A shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs for different concentrations of P-lactoglobulin ((3LG) (data points indicated as squares) versus 2.5 mg / ml AIbuMAX I (data point indicated as a circle). Data is shown as mean + SEM (standard error of the mean) based on 4 replicates. The horizontal dotted line indicates the population doublings obtained when no PLG or albumin sample was added. The horizontal dashdotted line indicates the population doublings obtained for 2.5 mg / ml A1buMAX I. Culture conditions were identical to those described for Fig. 1. PLG can be seen to replace approximately 90% of the effect of A1buMAX I. Not wishing to be bound by theory, but after the observation that PLG alone did not replace the full proliferative effect of A1buMAX I for the cell type used to obtain the data of Fig. 2 panel A (bovine FAPs), a hypothesis was formed based on the aal objective of a cell culture (in proliferation stage) which is to produce as many cells as possible, meaning that with every cell division a new cell membrane has to be formed, and cell membranes mainly consist of phospholipids, of which 70% are estimated to be phosphatidylcholine, and as such these types of lipid classes would be prime for experimentation. Lecithins, for example sunflower lecithin, are composed mainly of phospholipids, so plant-derived sunflower, rapeseed, and soy lecithin was supplied to the medium as a complement to f3LG. Optimally working sterile lecithin stock solutions can be obtained using conventional methods known to someone of common knowledge in the art (e.g. PBS-based solutions or autoclavable solutions such as described by Zhang et al. Lecithin promotes adipocyte differentiation and hepatic lipid accumulation, 2009, Int J Mol Med 23: 449-454). Fig. 2 panel B summarises these experiments and results, as explained below. Fig. 2 panel B shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs for different concentrations of PLG (data points indicated as squares) and different concentrations of plant-derived lecithins (sunflower, rapeseed, soy lecithin) on 13 / 25 top of 1.67 mg / ml PLG. Data is shown as mean + SEM based on 4 replicates and normalised to the 1.67 mg / ml PLG condition. The horizontal dashdotted line indicates the population doublings obtained for 1.67 mg / ml PLG without lecithin. Culture conditions were identical to those described for Fig. 1. It is clear that addition of sunflower lecithin (data points indicated as triangles with apex going down), but not rapeseed lecithin (data points indicated as triangles with apex going up) or soy lecithin (data points indicated as diamonds) to 1.67 mg / m113LG improves its performance. To better define the range of sunflower lecithin concentrations that, in combination with 13LG, enhance the proliferation-promoting effect, a concentration range of sunflower lecithin was tested as addition to 1.67 mg / ml PLG. Results of these tests are shown in Fig. 2, panel C. Fig. 2 panel C shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs for different concentrations of sunflower lecithin on top of 1.67 mg / ml PLG (data points indicated as circles). Sunflower lecithin concentrations were tested in the range of 0.00375 to 0.06 mg / ml which equals 0.22 to 3.6 % (w / w) on top of 1.67 mg / ml PLG. Data is shown as mean + SEM (standard error of the mean) based on 4 replicates and normalised to the condition without sunflower lecithin addition. The horizontal dashdotted line indicates the population doublings obtained when no sunflower lecithin was added. Culture conditions were identical to those described for Fig. 1. Sunflower lecithin can be seen to enhance proliferation effects of I3LG for the entire range of concentrations tested (0.22-3.6% w / w); the effect plateaus for concentrations of 1.8% (w / w) and higher. Having defined an improving effect of 0.03 mg / ml sunflower lecithin added to 1.67 mg / ml PLG (which equals 1.8% (w / w) of sunflower lecithin), this combination of I3LG and sunflower lecithin was tested for a wider concentration range to determine if the proliferation- promoting performance of albumin can be matched with or without adding sunflower lecithin to KG. In order to compare the proliferation-promoting potency of the albumin and I3LG molecules, molar concentrations are reported considering molecular weights of 66.5 kDa for albumin and 18.4 kDa for PLG. Fig. 3 shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs for different concentrations of A1buMAX I (data points indicated as circles), KG 30 (data points indicated as squares) and PLG + 1.8% w / w sunflower lecithin (data points indicated as triangles). Data is shown as mean + SEM (standard error of the mean) based on 4 14 / 25 replicates. The horizontal dotted line indicates the population doublings obtained when no f3LG or albumin sample was added. The horizontal dashdotted line indicates the population doublings obtained for 2.5 mg / ml A1buMAX I (2.5 mg / ml albumin equals 37.5 i_tM according to albumin's molecular weight of 66,500 Da). Culture conditions were identical to those described for Fig. 1. 3LG can be seen to be partially replace approximately 90% of the effect of A1buMAX I. It is clear that the addition of 1.8% (w / w) sunflower lecithin to I3LG improves the performance and yields an effect equal to albumin. With the albumin replacing potential of f3LG in adherent culture being proven, suspension cultures were then trialled, as suspension cultures are mostly used in industrial setting to create as many cells as possible for biotechnological applications, making this further check an important one in terms of evaluating real world applicability of the invention. 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 A1buMAX I (grey bar), without albumin or I3LG (white bar, labelled as 'neg ctrl'), 150 µ,M r3LG (single hatched bar) or 150 1.1M PLG + 1.8% w / w sunflower lecithin (double hatched bar). Data is shown as mean + SEM based on 4 replicates. Cells were seeded with a density of 25.000 eens / mi and cultured in DMEM / F12 supplemented with 1% PSA, 17.5 mM glucose, 2 mM L-alanyl-L-glutamine, 50 µg / m1 L-ascorbic acid 2-phosphate, 1 µg / m1 a- linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2µg / ml ethanolamine, 10 µg / ml insulin, 5.5µg / m1transferrin, 10 ng / ml FGF2, 10 ng / m1PDGF-BB, and 1 ng / ml TG931. It is clear that f3LG's performance is equipotent to or more potent than A1buMAX I and that the addition of sunflower lecithin to KG enhances this effect. Fig. 4 panel B shows 20x magnification microscopie images of the suspension cultures of the different conditions after 4 days of culture. These images display the higher abundance and increased size of cell aggregates for PLG-containing conditions compared to AlbuMAX I and the negative control condition. Addition of sunflower lecithin may prevent the occurrence of larger sized aggregates as observed for f3LG alone. After proving without doubt that, in both adherent and suspension cultures, f3LG and sunflower lecithin 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 PLG and sunflower lecithin can be isolated from the base media or the cell type used. 15 / 25 Fig. 5 shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs grown with 37.5 µM A1buMAX I (grey bars), no albumin or PLG (white bars) or 150 1.1.M PLG with 1.8% w / w sunflower lecithin (hatched bars) which were added to the medium described for Fig 1 containing DMEM / F12 (left 3 bars) or RMPI 1640 (right 3 bars) 5 as base. As RPMI 1640 already contained 2.1 mM L-glutamine and 11.1 mM glucose, no additional Glutamax or glucose was added. Data is shown as mean + SEM based on 4 replicates. It is clear that PLG with 1.8% w / w sunflower lecithin is equipotent to A1buMAX I, irrespective of the used base medium. After proving independence of the PLG with 1.8% w / w sunflower lecithin effect from 10 the basal media used, independence of cell type was checked. Growth for adherent and suspension cultures of bovine FAPs had been shown already in Figures 1 to 4. Fig. 6 shows the population doublings obtained in 3 days of adherent culture of primary bovine satellite cells (SCs) for different concentrations of A1buMAX I (data represented as circles), PLG (data represented as squares) and PLG with 1.8% w / w sunflower lecithin (data represented as triangles). Data is shown as mean + SEM based on 4 replicates. The cultures were performed in laminin-coated 96-well plates, or what is considered a 2D adherent culture, in the normai parlance of the cell cultivation technical area. Cells were seeded with a density of 5.000 cells / cm2 and cultured in DMEM / F12 supplemented with 1% PSA, 17.5 mM glucose, 2 mM L-alanyl-L-glutamine, 50 µg / m1 L-ascorbic acid 2-phosphate, 1 µg / m1 a-linolenic acid, 36 ng / ml hydrocortisone, 6.7 ng / ml sodium selenite, 2 iig / m1 ethanolamine, 10 µg / m1 insulin, 5.5 µg / mi transferrin, 10 ng / ml FGF2, 50 ng / ml HGF, and 20 ng / ml 3,3',5-Triiodo-L-thyronine. It can be seen that for the tested concentration range, PLG (and sunflower lecithin) can match the induced population doublings as obtained for A1buMAX I. Both these SCs in Fig. 6 and FAPs in Figs 1-5 are of bovine origin, so, at least in the bovine species there is sufficient proof 25 to say that F3LG (and sunflower lecithin) as a replacement to albumin is cell type independent. Clearly, bovine cells that benefit from the presence of albumin benefit in the same way, or more potently, from the presence of PLG (and sunflower lecithin). After proving independence of the PLG and sunflower lecithin effect from the cell type used, independence of cell species was checked. Growth for bovine FAPs had been shown 30 already in Figures 1 to 4. Fig. 7 shows figures for primary murine FAPs. 16 / 25 Fig. 7 shows the population doublings obtained in 3 days of culture of primary murine FAPs for different concentrations of A1buMAX I (data represented as circles), (3LG (data represented as squares) and 150 µM f3LG with the addition of a concentration range of sunflower lecithin (data represented as triangles). Data is shown as mean + SEM based on 4 replicates. The horizontal dashdotted line indicates the population doublings obtained for 150 µM (3LG without the addition of sunflower lecithin. Culture conditions were identical to those described for Fig. 1. The performance of A1buMAX I can be seen to be successfully replaced by [3LG without sunflower lecithin. The addition of low concentrations of sunflower lecithin to f3LG provides an improvement in performance. It should be noted that some differences in proliferation performance seem to exist comparing bovine FAPs versus murine FAPs. Firstly, while [3LG without sunflower lecithin is not sufficient to fully replace albumin for bovine FAPs (Fig. 2A and Fig. 3), (3LG without sunflower lecithin is equipotent to albumin for murine FAPs. However, the addition of sunflower lecithin to 150 [AM (3LG enhances the proliferative effect further for murine FAPs. Secondly, while bovine FAPs barely grow, but also do not die, in absence of AlbuMAX I or f3LG (horizontal dotted line in Fig 2A), murine FAPs do die when cultured without A1buMAX I or (3LG (horizontal dotted line in Fig 7). 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 murine FAPs which is also reflected by the difference in maximum number of population doublings achieved between bovine and murine FAPs. This proves that the effect of (3LG as an albumin replacement is independent of the species of the cell. Bovine (3LG exists as different genetic variants with variants A and B being the most prevalent. Milk-derived, purified (3LG usually comprises a mix of [3LG genetic variants depending on the genotype of the cows from which the milk is derived, also mainly comprising variants A and B according to these variants' prevalences. The inventor realized that the data from Fig 2-7 were obtained from a commercial milk-derived f3LG product that comprises an undefined mix of [3LG variants (mainly consisting of variants A and B). To determine if f3LG's growth-promoting and albumin-replacing effect depends on the (3LG variant and therefore amino acid sequence, pure [3LG variant A and pure (3LG variant B were tested for their proliferation-enhancing effect and potential variations therein. Fig. 8 shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs for 150 µM f3LG comprising an undefined mix of different variants (data point 17 / 25 indicated as square; labelled as (3LG-A / B) and different concentrations of PLG variant A (data points indicated as triangles; labelled as I3LG-A) and PLG variant B (data points indicated as diamonds (labelled as (3LG-B). Commercial product codes are indicated in the (gure legend. Data is shown as mean + SEM based on 4 replicates. The horizontal dotted line indicates the 5 population doublings obtained when no PLG was added. The horizontal dashdotted line indicates the population doublings obtained for 150 µM f3LG comprising an undefined mix of different variants. Culture conditions were identical to those described for Fig. 1. PLG variant A can be seen to be 10-fold more potent than the undefined mix of different PLG variants as seen by the lower concentration required to obtain the same population doublings. Data for PLG variants in Fig. 8 shows that PLG, whether sourced from milk, or milk- derived products like whey, or obtained recombinantly, is ideally to be used in the protein sequence variant A to replace the effects of albumin. Following up on the finding in Fig 3 that the addition of sunflower lecithin to f3LG increases the number of obtained population doublings to the level of A1buMAX I, but that this effect is not achieved with PLG alone, the difference in lipid composition of A1buMAX I, I3LG and PLG with sunflower lecithin was investigated by means of lipidomics. Since the sunflower lecithin used consist of 91% fat, it was investigated which lipid species are higher in quantity in A1buMAX I compared to 13LG but are present in equal amounts or higher than A1buMAX I upon adding sunflower lecithin to the PLG. The analysed conditions / media correspond to conditions shown in Fig 3; A1buMAX I (37.5 µM), PLG (150 µM) and pLG (150 µM) with 1.8% w / w sunflower lecithin. Lipidomic analysis of lipids higher in quantity in A1buMAX I compared to PLG but present in equal amounts or higher than A1buMAX I upon adding sunflower lecithin to the PLG is shown in Fig. 9. Figure 9 depicts the mean peak areas of the lipid species based on lipidomics analysis; data is shown as mean + SEM for 3 replicates. It should be noted that the area values for the 150 µM PLG condition are too small to be visualized. Strikingly, three specific lipid species, that were significantly enriched in A1buMAX I compared to PLG, were not different between A1buMAX I and f3LG + sunflower lecithin. These species are 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:2)), 1 -palmitoy1-2-linoleoyl- sn-glycero -3 -phosphocholine (PC(16:0 18:2)), and 1-stearoy1-2- linoleoyl-sn-glycero-3-phosphocholine (PC(18:0 18:2)). It is clear that (lyso)phosphatidylcholine species containing a linoleic acid are provided by sunflower lecithin to match levels of or achieve levels higher than those found in A1buMAX I. 18 / 25 Based on the observation that sunflower lecithin addition on f3LG improves the proliferation-promoting effect and that sunflower lecithin addition introduces lipid species that are absent in F3LG, it was investigated why sunflower lecithin, but not rapeseed lecithin enhanced the effect of 13LG (see also Fig. 2, panel B). Lipidomic analysis was performed on sunflower lecithin and rapeseed lecithin which revealed that both samples contain the same amounts of LPC(18:2), PC(16:0 18:2), and PC(18:0_18:2). Phospholipids with different polar heads can exert different biological functions. It was investigated, by means of lipidomics, if the identified lipid species in Fig. 9 but conjugated to the different polar heads (e.g. an ethanolamine or inositol instead of a choline) are present in different proportion between sunflower lecithin and rapeseed lecithin since the species with choline heads were not present in different amounts between sunflower and rapeseed lecithin. Fig. 10 reveals that for the lysophospholipid 18:2, phospholipid (16:0_18:2), and phospholipid (18:0_18:2), rapeseed lecithin is, compared to sunflower lecithin, more enriched in (lyso)phosphatidylethanolamines ((L)PE), phosphatidylinositols (PI) and / or phosphatidylglycerols (PG) versus (lyso)phosphatidylcholines ((L)PC) for the corresponding lipid species with the particular carbon chains. Of note, no (lyso)phosphatidylserine ((L)PS) species with the aforementioned carbon chains were detected in either of the lecithin samples. PC and PE species are most abundant in mammalian cell membranes and are also readily provided by lecithins like sunflower and rapeseed lecithin. However, the proportion of the provided (lyso)phospholipid classes with their specific carbon chains may be more suitable or favourable for the creation of new cell membranes during cell proliferation. (Lyso)phospholipids provided by rapeseed may present a suboptimal proportion which leads to incorporation of suboptimal lipid species; for example, relatively more PE species are incorporated than PC species or PE species have to be enzymatically converted into other required phospholipids which requires cellular energy that can hamper cell proliferation rates. Sunflower lecithin's higher relative proportion of PC species compared to PE, PI and / or PG may better reflect the cell membrane's composition, compared to rapeseed lecithin, and therefore be better suitable to be incorporated into a new cell membrane during cell proliferation. The lipids provided by sunflower lecithin comprise a mix of both saturated and unsaturated fatty acids in the form of (lyso)phospholipids. Specifically, the chain lengths of 16 to 18 carbons are well suited to be used for phospholipid membrane synthesis (Harayama and 19 / 25 Riezman. Understanding the diversity of membrane lipid composition. 2018, Nat Rev Mol Cell Biol, 19(5), 281-296.). Moreover, the linoleic acid containing phospholipids in sunflower lecithin, i.e. LPC(18:2), PC(16:0_18:2), and PC(18:0_18:2), provide a source of essential co6 fatty acids that can be used for the synthesis of other lipid species with biological actions. The use of (essential) fatty acids in cell culture is usually hampered by their low solubility. However, the use of (lyso)phospholipids increases solubility in water and makes them biologically more available. By forming micelles, these (lyso)phospholipids can readily interact and merge with existing cell membranes to facilitate the cell's use of them without requiring energy-demanding (enzymatic) synthesis, breakdown and / or conversion of free fatty acids into phospholipids. Requiring less energy for cell membrane creation would decrease the metabolic burden on cells allowing for more energy-efficient, quicker proliferation. Based on the lipidomic analyses shown in Fig. 9 and Fig. 10, the (lyso)phospholipid types to add to PLG are preferably those containing a linoleic acid, preferably with a choline as phospholipid polar head, and preferably with a palmitic or stearic acid in addition to the linoleic acid in case of a phospholipid. From the difference between sunflower and rapeseed lecithin lipidomics and proliferation, it seems preferable that the proportion of (lyso)phospholipids containing a linoleic acid preferably is high for (lyso)phosphatidylcholines compared to (lyso)phosphatidylethanolamines, phosphatidylglycerols and / or phosphatidylinositols. In this context, sunflower lecithin provided a more preferable combination of lipid species than rapeseed lecithin to promote cell proliferation. However, if a different lipid composition in the cells' membrane is desired and / or required, e.g. a higher phosphatidylethanolamine and / or phosphatidylinositol content, then another lecithin or lipid source can be considered. The fact that the type of phospholipids mix described above, as preferably provided by sunflower lecithin, in combination with I3LG has a more potent effect on the proliferation of cells than just by using PLG alone is an unexpected effect, showing a potential synergy between these two components, the I3LG and the phospholipids. The inventor also puts forward the hypothesis, about which there is no reason not to believe it is not true, that if another molecule or molecules exist that replace albumin in a cell culture, or when using albumin in itself, that the nature of the ((lyso)phospho)lipid mix may also enhance the function of that other molecule or molecule, or albumin, particularly the ((lyso)phospho)lipid mix presenting as sunflower lecithin. 20 / 25 PLG, PLG with (sunflower) lecithin or f3LG with specific lipids can be obtained by using recombinant filLG and adding sunflower lecithin or specific lipids separately or by sourcing 13LG from (commercial) whey (which may contain sunflower lecithin as an additive, i.e. as an emulsifier), or other milk-derived products. Besides all the technical effects and advantages disclosed so far as regards to PLG in the context of this invention, it is also noted that sourcing PLG is much more animal-friendly and scalable than albumin, 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. The media used to generate the data presented in this specification are serum free media. Serum free in this context means free of serums such as FBS (foetal bovine serum) or FHS (foetal horse serum) amongst others. These types of serums are non-chemically defined, usually contain albumin in varying dosages and are used mainly to improve the performance of cell cultures. There is no reason to assume the invention would not work in the presence of media with these types of serum, actually it would simply improve the performance of those media even further. In general, serum-free media are more appropriate at least to the field of cellular agriculture, where a reduction in compounds sourced from unborn butchered animals is desired. It is thus preferable, for the inventor, to use serum free media with this albumin replacement. It is also important to stress that, independent of, in albumin, the current lack of clarity from the scientific establishment of which function(s) is / are crucial to promote proliferation for specific cell types, PLG simply works to replace whatever that function(s) is( / are). Another important detail to stress is that even if all the testing was done with one type of cell per culture, cultures can be co-cultures, i.e. have different cells in the same culture vessel. For example, bovine SCs and FAPs could be cultured together. The proof point that f3LG (supplemented with sunflower lecithin / (lyso)phospholipids or not) is a viable substitute for albumin in FAPs and SCs, both of a mesenchymal lineage, in different species, shows that PLG can be seen to work as an albumin replacement for cells of a mesenchymal lineage in general. Exemplary Embodiments of the Invention There are a myriad of possible embodiments for this invention, since it can be applied in several types of cultures under many different conditions, but in basic terms a preferred embodiment of the invention is as, a medium for, or as an animal cell culture, comprising at least a basal medium, such as Minimum Essential Medium (MEM), Eagle Medium, Dulbecco's Modified Eagle Medium (DMEM), Ham's F-12, Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (DMEM F12), Roswell Park Memorial Institute (RPMI) 1640, etc; preferably with a carbon source, preferably with an appropriate growth factor, where the culture is of a cell type or types (the culture may be a co-culture mix of different cell types, or even from different species) that would benefit from the presence of albumin, asserted by an improvement in the culture outcome being verified experimentally when albumin is present, wherein the actual culture either has no albumin added to it or does not have enough albumin to substantially increase the performance of the culture, and wherein 13LG is added at a concentration that replaces the desired effect of albumin, usually at a relationship of 1:1 in molar concentration (1 M of(3LG for 1 M of albumin) or more (more than 1 M of (3LG for 1 M of albumin) or less, depending on the cell type. Usually, maximum performance of a culture is desired so the relationship should be the correct one that gives the best possible effect, as determined experimentally for the cell type, species or both. The culture may be run on any of several types of common culture vessels, in adherent or suspension cultures, such as well plates, spinner flasks or bioreactors. A further preferred embodiment is the replacement of albumin being done with the use of PLG with added sunflower lecithin, for improved effect with certain cell types, or more specifically with added (lyso)phospholipids, preferably those containing a linoleic acid, preferably with a choline as phospholipid polar head, and preferably with a palmitic or stearic acid in addition to the linoleic acid in case of a phospholipid, that may be provided by the aforementioned sunflower lecithin. A preferred embodiment is also, as explained throughout this specification, the straightforward claim of the use of PLG (with or without lecithin, or the phospholipid mix described above) 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 5 of the cells in the culture when compared to albumin (or equivalent molecules) absence.

Claims

1. A method to reduce the amount of albumin in a cell culture, characterized by the following steps: a) Providing a proliferation cell culture with at least one cell type that benefits from improved proliferation in the presence of albumin; b) The addition of (3-lactoglobulin to the cell culture medium in a concentration that offers the same or approximately the same proliferation improvement as albumin; c) Allowing the cells to proliferate in the P-lactoglobulin-containing growing medium.

2. A method according to conclusion 1, where the cell culture is of an animal cell type, more in particular of a mammalian cell type, more specifically of a mesenchymal lineage, more specifically a primary cell, more specifically a fibro- adipogenic precursor (FAP) or a satellite cell (SC), more specifically of a bovine origin.

3. A method according to conclusion 1 and / or 2 whereby P-lactoglobulin is added to the cell culture is added in a concentration that replaces the desired effect of albumin, usually in a preference ratio of 1:1 in molar concentration (1 M (3LG for 20 1 M albumin) or optionally more (more than 1 M(3LG for 1 M albumin) or optionally less, depending on the cell type.

4. A method according to conclusion 3 whereby P-lactoglobulin is supplemented with sunflower lecithin, to assess the effectiveness of replacing albumin for p- to improve lactoglobulin or surpass the effect of albumin. 25 5. A method according to claim 4 whereby the sunflower lecithin is added in a concentration of ideally 1.8% (w / w).

6. A method according to one of the preceding conclusions in which no albumin is added to the cell culture medium or whereby an amount of albumin that not is sufficient to maximize the proliferation potential of the culture, becomes added to the cell culture medium.

7. A method according to one of the preceding conclusions, whereby 13-lactoglobulin in the culture medium is present in a concentration of approximately 0.3 µM to approximately 150 µM.

8. A method according to one of the preceding conclusions, whereby the medium a is serum-free medium.

9. A method according to one of the preceding claims, whereby P-lactoglobulin from is of bovine origin, preferably the genetic variant A.

10. A method according to one of the preceding conclusions, whereby P-lactoglobulin is supplemented with (lyso)phospholipids, preferably those containing linoleic acid, preferably with a choline as the polar head of the (lyso)phospholipid, and preferably with a palmitic or stearic acid alongside the linoleic acid in the case of a phospholipid.

11. A medium for a cell culture characterized by the inclusion of a basal medium, where the cultivation of a cell type or cell types is that would benefit from the presence of albumin, confirmed by an improvement in the proliferation outcome which is experimentally verified when albumin is present, whereby the actual culture either has not added albumin or does not have enough albumin to substantially increase the proliferation performance of the breeding, and where f3- Lactoglobulin has been added in a concentration that provides the desired effect of albumin. replaces, or where P-lactoglobulin is supplemented with sunflower lecithin added in a concentration that replaces the desired effect of albumin.

12. The cell culture medium of conclusion 11, in which P-lactoglobulin is present in a concentration of approximately 0.3 µM to approximately 150 µM.

13. The cell culture medium according to one of the conclusions 11 to 12, whereby the p- is lactoglobulin of bovine origin, preferably the genetic variant A.

14. The cell culture medium according to one of the conclusions 11 to 13, where the 0- Lactoglobulin is supplemented with (lyso)phospholipids, preferably those containing linoleic acid contain, preferably with a choline as the polar head of the (lyso)phospholipid, and at preference with a palmitic or stearic acid alongside the linoleic acid in the case of a phospholipid.

15. Use of 13-lactoglobulin as an albumin substitute in a cell culture.

16. A cell culture medium containing an albumin or an albumin substitute contains, supplemented with (lyso)phospholipids, preferably those containing linoleic acid, preferably with a choline as the polar head of the (lyso)phospholipid, and preferably with a palmitic or stearic acid alongside the linoleic acid in the case of a phospholipid.

17. Medium according to claim 16, where the source of the (lyso)phospholipids sunflower lecithin is. 1 / 7 Fig. 1

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