Reduction of albumin in a cell culture medium

Abstract

The present invention discloses a method for reducing or eliminating the use of albumin in cell culture media by replacing it with β-lactoglobulin (βLG), optionally supplemented with sunflower lecithin. The invention demonstrates that βLG 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

[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 Human albumin can also be used, which is similarly obtained from withdrawn blood.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] In Nilausen, K, Role of Fatty Acids in Growth-promoting Effect of Serum Albumin on Hamster Cells In Vitro, Journal of Cellular Physiology, issue 96, nr.1, 1978-07-01, a comparison is performed between serum albumin and P-lactoglobulin (PLG). All cultures were performed in the presence of 10% FBS (page 2, column 1, Cells and Medium section), which makes this investigation one done in a non-serum free medium, where the effects of exogenous albumin or P-lactoglobulin are difficult to assess, and the results actually indicate that P- lactoglobulin containing medium as having worse performance and as such that P-lactoglobulin cannot replace albumin (fig. 7) leading one away from thinking P-lactoglobulin could replace albumin in a serum free medium.

[0014] In Capiaumont J. el al, Whey and fl-lactoglobulin: 2 milk by-products which can replace fetal calf serum in mouse hybridoma cell culture, Dairy Science and Technology, issue 74, nr.2, 1994-01-01, P-lactoglobulin is investigated as a replacement for albumin, but, not in a serum free medium, as can be seen on page 135: “Nevertheless, 1% of serum remains necessary for good cell growth and secretion.” As such, this paper shows the effect of combining P- lactoglobulin with serum, giving no insight of the potential potency of P-lactoglobulin in the absence of serum.

[0015] WO2023225687 discloses an albumin and serum free media containing plant proteins, and specifically in paragraph 136 that P-lactoglobulin could be evaluated in the future - meaning, this publication shows absolutely no data on P-lactoglobulin.

[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

[0019] Providing a serum-free medium, comprising a basal medium; Providing the cell culture with at least a cell type that benefits from enhanced proliferation in the presence of albumin; Adding P-lactoglobulin to the cell culture medium in a concentration that provides the same or approximately the same proliferation improvement as albumin, preferably in a range of P- lactoglobulin concentration in the cell culture of 0.3 pM to about 150 pM; Allowing the cells to proliferate in the P-lactoglobulin containing culture medium.

[0020] In a further preferred embodiment, the invention is a method characterised by the cell types being vertebrate animal cells, or eukaryote, for example fish cells, for example avian cells, for example mammalian cells, 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 characterised by P- lactoglobulin being added to the cell culture at a concentration that replaces the desired effect of albumin, usually at a preferable relationship of 1 : 1 in molar concentration (1 M of PLG for 1 M of albumin) or optionally more (more than 1 M of PLG for 1 M of albumin) depending on the cell type.

[0022] In a further preferred embodiment, the invention is a method according to any of the preceding claims wherein P-lactoglobulin is supplemented with sunflower lecithin.

[0023] In a further preferred embodiment, the invention is a method wherein the sunflower lecithin is added at a concentration in the range 0.22-3.6% w / w, more preferably 1.8% w / w.

[0024] In a further preferred embodiment, the invention is a method wherein no supplemental albumin is added to the cell culture medium or wherein an amount of albumin is not enough to maximise the proliferation potential of the culture is added to the cell culture medium.

[0025] In a further preferred embodiment, the invention is a method wherein the P- lactoglobulin is present in the culture medium at a concentration of about 0.3 pM to about 150 pM, preferably 10 to 150 pM, more preferably 50 to 150 pM

[0026] In a further preferred embodiment, the invention is a method wherein the P- lactoglobulin is of bovine origin, preferably the genetic variant A.

[0027] In a further preferred embodiment, the invention is a wherein the P-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.

[0028] In another preferred embodiment, the invention is a serum-free medium for a cell culture characterised by comprising a base medium, P-lactoglobulin and no supplemental albumin, and wherein P-lactoglobulin is added at a concentration that replaces the desired effect of albumin, the medium optionally supplemented with sunflower lecithin. In a further preferred embodiment, the invention is a cell culture medium, wherein the culture medium either has no supplemental albumin added to it or does not have enough albumin to substantially increase the proliferation performance of the culture.

[0029] In a further preferred embodiment, the invention is a cell culture medium wherein the P-lactoglobulin is present at a concentration of about 0.3 pM to about 150 pM.

[0030] In a further preferred embodiment, the invention is a cell culture medium wherein the P-lactoglobulin is of bovine origin, preferably the genetic variant A.

[0031] In a further preferred embodiment, the invention is a cell culture wherein the P- 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.

[0032] In a further preferred embodiment, the invention is a medium wherein the source of the (lyso)phospholipids is sunflower lecithin.

[0033] In another preferred embodiment, the invention is the use of P-lactoglobulin as an albumin replacement in a cell culture.

[0034] In a further preferred embodiment, the invention is the use of P-lactoglobulin wherein the P-lactoglobulin is present at a concentration of about 0.3 pM to about 150 pM.

[0035] 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 P-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 P-lactoglobulin for 1 g of albumin) or optionally more (more than 1 g of P-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 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 P-lactoglobulin is present in the culture medium at a concentration of about 0.3 pM to about 150 pM, more preferably a method wherein the medium is a serum free medium; more preferably a method wherein the P-lactoglobulin is of bovine origin, preferably the genetic variant A; more preferably a method wherein the P-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.

[0036] In another preferred embodiment the invention is a medium for a cell culture characterised by comprising a base medium, 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 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 pM to about 150 pM; more preferably a cell culture medium, wherein the P-lactoglobulin is of bovine origin, preferably the genetic variant A; more preferably a cell culture medium wherein the P-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.

[0037] In another preferred embodiment the invention is the use of P-lactoglobulin as an albumin replacement in a cell culture.

[0038] 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 phospholipid; more preferably a medium wherein the source of the (lyso)phospholipids is sunflower lecithin.

[0039] DESCRIPTION OF THE DRAWINGS

[0040] 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 serum-free medium containing different albumins. Manufacturer-to-manufacturer and batch-to-batch performance variations are demonstrated.

[0041] Fig. 2 - Panel A shows the population doublings obtained in 3 days of serum-free culture of primary bovine FAPs for different concentrations of AlbuMAX I and P- Lactoglobulin (PLG). Panel B shows the normalized population doublings obtained in 3 days of serum-free culture of primary bovine FAPs for different concentrations of PLG and different lecithins. The successful replacement of albumin with PLG or PLG supplemented with lecithin is proven. Panel C shows the normalized population doublings obtained in 3 days of serum- free culture of primary bovine FAPs for different concentrations of sunflower lecithin added to 1.67 mg / ml PLG.

[0042] Fig. 3 - Population doublings obtained in 3 days of adherent serum-free culture of primary bovine FAPs for different concentrations of AlbuMAX 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).

[0043] Fig. 4 - Panel A shows the population doublings obtained in 4 days of serum-free culture of primary bovine FAPs grown in suspension in well plates with AlbuMAX I (grey bar), without albumin or PLG (white bar, labelled as ‘neg Ctrl’), 150 pM PLG (single hatched bar) or 150 pM 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.

[0044] Fig. 5 - Population doublings obtained in 3 days of serum-free adherent culture of primary bovine FAPs grown with 37.5 pM AlbuMAX I (grey bars), no albumin or PLG (white bars) or 150 pM PLG with 1.8% w / w sunflower lecithin (hatched bars) in different base media.

[0045] Fig. 6 - Panel A shows the population doublings obtained in 3 days of serum-free adherent culture of primary bovine satellite cells (SCs) for different concentrations of AlbuMAX I (data represented as circles), PLG (data represented as squares) and PLG with 1.8% w / w sunflower lecithin (data represented as triangles). Panel B shows the population doublings obtained in 3 days of serum-free adherent culture of primary bovine dermal fibroblasts for different concentrations of AlbuMAX I (data represented as circles), PLG (data represented as squares) and PLG with 1.8% w / w sunflower lecithin (data represented as triangles). Panel C shows the population doublings obtained in 3 days of serum-free adherent culture of primary bovine endothelial cells for different concentrations of AlbuMAX I (data represented as circles) and PLG (data represented as squares). Panel D shows the population doublings obtained in 3 days of serum-free adherent culture of adipose-derived bovine stromal vascular fraction for different concentrations of AlbuMAX I (data represented as circles) and PLG (data represented as squares).

[0046] Fig. 7 - Panel A shows the population doublings obtained in 3 days of serum-free adherent culture of primary ovine dermal fibroblasts for different concentrations of AlbuMAX I (data represented as circles) and PLG (data represented as squares). Panel B shows the population doublings obtained in 3 days of serum-free adherent culture of adipose-derived porcine stromal vascular fraction for different concentrations of AlbuMAX I (data represented as circles) and PLG (data represented as squares). Panel C shows the population doublings obtained in 3 days of serum-free adherent culture of primary porcine endothelial cells for different concentrations of AlbuMAX I (data represented as circles) and PLG (data represented as squares).

[0047] Fig. 8 - Panel A shows the population doublings obtained in 3 days of serum-free adherent culture of human bone marrow-derived mesenchymal stem cells (BM-MSCs) for different concentrations of AlbuMAX I (data represented as circles) and PLG (data represented as squares). Panel B shows the population doublings obtained in 3 days of serum-free adherent culture of human embryonic kidney cells for different concentrations of AlbuMAX I (data represented as circles), PLG (data represented as squares) and PLG with 1.8% w / w sunflower lecithin (data represented as triangles). Panel C shows the population doublings obtained in 3 days of serum-free adherent culture of African monkey epithelial kidney cells for different concentrations of AlbuMAX I (data represented as circles) and PLG (data represented as squares). Panel D shows the population doublings obtained in 3 days of serum-free adherent culture of human cervical epithelial cells for different concentrations of AlbuMAX I (data represented as circles) and PLG (data represented as squares). Panel E shows the population doublings obtained in 3 days of serum-free adherent culture of human retinal epithelial cells for different concentrations of AlbuMAX I (data represented as circles) and PLG (data represented as squares).

[0048] Fig. 9 - Panel A shows the population doublings obtained in 3 days of serum-free adherent culture of murine fibro-adipogenic progenitors (FAPs) for different concentrations of AlbuMAX I (data represented as circles) and PLG (data represented as squares). Panel B shows the population doublings obtained in 3 days of serum-free adherent culture of murine dermal fibroblasts for different concentrations of AlbuMAX I (data represented as circles), PLG (data represented as squares) and PLG with 1.8% w / w sunflower lecithin (data represented as triangles). Panel C shows the population doublings obtained in 3 days of serum-free adherent culture of Chinese hamster ovary cells for different concentrations of AlbuMAX I (data represented as circles) and PLG (data represented as squares).

[0049] Fig. 10 - Panel A shows the population doublings obtained in 3 days of serum-free adherent culture of embryonic chicken fibroblasts for different concentrations of AlbuMAX I (grey bar), without albumin or PLG (white bar, labelled as ‘negative Ctrl’), 150 pM PLG (single hatched bar) or 150 pM PLG + 1.8% w / w sunflower lecithin (double hatched bar). Panel B shows the population doublings obtained in 3 days of serum-free adherent culture of embryonic zebrafish fibroblasts for different concentrations of AlbuMAX I (grey bar), without albumin or PLG (white bar, labelled as ‘negative Ctrl’), 150 pM PLG (single hatched bar) or 150 pM PLG + 1.8% w / w sunflower lecithin (double hatched bar).

[0050] Fig. 11 - 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 PLG in presence of different ‘baseline’ concentrations of AlbuMAX I.

[0051] Fig. 12 - Population doublings obtained in 3 days of adherent culture of primary bovine FAPs for 150 pM PLG comprising an undefined 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).

[0052] Fig. 13 - Population doublings obtained in 3 days of serum-free adherent culture of primary bovine FAPs with 150 pM milk-derived PLG (grey bar), without albumin or PLG (white bar, labelled as ‘negative Ctrl’), or 150 pM recombinant PLG (single hatched bar).

[0053] Fig. 14 - Lipidomics analysis results of AlbuMAX I, PLG and PLG supplemented with sunflower lecithin. The mean peak areas of the lipid species are plotted.

[0054] Fig. 15 - Lipidomics results for (lyso)phospholipid species present in sunflower lecithin and rapeseed lecithin.

[0055] DETAILED DESCRIPTION OF THE INVENTION

[0056] 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.

[0057] 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. 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 6, panel C). PLG 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 PLG (with sunflower lecithin) used instead of albumin.

[0058] 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.

[0059] 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.

[0060] 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. 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).

[0061] 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.

[0062] 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.

[0063] 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).

[0064] 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 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. P-Lactoglobulin (PLG) 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 rodent milk. In several species PLG exists as several genetic variants (Godovac-Zimmermann et al. Genetic Variants of Bovine f-Lactoglobulin. A Novel Wild-type f-Lactoglobulin W and 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 PLG is not fully understood.

[0065] 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 vertebrate animal cells (or eukaryote), for example fish cells, for example avian cells, for example of mammalian cells, for example of a mesodermal or ectodermal lineage, for example of a typical farm animal, for example of bovine, ovine or porcine origin, for example 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 P-Lactoglobulin (PLG). This is to say, a reduction (reduction interpreted to include elimination, so, reduction to zero) of supplemental albumin is achieved by using PLG in its stead. In short, the inventor tested several cell types from several species.

[0066] 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 PLG from other species will have similar effects, especially ones with higher homology to and similar functionality as bovine PLG. As such, PLG in this specification may be read as “bovine PLG” or “PLG from other species with similar performance as, or homologous sequence to, bovine PLG”.

[0067] 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 culture, in the normal parlance of the cell cultivation technical area. Cells were seeded with a density of 5.000 cells / cm2and 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, 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 P-Lactoglobulin (PLG) is compared. So, in this specification, AlbuMAX I should be read as a synonym to “best performing albumin”.

[0068] 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 of each indicated by the dashed and the dotted line).

[0069] The full list of albumins tested is as follows:

[0070] 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 (PLG) (data points indicated as squares) versus 2.5 mg / ml AlbuMAX 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 AlbuMAX I. Culture conditions were identical to those described for Fig. 1. PLG can be seen to replace approximately 90% of the effect of AlbuMAX I. Not wishing to be bound by theory, but after the observation that PLG alone did not replace the full proliferative effect of AlbuMAX I for the cell type used to obtain the data of Fig. 2 panel A (bovine FAPs), a hypothesis was formed based on the final 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 PLG. 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.

[0071] 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 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 / ml PLG improves its performance.

[0072] To better define the range of sunflower lecithin concentrations that, in combination with PLG, 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.

[0073] 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 PLG 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.

[0074] 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 PLG and sunflower lecithin was tested for a wider concentration range to determine if the proliferationpromoting performance of albumin can be matched with or without adding sunflower lecithin to PLG. In order to compare the proliferation-promoting potency of the albumin and PLG molecules, molar concentrations are reported considering molecular weights of 66.5 kDa for albumin and 18.4 kDa for PLG.

[0075] Fig. 3 shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs for different concentrations of AlbuMAX 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). 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 AlbuMAX I (2.5 mg / ml albumin equals 37.5 pM according to albumin’s molecular weight of 66,500 Da). Culture conditions were identical to those described for Fig. 1. PLG can be seen to be partially replace approximately 90% of the effect of AlbuMAX I. It is clear that the addition of 1.8% (w / w) sunflower lecithin to PLG improves the performance and yields an effect equal to albumin.

[0076] With the albumin replacing potential of PLG 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.

[0077] 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), without albumin or PLG (white bar, labelled as ‘neg Ctrl’), 150 pM PLG (single hatched bar) or 150 pM 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 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, 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 PLG’s performance is equipotent to or more potent than AlbuMAX I and that the addition of sunflower lecithin to PLG enhances this effect. Fig. 4 panel B shows 20x magnification microscopic 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 PLG alone.

[0078] After proving without doubt that, in both adherent and suspension cultures, PLG 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.

[0079] Fig. 5 shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs grown with 37.5 pM AlbuMAX I (grey bars), no albumin or PLG (white bars) or 150 pM PLG with 1.8% w / w sunflower lecithin (hatched bars) which were added to the medium described for Fig 1 containing DMEM / F12 (left three bars) or RPMI 1640 (right three bars) as base. As RPMI 1640 already contained 2.1 mM L-glutamine and 11.1 mM glucose, no additional Glutamax, L-glutamine 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 AlbuMAX I, irrespective of the used base medium.

[0080] After proving independence of the effect of PLG (with 1.8% w / w sunflower lecithin) from the basal media 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.

[0081] Fig. 6 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), 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 horizontal dotted line indicates the population doublings obtained when no albumin or PLG 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 / cm2and 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 PLG (with 1.8% w / w sunflower lecithin) induces the same as or more population doublings than AlbuMAX I. Both these SCs in Fig. 6 panel A and FAPs in Figures 1 to 5 are of bovine origin, so, at least in the bovine species there is sufficient proof to say that PLG (with 1.8% w / w sunflower lecithin) 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 PLG (with 1.8% w / w sunflower lecithin).

[0082] Fig. 6 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 PLG (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 PLG sample was added. Culture conditions were identical to those described for Fig. 1. It can be seen that for the tested concentration range, PLG can induce the same population doublings as AlbuMAX I. Both these dermal fibroblasts in Fig. 6, and FAPs (Figures 1 to 5) and SCs (Figure 6, panel A) are of bovine origin, so, at least in the bovine species there is sufficient proof to say that PLG 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 PLG.

[0083] Fig. 6 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 PLG (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 PLG 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 / cm2and 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 PLG 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 PLG 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 PLG. The absence of net positive population doublings is likely a result of using a suboptimal medium lacking crucial mitogenic components. An expert in the art would be able to define these mitogenic factors if needed. The fact that PLG 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 PLG is cell type and cell morphology -type independent.

[0084] Considering the albumin-replacing effect of PLG (with 1.8% w / w sunflower lecithin) for bovine mesenchymal and endothelial cells, it was tested if albumin can be replaced by PLG (with 1.8% w / w sunflower lecithin) in co-cultures containing mixtures of aforementioned cells. Therefore, stromal vascular fractions were obtained from bovine adipose tissue and cultured.

[0085] Fig. 6 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 PLG (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 PLG 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 / cm2and 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 PLG 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 bovine mesodermal cell types, PLG acts as an albumin replacement in serum-free media.

[0086] After proving independence of the PLG (with 1.8% w / w sunflower lecithin) 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 6. Figure 7 shows results for ovine and porcine cells; specifically primary ovine dermal fibroblasts, porcine adipose-derived stromal vascular fraction and primary porcine endothelial cells.

[0087] Fig. 7 panel A shows the population doublings obtained in 3 days of culture of primary ovine dermal fibroblasts for different concentrations of AlbuMAX I (data points represented as circles) and PLG (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 PLG sample was added. Culture conditions were identical to those described for Fig. 1, except for the seeding density, which was 3.200 cells / cm2for ovine dermal fibroblasts. It can be seen that for the tested concentration range PLG induces the same population doublings as AlbuMAX I. This proves that the effect of PLG as an albumin replacement is independent of the species of the cell within the mammalian order of Artiodactyla.

[0088] Fig. 7 panel B shows the population doublings obtained in 3 days of culture of adipose- derived porcine stromal vascular fractions for different concentrations of AlbuMAX I (data represented as circles) and PLG (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 PLG 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 / cm2and 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 PLG induces the same population doublings as AlbuMAX I.

[0089] Fig. 7 panel C 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 PLG (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 PLG 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 / cm2and 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 PLG 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 PLG 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 PLG. 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.

[0090] Figure 6, panels A to D and figure 7, panels A to C, prove the effect of PLG (with 1.8% w / w sunflower lecithin) 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 PLG (with 1.8% w / w sunflower lecithin) 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 7. More specifically, human bone marrow-derived mesenchymal stem cells, human kidney epithelial cells, African green monkey kidney epithelial cells, human cervical epithelial and human retinal epithelial cells were tested.

[0091] Fig. 8 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 PLG (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 PLG 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 / cm2and 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 PLG 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 PLG.

[0092] Fig. 8 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 points indicated as circles), PLG (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 replicates. The horizontal dotted line indicates the population doublings obtained when no PLG or albumin 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 / cm2and 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, 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. PLG can be seen to be partially replace approximately 90% of the effect of AlbuMAX I. It is clear that the addition of 1.8% (w / w) sunflower lecithin to PLG improves the performance and yields an effect equal to albumin proving that human mesodermal cells and cells exhibiting an epithelial phenotype that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of PLG (and sunflower lecithin).

[0093] Fig. 8 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 PLG (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 PLG 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 / cm2and 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 PLG induces the same as or more population doublings than AlbuMAX I. Clearly, based on figure 8 panels B and C, 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 PLG.

[0094] Fig. 8 panel D shows the population doublings obtained in 3 days of culture of human cervical cancer cells (HeLa) for different concentrations of AlbuMAX I (data points indicated as circles) and PLG (data points indicated as squares). 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 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 / cm2and 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, 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 10 ng / ml EGF (epidermal growth factor). Clearly, human mesodermal cells and cells exhibiting an epithelial phenotype that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of PLG.

[0095] Fig. 8 panel E 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 PLG (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 PLG 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 / cm2and 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 PLG 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 PLG.

[0096] After establishing PLG (with 1.8% w / w sunflower lecithin) 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 9, panels A, B, and C display results for primary murine FAPs, primary murine dermal fibroblasts, and Chinese hamster (Cricetulus griseus) ovary cells (CHO-K1).

[0097] Fig. 9 panel A shows the population doublings obtained in 3 days of culture of primary murine FAPs for different concentrations of AlbuMAX I (data represented as circles), and PLG (data represented as squares). Data is shown as mean + SEM based on 4 replicates. Culture conditions were identical to those described for Fig. 1. It can be seen that for the tested concentration range PLG induces the same as or more population doublings than AlbuMAX I. It should be noted that some differences in proliferation performance seem to exist comparing bovine FAPs (Figure 3) versus murine FAPs (Figure 9 panel A). Firstly, while PLG without sunflower lecithin is not sufficient to fully replace albumin for bovine FAPs (Fig. 2A and Fig. 3), PLG without sunflower lecithin is equipotent to albumin for murine FAPs (Fig. 9 panel A). Secondly, while bovine FAPs barely grow, but also do not die, in absence of AlbuMAX I or PLG (horizontal dotted line in Fig 2A), murine FAPs do die when cultured without AlbuMAX I or PLG (horizontal dotted line in Fig 9 panel A). 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. Clearly, primary murine FAPs that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of PLG. This proves that the effect of PLG as an albumin replacement is independent of the species of the cell from different mammalian orders.

[0098] Fig. 9 panel B shows the population doublings obtained in 3 days of culture of primary murine dermal fibroblasts for different concentrations of AlbuMAX I (data represented as circles) and PLG (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 PLG sample was added. Culture conditions were identical to those described for Fig. 1. It can be seen that for the tested concentration range PLG 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 PLG. 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 PLG 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 PLG. 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.

[0099] Fig. 9 panel C 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 PLG (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 PLG 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 / cm2and 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 PLG 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 PLG.

[0100] From Figures 1 to 9 it is clear that cultures of mammalian cell types from different embryonic lineages (i.e. mesodermal and ectodermal) exhibiting different morphologies (i.e. endothelial, mesenchymal and epithelial) that benefit from albumin benefit in the same way, or very similarly, to the presence of PLG (sometimes with sunflower lecithin). In Figures 1 to 9, optimal performance was consistently found when cultures were supplemented with 150 pM PLG (sometimes with sunflower lecithin). To extend and translate the albumin-replacing potential of 150 pM PLG (sometimes with sunflower lecithin) further from the mammalian bovine, ovine and murine fibroblasts, it was investigated if 150 pM PLG (sometimes with sunflower lecithin) can also replace albumin in cultures of fibroblasts from species belonging to other, non-mammalia, taxonomic classes within the phylum of Chordata and more specifically the subphylum of Vertebrate (i.e. vertebrates). Specifically, embryonic fibroblasts from chicken (class: Aves) and zebrafish (class: Actinopterygii) were tested and results are shown in Figure 10.

[0101] Fig. 10 panel A shows the population doublings obtained in 3 days of culture of embryonic chicken fibroblasts (SL-29) with 37.5 pM AlbuMAX I (grey bar), without albumin or PLG (white bar, labelled as ‘negative Ctrl’), 150 pM PLG (single hatched bar) or 150 pM PLG + 1.8% w / w sunflower lecithin (double hatched bar)). Data is shown as 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 40.000 cells / cm2and 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, 30 ng / ml FGF-2, and 100 ng / ml LR3-IGF1. It can be seen that PLG with sunflower lecithin induces the same population doublings as AlbuMAX I. Clearly, avian cells that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of PLG (with sunflower lecithin).

[0102] Fig. 10 panel B shows the population doublings obtained in 3 days of culture of embryonic zebrafish fibroblasts (ZF4) with 37.5 pM AlbuMAX I (grey bar), without albumin or PLG (white bar, labelled as ‘negative Ctrl’), 150 pM PLG (single hatched bar) or 150 pM PLG + 1.8% w / w sunflower lecithin (double hatched bar)). Data is shown as mean + SEM based on 4 replicates. 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 20.000 cells / cm2and 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 FGF-2 and 10 ng / ml LR3-IGF1. It can be seen that PLG (with sunflower lecithin) 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 white bar, both AlbuMAX I and PLG (with sunflower lecithin) improve survival to the same extent. Clearly, embryonic zebrafish fibroblasts that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of PLG (with sunflower lecithin). 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. Clearly, ray-finned fish cells that benefit from the presence of albumin benefit in the same way, or very similarly, to the presence of PLG (with sunflower lecithin).

[0103] Figures 1 to 10 clearly indicate that PLG (sometimes with sunflower lecithin) can act as a replacement for albumin in serum-free media to culture vertebrate cells exhibiting endothelial, mesenchymal or epithelial phenotypes.

[0104] The vertebrate 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:

[0105] • Bovine / ovine / murine fibro-adipogenic progenitor (FAP) cells o Obtained by isolation from muscle tissue as described by Messmer et al., Single-cell analysis of bovine muscle-derived cell types for cultured meat production, Front. Nutr., 13 September 2023

[0106] • 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.

[0107] • Bovine satellite cells (SC) o Obtained by isolation from muscle tissue as described by Messmer et al., Single-cell analysis of bovine muscle-derived cell types for cultured meat production, Front. Nutr., 13 September 2023

[0108] • Bovine / porcine 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, iScience, Volume 26, Issue 1, 20 January 2023.

[0109] • Bovine / porcine endothelial cells o Obtained by isolation from muscle tissue as described by Messmer et al., Single-cell analysis of bovine muscle-derived cell types for cultured meat production, Front. Nutr., 13 September 2023.

[0110] • 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), SL-29 (CRL-1590), ZF4 (CRL- 2050), bone marrow-derived human mesenchymal stem cells (PCS-500- 012)

[0111] 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.

[0112] Based on the similarity in curve shapes for albumin and PLG (sometimes with sunflower lecithin) in the aforementioned figures, the survival and / or proliferative response to either albumin or PLG (sometimes with sunflower lecithin)may be based on a similar mechanism. As the observations in aforementioned figures indicated that the full effect of albumin can be replaced by PLG (sometimes with sunflower lecithin), it was investigated whether partial substitution of albumin by PLG would add up to the effect that would be elicited by just albumin. Figure 11 shows the population doublings obtained in 3 days of culture of primary bovine FAPs for different concentrations of PLG on top of different ‘baseline’ concentrations of AlbuMAX I. Presence of AlbuMAX I in the concentration of 45.112 pM is represented as circles, 11.28 pM is represented as squares, 2.82 pM is represented as triangles with apex pointing up, 0.705 pM is represented as triangles with apex pointing down, 0.176 pMis represented as hexagons, and 0 pM 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 PLG sample was added on top of the AlbuMAX I concentration (0-11.28 pM). The horizontal dashed line indicates the population doublings obtained when no PLG sample was added on top of 45.112 pM AlbuMAX I. Culture conditions were identical to those described for Fig. 1. It can be clearly seen that adding PLG protein in concentrations up to 150 pM on top of AlbuMAX I concentrations can promote population doublings up to and exceeding the effect obtained by 45.112 pM AlbuMAX I alone (dashed horizontal line). This shows that the effect elicited by a concentration of albumin can be matched by fully or partially replacing albumin with PLG. It should be noted that 150 pM PLG in absence of AlbuMAX I reaches about 90% of the effect of 45.112 pM AlbuMAX I alone, repeating the observation described in Figure 2 panel A. This effect can be improved further by adding sunflower lecithin as described for Figure 2 panels B and C and Figure 3.

[0113] Bovine PLG exists as different genetic variants with variants A and B being the most prevalent. Milk-derived, purified PLG usually comprises a mix of PLG 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 PLG product that comprises an undefined mix of PLG variants (mainly consisting of variants A and B). To determine if PLG’s growth-promoting and albumin-replacing effect depends on the PLG variant and therefore amino acid sequence, pure PLG variant A and pure PLG variant B were tested for their proliferation-enhancing effect and potential variations therein.

[0114] Fig. 12 shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs for 150 pM PLG comprising an undefined mix of different variants (data point indicated as square; labelled as PLG-A / B) and different concentrations of PLG variant A (data points indicated as triangles; labelled as PLG- A) and PLG variant B (data points indicated as diamonds (labelled as PLG-B). Commercial product codes are indicated in the figure legend. Data is shown as mean + SEM based on 4 replicates. The horizontal dotted line indicates the population doublings obtained when no PLG was added. The horizontal dashdotted line indicates the population doublings obtained for 150 pM PLG 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.

[0115] All experiments shown used PLG derived from milk. In addition to purification from milk, PLG can be obtained from recombinant origin. In Figure 13, the effect of milk-derived PLG is compared to recombinant PLG. Use of recombinant PLG allows for animal-free media formulations.

[0116] Fig. 13 shows the population doublings obtained in 3 days of adherent culture of primary bovine FAPs for 150 pM PLG derived from bovine milk (grey bar), no PLG (white bar) and 150 pM PLG from recombinant origin (hatched bar). Data is shown as mean + SEM based on 4 replicates. Culture conditions were identical to those described for Fig. 1. It is clear that PLG from any origin, either from milk or recombinantly produced, has the same proliferation-enhancing effect. Medium supplementation with recombinant PLG would allow for animal-free medium formulations.

[0117] Data for PLG in Fig. 12 and Fig. 13 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.

[0118] Following up on the finding in Fig 3 that the addition of sunflower lecithin to PLG increases the number of obtained population doublings to the level of AlbuMAX I, but that this effect is not achieved with PLG alone, the difference in lipid composition of AlbuMAX I, PLG and PLG with sunflower lecithin was investigated by means of lipidomics. Since the sunflower lecithin used consists of 91% fat, it was investigated which lipid species are higher in quantity in AlbuMAX I compared to PLG but are present in equal amounts or higher than AlbuMAX I upon adding sunflower lecithin to the PLG. The analysed conditions / media correspond to conditions shown in Fig 3; AlbuMAX I (37.5 pM), PLG (150 pM) and PLG (150 pM) with 1.8% w / w sunflower lecithin. Lipidomic analysis of lipids higher in quantity in AlbuMAX I compared to PLG but present in equal amounts or higher than AlbuMAX I upon adding sunflower lecithin to the PLG is shown in Fig. 14. Figure 14 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 pM PLG condition are too small to be visualized. Strikingly, three specific lipid species, that were significantly enriched in AlbuMAX I compared to PLG, were not different between AlbuMAX I and PLG + sunflower lecithin. These species are l-linoleoyl-2-hydroxy-sn-glycero-3 -phosphocholine (LPC(18:2)), l-palmitoyl-2-linoleoyl-sn-glycero-3 -phosphocholine (PC(16:0_18:2)), and l-stearoyl-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 AlbuMAX I.

[0119] Based on the observation that sunflower lecithin addition on PLG improves the proliferation-promoting effect in some cases and that sunflower lecithin addition introduces lipid species that are absent in PLG, it was investigated why sunflower lecithin, but not rapeseed lecithin enhanced the effect of PLG (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).

[0120] Phospholipids with different polar heads can exert different biological functions. It was investigated, by means of lipidomics, if the identified lipid species in Fig. 14 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. 15 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.

[0121] 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.

[0122] 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 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.

[0123] Based on the lipidomic analyses shown in Fig. 14 and Fig. 15, 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.

[0124] 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 PLG has a more potent effect on the proliferation of certain cell types than just by using PLG alone is an unexpected effect, showing a potential synergy between these two components, the PLG 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.

[0125] PLG, PLG with (sunflower) lecithin or PLG with specific lipids can be obtained by using recombinant PLG and adding sunflower lecithin or specific lipids separately or by sourcing PLG from (commercial) whey (which may contain sunflower lecithin as an additive, i.e. as an emulsifier), or other milk-derived products.

[0126] 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.

[0127] 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.

[0128] 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” (http s : / / documents . th errnofi slier. com / TF S - Assets / LSG / manuals / 3117.pdf) which corresponds to effective concentrations found in Figures 1 to 10 for a variety of cell types.

[0129] 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. In other words, the medium does not contain supplemented albumin in an amount effective to provide the functions and / or results desired.

[0130] To recap, the media used in this invention is serum-free and free of supplemented albumin.

[0131] 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 or survival for specific cell types, PLG simply works to replace whatever that function(s) is( / are).

[0132] 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 6 panel D and Figure 7 panel B which feature cultures of stromal vascular fractions of bovine and porcine origin, respectively, and which comprises different cell types (e.g. mesenchymal stem cells, fibroblasts and endothelial cells).

[0133] The proof point that PLG (sometimes supplemented with sunflower lecithin / (lyso)phospholipids or not) is a viable substitute for albumin in vertebrate cells in general. As a final comment, the lack of growth for some cell types (negative PDs) indicates that even in the absence of an optimized medium lacking crucial components for growth, PLG (sometimes supplemented with sunflower lecithin) can replace albumin, in this case for its survival-enhancing effect, thus, one can infer that the effect of PLG is, in addition to, as previously shown, base medium independent (Figure 5), also independent of further medium components, especially since many different cell types and medium compositions were tested here. In short, the inventor states that the minimum medium composition that is to be supplemented required for obtaining an albumin replacing effect of PLG (with or without sunflower lecithin) is simply a basal medium.

[0134] 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, PLG can be used in smaller concentrations for the same effect (proliferation, survival), also an even better effect can be achieved when using PLG , since it performs at par with the best albumin. This also means that, at any point when replacing albumin with PLG , the same performance would be achieved with less PLG 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.

[0135] Exemplary Embodiments of the Invention

[0136] 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 Fl 2), 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 PLG 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 PLG for 1 M of albumin) or more (more than 1 M of PLG for 1 M 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.

[0137] The preferred dosage of PLG in the cell culture media based on the results obtained in all the trials is between 0.3 pM to 150 pM, simply because above 150 pM no better effect should be detectable, which is not to say more whey couldn’t be added; preferably 10 to 150 pM, more preferably 50 to 150 pM.

[0138] 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. The concentration of sunflower lecithin is preferably within the range 0.22-3.6% vi / vi, more preferably 1.8%.

[0139] 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 or survival of the cells in the culture when compared to albumin (or equivalent molecules) absence.

[0140] Further preferred embodiments may be taken from the specification as a whole, and this section should not be considered as limiting.

Claims

Claims1. 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 cell type that benefits from enhanced proliferation in the presence of albumin; c) Adding P-lactoglobulin to the cell culture medium in a concentration that provides the same or approximately the same proliferation improvement as albumin, preferably in a range of P-lactoglobulin concentration in the cell culture of 0.3 pM to about 150 pM; d) Allowing the cells to proliferate in the P-lactoglobulin containing culture medium.

2. A method according to claim 1 characterised by the cell types being vertebrate animal cells, or eukaryote, for example fish cells, for example avian cells, for example mammalian cells, preferably of mesodermal (exhibiting endothelial, mesenchymal, or epithelial morphology) or ectodermal origin (exhibiting epithelial morphology).

3. A method according to any of the preceding claims characterised by P-lactoglobulin being added to the cell culture at a concentration that replaces the desired effect of albumin, usually at a preferable relationship of 1 : 1 in molar concentration (1 M of PLG for 1 M of albumin) or optionally more (more than 1 M of PLG for 1 M of albumin) depending on the cell type.

4. A method according to any of the preceding claims wherein P-lactoglobulin is supplemented with sunflower lecithin.

5. A method according to claim 4 wherein the sunflower lecithin is added at a concentration in the range 0.22-3.6% w / w, more preferably 1.8% w / w.

6. A method according to any of the preceding claims wherein no supplemental albumin is added to the cell culture medium or wherein an amount of albumin is not enough to maximise the proliferation potential of the culture is added to the cell culture medium.

7. A method according to any of the preceding claims, wherein the P-lactoglobulin is present in the culture medium at a concentration of about 0.3 pM to about 150 pM, preferably 10 to 150 pM, more preferably 50 to 150 pM8. A method according to any of the preceding claims wherein the P-lactoglobulin is of bovine origin, preferably the genetic variant A.

9. A method according to any of the preceding claims wherein the P-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.

10. A serum -free medium for a cell culture characterised by comprising a base medium, P- lactoglobulin and no supplemental albumin, and wherein P-lactoglobulin is added at a concentration that replaces the desired effect of albumin, the medium optionally supplemented with sunflower lecithin.

11. The cell culture medium of claim 10, wherein the culture medium either has no supplemental albumin added to it or does not have enough albumin to substantially increase the proliferation performance of the culture.

12. The cell culture medium of any of claims 10 to 11, wherein the P-lactoglobulin is present at a concentration of about 0.3 pM to about 150 pM.

13. The cell culture medium of any of claims 10 to 12, wherein the P-lactoglobulin is of bovine origin, preferably the genetic variant A.

14. The cell culture medium of any of claims 10 to 13, wherein the P-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.

15. A medium according to claim 14 wherein the source of the (lyso)phospholipids is sunflower lecithin.

16. Use of P-lactoglobulin as an albumin replacement in a cell culture.

17. Use of P-lactoglobulin according to claim 16, wherein the P-lactoglobulin is present at a concentration of about 0.3 pM to about 150 pM.

Images