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WO-2026093215-A1 - REDUCTION OF ALBUMIN IN A CELL CULTURE MEDIUM

WO2026093215A1WO 2026093215 A1WO2026093215 A1WO 2026093215A1WO-2026093215-A1

Abstract

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

Inventors

  • PAES, Dean

Assignees

  • Mosa Meat B.V.

Dates

Publication Date
20260507
Application Date
20251027
Priority Date
20241029

Claims (13)

  1. 38/39 Claims
  2. 2. A method to improve cell proliferation or improve the survival of cells in a cell culture characterised by the steps of: a. Providing a serum-free medium, comprising a basal medium; b. Providing the cell culture with at least a mammalian cell type that benefits from enhanced proliferation in the presence of albumin; c. Supplementing the serum-free medium with whey in a concentration that provides the same cell proliferation or cell survival improvement as albumin, for a specific mammalian cell type, preferably in a range of whey concentration in the cell culture media of 0.1 to 10 mg/ml. d. Allowing the cells to proliferate in the whey containing cell culture.
  3. 3. A method according to claim 1 wherein the whey is a whey protein isolate or whey protein concentrate.
  4. 4. A method according to any of the preceding claims characterised by the cell types being of mammalian origin, preferably of mesodermal (exhibiting endothelial, mesenchymal, or epithelial morphology) or ectodermal origin (exhibiting epithelial morphology).
  5. 5. A method according to any of the preceding claims wherein the cell culture is of an animal cell type, more particularly of a mammalian cell type, more particularly of a mesenchymal lineage, more particularly a primary cell, more particularly a fibro adipogenic progenitor (FAP) or a satellite cell (SC), more particularly of a bovine origin.
  6. 6. A method according to any of the preceding claims wherein no supplemental albumin is added to the serum-free cell culture medium.
  7. 7. A method according to any of the preceding claims, wherein the whey concentration is 0.1 mg/ml to about 10 mg/ml, preferably 1 to 10 mg/ml, 39/39 preferably 2.5 to 10 mg/ml, preferably 3 to 10 mg/ml, more preferably 2.5 or 3 mg/ml.
  8. 8. A method according to any of the preceding claims, wherein the culture medium comprises besides a basal medium, preferably a carbon source, and preferably a growth factor.
  9. 9. A serum-free medium for a cell culture characterised by comprising a base medium, whey and no supplemental albumin, and wherein whey is added at a concentration that replaces the desired effect of albumin.
  10. 10. A serum-free medium according to claim 8, wherein the concentration of whey is 0.1 mg/mL to about 10 mg/ml, preferably 1 to 10 mg/ml of whey, preferably 2.5 to 10 mg/ml, preferably 3 to 10 mg/ml, more preferably 2.5 or 3 mg/ml.
  11. 11. A medium according to claims 8 or 9, further comprising: a. A carbon source; b. A growth factor; and c. Whey protein isolate or whey protein concentrate as a substitute for albumin.
  12. 12. Use of whey as an albumin replacement in a cell culture.
  13. 13. Use of whey according to claim 11, in a concentration of 0.1 mg/mL to about 10 mg/ml, preferably 1 to 10 mg/ml of whey, preferably 2.5 to 10 mg/ml, preferably 3 to 10 mg/ml, more preferably 2.5 or 3 mg/ml.

Description

DESCRIPTION TITLE: Reduction of albumin in a cell culture medium. TECHNICAL FIELD The invention is in the general field of biotechnology, specifically in the sub-field of media for cell cultures. BACKGROUND OF THE INVENTION It is known that in cell cultures of many animal, or more broadly eukaryote cell types, albumin (blood-derived or from recombinant origin) may be used as a component of the culture media to promote, or enhance, the proliferation of the cells. The use of blood-derived and/or recombinant albumin as a cell culture supplement is not ideal or undesirable considering a number of aspects. Blood-derived albumin poses ethical issues considering the fact that blood has to be drawn (repeatedly) from animals or animals have to be slaughtered to obtain the blood from which albumin can be purified. Human albumin can 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. 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. Different methods exist to purify both blood-derived and recombinant albumin. These methods can differentially affect the albumin protein and the bound molecules (e.g. lipids being removed during ethanol precipitation), which impacts its biological effect and the variation in biological activity across suppliers, products and batches. Or, in other words, the variation in lipids, oxidation and glycation affect the structure of the albumin protein, and the structure affects the function of the albumin. Many functions have been ascribed to albumin. For example, albumin can act as an antioxidant, in part owing to (the oxidation status of) its free cysteine residue, and can bind 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., Interaction