A cell culture medium and preparation method therefor

Abstract

The present invention relates to a cell culture medium comprising a hydrolysate composition. In particular, the hydrolysate composition is a plant protein hydrolysate, prepared from soy or wheat enzymatically hydrolysed with alcalase, flavourzyme, neutrase, or protamex. The cell culture medium is suited for culturing animal-derived cells in cultivated meat production, where it offers a cost-effective and sustainable alternative to conventional formulations, facilitating broader commercial adoption. Also disclosed is a method of preparing the cell culture medium.

Description

DESCRIPTIONTITLE OF INVENTION: [A CELL CULTURE MEDIUM AND PREPARATION METHOD THEREFOR]TECHNICAL FIELD

[0001] The present invention relates to the technical field of cell culture media, and more particularly to a cell culture medium comprising a hydrolysate composition, such as a plant protein hydrolysate (PPH).BACKGROUND

[0002] The following discussion of the background to the invention is intended to facilitate an understanding of the present invention only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was published, known or part of the common general knowledge of the person skilled in the art in any jurisdiction as at the priority date of the invention.

[0003] There is a growing demand for large-scale industrial cultivation of animal cells for a variety of applications, including vaccine production, biopharmaceutical manufacturing, and cell-based therapies. More recently, interest in cultivated meat has significantly increased due to global concerns surrounding food security and the sustainability of conventional animal agriculture. This has further intensified the need for scalable, cost-effective systems for producing animal cell biomass.

[0004] Currently, in vitro cultivation of animal cells relies heavily on animal-derived components, such as fetal bovine serum (FBS). However, FBS is costly due to limited supply and poses potential risks of contamination from animal-borne pathogens. In response, various research groups and commercial entities have developed serum-free and animal-component-free media, often comprising chemically defined components and recombinant growth factors produced via microbial expression systems. While these formulations offer a high degree of purity and reduce the risk of contamination, they are often prohibitively expensive and typically optimized for specific cell types. This limits their practical application in broader cell culture scenarios, such as the production of various cell types used in cultivated meat.

[0005] Accordingly, there remains a need for the development of cell culture media that are free from animal-derived components, or that reduce reliance on animal serum, while being cost-effective, broadly applicable to a wide range of cell types, and scalable for industrial production, addressing at least one of the limitations associated with conventional cell culture formulations, such as high cost, ethical concerns, variability, and limited supply.SUMMARY

[0006] Specifically, to address the above-mentioned technical problems, the present invention specifically uses the following technical solutions:

[0007] Accordingly, an aspect of the invention refers to a cell culture medium comprising a hydrolysate composition, wherein the hydrolysate composition is selected from the group consisting of a plant protein hydrolysate, a yeast protein hydrolysate, a microalgae protein hydrolysate, an insect protein hydrolysate, and any combination thereof.

[0008] In some embodiments, the plant protein hydrolysate is derived from a plant source, wherein the plant source is selected from the group consisting of Pisum sativum (pea), Sorghum bicolor (sorghum), Oryza sativa (rice), Triticum aestivum (wheat), Cannabis sativa (hemp), Chenopodium quinoa (quinoa), Glycine max (soybean), Zea mays (corn), Vigna radiata (mung bean), Vicia faba (fava bean), Cicer arietinum (chickpea), Lens culinaris (lentil), Arachis hypogaea (peanut), Prunus dulcis (almond), Salvia hispanica (chia seed), Solanum tuberosum (potato), seitan (wheat gluten), Avena sativa (oat), Hordeum vulgare (barley), brewer’s spent grain, and any combination thereof.

[0009] In some embodiments, the plant protein hydrolysate is produced by enzymatic hydrolysis.

[0010] In some embodiments, the enzymatic hydrolysis is carried out using one or more enzymes selected from the group consisting of protease, amylase, and multienzyme complex.

[0011] In some embodiments, the protease is selected from the group consisting of Alcalase, Flavourzyme, Neutrase, Protamex, Protana, Trypsin, Papain, Formea, and anycombination thereof.

[0012] In some embodiments, the amylase is selected from the group consisting of Termamyl, Maltogenase, Fungamyl, and any combination thereof.

[0013] In some embodiments, the multi-enzyme complex is selected from the group consisting of Vertera, Viscozyme, and any combination thereof.

[0014] In some embodiments, the hydrolysate composition is present in an amount ranging from 0.0000001 % to 99% weight by volume.

[0015] In some embodiments, the hydrolysate composition is present in an amount ranging from 0.001 % to 2% weight by volume.

[0016] In some embodiments, the cell culture medium further com prises animal serum.

[0017] In some embodiments, the animal serum is selected from the group consisting of fetal bovine serum, calf bovine serum, adult bovine serum, horse serum, porcine serum, goat serum, rabbit serum, fish serum, and any combination thereof.

[0018] In some embodiments, the basal medium (BM) is selected from the group consisting of Eagle's Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle Medium (DMEM), RPMI 1640, Ham’s F-10, Ham’s F-12, DMEM / F-12, and any combination thereof.

[0019] In some embodiments, the cell culture medium further com prises non-essential amino acids.

[0020] In some embodiments, the cell culture medium further comprises growth factor and / or hormone; wherein the growth factor optionally comprises fibroblast growth factor.

[0021] In some embodiments, the cell culture medium is suitable for culturing adherent cells, suspension cells, or a combination thereof.

[0022] In some embodiments, wherein the cell culture medium is suitable for culturing animal-derived cells for cultivated meat production.

[0023] In some embodiments, the animal-derived cells are derived from terrestrialanimals, wherein the terrestrial animals comprise bovine, porcine, ovine, caprine, and avian species.

[0024] In some embodiments, the animal-derived cells are derived from aquatic animals, wherein the aquatic animals comprise fish, shellfish, shrimp, and crustaceans.

[0025] Another aspect of the invention refers to a method of preparing cell culture medium, comprising the steps of: S100, preparing a hydrolysate composition; and S200, dissolving the hydrolysate composition in a basal medium (BM) to obtain the cell culture medium for culturing cell; wherein the step S100 comprises: S101 , enzymatically hydrolyzing a protein source using one or more enzymes to obtain a hydrolysis mixture; S102, inactivating the one or more enzymes in the hydrolysis mixture; and S103, filtering the hydrolysis mixture to remove insoluble components to obtain the hydrolysate composition.

[0026] In some embodiments, the protein source is selected from the group consisting of plant biomass, yeast biomass, microalgae biomass, insect biomass, protein extracts derived therefrom, and any combination thereof.

[0027] In some embodiments, the plant biomass is selected from the group consisting of Pisum sativum (pea), Sorghum bicolor (sorghum), Oryza sativa (rice), Triticum aestivum (wheat), Cannabis sativa (hemp), Chenopodium quinoa (quinoa), Glycine max (soybean), Zea mays (corn), Vigna radiata (mung bean), Vicia faba (fava bean), Cicer arietinum (chickpea), Lens culinaris (lentil), Arachis hypogaea (peanut), Prunus dulcis (almond), Salvia hispanica (chia seed), Solanum tuberosum (potato), seitan (wheat gluten), Avena sativa (oat), Hordeum vulgare (barley), brewer’s spent grain, and any combination thereof.

[0028] In some embodiments, the one or more enzymes are selected from the group consisting of protease, amylase, and multi-enzyme complex.

[0029] In some embodiments, the protease is selected from the group consisting of Alcalase, Flavourzyme, Neutrase, Protamex, Protana, Trypsin, Papain, Formea, and any combination thereof.

[0030] In some embodiments, the amylase is selected from the group consisting ofTermamyl, Maltogenase, Fungamyl, and any combination thereof.

[0031] In some embodiments, the multi-enzyme complex is selected from the group consisting of Vertera, Viscozyme, and any combination thereof.

[0032] In some embodiments, in the step S101 , enzymatical hydrolysis is conducted at a temperature in the range of 0 °C to 80 °C.

[0033] In some embodiments, in the step S102, the one or more enzymes are inactivated by heat inactivation.

[0034] In some embodiments, the heat inactivation is conducted at a temperature in the range of 85 °C to 130 °C.

[0035] In some embodiments, in the step S103, the hydrolysis mixture is filtered by centrifugation, membrane separation, or a combination thereof.

[0036] In some embodiments, the centrifugation is conducted at a centrifugal force in the range of 100 x g to 30,000 x g for a duration of 1 min to 1 ,440 mins.

[0037] In some embodiments, in the step S100, the preparation of the hydrolysate composition is performed at a pH in the range of 2 to 12.

[0038] In some embodiments, the preparation of the hydrolysate composition is performed at a pH in the range of 4 to 8.

[0039] In some embodiments, the step S101 is conducted for a duration in the range of 1 min to 1 ,440 mins.

[0040] In some embodiments, the step S200 further comprises step S201 , comprising removing precipitate from the cell culture medium after dissolving the hydrolysate composition in the basal medium (BM); wherein the cell culture medium is allowed to stand undisturbed to permit the precipitate to settle prior to removal.

[0041] In some embodiments, the step S100 further comprises step S104, in which the hydrolysate composition is subjected to a drying process.

[0042] In some embodiments, the drying process is selected from the group consistingof freeze drying, spray drying, vacuum drying, and any combination thereof.

[0043] Another aspect of the invention refers to a method of culturing a cell, comprising plating the cell in a cell culture medium according to the present invention.

[0044] In some embodiments, the method further comprises one or more of the steps of growing, passaging, expanding, splitting, or adapting the cell using the cell culture medium.

[0045] In some embodiments, the cell is animal-derived cells derived from terrestrial animals, wherein the terrestrial animals comprise bovine, porcine, ovine, caprine, and avian species.

[0046] In some embodiments, cell is animal-derived cells derived from aquatic animals, wherein the aquatic animals comprise fish, shellfish, shrimp, and crustaceans.BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In the figures, which illustrate, by way of non-limiting examples only, embodiments of the present invention,

[0048] [FIG. 1a - 1d]: illustrates the relative fold change of confluency of PSC in the presence of soy PPHs, commercial soy PPH, and unhydrolyzed protein without FBS after 3 days; In the 'All potential concentration' group, except for S14, the significance of the differences between the 13 PPHs and the negative group is indicated.

[0049] [FIG. 2a - 2d]: illustrates the relative fold change of confluency of PSC in the presence of wheat PPHs, commercial wheat PPH, and unhydrolyzed protein without FBS after 3 days; In the 'All tested concentration' group, except for W14, the significance of the differences between the 13 PPHs and the negative group is indicated.

[0050] [FIG. 3a - 3d]: illustrates the relative fold change of confluency of C2C12 in the presence of soy PPHs, commercial soy PPH, and unhydrolyzed protein without FBS after 3 days; In the 'All potential concentration' group, except for S14, the significance of the differences between the 13 PPHs and the negative group is indicated.

[0051] [FIG. 4a - 4d]: illustrates the relative fold change of confluency of C2C12 inthe presence of wheat PPHs, commercial wheat PPH, and unhydrolyzed protein without FBS after 3 days; In the 'All potential concentration' group, except for W14, the significance of the differences between the 13 PPHs and the negative group is indicated.

[0052] [FIG. 5a - 5b]: illustrates the relative fold change of PSC in the presence of 13 soy PPHs with 5% FBS (FIG. 5a, a) and 1 % FBS (FIG. 5a, b); 13 wheat PPHs with 5% FBS (FIG. 5b, a) and 1 % FBS (FIG. 5b, b) in 4 days.

[0053] [FIG. 6a - 6b]: illustrates the relative fold change of C2C12 in the presence of 13 soy PPHs with 5% FBS (FIG. 6a, a) and 1 % FBS (FIG. 6a, b); 13 wheat PPHs with 5% FBS (FIG. 6b, a) and 1 % FBS (FIG. 6b, b) in 3 days.

[0054] [FIG. 7]: illustrates the cell images of PSC after 6 days of differentiation.

[0055] [FIG. 8]: illustrates a schematic diagram of the PPH production process according to some embodiments of the present invention.

[0056] [FIG. 9]: illustrates a schematic diagram providing a conceptual summary of the bioactivities of PPHs and growth factors that mimic the functional roles of serum.

[0057] DETAILED DESCRIPTION

[0058] Throughout this document, unless otherwise indicated to the contrary, the terms “comprising”, “consisting of’, “having” and the like, are to be construed as non-exhaustive, or in other words, as meaning “including, but not limited to”.

[0059] Furthermore, throughout the document, unless the context requires otherwise, the word “include” or variations such as “includes” or “including” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0060] Throughout this document, the term “protein hydrolysate” refers to a composition comprising peptides, free amino acids, and partially hydrolyzed proteins, produced by subjecting protein-containing materials to enzymatic, acidic, alkaline, or other suitable hydrolysis processes. The protein hydrolysate may be prepared either directly from raw biological materials, such as whole plant matter (e g., soybean, wheat), yeast biomass, microalgae, or insects, or from protein extracts derived from thesematerials prior to hydrolysis. In the context of the present invention, hydrolysates prepared from plant sources are referred to as “plant protein hydrolysates (PPH)”, those from yeast as “yeast protein hydrolysates", from microalgae as “microalgae protein hydrolysates”, and from insects as “insect protein hydrolysates”. The definition encompasses hydrolysates obtained through direct hydrolysis of intact biomass as well as those prepared from isolated or partially purified protein fractions. The term also encompasses both crude and purified hydrolysates, and includes hydrolysates that have been subjected to further processing steps such as centrifugation, filtration, lyophilization, or pH adjustment to remove insoluble fractions or concentrate bioactive peptides.

[0061] An aspect of the present invention relates to an improved cell culture medium comprising a hydrolysate composition. The hydrolysate composition is selected from the group consisting of a plant protein hydrolysate, a yeast protein hydrolysate, a microalgae protein hydrolysate, an insect protein hydrolysate, and any combination thereof. Plant, yeast, microalgae, and insect biomasses are widely available in nature and are known to be rich sources of proteins containing essential amino acids and conserved peptide motifs. When subjected to appropriate hydrolysis methods, such as enzymatic hydrolysis as described in later sections, these protein sources can yield a protein hydrolysate, comprising a mixture of small peptides and free amino acids. These protein hydrolysates not only provide nutritional support but may also exert bioactive effects that promote cell growth and regulate cellular metabolism. Accordingly, these protein lysates could serve as functional components in the cell culture medium, supporting cell proliferation and maintenance while reducing or eliminating the need for animal-derived additives such as serum.

[0062] More specifically, the present invention attempts to alleviate the challenge of reducing or replacing serum and other animal-derived components in the formulation of animal cell culture media. To achieve this, the present invention provides a method for producing PPHs suitable for use in cell culture (i.e., as a supplement for cell culture medium). As shown in FIG. 8, the method of the present invention involves enzymatic hydrolysis of protein sources (e.g., whole plant matter (e.g., soybean, wheat), yeast biomass, microalgae, or insects, or from protein extracts derived from these materials), with process parameters including the choice of proteolytic enzymes, the functional temperature range for enzymatic activity, the duration of hydrolysis, and the selection ofplant protein sources.

[0063] In various embodiments, the enzymatic system comprises endoproteases, exoproteases, or enzymes exhibiting both types of proteolytic activity, in order to enhance hydrolysis efficiency and produce a diverse peptide profile. In some embodiments, the enzymatic hydrolysis is carried out using one or more enzymes selected from the group consisting of protease, amylase, and multi-enzyme complex: wherein the protease is selected from the group consisting of Alcalase, Flavourzyme, Neutrase, Protamex, Protana, Trypsin, Papain, Formea, and any combination thereof; the amylase is selected from the group consisting of Termamyl, Maltogenase, Fungamyl, and any combination thereof; and, the multi-enzyme complex is selected from the group consisting of Vertera, Viscozyme, and any combination thereof.

[0064] The hydrolysis process is designed to operate across a broad temperature range of 0 °C to 100 °C, provided that the selected enzyme remains active within the specified range. The reaction may be carried out for a duration of up to 24 hours (i.e. , 1 ,440 minutes), depending on the enzyme characteristics and desired degree of hydrolysis.

[0065] A variety of protein sources can be utilized as starting materials to produce hydrolysates with amino acid profiles and functional characteristics. For plant protein source, examples include Pisum sativum (pea), Sorghum bicolor (sorghum), Oryza sativa (rice), Triticum aestivum (wheat), Cannabis sativa (hemp), Chenopodium quinoa (quinoa), Glycine max (soybean), Zea mays (corn), Vigna radiata (mung bean), Vicia faba (fava bean), Cicer arietinum (chickpea), Lens culinaris (lentil), Arachis hypogaea (peanut), Prunus dulcis (almond), Salvia hispanica (chia seed), Solanum tuberosum (potato), seitan (wheat gluten), Avena sativa (oat), Hordeum vulgare (barley), brewer’s spent grain, and any combination thereof.

[0066] In some embodiments of the formulated media, the concentration of plant protein hydrolysates (PPHs) may range from 0.01 % to 5% (w / v), depending on the target cell type and application. In certain embodiments, the concentration of the hydrolysate composition may be broader, ranging from 0.0000001 % to 99% (w / v). In yet other embodiments, the hydrolysate composition may be present in a range from 0.001 % to 2% (w / v), to balance cost-effectiveness and functional performance in supporting cell growth.

[0067] In some embodiments of the present invention, the hydrolysate composition incorporated into the cell culture medium may function in a manner analogous to growth factors, which are typically effective at nanogram-per-milliliter (ng / mL) concentrations. Accordingly, the hydrolysate composition may be used in very small amounts (similar to growth factors) to achieve functional support of cell growth. For example, plant protein hydrolysates (PPHs) may be included at concentrations as low as 0.0000001 % (w / v) (i.e. , 1 x 1 O"7%) in the cell culture medium.

[0068] In other embodiments, the hydrolysate composition may constitute the major component of the cell culture medium. When coupled with enzymatic treatments (e.g., amylase hydrolysis), plant protein hydrolysates can serve as a primary nutritional source by supplying both amino acids and carbohydrates. For instance, typical plant protein products comprising approximately 80-90% protein and 10% carbohydrate, while many plant seeds naturally contain 30-40% protein and 20-30% carbohydrate. As such, even a cell culture medium composed primarily of protein lysates, including PPHs at concentrations approaching 99%, may be capable of sustaining cell growth by fulfilling essential nutritional requirements. This flexible formulation enables either partial or complete replacement of serum, depending on the specific requirements of the target cell type or the intended production process (see FIG. 9). The resulting media offer a cost- effective, scalable, and animal-component-reduced alternative to conventional serumcontaining formulations, thereby enhancing their applicability across both research and industrial settings.

[0069] Thus, the present invention provides a plant-based cell culture medium that reduces or eliminates the need for animal-derived supplements such as fetal bovine serum (FBS), which is widely used in conventional cell culture. FBS is derived from the blood of animal fetuses, raising significant ethical concerns, and is limited in supply. It also carries inherent risks of contamination by animal-borne pathogens. While synthetic growth factors and chemically defined media have been explored as alternatives, these formulations are often prohibitively expensive to manufacture at scale and are typically tailored to specific cell types, thereby limiting their broader applicability.

[0070] In contrast, the cell culture medium disclosed in the present invention incorporates plant-, yeast-, microalgae-, or insect-derived protein hydrolysates produced via controlled enzymatic hydrolysis. These hydrolysates offer a more sustainable, safer, toand cost-effective alternative to animal-based components. Functionally, they support cell proliferation and viability, enabling significant reductions in the use of both serum and recombinant growth factors. This is particularly advantageous in the context of cultivated meat (CBM) production, where the high cost, scalability limitations, and ethical concerns associated with FBS and synthetic proteins have been major barriers to commercial viability. The improved medium directly addresses these limitations, facilitating the development of scalable, economically feasible CBM manufacturing processes.

[0071] Beyond cultivated meat, the advantages of the present invention extend to a wide range of cell manufacturing applications, including vaccine development, recombinant protein expression, and the production of therapeutic biologies. In academic and industrial research environments, the reduced-cost, animal-component-free formulation may lower the barriers to access for advanced cell culture technologies. Overall, the invention provides a robust, flexible, and ethically aligned platform for diverse biotechnology applications, advancing both economic and sustainability goals in modern life science industries.

[0072] EMBODIMENT 1

[0073] Preparation of PPHs

[0074] In one embodiment, soy- and wheat-based PPHs were prepared using enzyme- assisted aqueous extraction. Hydrolysis was performed using a range of proteolytic enzymes, with pH and temperature conditions selected based on manufacturer- recommended specifications, as summarized in Table 1. The reaction pH was adjusted using 0.1 M NaOH or 0.1 M HCI, and temperature control was maintained using a temperature-controlled water bath (Julabo SW22, Julabo, Pennsylvania, USA) under continuous agitation. Each hydrolysis reaction was conducted by combining 10 g of soy or wheat protein (5% w / w) with 0.2 g of enzyme (2% w / w relative to protein mass) in 200 mL of deionized water. Four different enzymes were used, and each reaction was incubated for one of three durations: 0.5 hours, 2 hours, or 24 hours. Enzymatic activity was terminated by heating the mixture to 95 °C for 15 minutes.Table.1 Details of Enzymes And The Hydrolysis Conditions Used.

[0075] The hydrolysate mixtures were subsequently centrifuged at 3000 x g for 1 hour at 4 °C. The resulting supernatants, which contain the soluble protein hydrolysates, were collected and lyophilized for long-term storage at -20 °C. The designations for each PPH supernatant prepared under the various enzyme and incubation conditions are summarized in Table 2.

[0076] Specifically, various soy-based and wheat-based protein hydrolysates were prepared using different proteolytic enzymes and hydrolysis durations. Specifically, samples S1 to S3 are soy-based protein hydrolysates produced via enzymatic hydrolysis with Alcalase, with hydrolysis times ranging from 30 minutes to 24 hours. Similarly, S4 to S6 were prepared using Flavourzyme, S7 to S9 using Neutrase, and S10 to S12 using Protamex, each with hydrolysis durations ranging from 30 minutes to 24 hours.

[0077] In parallel, wheat-based protein hydrolysates were prepared using the same enzymes under similar conditions: W1 to W3 were hydrolyzed with Alcalase, W4 to W6 with Flavourzyme, W7 to W9 with Neutrase, and W10 to W12 with Protamex, each group also processed at hydrolysis times ranging from 30 minutes to 24 hours.

[0078] For comparison, S13 and W13 represent commercially available soy-based and wheat-based protein hydrolysates, respectively, while S14 and W14 refer to unhydrolyzed plant proteins derived from soy and wheat.Table.2 The Abbreviations of All PPHs Lyophilized Powers.

[0079] Characterisation of PPHs

[0080] Each PPH was subjected to proximate analysis to determine its protein, fat, ash, carbohydrate, and moisture content. Total protein content was measured using the Kjeldahl method (VAPODEST 20, Gerhardt GmbH & Co. KG, Germany) in accordance with the Association of Official Analytical Chemists (AOAC, 1995) procedures. Crude fat content was extracted using hexane and quantified via a Soxtherm rapid Soxhlet system (SOXTHERM, C. Gerhardt GmbH & Co. KG, Germany), following AOAC (1995) guidelines. Ash content was determined using a muffle furnace (Vulcan 3-1750, NeyTech Co. Ltd., Burlington, USA) in accordance with AOAC (1995) protocols. Moisture content was measured using a moisture balance (MOC-120H, Shimadzu Co. Ltd., Kyoto, Japan).

[0081] Statistical Analysis

[0082] All analyses were conducted in triplicate (n = 3). Data are presented as mean ± standard deviation, calculated using the AVERAGE and STDEVA functions in Microsoft Excel (version 2306). Statistical analysis was performed using GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, CA, USA).

[0083] Results and AnalysisTable.3 Compositional analysis of PPHs of soy protein hydrolyzed by Alcalase.Table 9. Compositional analysis of PPHs of wheat protein hydrolyzed by Neutrase.Hydrolysis Carbohydrate

[0084] The analysis of all tested hydrolysates showed that the composition fell within the following ranges: protein content ranged from 75% to 95%, ash content from 0.01 % to 5%, fat content from 0.01 % to 5%, and carbohydrate content from 0.01 % to 20%.

[0085] EMBODIMENT 2

[0086] Preparation of Cells

[0087] All the cells were grown in a standard growth medium (SGM). SGM provides cells with nutrients to achieve stable, reproducible, and relatively fast growth. The formulation of SGM is listed in Table 1 1 . For general cell maintenance, SGM was replaced daily and vessels were incubated at 37 °C in a 5% CO2atmosphere. Cells were grown to 70%-80% confluence as inspected visually, and the cell count was assessed using a haemocytometer. The cells were either passaged by treatment with 0.5% trypsin-EDTA and incubated for at least 3 minutes, or stored for long-term preservation in liquid nitrogen with 10% dimethyl sulfoxide in SGM. C2C12 cells were purchased from Accegen (#ABC- TC0091 ; Fairfield, NJ, USA). Porcine satellite cells (PSC) were isolated in-house following the method described by Zhu et al. (2022). C2C12 cells were cultured on regular cell culture plates without coating. Primary cells were cultured in cell culture plates (#20101 ,#30006, and #30096; SPL LIFE SCIENCES, Korea) coated with Matrigel (#354234; Corning, NY, USA).

[0088] Preparation of media supplied with PPHs

[0089] To supplement PPHs in cell culture media (CCM), each PPH was first dissolved in basal media (BM) at a concentration of 10% (w / v) to prepare a stock solution. The pH was adjusted to 7.4 using 0.1 M NaOH or 0.1 M HCI. The stock solutions were then centrifuged at 8000 x g for 1 hour at room temperature to remove any undissolved components. The formulation of BM is listed in Table 1 1.

[0090] Prior to each experiment, the wheat-based PPH stock solution was diluted to the required concentration using BM. The resulting PPH-containing medium was then mixed with SGM to prepare the final CCM containing 1 % or 5% FBS and the desired concentration of PPHs.

[0091] For soy-based PPHs, the stock solution was incubated in the dark at room temperature overnight, followed by centrifugation at 10000 x g for 1 hour to remove insoluble components. The supernatant was then diluted with SGM to obtain the required concentration.

[0092] All PPH solutions were sterilized using 0.22 pm polyethersulfone syringe filters (Minisart, Sartorius, Goettingen, Germany).

[0093] Growth Analysis of Cells

[0094] Cell growth was analyzed using cells harvested with 0.5% trypsin-EDTA after reaching 70%-80% confluence. The detached cells were seeded into 96-well plates at a density of 3000 cells / cm2in 100 pL of SGM per well. After 24 hours, the SGM was removed, and cells were washed once with 200 pL of 1 x PBS to eliminate residual media. Subsequently, fresh test media, comprising PPHs diluted in basal media (BM) at the required concentration, was added to each well.

[0095] Cell confluency was monitored every 6 hours using the IncuCyte® S3 Live-Cell Analysis System (Sartorius, Gottingen, Germany) at 4x magnification. At each time point, five brightfield images were captured from different locations within each well. Images were analyzed using IncuCyte S3 v2017A software. The analysis parameters weremanually adjusted based on cell morphology to ensure accurate segmentation of cells from background, using the following settings: segmentation adjustment = 0.2; hole fill = 500 pm2; adjust size = 4 pixels; minimum cell width = 1000 pm.

[0096] Statistical Analysis

[0097] Data from each group were presented as mean ± standard deviation, calculated using the AVERAGE and STDEVA functions in Microsoft Excel (version 2306). Statistical analyses were performed using GraphPad Prism 9.5.1 (GraphPad Software, San Diego, GA, USA). One-way ANOVA followed by Tukey’s post hoc test was used to evaluate statistical differences. A p-value of <0.05 was considered statistically significant. Significance levels are indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

[0098] The total serum replacement experiment was conducted using two biological replicates for each PPH and two technical replicates per cell line (n = 4).Table 10. Compositional analysis of PPHs of wheat protein hydrolyzed by Protamex

[0099] Results and Analysis

[0100] The effect of various PPHs on porcine satellite cell (PSC) growth in the absence of FBS was evaluated. Thirteen different soy PPHs and one unhydrolyzed soy protein sample were individually supplemented into SGM without FBS, and the relative fold change in cell confluency was measured over a 3-day period. The results are summarized in FIG. 1a - 1 d. The confluency of PSCs cultured in complete SGM (with FBS) for 3 days was used as a normalization reference (set to 1 ) for evaluating the performance of each PPH under serum-free conditions.

[0101] Most PPHs exhibited a concentration-dependent trend in which higher concentrations were detrimental to cell proliferation. Several PPHs also showed reducedsupport for growth at specific concentrations. For example, soy hydrolysate S1 induced a relative fold change in confluency of 23 ± 9% after 3 days, comparable to the negative control (basal medium (BM) only, 14 ± 3%). Supplementation of S1 at 0.5%, 1%, and 2% concentrations did not yield significant improvements in PSC growth. However, supplementation with 0.1 % S1 significantly increased confluency to 32 ± 6%, indicating a concentration-specific effect. Among all tested soy PPHs, 0.1 % S4 showed the highest relative fold change at 42 ± 2%.

[0102] In contrast, unhydrolyzed soy protein did not significantly improve PSC confluency at any concentration tested. Similarly, unhydrolyzed wheat protein failed to support cell growth across all doses. As shown in FIG. 2a - 2d (“All concentrations”), the optimal concentrations of each PPH resulted in comparable relative fold changes in confluency. However, the level of statistical significance relative to the negative control varied, suggesting that different PPHs may exert their effects through distinct mechanisms.

[0103] Wheat-derived PPHs demonstrated similar dose-dependent effects on PSC confluency. The highest confluency observed among wheat PPHs was 38 ± 6%, achieved with 0.5% W5.

[0104] A similar trend was observed when the PPHs were tested on C2C12 mouse myoblast cells (FIG. 3a - 3d and FIG. 4a - 4d). The highest confluency was recorded at 46 ± 11 % with 2% W6, followed by 35 ± 8% with 0.5% S8. Notably, 0.1 % S4 also induced a significant increase in confluency (35 ±0.9%), further supporting the observation that low concentrations of specific PPHs promote cell growth.

[0105] Among the tested PPHs, those prepared using Flavourzyme consistently supported higher proliferation in both PSC and C2C12 cells compared to other enzyme treatments.

[0106] In summary, unhydrolyzed soy and wheat proteins were not effective in sustaining PSC growth in the absence of FBS. In contrast, specific soy and wheat PPHs significantly enhanced cell confluency under serum-free conditions, confirming their potential as effective and scalable alternatives to animal-derived serum supplements in cell culture media.

[0107] EMBODIMENT S

[0108] Preparation of Cells

[0109] All cells were cultured in SGM, which provides essential nutrients to support stable, reproducible, and relatively rapid cell growth. The formulation of SGM is listed in Table 11. For routine maintenance, the SGM was replaced daily, and culture vessels were incubated at 37 °C in a humidified incubator containing 5% CO2.

[0110] Cells were maintained until reaching 70%-80% confluence, as determined by visual inspection. Cell numbers were assessed using a haemocytometer. For subculturing, cells were treated with 0.5% trypsin-EDTA and incubated for at least 3 minutes at 37 °C. Cells were either passaged for continued culture or cryopreserved in liquid nitrogen using SGM supplemented with 10% dimethyl sulfoxide (DMSO).

[0111] C2C12 mouse myoblast cells were purchased from Accegen (#ABC-TC0091 ;Fairfield, NJ, USA) and cultured on standard, uncoated tissue culture plates. Porcine satellite cells (PSCs) were isolated in-house following the protocol described by Zhu et al. (2022). Primary cells were cultured in Matrigel-coated culture plates (#20101 , #30006, and #30096; SPL Life Sciences, Korea) using Matrigel (#354234; Corning, NY, USA) as the extracellular matrix.

[0112] Preparation of Media Supplemented with PPHs

[0113] To prepare cell culture media (CCM) supplemented with PPHs, each PPH was first dissolved in basal medium (BM) at a concentration of 10% (w / v) to form a stock solution. The pH was adjusted to 7.4 using either 0.1 M NaOH or 0.1 M HCI. The stock solutions were then centrifuged at 8000 x g for 1 hour at room temperature to remove undissolved components. The formulation of BM is provided in Table 11 .

[0114] Prior to each experiment, wheat-based PPH stock solutions were diluted to the required concentration using BM. The resulting PPH-containing medium was then mixed with SGM to generate the final CCM, containing either 1 % or 5% FBS and the desired PPH concentration.

[0115] For soy-based PPHs, the stock solution was incubated in the dark at room temperature overnight, followed by centrifugation at 10000 x g for 1 hour to removeinsoluble components. The supernatant was then diluted with SGM to reach the final working concentration.

[0116] All PPH-containing solutions were sterilized using 0.22 pm polyethersulfone syringe filters (Minisart, Sartorius, Goettingen, Germany) prior to use in cell culture.

[0117] Growth Analysis of Cells

[0118] Cell growth analysis was performed using cells harvested at 70%-80% confluence via treatment with 0.5% trypsin-EDTA. The detached cells were seeded into 96-well plates at a density of 3000 cells / cm2in 100 pL of SGM per well. After 24 hours of incubation, the SGM was removed, and the cells were washed once with 200 pL of 1 * PBS to eliminate residual medium. Fresh test media, comprising PPHs diluted in basal medium (BM) to the desired concentrations, was then added.

[0119] Cell confluency was monitored every 6 hours using the IncuCyte® S3 Live-Cell Analysis System (Sartorius, Gottingen, Germany) under 4* magnification. At each time point, five brightfield images were captured from different locations within each well and analyzed using IncuCyte S3 software (version 2017A).

[0120] To ensure accurate segmentation of cells from the background, image analysis parameters were manually adjusted based on cell morphology. The settings applied were: segmentation adjustment = 0.2; hole fill = 500 pm2; size adjustment = 4 pixels; and minimum cell width = 1000 pm.

[0121] Statistical Analysis

[0122] All data are presented as mean ± standard deviation (SD), calculated using the AVERAGE and STDEVA functions in Microsoft Excel (version 2306). Statistical analyses were conducted using GraphPad Prism software (version 9.5.1 ; GraphPad Software, San Diego, CA, USA).

[0123] Group differences were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p-value of less than 0.05 was considered statistically significant. Statistical significance is indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

[0124] The partial serum replacement experiment was conducted using two independent PPH replicates and three biological cell replicates (n = 6).

[0125] Results and Analysis

[0126] FIG. 5a - 5b presents the results of soy and wheat PPHs supplemented into cell culture media (COM), demonstrating their ability to support porcine satellite cell (PSC) growth under reduced serum conditions. As expected, PSC proliferation increased in a dose-dependent manner with rising concentrations of FBS. In CCM containing only 1 % FBS, PSC growth was minimal. With 5% FBS, moderate growth was observed, but the results were poorly reproducible.

[0127] Notably, when basal medium (BM) was supplemented with specific soy PPHs, namely S4, S5, and S10, PSC confluency increased significantly compared to SGM. Among these, 0.1 % S4 induced the highest relative fold change in confluency, reaching 157 ±9%, suggesting that S4 has a strong capacity to enhance PSC growth and potentially reduce or replace FBS supplementation.

[0128] Under 1 % FBS conditions, several soy-derived PPHs also significantly improved PSC proliferation. Specifically, S2, S3, S4, S7, S8, S10, and S12 resulted in significantly higher confluency relative to 1 % FBS alone. In fact, the growth-promoting effects of these PPHs were comparable to those observed with 5% FBS. Among these, 0.1% S10 achieved the highest fold change in confluency at 40 ± 12%, while 0.1% S4 also performed well, yielding a 36 ± 25% increase.

[0129] Wheat-derived PPHs exhibited similar trends. At 5% FBS, 0.05% W4 induced the highest relative fold change in confluency (191 ± 58%). Under 1 % FBS, W4 again demonstrated strong efficacy, with a fold change of 44 ±24%, indicating its potential as a serum-reducing additive.

[0130] Similar results were observed in C2C12 cells, as shown in FIG. 6a - 6b. Both soy and wheat PPHs enhanced C2C12 cell growth under low-serum conditions in a manner consistent with the results obtained for PSCs.

[0131] These findings collectively highlight the potential of soy and wheat PPHs as effective supplements for reducing FBS concentrations in cell culture media, therebyoffering a scalable strategy for at least partial serum replacement.

[0132] EMBODIMENT 4

[0133] Preparation of Cells

[0134] All cells were cultured in SGM, which provides essential nutrients to support stable, reproducible, and relatively rapid cell proliferation. The formulation of SGM is listed in Table 11. For routine maintenance, the SGM was replaced daily, and culture vessels were incubated at 37 °C in a humidified atmosphere containing 5% CO2.

[0135] Cells were maintained until reaching 70%-80% confluence, as determined by visual inspection. Cell density was assessed using a haemocytometer. For passaging, cells were treated with 0.5% trypsin-EDTA and incubated for at least 3 minutes at 37 °C. After detachment, cells were either subcultured or cryopreserved in liquid nitrogen using SGM supplemented with 10% dimethyl sulfoxide (DMSO).

[0136] Porcine satellite cells (PSCs) were isolated in-house following the protocol described by Zhu et al. (2022). Primary cells were seeded into Matrigel-coated culture plates (#20101 , #30006, and #30096; SPL Life Sciences, Korea) using Matrigel (#354234; Corning, NY, USA) as the extracellular matrix.

[0137] Preparation of Media Supplemented with PPHs

[0138] To prepare cell culture media (CCM) supplemented with PPHs, each PPH was first dissolved in basal medium (BM) at a concentration of 10% (w / v) to form a stock solution. The pH was adjusted to 7.4 using 0.1 M NaOH or 0.1 M HCI. The stock solutions were then centrifuged at 8000 x g for 1 hour at room temperature to remove any undissolved components. The formulation of BM is provided in Table 11 .

[0139] Before each experiment, the wheat-based PPH stock solution was diluted to the desired concentration using BM. The resulting solution was then mixed with SGM to prepare the final CCM containing either 1 % or 5% FBS and the required concentration of PPHs.

[0140] For soy-based PPHs, the stock solution was incubated overnight in the dark at room temperature, followed by centrifugation at 10000 x g for 1 hour to remove insolublecomponents. The clarified supernatant was then diluted in SGM to the desired working concentration.

[0141] All prepared PPH solutions were sterilized using 0.22 pm polyethersulfone syringe filters (Minisart, Sartorius, Goettingen, Germany) prior to use in cell culture.

[0142] Differentiation of Cells

[0143] Cell differentiation was carried out as follows: Cells were seeded at a density of 6000 cells / cm2in 3 mL of SGM in 6-well cell culture plates. After 24 hours, the SGM was removed, and cells were washed once with 2 mL of 1 x PBS. Test media, comprising PPHs diluted in basal medium (BM) to the desired concentrations, was then added.

[0144] Cells were cultured in the test media until reaching 70%-80% confluence, followed by two additional PBS washes. Differentiation was then induced by replacing the culture medium with differentiation medium consisting of 2% horse serum (#16050130; ThermoFisher Scientific, MA, USA) in DMEM / F12. During the differentiation process, 30% of the medium was replaced with fresh differentiation medium every 2 days. After 6 days of differentiation, the cells were imaged.

[0145] Results and Analysis

[0146] FIG. 7 shows representative images of cells differentiated under various culture conditions and imaged after 6 days. Cells cultured in SGM served as the positive control and exhibited successful differentiation, as evidenced by the formation of multinucleated myotubes, indicative of myogenic fusion events.

[0147] Cells cultured in low-serum media supplemented with plant protein hydrolysates (PPHs), specifically S4 (soy-based, hydrolyzed with Flavourzyme for 30 minutes), S5 (soy-based, hydrolyzed with Flavourzyme for 2 hours), W4 (wheat-based, hydrolyzed with Flavourzyme for 30 minutes), and W11 (wheat-based, hydrolyzed with Protamex for 30 minutes), also exhibited successful differentiation, with visible formation of myotubes similar to those in the SGM control (see FIG. 7, the middle and right panels). These results indicate that the presence of PPHs in combination with low serum concentrations does not impair the differentiation capacity of porcine satellite cells (PSCs), supporting the compatibility of PPH-supplemented media for both proliferation anddifferentiation stages.

[0148] In addition to the PPHs illustrated in FIG. 7, other plant protein hydrolysates, such as S2 (soy-based, hydrolyzed with Alcalase for 2 hours), S7 (soy-based, hydrolyzed with Neutrase for30 minutes), S10 (soy-based, hydrolyzed with Protamexfor30 minutes), W3 (wheat-based, hydrolyzed with Alcalase for 24 hours), and W7 (wheat-based, hydrolyzed with Neutrase for 30 minutes), also exhibited similar capabilities in promoting cellular differentiation, as evidenced by the visible formation of myotubes. These PPHs may therefore be suitably used as the hydrolysate composition in the formulation of cell culture media as well.

[0149] EMBODIMENT S

[0150] An aspect of the present invention relates to a composition of plant protein hydrolysate or hydrolysates that can be used as a supplement to reduce the serum amount required in animal cell culture.

[0151] In some embodiments, the serum includes the fetal bovine serum, fetal serum, horse serum, porcine serum, goat serum, rabbit serum, fish serum or any combination thereof.

[0152] In some embodiments, the composition that can be used as a supplement to reduce the serum amount required in the culture of bovine, porcine, sheep, chicken cells, or any combination thereof.

[0153] In some embodiments, the composition that can be used as a supplement to reduce the serum amount required in the culture of fish, shellfish, shrimp cells, or any combination thereof.

[0154] In some embodiments, the composition of the present invention does not affect the general properties of cells, with its ability to proliferate, differentiate, and sternness.

[0155] In some embodiments, the composition of the present invention may reduce the cells' initial "lag" phase before their exponential growth.

[0156] In some embodiments, the composition of the present invention may mitigate the effect of toxic waste products in the spent media.

[0157] In some embodiments, the composition of the present invention may enhance cell yield and specific protein expression.

[0158] In some embodiments, the composition of the present invention comprises an amount of plant protein hydrolysates, yeast hydrolysates, microalgae hydrolysates, or any combination thereof.

[0159] In some embodiments, the plant protein hydrolysates are prepared from Pea, Sorghum, Rice, Wheat, Hemp, Quinoa, Soybean, Corn, Mung bean, Fava bean, Chickpeas, Lentils, Peanuts, Almonds, Chia seeds, Potatoes, Seitan, Oat, Barley, Brewers Spent Grain, or any combination thereof.

[0160] In some embodiments, the enzyme used in plant protein hydrolysate is Alcalase®, Flavourzyme®, Formea®, Fungamyl®, Maltogenase®, Neutrase®, Protamex®, Protana®, Termamyl®, Vertera®, Viscozyme®, Trypsin, Papain, Amylase, or any combination thereof.

[0161] In some embodiments, the hydrolysis is performed in the pH between 2-12.

[0162] In some embodiments, the hydrolysis is performed at the temperature between0-90 °C.

[0163] In some embodiments, where the inactivation of enzyme is performed in the temperature between 0-130 °C.

[0164] In some embodiments, the hydrolysis is performed in the time between 1 -1440 min.

[0165] In some embodiments, the separation of protein hydrolysates includes but is not limited to centrifuge and membrane separation.

[0166] In some embodiments, the centrifuge is performed in the centrifugal force between 100* g-30000x g for 0-24 h.

[0167] Another aspect of the present invention relates to a method of cell culture comprising: culturing one or more species of cells use the composition of the present invention.

[0168] In some embodiments, the culturing method comprises, plating, growing, passaging, expanding, splitting, adapting, or any combination thereof one or more times.

[0169] In some embodiments, in the method of the present invention, the cells are cultured in 2D cell culture plates, microcarriers, 3D scaffolds, 3D cell culture vessels, or any combination thereof one or more times.

[0170] In some embodiments, the cells used are adherent cells, suspension cells, or any combination thereof.

[0171] In some embodiments, the composition of the present invention is in the range of 0.0000001 %-100%.

[0172] In some embodiments, the composition of serum is in the range of 0%-30%.

[0173] In some embodiments, the serum includes the fetal bovine serum, bovine calf serum, adult bovine serum, horse serum, porcine serum, goat serum, rabbit serum, fish serum or any combination thereof.

[0174] In some embodiments, the composition of the basal media is in the range of 0.0000001 %-99.99999%.

[0175] In some embodiments, the composition of basal media includes but not limited to Eagle minimal essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), RPMI 1640, Ham F10, Ham F12, DMEM / F12, or any combination thereof.

[0176] In some embodiments, the composition of non-essential amino acids is in the range of 0.00001 %-10%.

[0177] In some embodiments, the composition of fibroblast growth factor, any other growth factors, or any hormones, are in the range of 0.00000000001 %-10%.

[0178] Another aspect of the present invention relates to a method of preparing a culture media using composition of the present invention: a) blending compositions of statement 18; b) adjust the pH; c) remove the insoluble portion; d) keep the supernatant fora certain time; e) remove the insoluble portion and keep the supernatant for cell culture.

[0179] In some embodiments, wherein (a) is in the ratio of 0.00001 %-100%.

[0180] In some embodiments, wherein (b) is performed at a pH in the range of between 2-12.

[0181] In some embodiments, wherein (c) is the separation of insoluble portions includes but is not limited to centrifuge and membrane separation.

[0182] In some embodiments, the centrifuge is performed in the centrifugal force between 100x g-30000x g for 0-24 h.

[0183] In some embodiments, wherein (d) is for 0 h-144 h.

[0184] In some embodiments, the temperature is for 4-60C.

[0185] In some embodiments, wherein (e) is the separation of insoluble portions includes but is not limited to centrifuge and membrane separation in 100x g-30000x g centrifuge force.

[0186] In some embodiments, the centrifuge is performed in the centrifugal force between 100x g-30000x g for 0-24 h.

[0187] As illustrated in FIG. 9, the present invention provides an improved cell culture medium comprising a hydrolysate composition, such as plant protein hydrolysates, which serve as a cost-effective and environmentally friendly alternative to animal-derived serum and synthetic growth factors commonly used in conventional cell culture media. Conventional cell culture media, including those used in cultivated meat (CBM) production and other biomanufacturing applications, rely heavily on FBS, a component derived from living animal fetuses, or highly purified synthetic growth factors, both of which are expensive, ethically controversial, and limited in supply. These components also pose risks of animal-borne contamination. By contrast, the plant protein hydrolysates produced through the enzymatic hydrolysis of plant proteins in the present invention can partially replace or reduce the need for FBS and growth factors in cell culture. This could help lower the cost of cultivated meat production and enables broader adoption by improving economic and ethical sustainability. Furthermore, the cell culture medium of the present invention offers substantial cost benefits across a wide range of applications, including biopharmaceutical manufacturing, protein synthesis, vaccine production, and academic or industrial research involving cell culture.

[0188] It should be further appreciated by the person skilled in the art that variations and combinations of features described above, not being alternatives or substitutes, may be combined to form yet further embodiments falling within the intended scope of the invention. As would be understood by a person skilled in the art, each embodiment, may be used in combination with other embodiment or several embodiments.

Claims

CLAIMSClaim 1 . A cell culture medium comprising a hydrolysate composition, wherein the hydrolysate composition is selected from the group consisting of a plant protein hydrolysate, a yeast protein hydrolysate, a microalgae protein hydrolysate, an insect protein hydrolysate, and any combination thereof.Claim 2. The cell culture medium according to Claim 1 , wherein the plant protein hydrolysate is derived from a plant source, wherein the plant source is selected from the group consisting of Pisum sativum (pea), Sorghum bicolor (sorghum), Oryza sativa (rice), Triticum aestivum (wheat), Cannabis sativa (hemp), Chenopodium quinoa (quinoa), Glycine max (soybean), Zea mays (corn), Vigna radiata (mung bean), Vicia faba (fava bean), Cicer arietinum (chickpea), Lens culinaris (lentil), Arachis hypogaea (peanut), Prunus dulcis (almond), Salvia hispanica (chia seed), Solanum tuberosum (potato), seitan (wheat gluten), Avena sativa (oat), Hordeum vulgare (barley), brewer’s spent grain, and any combination thereof.Claim 3. The cell culture medium according to Claim 1 or 2, wherein the plant protein hydrolysate is produced by enzymatic hydrolysis.Claim 4. The cell culture medium according to Claim 3, wherein the enzymatic hydrolysis is carried out using one or more enzymes selected from the group consisting of protease, amylase, and multi-enzyme complex.Claim 5. The cell culture medium according to Claim 4, wherein the protease is selected from the group consisting of Alcalase, Flavourzyme, Neutrase, Protamex, Protana, Trypsin, Papain, Formea, and any combination thereof.Claim 6. The cell culture medium according to Claim 4, wherein the amylase is selected from the group consisting of Termamyl, Maltogenase, Fungamyl, and any combination thereof.Claim 7. The cell culture medium according to Claim 4, wherein the multi-enzyme complex is selected from the group consisting of Vertera, Viscozyme, and anycombination thereof.Claim 8. The cell culture medium according to any one of the preceding claims, wherein the hydrolysate composition is present in an amount ranging from 0.0000001 % to 99% weight by volume.Claim 9. The cell culture medium according to Claim 8, wherein the hydrolysate composition is present in an amount ranging from 0.001 % to 2% weight by volume.Claim 10. The cell culture medium according to any one of the preceding claims, further comprising animal serum.Claim 11. The cell culture medium according to Claim 10, wherein the animal serum is selected from the group consisting of fetal bovine serum, calf bovine serum, adult bovine serum, horse serum, porcine serum, goat serum, rabbit serum, fish serum, and any combination thereof.Claim 12. The cell culture medium according to any one of the preceding claims, further comprising a basal medium, wherein the basal medium is selected from the group consisting of Eagle’s Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle Medium (DMEM), RPMI 1640, Ham’s F-10, Ham’s F-12, DMEM / F-12, and any combination thereof.Claim 13. The cell culture medium according to any one of the preceding claims, wherein further comprising non-essential amino acids.Claim 14. The cell culture medium according to any one of the preceding claims, further comprising growth factor and / or hormone; wherein the growth factor optionally comprises fibroblast growth factor.Claim 15. The cell culture medium according to any one of the preceding claims, wherein the cell culture medium is suitable for culturing adherent cells, suspension cells, or a combination thereof.Claim 16. The cell culture medium according to any one of the preceding claims, wherein the cell culture medium is suitable for culturing animal-derived cells for cultivated meat production.Claim 17. The cell culture medium according to Claim 16, wherein the animal-derived cells are derived from terrestrial animals, wherein the terrestrial animals comprise bovine, porcine, ovine, caprine, and avian species.Claim 18. The cell culture medium according to Claim 16, wherein the animal-derived cells are derived from aquatic animals, wherein the aquatic animals comprise fish, shellfish, shrimp, and crustaceans.Claim 19. A method of preparing cell culture medium, comprising the steps of:5100, preparing a hydrolysate composition; andS200, preparing the hydrolysate composition to obtain the cell culture medium for culturing cell; wherein the step S100 comprises:5101 , enzymatically hydrolyzing a protein source using one or more enzymes to obtain a hydrolysis mixture;5102, inactivating the one or more enzymes in the hydrolysis mixture; and5103, filtering the hydrolysis mixture to remove insoluble components to obtain the hydrolysate composition.Claim 20. The method of preparing cell culture medium according to Claim 19, wherein step S200 comprises dissolving the hydrolysate composition in a basal medium.Claim 21 . The method of preparing cell culture medium according to Claim 19 or 20, wherein the protein source is selected from the group consisting of plant biomass, yeast biomass, microalgae biomass, insect biomass, protein extracts derived therefrom, and any combination thereof.Claim 22. The method of preparing cell culture medium according to Claim 21 , whereinthe plant biomass is selected from the group consisting of Pisum sativum (pea), Sorghum bicolor (sorghum), Oryza sativa (rice), Triticum aestivum (wheat), Cannabis sativa (hemp), Chenopodium quinoa (quinoa), Glycine max (soybean), Zea mays (corn), Vigna radiata (mung bean), Vicia faba (fava bean), Cicer arietinum (chickpea), Lens culinaris (lentil), Arachis hypogaea (peanut), Prunus dulcis (almond), Salvia hispanica (chia seed), Solanum tuberosum (potato), seitan (wheat gluten), Avena sativa (oat), Hordeum vulgare(barley), brewer’s spent grain, and any combination thereof.Claim 23. The method of preparing cell culture medium according to any one of Claims 19 - 22, wherein the one or more enzymes are selected from the group consisting of protease, amylase, and multi-enzyme complex.Claim 24. The method of preparing cell culture medium according to Claim 23, wherein the protease is selected from the group consisting of Alcalase, Flavourzyme, Neutrase, Protamex, Protana, Trypsin, Papain, Formea, and any combination thereof.Claim 25. The method of preparing cell culture medium according to Claim 23, wherein the amylase is selected from the group consisting of Termamyl, Maltogenase, Fungamyl, and any combination thereof.Claim 26. The method of preparing cell culture medium according to Claim 23, wherein the multi-enzyme complex is selected from the group consisting of Vertera, Viscozyme, and any combination thereof.Claim 27. The method of preparing cell culture medium according to any one of Claims 19 to 26, wherein, in the step S 101 , enzymatical hydrolysis is conducted at a temperature in the range of 0 °C to 80 °C.Claim 28. The method of preparing cell culture medium according to any one of Claims 19 to 27, wherein, in the step S102, the one or more enzymes are inactivated by heat inactivation.Claim 29. The method of preparing cell culture medium according to Claim 28, whereinthe heat inactivation is conducted at a temperature in the range of 85 °C to 130 °C.Claim 30. The method of preparing cell culture medium according to any one of Claims 19 to 29, wherein, in the step S103, the hydrolysis mixture is filtered by centrifugation, membrane separation, or a combination thereof.Claim 31. The method of preparing cell culture medium according to Claim 30, wherein the centrifugation is conducted at a centrifugal force in the range of 100 x g to 30,000 x g for a duration of 1 min to 1 ,440 mins.Claim 32. The method of preparing cell culture medium according to any one of Claims 19 to 31 , wherein, in the step S100, the preparation of the hydrolysate composition is performed at a pH in the range of 2 to 12.Claim 33. The method of preparing cell culture medium according to Claim 31 , wherein the preparation of the hydrolysate composition is performed at a pH in the range of 4 to 8.Claim 34. The method of preparing cell culture medium according to any one of Claims 19 to 33, wherein step S101 is conducted for a duration in the range of 1 min to 1 ,440 mins.Claim 35. The method of preparing cell culture medium according to any one of Claims 19 to 34, wherein step S200 further comprises step S201 , comprising removing precipitate from the cell culture medium after dissolving the hydrolysate composition in the basal medium; wherein the cell culture medium is allowed to stand undisturbed to permit the precipitate to settle prior to removal.Claim 36. The method of preparing cell culture medium according to any one of Claims 19 to 35, wherein step S100 further comprises step S104, in which the hydrolysate composition is subjected to a drying process.Claim 37. The method of preparing cell culture medium according to Claim 36, whereinthe drying process is selected from the group consisting of freeze drying, spray drying, vacuum drying, and any combination thereof.Claim 38. A method of culturing a cell, comprising plating the cell in a cell culture medium according to any one of Claims 1 to 18.Claim 39. The method of Claim 38, further comprising one or more of the steps of growing, passaging, expanding, splitting, or adapting the cell using the cell culture medium.Claim 40. The cell culture method according to Claims 38 or 39, wherein the cell is animal- derived cells derived from terrestrial animals, wherein the terrestrial animals comprise bovine, porcine, ovine, caprine, and avian species.Claim 41. The cell culture medium according to Claims 38 or 39, wherein the cell is animal-derived cells derived from aquatic animals, wherein the aquatic animals comprise fish, shellfish, shrimp, and crustaceans.

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