Media compositions comprising microorganismal lysates and systems and methods for using the same

Abstract

Disclosed are cell culture media using microorganismal, e.g., bacterial, fungal, lysates as a replacement for serum that are effective for short-term and long-term cultivation of animal cells, e.g., generation of cultivated meat. Also disclosed are systems for culturing animal cells using the media, methods of making the media, methods of detecting suitable serum replacements, and kits.

Description

MEDIA COMPOSITIONS COMPRISING MICROORGANISMAL LYSATES AND SYSTEMS AND METHODS FOR USING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No.63 / 712.061 that was filed October 25, 2024. The entire contents of which are hereby incorporated by reference.SEQUENCE LISTING

[0002] A Sequence Listing accompanies this application and is submitted as an xml file of the sequence listing named “166118 01559. xml” which is 8,435 bytes in size and was created on October 21, 2025. The sequence listing is electronically submitted via Patent Center and is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grant number 2021-05678 awarded by the United States Department of Agriculture. The government has certain rights in the invention.BACKGROUND

[0004] The environmental impact of meat consumption is expected to continue to rise with increasing demand and the increasing global population1. The production of beef using conventional livestock agriculture is unsustainable due to greenhouse gas emissions, eutrophication, deforestation, overuse of water, and the spread of zoonotic disease2, 3. Similarly, modem fishing practices have resulted in 90% of the world’s fish stocks to be overexploited or collapsed4’5.

[0005] Cultivated meat - meat products generated from in vitro cultures of animal cells -represents a highly sustainable alternative to conventional meat and fish production, reduces negative externalities, and benefits animal welfare6, 7Much progress has been made recently indeveloping stable immortalized lines of both bovine8and mackerel9muscle satellite cells, leading to the creation of "iBSC" (Immortalized Bovine Satellite Cell) and “Mackl” cell lines, respectively. However, mammalian and marine cell cultures still largely depend on the use of fetal bovine serum (FBS), an expensive and animal-based ingredient with significant lot variability10and risks of contamination factors11, making its use in cultivated meat infeasible. With 90% of the cost of cultivated meat production stemming from growth media12, inexpensive FBS alternatives are needed to bring cultivated meat towards commercial production.SUMMARY

[0006] In an aspect of the current disclosure, cell culture media is provided. In some embodiments, the cell culture medium comprises an amount of a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Bacillus subtilis, Lactobacillus lactis, Latilactobacillus sakei, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, and Lactobacillus gasseri.

[0007] In an aspect of the current disclosure, aqueous solutions are provided. In some embodiments, the aqueous solutions comprise a serum replacement, wherein the serum replacement comprises a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Lactobacillus lactis, Bacillus subtilis, and Lactobacillus plantarum, and one or more of sodium selenite, albumin, transferrin, transforming growth factor beta, L-ascorbic acid, insulin, neuregulin 1 (NRG1), fibroblast growth factor 2 (FGF-2), DMEM, HEPES, or sodium bicarbonate.

[0008] In an aspect of the current disclosure, systems are provided. In some embodiments, the systems are systems for generating cultivated meat and comprise: (a) a cell culture medium comprising an amount of a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Bacillus subtilis, Lactobacillus lactis, Latilactobacillus sakei, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, and Lactobacillus gasseri.' and (b) an animal cell.

[0009] In an aspect of the current disclosure, methods of making the cell culture medium are provided. In some embodiments, the methods comprise: (a) generating a lysate from a microbe; (b) combining an amount of the lysate from the microbe with a base medium, thereby generating the cell culture medium.

[0010] In an aspect of the current disclosure, methods of generating cultivated meat are provided. In some embodiments, the methods comprise culturing an animal cell in a medium comprising a microorganismal lysate for a sufficient time to generate cultivated meat.

[0011] In an aspect of the current disclosure, methods of detecting a suitable microorganismal lysate for use as a mammalian serum replacement for animal cell culture are provided. In some embodiments, the methods comprise: (a) preparing a lysate from a sample of a species of cultured microorganisms; (b) culturing (1) a test plurality of animal cells in a test medium, wherein the test medium comprises the lysate prepared in step (a), and (2) a control plurality7of animal cells in a control medium; (c) analyzing the growth of the test plurality of animal cells and the control plurality of animal cells; (d) detecting the lysate as a suitable microorganismal lysate for use as a mammalian serum replacement for animal cell culture wherein growth of the test plurality of animal cells is about similar or greater than the control plurality7of animal cells.

[0012] In an aspect of the current disclosure, kits, systems, and platforms are provided. In some embodiments, the kits, systems or platforms comprise a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Bacillus subtilis, Lactobacillus lactis, Latilactobacillus sakei, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, and Lactobacillus gasseri.

[0013] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0014] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 20 is understood to include any number, combination ofnumbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.BRIEF DESCRIPTION OF THE FIGURES

[0015] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] FIGs. 1A, IB, and 1C show SDS-PAGE analysis of proteins from various microbial lysates. A) Overview of lysate generation and screening by culturing the microbes, obtaining the lysate by sonicating it. After filter sterilization, the lysate is added to basal media, which is used for culturing iBSCs, which is followed by estimating cell proliferation and differentiation. B) SDS-PAGE profile of seven lysates used in the initial screening. (L-R) Ladder, B. subtilis 168, E. coli Nissle, V. natriegens, E. coli BL21, L. lactis 1635, S. cerevisae CEN. PK andL. plantarum WCFS1).

[0017] FIGs. 2A, 2B, and 2C show Short-term proliferation assay of iBSCs and Mackl cells using various microbial lysates. A) iBSC (not serum-free adapted) proliferation screen (5 d incubation) on microbial lysate-based serum-free media shows VN and LP lysates performing similarly to Beefy-9 media (positive control). B8 media (Beefy-9 without rAlbumin) used as negative control. Lysate-based media created through addition of respective lysates to B8 media. Data is from Cy Quant DNA staining kit. n=4 (experimental groups) or 10 (controls). B) Mackl (not serum-free adapted ) proliferation screen using an L-15 media supplemented with 2.5% FBS (negative control) and additional microbial lysates. n=3. C) iBSC (4x serum-free Beefy-9 passages) proliferation screen using selected lysates at lower concentrations. n=4 (experimental groups) or 10 (controls). Lysate formulations were compared to one another to determine candidates for long-term growth studies, showing V. natriegens as top contender. All presented data is mean values ± SD. Statistical analysis by unpaired t-test with Welch’s correction in which p > 0.05 (NS), p < 0.05 (*), 0.01 (**), and 0.001(***).

[0018] FIGs. 3A. 3B, 3C, 3D, 3E, 3f, and 3G show Long-term growth of iBSCs in VN40 and Beefy-9. A) iBSCs grown over multiple passages in B8 medium containing increasingly concentrated V. natriegens lysate (corresponding to 10, 20, 40 pg / mL protein concentrations).The cells were grow n in T-25 flasks and passaged at 80-90 % confluence. Cells counted by direct cell count via automated cell counter. Dotted line representing doubling times in Beefy-9 & iBSCGM (General Media for iBSCs containing 20 % FBS) collected throughout this study. B) iBSC adaptation to V. natriegens lysate-containing media beginning with 20 pg / mL protein concentration. C) iBSCs grown continuously in VN40 and Beefy-9 media with cells counted at each passage to determine cumulative cell doublings. Cells grown in triplicates in 6 well plates for each condition. Significance indicated for final cumulative cell doublings. All presented data is mean values ± SD. Statistical analysis by unpaired t-test with Welch’s correction in which p < 0.001 (***). Cells counted by direct cell count via automated cell counter. D-E) Cells after the final experimental passage (passage 82 overall, passage 22 in serum-free media) grown for 3 days and imaged by phase contrast microscopy, showing improved growth of VN40 (D) compared to Beefy-9 (E). Scale bar = 200 pm. F-G) Magnification increased to show additional detail, with orange arrows indicating increased cytoplasmic lipid accumulation in cells grown in Beefy-9 (G) compared to VN40 (F). Scale bar = 100 pm.

[0019] FIGs. 4A and 4B show the role of HMW and LMW Vibrio natriegens lysate fractions on iBSC growth. A) The growth of iBSCs using a short-term screen (5 days) demonstrates that whole-cell lysates support growth more effectively than either the >3 kDa (HMW) or <3 kDa (LMW) fractions alone. HMW and LMW samples represent retentate and filtrate, respectively, that were added to B8 media at protein concentrations indicated (<3kDa concentration assessed by adding low molecular weight fraction representative of corresponding whole cell lysate). Beefy-9 media used as positive control, B8 media used as negative control. Data is from CyQuant DNA staining kit. B) iBSC proliferation in whole-cell VN40 medium compared to the sum of HMW and LMW growth indicates additivity of their relative contributions to iBSC growth. All presented data is mean values ± SD. Statistical analysis by unpaired t-test with Welch’s correction in which p > 0.05 (NS), p < 0.01 (**).

[0020] FIGs. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 51, 5J, and 5K show the sternness and differentiation of VN40-adapted iBSCs. A-B) fmmunofluorescent staining of iBSCs before (A) and after (B) adaptation and serial passaging (22 passages) in VN40 media show no change in satellite cell phenofype after VN40 adaptation. Cells were stained with phalloidin (green) to indicate actin expression and anti-Pax7 (orange). Scale bar = 100 pm. C) Quantification of Pax7+ cells before and after adaptation and serial passaging (22 passages) in VN40 media. % Cells Pax7+ determined by (number of Pax7+ cells) / (number of actin+ cells). Statistical analysis by unpaired t-test with Welch’s correction in which p>0.05 (NS). D-G) Immunofluorescent staining of gradually adapted iBSCs (p98) after 18 days differentiation in differentiation media (2% horse serum, 50 pg / mL rAlbumin, 0.5 pM LDN-193189, ITS-X). H-K) Immunofluorescent staining of rapidly adapted iBSCs (p70) after 13 days differentiation in differentiation media. Results show successful differentiation of cells expressing early-stage (Desmin, green) and late-stage (myosin heavy chain, red) differentiation markers, with more robust differentiation of rapidly adapted cells. Cells were stained with anti-desmin, MF20 (for myosin heavy chain), and DAPI (blue) to stain nuclei. D,F,H,J: low magnification, Scale bar = 200 pm; E,G,I,K: high magnification, Scale bar = 100 pm.

[0021] FIG. 6 shows that VN lysate is less expensive than FBS, making VN40 media more economically sustainable than BSCGM. Media costs for BSC-GM, Beefy-9 and VN40 were assessed for reagents purchased at USD / L. VN media is the most economical followed by B9 and BSC-GM. FBS in BSCGM and rAlbumin in B9 are major cost-drivers for respective media compared negligible cost of VN lysate in VN media.

[0022] FIG. 7 shows iBSCs fed serum-free media with V. natriegens lysate at 40 (VN40), 60, and 80 pg / mL, and resultant doubling times.

[0023] FIG. 8 shows a Venn diagram indicating the number of metabolites detected by each method (HILIC (-), HILIC (+), RP (+) and RP (-) and the common metabolites among the method groups. A total of four methods was applied to each sample using either a hydrophilic interaction chromatography (HILIC) column (Phenomenex Luna NH2, Torrance, CA, USA) or a reversephase column (Phenomenex Synergi Hydro-RP, Torrance, CA, USA) in both positive and negative ionization modes.

[0024] FIGs. 9A and 9B show short-term screens using lysates from lactic acid bacteria (A) and fungal strains (B), along with B8 media (negative control) and Beefy 9 media (serum-free positive control) after 5 days of incubation. Lysate-based media created through addition of respective lysates to B8 media. Data is from CyQuant DNA staining kit. n=4 (experimental groups) or 8 (controls). All presented data is mean values ± SD. Statistical analysis by unpaired t-test withWelch’s correction to compare growth to Beefy 9, p>0.05 (NS). Negative values represent data for which lysates have higher autofluorescence than cells.

[0025] FIG. 10 shows an SDS-PAGE gel demonstrating expression of bovine FGF in V. natriegens protein lysate (lane 3), molecular weight ladder (lane 1), and V. natriegens lysate containing the empty vector (lane 2).

[0026] FIG. 11 shows that lysate from V. natriegens expressing FGF-2 leads to equivalent growth of mammalian cells grown in V. natriegens lysate supplemented with exogenous FGF-2.

[0027] FIG. 12 shows brightfield images of iBSCs grown in VN40 and VN40FGF media for 4 days to -80-90% confluence, showing visually similar growth rate and cell morphology. SB = 100 pm. VN40: Serum-free media containing V. natriegens lysate 40 pg / mL protein and 40 ng / mL added pure FGF (purchased). VN40FGF: Serum-free media containing FGF-expressing engineered V. natriegens 40 pg / mL protein, no added FGF.

[0028] FIG. 13 shows iBSCs adapted to VN40FGFmaintain differentiation capacity, with expression of muscle differentiation markers Desmin and Myosin Heavy Chain (MHC) after subjecting to differentiation media.

[0029] FIG. 14 show s iBSCs grown in VN40FGF media maintain their sternness (indicated by Pax7 expression, a marker for satellite cell phenoty pe) and ability to differentiate (as shown by myosin heavy chain (MHC) and desmin expression).

[0030] FIG. 15 shows V. natriegens grown in fresh media vs. spent culture media. The fresh medium is comprised of Luria Broth (LB) + 2% NaCl. The spent medium is autoclaved for a 40 minute cycle and supplemented with 3% NaCl, by w eight.

[0031] FIG. 16 shows Table 3: cost comparison of itemized media components and total cost of iBSCGM and B9 with VN40 medium.DETAILED DESCRIPTION

[0032] The inventors tested multiple lysates from Gram-positive and Gram-negative bacteria, as w ell as ayeast, to determine their suitability as replacements for FBS in animal cell, e.g., bovineand fish cell, culture media. The inventors discovered that, surprisingly, several different lysates, e.g., lysates derived from the gram-negative bacterium Vibrio natriegens, could serve as an alternative to FBS for iBSCs. A similar screen also identified that lysates from Saccharomyces cerevisiae could support growth of Mack 1 cells in reduced serum conditions. The inventors show that iBSCs can be adapted to grow rapidly in Vibrio natriegens lysate-containing media by longterm and short-term serially passaging. Cells cultured in this microbial lysate media (called VN40) demonstrate robust growth, preservation of satellite cell phenotype, and an ability’ to differentiate into multinucleated myotubes. These results highlight the promise of the whole-cell lysate derived from microorganisms, e.g., the fast-growing non-pathogenic marine bacterium, V. natriegens, as an easily producible, low-cost supplement for long-term serum-free growth of iBSCs for cultivated meat. Further, the inventors’ approach may be generalizable to help identify novel microbial lysates as FBS substituents for different cells, e.g., cultivated meat cell lines.

[0033] Here, the inventors describe the use of whole-cell lysates from microbes as a substitute for serum in media which may be used, for instance, to produce cultivated meat. Lysates of bacterial, fungal, and yeast origins may be used to create completely serum-free media (SFM). In some embodiments, the lysates may be used as a supplement to serum-containing media. Lysates can be rich in proteins, metabolites, and other compounds beneficial for cell growth. “Media” refers to a solution of compounds useful to aid in the maintenance, proliferation, and / or differentiation of cells. Certain lysates were found to perform equally if not better than previously established SFM formulations in stimulating short-term and long-term growth of cultivated meat cells. Lysates have been found to aid cell proliferation at concentrations of 1 mg / ml - 200 mg / ml, depending on microbial species. Lysate preparation is straightforward, requiring only steps of sonication and filtration, without the need of further purification. Microbial lysates are advantageous to other SFM formulations as they are inexpensive, non-toxic. simple to produce, and derived from rapidly-duplicating organisms. Overall, the disclosure presents a simple to produce, cost-saving solution to arriving at serum-free media for cultivated meat.

[0034] Problems addressed by the innovation

[0035] Media costs currently account for 90% of the cost to produce cultivated meat [1], Fetal bovine serum (FBS)-containing media is still the gold standard for mammalian cell cultivation, but its use is antithetical to cultivated meat’s goals of producing animal-free meat alternatives.Further, FBS is expensive (~$1,OOO / L) and comes with significant lot variability [2] and risks of contamination factors [3], Many solutions have been developed for creating serum-free media (SFM) for cultivated meat, such as adding purified recombinant grow th factors like albumin [4] [5], or other mitogenic proteins like fetuin [6], to existing media formulations. Approaches using crude extracts or hydrolysates from plant [7] or microalgal [8,9] sources have been explored to reduce costs. Recent work exploring microbes such as yeast, bacteria, and algae have shown initial success in reducing serum for cultivated meat media but have not eliminated FBS entirely for sustained cell proliferation [9,13,14,15], The inventors have found that by supplementing lysates into existing formulations containing small quantities of growth factors and other mitogenic compounds, the inventors can create fully formulated serum-free media for cultivated meat production. Unlike previously discovered formulations, these microbial lysate-based formulations are inexpensive, simple to produce, and completely serum-free.

[0036] Developing serum-free media for cell culture has been of interest for decades

[0016] , Many recent attempts to reduce or replace serum in cultivated meat production have been explored. Of note is recent work adapting serum-freeL‘B8??media designed for human iPSC culture

[0017] to the culture of bovine satellite cells [5] via addition of recombinant albumin, leading to the creation of “Beefy-9” media. Efforts like these and others using supplemental cytokines and growth factors [4,6] require expensive purified recombinant protein on the g / L scale. Current estimates show cultivated meat may require as much as 42 liters of media per kilogram to produce

[0018] , making the dependence on such quantities of recombinant proteins economically infeasible. Attempts using plant and algal extracts often require laborious and chemically-intensive extraction protocols. Further, plant and algae grow' slowly and are resource-intensive to cultivate, adding additional cost and labor burden to the cultivated meat production supply chain. Rarely have these innovations resulted in long-term cell growth and differentiation. Past attempts to use microbial extracts have failed to show growth under serum-free conditions [14,19,20]

[0037] The disclosed innovations use microbial lysates to show complete replacement of fetal bovine serum in media used for cultivated meat cell growth. The inventors have shown that several microbial lysates stimulate cell growth at levels on par with or above previously established serum-free media. The inventors have demonstrated that certain lysates stimulate long-term cell growth over many passages and maintain cellular phenotype and myogenicpotential (see attached manuscript draft). Due to low-cost, simple production, rapidly replicating nature, ability to completely replace FBS, and support of long-term cell health, microbial lysates represent a novel invention in the field of serum-free cultivated meat media.

[0038] Consumers of these novel media formulae may be cultivated meat and seafood companies, who represent a $3.1 billion industry. For ethical and economic reasons, serum-free media development is a foremost priority of the industry’ as a whole. With 185 companies in this space, and over 48 cultivated meat production facilities currently opened or announced, this represents a sizeable consumer base for microbial lysate-based serum-free media

[0026] , Many cultivated meat and seafood companies create media formulations in-house, and microbial lysates are a promising option for rapid and inexpensive on-site media production. Consumers can test innumerable microbial lysate options to find optimal ingredients for their specific conditions and cell ty pes. Academic labs pursuing cultivated meat research may also find microbial lysates useful for their work. With over 500 academics active in cultivated meat research globally, microbial lysates can accelerate academic progress on cell line and media development. Ultimately, microbial lysates would be of great utility, as purchasable products or licensable technologies, for industry' and academia alike.Cell culture medium

[0039] The inventors discovered that microbial lysates, e.g., lysates from the organisms Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Bacillus subtilis, Lactobacillus lactis, Latilactobacilhis sakei. Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, or Lactobacillus gasseri may be used in the culture of animal cells, e.g., as a replacement for serum, e.g., bovine serum, e.g., fetal bovine serum.

[0040] Accordingly, in an aspect of the current disclosure, cell culture media is provided. In some embodiments, the cell culture media comprises an amount of a lysate from a microorganism, e.g., the microorganism is selected from the group consisting of Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Bacillus subtilis, Lactobacillus lactis, Latilactobacillus sakei. Lacticaseibacillus rhamnosus. Limosilactobacillus reuteri, and Lactobacillus gasseri.

[0041] “Lysates” may be generated, in one example, from microorganisms by physically disrupting the microorganisms, e.g., by sonication, then clearing of the lysates by centrifugation, followed by filtration of the lysates. However, the term “lysate,” as used herein, refers to a soluble fraction of lysed microorganisms, where “lysed,” and grammatical variations thereof, refers to disruption of the cell membrane and / or wall releasing cytoplasmic contents. There are a variety of methods that may be used to “lyse” microorganisms to generate lysates including, but not limited to, physical methods, e.g.. sonication, bead beating, centrifugation, chemical methods, e.g., acid exposure, base exposure, detergent exposure.

[0042] The microorganism may be Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Bacillus subtilis. Lactobacillus lactis, Latilactobacillus sakei, Lacticaseibacillus rhamnosus. Limosilactobacillus reuteri, or Lactobacillus gasseri.

[0043] As used herein, the amount of the lysate from the microorganism in the media may be about 0.01 pg / ml to about 1000 pg / ml. any amount within a range bounded by about 0.01 pg / ml on the low end to about 1000 pg / inl on the high end, inclusive of the end points, or may be about 1 pg / ml, about 10 pg / ml. about 20 pg / ml. about 30 pg / ml. about 40 pg / ml. about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, about 100 pg / ml, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45. 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57. 58. 59. 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71. 72. 73. 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 pg / ml or more. The amount of the lysate may be about 10 mg / ml to about 100 mg / ml, about 10 mg / ml to about 50 mg / ml, about 10 mg / ml to about 40 mg / ml, about 40 mg / ml, or about 20 mg / ml.

[0044] The media may comprise a basal media (e g., DMEM, DMEM / F 12, etc.) containing one or more of the following non-limiting elements: sugars (e.g., glucose) at concentrations ranging from 0.01-10 g / L; amino acids (e.g., glutamine, lysine) at concentrations ranging from 0.001-5 g / L, vitamins (e.g., folate, niacin) at concentrations ranging from 0.001-1 g / L; minerals (e.g.. NaCl) at concentrations ranging from 0-15 g / L; and trace elements (e.g., iron, selenium) at concentrations ranging from 0.0001-10 mg / L. The basal media can be supplemented with growth-stimulating or cell-signaling factors (e.g., insulin, fibroblast growth factor, transforming growth factor, etc.) at concentrations ranging from 0.01-100,000 ng / mL and by carrying proteins (e.g., transferrin) at concentrations ranging from 0.01-1 g / L. The basal media may be adapted to suit a particular cell type according to methods known in the art.

[0045] B8 medium is a defined medium known in the art comprising the following elements: insulin, sodium selenite, FGF2. DMEM / F12. TGF-(31, ascorbic acid 2-phosphate, transferrin, and NRG1. See also Lyra-Leite et al. “An updated protocol for the cost-effective and weekend-free culture of human induced pluripotent stem cells’’ STAR Protoc. 2021 Feb 3;2(l):100213, which is incorporated by reference herein in its entirety.

[0046] B9 medium refers to an improved version of B8 medium further comprising, e.g., albumin or recombinant albumin. See, e.g., Stout et al. “Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat” Commun Biol. 2022 Jun 2;5(1):466, which is incorporated by reference herein in its entirety.

[0047] The disclosed media may be used for the culture of cells, e.g., animal cells, e.g., muscle cells, e.g., muscle cells derived from the following non-limiting list of organisms: fish (e.g., salmon, tuna, tilapia, perch, mackerel, cod, sardine, trout, etc.), shellfish (e.g., clams, mussels, and oysters); crustaceans (e.g., lobsters, shrimp, prawns, and crayfish), echinoderms (e.g., sea urchins and sea cucumbers), cow, chicken, sheep, pig, horse, mouse, goat, or rabbit. The cells may be of bovine, galline, ovine, porcine, equine, murine, caprine, lapine, or piscine origin. The disclosed media, aqueous solutions, systems, etc. may be used to for the short-term or long-term culture of a variety of cells including cells intended for food production and other agricultural relevance, e.g., muscle and / or adipose cells. The cells may be chicken fibroblast cells, e.g., DF1 chicken fibroblast cells. The cells may be bovine fat cells, e.g., primary bovine fat cells.

[0048] The disclosed media may support either short-term or long-term growth of animal cells. As used herein, “short-term growth” refers to growth of the subject cells for about 1, about 2, about 3, about 4, about 5, about 6, or about 7 days after being introduced to a particular culture condition, e.g., a new medium.

[0049] “Long-term growth,” as used herein, refers to growth of the subject cells for about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days,about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, or more, about 2 weeks, about 3 weeks about 4 weeks, or more, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months or more. Long-term grow th may be measured in a period marked by the number of cell passages, where the cells are passaged when media needs to be replenished, e.g., when media becomes acidified, and may be defined as 5 passages. 6 passages, 7 passages, 8 passages, 9 passages, 10 passages, 20 passages, 30 passages, 40 passages, 50 passages, 60 passages, 70 passages, 80 passages, 90 passages, 100 passages or more.

[0050] The inventors performed tandem high performance liquid chromatography-mass spectrometry (LC-MS) to detect compounds present in V. natriegens lysate and, in four experiments using different liquid chromatography techniques, found that the lysate comprised the following compounds and in the following relative concentrations (see also Table 1):

[0051] (1) the following compounds present at about the relative concentration to uracil listed below' or the absolute concentrations listed below . The concentrations are provided in ranges, e.g., 1-1000 ng / ml for uracil, however, the ranges are to be interpreted to be about 1 to about 1000 ng / ml, in the case of uracil.Cone. RangeRelative (ng / mL)Compoundconcentration1 - 1,000Uracil 10.26 - 260Hypoxanthine 0.26Xanthine 0.17 0.17 - 170L- 0.14 - 1400.14PhenylalanineGuanine 0.05 0.05 - 500.05 - 50L-Arogenate 0.050.04 - 40L-Glutamate 0.04L-Citrulline 0.03 0.03 - 300.02 - 20D-Xylulose0.025 -phosphate0.02 - 20L-Methionine 0.02

[0052] (2) the following metabolites present at about the relative concentration to 5-aminopentanoate listed belowCone. RangeRelativeCompound (ng / mL)concentration5- 25 - 25,0001Aminopentanoate1 - 1,000Uracil 0.04Cytosine 0.003 0.075 - 750.05 - 50L-Proline 0.002Inosine 0.002 0.05 - 500.025 - 25L-Serine 0.0010.025 - 25Benzonitrile 0.001Portulacaxanthin 0.0075 - 7.50.0003II

[0053] (3) the following metabolites present at about the relative concentration to xanthine listed belowCone. RangeRelativeCompound (ng / mL)concentration2.25 - 2250Xanthine 1L- 2 - 20000.87PhenylalanineInosine 0.55 1.25 - 12501 - 1000Uracil 0.440.7 - 700Hypoxanthine 0.310.5 - 500L-Tyrosine 0.260.5 - 500L-Tryptophan 0.260.5 - 500Guanosine 0.250.4 - 400Xanthosine 0.190.3-300L-Citrulline 0.14

[0054] or (4) the following metabolites present at about the relative concentration to L-phenylalanine listed belowCone. RangeRelative (ng / mL)CompoundconcentrationL- 3 - 30001Phenylalanine2 - 2000L-Tyrosine 0.56Guanine 0.55 2 - 20001.25 - 1250Hypoxanthine 0.37Uracil 0.3 1 - 10001 - 1000Guanosine 0.291 - 1000L-Methionine 0.28Xanthine 0.17 0.5 - 5000.5 - 500Guanine 0.150.3 - 300Inosine 0.11

[0055] Thus, the lysate or medium may comprise one or more of the following compounds: uracil, hypoxanthine, xanthine, L-phenylalanine, guanine, L-arogenate, L-glutamate, L-citrulline, D-xylulose 5-phosphate, L-methionine, 5-aminopentanoate, cytosine, L-proline, inosine, L-serine, benzonitrile, portulacaxanthin II, L-tyrosine, L-tryptophan, guanosine, or xanthosine. The lysate may comprise the following compounds: uracil, hypoxanthine, xanthine, L-phenylalanine, guanine, L-arogenate, L-glutamate, L-citrulline, D-xylulose 5-phosphate, L-methionine, 5-aminopentanoate, cytosine, L-proline, inosine, L-serine, benzonitrile, portulacaxanthin II, L-tyrosine, L-tryptophan, guanosine, and xanthosine. The lysate or medium may comprise one or more of the compounds selected from (1), (2), (3), or (4) and the compounds may be in the relative concentration listed above in (1). (2), (3), or (4).

[0056] In some embodiments, the medium is serum free / does not comprise serum.Aqueous solution comprising microorganismal lysate

[0057] In an aspect of the current disclosure, aqueous solutions comprising a serum replacement are provided. In some embodiments, the serum replacement comprises a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrionatriegens, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Lactobacillus lactis, Bacillus subtilis, and Lactobacillus plantarum.

[0058] In some embodiments, the serum replacement comprises one or more of the following compounds: uracil, hypoxanthine, xanthine, L-phenylalanine, guanine, L-arogenate, L-glutamate, L-citrulline, D-xylulose 5 -phosphate, L-methionine, 5-aminopentanoate, cytosine, L-proline, inosine. L-serine, benzonitrile, portulacaxanthin II, L-tyrosine. L-tryptophan, guanosine, or xanthosine. The serum replacement may comprise the following compounds: uracil, hypoxanthine, xanthine, L-phenylalanine, guanine, L-arogenate, L-glutamate, L-citrulline, D-xylulose 5-phosphate, L-methionine, 5-aminopentanoate, cytosine, L-proline, inosine, L-serine, benzonitrile, portulacaxanthin IL L-tyrosine. L-tryptophan, guanosine, and xanthosine.

[0059] In some embodiments, the serum replacement comprises the following metabolites (one of (1), (2), (3), or (4)) and may further comprise the following metabolites in the indicated relative concentrations or the indicated range of absolute concentrations:

[0060] (1) the following compounds present at about the relative concentration to uracil listed below' or the absolute concentrations listed below'. The concentrations are provided in ranges, e.g., 1-1000 ng / ml for uracil, however, the ranges are to be interpreted to be about 1 to about 1000 ng / ml, in the case of uracil.Cone. RangeRelativeCompound (ng / mL)concentration1 - 1,000Uracil 10.26 - 260Hypoxanthine 0.26Xanthine 0.17 0.17 - 170L- 0.14 - 1400.14PhenylalanineGuanine 0.05 0.05 - 500.05 - 50L-Arogenate 0.050.04 - 40L-Glutamate 0.04L-Citrulline 0.03 0.03 - 300.02 - 20D-Xylulose0.025 -phosphate0.02 - 20L-Methionine 0.02

[0061] (2) the following metabolites present at about the relative concentration to 5-aminopentanoate listed belowCone. RangeRelativeCompound (ng / mL)concentration5- 25 - 25,0001Aminopentanoate1 - 1,000Uracil 0.04Cytosine 0.003 0.075 - 750.05 - 50L-Proline 0.002Inosine 0.002 0.05 - 500.025 - 25L-Serine 0.0010.025 - 25Benzonitrile 0.001Portulacaxanthin 0.0075 - 7.50.0003II

[0062] (3) the following metabolites present at about the relative concentration to xanthine listed belowCone. RangeRelativeCompound (ng / mL)concentration2.25 - 2250Xanthine 1L- 2 - 20000.87PhenylalanineInosine 0.55 1.25 - 12501 - 1000Uracil 0.44Hyp x n h 0.31 0.7 - 700o a t ine0.5 - 500L-Tyrosine 0.260.5 - 500L-Tryptophan 0.260.25 0.5 - 500Guanosine0.4 - 400Xanthosine 0.190.3-300L-Citrulline 0.14

[0063] or (4) the following metabolites present at about the relative concentration to L-phenylalanine listed belowCone. RangeRelativeCompound (ng / mL)concentrationL- 3 - 30001Phenylalanine2 - 2000L-Tyrosine 0.56Guanine 0.55 2 - 20001.25 - 1250Hypoxanthine 0.37Uracil 0.3 1 - 10001 - 1000Guanosine 0.291 - 1000L-Methionine 0.28Xanthine 0.17 0.5 - 5000.5 - 500Guanine 0.150.3 - 300Inosine 0.11

[0064] The relative concentrations of the indicated metabolites may be measured by methods known in the art, e.g., LC-MS.

[0065] In some embodiments, the aqueous solutions are serum free / do not comprise serum.Systems

[0066] In an aspect of the current disclosure, systems are provided, e.g., systems for generating cultivated meat. In some embodiments, the systems comprise (a) a cell culture medium comprising an amount of a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Lactobacillus lactis, Bacillus subtilis, and Lactobacillus plantarum or any of the the aqueous solutions of this disclosure; and (b) an animal cell.

[0067] The animal cells may be, e.g., muscle cells, e.g., muscle cells derived from the following non-limiting list of organisms: seafood such as fish (e.g., salmon, tuna, tilapia, perch, mackerel, cod, sardine, trout, etc.), shellfish (e.g., clams, mussels, and oysters); crustaceans (e.g., lobsters, shrimp, prawns, and crayfish), echinoderms (e.g., sea urchins and sea cucumbers), cow, chicken,sheep, pig, horse, mouse, goat, or rabbit. The cells may be of bovine, galline, ovine, porcine, equine, murine, caprine, lapine, or piscine origin. The animal cells may be adipose cells or adipose precursors, e.g., from any of the preceding organisms. The animal cells may be cells of any tissue origin, e.g., from animals of the preceding list.

[0068] The animal cell may be a bovine satellite cell, a fish cell, e.g., a mackerel cell, e.g., a mackerel satellite cell.

[0069] In some embodiments, the amount of the lysate is an “effective amount” of the lysate which refers to an amount of the lysate that is sufficient to allow short-term growth or long-term growth of the cells, when cultured in an appropriate environment, e.g., a cell culture incubator set to known appropriate conditions for the cell. In some embodiments, an effective amount is an amount of the lysate that is sufficient to allow long-term growth of the cells.Methods of making cell culture media

[0070] In an aspect of the current disclosure, methods of making cell culture media are provided. In some embodiments, the methods comprise (a) generating a lysate from a microbe; (b) combining an amount of the lysate from the microbe with a base medium, thereby generating the cell culture medium.

[0071] The microorganism may be selected from Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), or Escherichia coli BL21 (BL21).

[0072] As described above, there are a variety of methods that may be used to “lyse” microorganisms to generate lysates including, but not limited to, physical methods, e.g., sonication, bead beating, centrifugation, chemical methods, e.g., acid exposure, base exposure, detergent exposure.

[0073] The lysates may be centrifuged and / or filtered, subsequent to lysing the microorganismal cells, to remove solid particles. Methods for centrifuging and filtering are routine in the art.

[0074] an amount of the lysate from the microorganism may be about 0.01 pg / ml to about 1000 [tg / ml, any amount within a range bounded by about 0.01 pg / ml on the low end to about 1000|lg / ml on the high end, about 1 pg / ml. about 10 pg / ml, about 20 pg / ml. about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, about 60 pg / ml. about 70 pg / ml. about 80 pg / ml. about 90 pg / ml. about 100 pg / ml, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47. 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61. 62. 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73. 74. 75. 76. 77. 78, 79, 80, 81, 82, 83, 84, 85, 86, 87. 88. 89. 90.91, 92, 93, 94, 95, 96, 97, 98, 99, 100 pg / ml or more.

[0075] The microorganism may be Vibrio natriegens and the amount may be about 20 pg / ml.

[0076] The microorganism may be Saccharomyces cerevisiae and the amount may be about 10 pg / ml to about 100 pg / ml.

[0077] The microorganism may be Escherichia coli Nissle 1917 (EcN) and the amount may be about 10 pg / ml.Methods of generating cultivated meat

[0078] In an aspect of the current disclosure, methods of generating cultivated meat are provided. In some embodiments, the methods of generating cultivated meat comprise culturing an animal cell in the medium of the instant disclosure or the aqueous solutions of the instant disclosure for a sufficient time to generate cultivated meat.

[0079] As used herein, “cultivated meat” refers to a composition comprising a plurality of muscle cells that are connected, e.g., about IxlO3muscle cells to about IxlO20muscle cells per kilogram, or any number in the preceding range, inclusive of the endpoints. Cultivated meat may bear an aesthetic resemblance to conventionally grown and harvested meat. The muscle cells may be any suitable muscle cell, as disclosed herein. Further, the cultivated meat may comprise adipose cells or connective tissue cells.

[0080] In some embodiments, the cultivated meat is cultured on a “scaffold” which may refer to any material which serves as a substrate for cell growth, which is later included in the cultured meat product, or otherwise separated from the cells. Such “scaffolds” may refer to microcarriers,porous sponges, fibers, hydrogels, decellularized tissue, 3D printed constructs, or films, made from synthetic or bio-based materials.

[0081] In some embodiments, the method causes cell doubling at a similar or greater rate than culturing the animal cells in B9 medium. As used herein, "doubling." “rate of doubling,” or grammatical variations thereof, refer to the rate at which a population of live cells doubles in absolute number. Doubling may be measured by methods known in the art, e.g., live-cell dye exclusion and counting or automated systems for accomplishing the same, over a period of time. Doubling may be referred to in units of minutes, hours, days, weeks, etc.

[0082] In some embodiments, the method results in a similar or greater level of Pax7+cells than culturing the animal cells in B9 medium. Pax7 is a marker of sternness in muscle satellite cells, thus, measuring the level of Pax 7 in satellite cells acts as a surrogate marker for sternness of the cells. The inventors demonstrated that the disclosed media are capable of maintaining the Pax7 expression in bovine satellite cells on a long-term basis.Methods of detecting a suitable microorganismal lysate for use as a mammalian serum replacement for animal cell culture

[0083] In an aspect of the current disclosure, methods of detecting a suitable microorganismal lysate for use as a mammalian serum replacement for animal cell culture are provided. In some embodiments, the methods comprise (a) preparing a lysate from a sample of a species of cultured microorganisms; (b) culturing (1) a test plurality of animal cells in a test medium, wherein the test medium comprises the lysate prepared in step (a), and (2) a control plurality of animal cells in a control medium; (c) analyzing the growth of the test plurality of animal cells and the control plurality of animal cells; (d) detecting the lysate as a suitable microorganismal lysate for use as a mammalian serum replacement for animal cell culture wherein growth of the test plurality' of animal cells is about similar or greater than the control plurality’ of animal cells.

[0084] The cells may be any suitable cell, as disclosed above.

[0085] A “control medium” as used herein, refers to any medium for animal cell culture known in the art, e.g., B8, B9, DMEM, RPMI, etc.

[0086] As used herein, “test medium” refers to a medium that is identical to the “control medium” but that the test medium comprises the lysate prepared in step (a) of the method and, if the control medium comprises serum, the test medium does not comprise the serum. In other words, the control medium is used as an experimental control for the test medium to determine the suitability' of the lysate for the growth of the subject cells. For example, the control medium may by B8 medium, and the test medium may be B8 medium further comprising the lysate; the control medium may be B8 medium with serum and the test medium would be B8 medium further comprising the lysate but not comprising serum; or the control medium may be B9 medium and the test medium may be B9 medium further comprising the lysate or B9 medium without albumin or recombinant albumin, depending on the desired method to be tested.

[0087] As used herein, “analyzing the growth” of the test plurality of animal cells and the control plurality of animal cells may comprise analyzing the doubling time of the test plurality of animal cells and the control plurality of animal cells or analyzing a number of successful passages of the test plurality of animal cells and the control plurality' of animal cells.

[0088] As used herein, “the number of successful passages” comprises the greatest passage number x where the doubling time of passage number x-1 is about equal or greater than the doubling time of passage number x. In other words, if the control plurality of animal cells doubled in about 2 days at passage 9, doubled in about 2 days at passage 10. and doubled in about 3 days at passage 11, the number of successful passages would be 10.

[0089] The methods may further comprise cultivating a plurality' of the animal cells in medium comprising the microorganismal lysate.Kits, systems, and platforms

[0090] In an aspect of the current disclosure, kits, systems, and platforms are provided. In some embodiments, the kits, systems, or platforms comprise a lysate from a microorganism. The lysate from the microorganism may be selected from the group consisting of Vibrio natriegens, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Lactobacillus lactis, Bacillus subtilis, and Lactobacillus plantarum.

[0091] The lysate may be packaged as a unit dose wherein the unit dose comprises an amount of the lysate for use as a substitute for serum in animal cell culture. The unit dose may comprise an amount of the lysate for use as a substitute for serum in animal cell culture when dissolved in a predetermined amount of a base medium.

[0092] The kits, sy stems, or platforms may further comprise one or more of sodium selenite, albumin, transferrin, transforming growth factor beta. L-ascorbic acid, insulin, neuregulin 1 (NRG1), fibroblast growth factor 2 (FGF-2), DMEM, HEPES, or sodium bicarbonate. The kits systems or platforms may comprise a liquid form of a base medium, e.g., DMEM, RPMI, B8, etc., or may comprise a powdered formulation of the same. The lysate may be stored separately from the medium, e.g., the lysate may be cryogenically stored at, e.g., -80 C, -20 C, etc. and the medium stored at room temperature.

[0093] Methods for recycling spent media

[0094] The inventors discovered that the microbes used to generate lysates of the disclosure may be grown in spent media from a previous round of cell culture, e.g., cells grown for cellular agriculture, like muscle or fat cells. This way, the cost of growing cells for consumption or recombinant protein production, for example, can further be reduced by the disclosed methods.

[0095] Therefore, in an aspect of this disclosure, methods for recycling spent media are provided. In some embodiments, the methods comprise culturing a plurality of cells on a cell culture medium; removing the plurality of cells from the cell culture medium to generate a cell-free spent culture medium; and culturing microorganisms on the cell free spent culture medium.

[0096] The cell culture medium used for initial culture of the cells may comprise or consist of the cell culture medium of this disclosure, i.e., cell culture media comprising an amount of a lysate from a microorganism, e.g., the microorganism is selected from the group consisting of Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Bacillus subtilis, Lactobacillus lactis, Latilactobacillus sakei, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, and Lactobacillus gasseri.

[0097] The method may further comprise generating a lysate from the microorganisms cultured on the cell free spent cultured medium. The lysate generated may be used in a further round of culturing cells, e.g., muscle or fat cells for cellular agriculture.Compositions comprising spent cell culture media comprising a lysate from a microorganism

[0098] As noted above, the inventors discovered that spent cell culture media may be recycled to grow a round of microorganisms that may be used to generate a lysate for another round of culturing cells, e.g., muscle or fat cells.

[0099] Accordingly, in an aspect of this disclosure, compositions are provided. In some embodiments, the compositions comprise spent cell culture media comprising a lysate from a microorganism. The microorganism may comprise any of the disclosed microorganisms, e.g., Vibrio natriegens, Lactobacillus plantarum. Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), o Escherichia coli BL21 (BL21).

[0100] Spent culture media has been shown to have increased concentrations of metabolic byproducts, e.g., lactate and ammonium, after culturing cells, e.g., muscle cells. See, e.g., FIG. 3 of Arian Amirvaresi and Reza Ovissipour "Evaluating the Efficacy of Serum-Free Media Supplemented with Protein Isolates for Bovine Satellite Cell Proliferation: A Sustainable Approach for Cultivated Meat Production” BioRxiv, 2024 doi.org / 10.1101 / 2024.08.23.609451 or O’Neill et al., “The effect of serum-free media on the metabolic yields and growth rates of C2C12 cells in the context of cultivated meat production” Future Foods, vol. 7. June 2023, 100226, O’Neill et al. “Spent media analysis suggests cultivated meat media will require species and cell type optimization” NPJ Sci Food. 2022 Sep 29;6:46, and Freund et al. “A Simple Method to Reduce both Lactic Acid and Ammonium Production in Industrial Animal Cell Culture” Int. J. Mol. Sci. 2018. 19(2), 385. each of which are incorporated by reference herein.

[0101] The spent culture media may comprise at least about 0.3 g / ml lactate, e.g., at least about 0.3, at least about 0.4, at least about 0.5 at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1.0 g / ml, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10 g / ml or more lactate or about 0.3 to about 10 g / ml lactate.

[0102] The spent cell culture may comprise at least about 0.4 mM ammonium, e.g., at least about 0.5 at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1.0. at least about 2, at least about 3, at least about 4, at least about 5. at least about 6, at least about 7, at least about 8, at least about 9, at least about 10 g / ml or more lactate or about 0.3 to about 10 mM ammonium or more or about 0.4 to about 20 mM ammonium.

[0103] Methods of measuring lactate ammonium or other metabolites in spent media are known in the art, e.g., in Arian Amirvaresi and Reza Ovissipour, supra,

[0104] The compositions may further comprise ingredients added to the spent media and / or sterilization of the spent media, e.g., by autoclaving. The ingredients added may comprise any suitable ingredient for cell culture media, as discussed above, e.g., NaCl, e.g., 0.1% to about 10% NaCl, or any subrange or value therein, or about 3% NaCl.Definitions

[0105] The disclosed subj ect matter may be further described using definitions and terminology as follows. The definitions and terminology' used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0106] As used in this specification and the claims, the singular forms “a,” ‘‘an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0107] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0108] As used herein, the terms “include” and “including” have the same meaning as the terms ‘comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted asbeing “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0109] The phrase "such as” should be interpreted as "for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0110] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary' skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone. A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0111] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0112] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspectof a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb "may" has the same meaning and connotation as the auxiliary verb "can."Illustrative Embodiments1. A cell culture medium comprising an amount of a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Bacillus subtilis, Lactobacillus lactis, Latilactobacillus sakei, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, and Lactobacillus gasseri.2. The cell culture medium of embodiment 1, wherein the microorganism is Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), ox Escherichia coli BL21 (BL21).3. The cell culture medium of any one of embodiments 1-2, wherein the microorganism is Vibrio natriegens.4. The cell culture medium of any one of embodiments 1-3, wherein the amount is about 1 pg / ml to about 100 pg / ml.5. The cell culture medium of any one of embodiments 1-3, wherein the amount is about 10 pg / ml to about 100 pg / ml.6. The cell culture medium of any one of embodiments 1-3, wherein the amount is about 10 pg / ml to about 50 pg / ml.7. The cell culture medium of any one of embodiments 1-3, wherein the amount is about 10 pg / ml to about 40 pg / ml.8. The cell culture medium of any one of embodiments 1-3, wherein the amount is about 40 pg / ml.9. The cell culture medium of any one of embodiments 1-3, wherein the amount is about 20 pg / ml.10. The cell culture medium of any one of embodiments 1-3, wherein the microorganism is Vibrio natriegens and an amount is about 20 pg / ml.11. The cell culture medium of any one of embodiments 1-3, wherein the microorganism is Saccharomyces cerevisiae and an amount is about 10 pg / ml to about 100 pg / ml.12. The cell culture medium of any one of embodiments 1-3, wherein the microorganism is Escherichia coll Nissle 1917 (EcN) and an amount is about 10 p.g / ml.13. The cell culture medium of any one of embodiments 1-12, wherein the cell culture medium comprises one or more of sodium selenite, albumin, transferrin, transforming growth factor beta, L-ascorbic acid, insulin, neuregulin 1 (NRG1), or fibroblast growth factor 2 (FGF-2).14. The cell culture medium of any one of embodiments 1-13, wherein the cell culture medium comprises DMEM, HEPES, and / or sodium bicarbonate.15. The cell culture medium of any one of embodiments 1-14, wherein the cell culture medium supports short or long term growth or differentiation of cells, w herein the cells are optionally mammalian, fish, and / or insect muscle or adipose cells.16. The cell culture medium of any one of embodiments 1-14, wherein the cell culture medium supports short or long term growth or differentiation of bovine satellite cells or mackerel cells (Mackl).17. The cell culture medium of any one of embodiments 1-16, wherein the medium does not contain serum.18. The cell culture medium of any one of embodiments 1-17, wherein the medium comprises one or more of the following compounds: uracil, hypoxanthine, xanthine, L-phenylalanine, guanine, L-arogenate, L-glutamate, L-citrulline, D-xylulose 5-phosphate, L-methionine, 5-aminopentanoate, cytosine, L-proline. inosine. L-serine, benzonitrile, portulacaxanthin II, L-tyrosine, L-tryptophan, guanosine, or xanthosine.19. The cell culture medium of any one of embodiments 1-18, wherein the medium comprises the following compounds: uracil, hypoxanthine, xanthine, L-phenylalanine, guanine, L-arogenate, L-glutamate, L-citrulline, D-xylulose 5-phosphate, L-methionine, 5-aminopentanoate, cytosine, L-proline, inosine, L-serine, benzonitrile, portulacaxanthin II, L-tyrosine, L-tryptophan, guanosine, and xanthosine.20. The cell culture medium of any one of embodiments 1-19, wherein the medium comprises one or more of (1), (2), (3), or (4), wherein:(1) the following compounds present at about the relative concentration to uracil listed below' or the absolute concentrations listed belowCone. RangeRelativeCompound (ng / mL)concentration1 - 1,000Uracil 10.26 - 260Hypoxanthine 0.26Xanthine 0.17 0.17 - 170L- 0.14 - 1400.14PhenylalanineGuanine 0.05 0.05 - 500.05 - 50L-Arogenate 0.050.04 - 40L-Glutamate 0.04L-Citrulline 0.03 0.03 - 300.02 - 20D-Xylulose0.025 -phosphate0.02 - 20L-Methionine 0.02(2) the following metabolites present at about the relative concentration to 5-aminopentanoate listed belowCone. RangeRelativeCompound (ng / mL)concentration5- 25 - 25,0001Aminopentanoate1 - 1,000Uracil 0.04Cytosine 0.003 0.075 - 750.05 - 50L-Proline 0.0020 0.05 - 50Inosine .0020.025 - 25L-Serine 0.0010.025 - 25Benzonitrile 0.001Portulacaxanthin 0.0075 -7.50.0003II(3) the following metabolites present at about the relative concentration to xanthine listed belowCone. RangeRelativeCompound (ng / mL)concentration2.25 - 2250Xanthine 1L- 2 - 20000.87PhenylalanineInosine 0.55 1.25 - 12501 - 1000Uracil 0.44Hypoxanthine 0.31 0.7 - 7000.5 - 500L-Tyrosine 0.260.5 - 500L-Tryptophan 0.26Guanosine 0.25 0.5 - 5000.4 - 400Xanthosine 0.190.3-300L-Citrulline 0.14or (4) the following metabolites present at about the relative concentration to L- phenylalanine listed belowCone. RangeRelativeCompound (ng / mL)concentrationL- 3 - 30001Phenylalanine2 - 2000L-Tyrosine 0.56Guanine 0.55 2 - 20001.25 - 1250Hypoxanthine 0.37Uracil 0.3 1 - 10001 - 1000Guanosine 0.291 - 1000L-Methionine 0.28Xanthine 0.17 0.5 - 5000.5 - 500Guanine 0.150.3 - 300Inosine 0.1121. An aqueous solution comprising a serum replacement, wherein the serum replacement comprises a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Lactobacillus lactis, Bacillus subtilis, and Lactobacillus plantarum, and one or more of sodium selenite, albumin,transferrin, transforming growth factor beta, L-ascorbic acid, insulin, neuregulin 1 (NRG1), fibroblast growth factor 2 (FGF-2), DMEM, HEPES, or sodium bicarbonate.22. The aqueous solution of embodiment 21, wherein the microorganism is Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), ox Escherichia coli BL21 (BL21).23. The aqueous solution of any one of embodiments 21 -22, wherein the microorganism is Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), ox Escherichia coli BL21 (BL21).24. The aqueous solution of any one of embodiments 21 -23, wherein the microorganism is Vibrio natriegens.25. A system for generating cultivated meat, the system comprising:(a) the cell culture medium of any one of embodiments 1-20; and(b) an animal cell.26. The system of embodiment 25, wherein the animal cell is a multipotent animal cell.27. The system of embodiment 25, wherein the animal cell is a satellite cell.28. The system of embodiment 25, wherein the animal cell is a bovine satellite cell. 29. The system of embodiment 25, wherein the animal cell is a fish cell.30. The system of embodiment 25, wherein the animal cell is a mackerel cell.31. The system of embodiment 25, wherein the animal cell is a mackerel satellite cell.32. A method of making the cell culture medium of embodiment 1, the method comprising:(a) generating a lysate from a microbe; and(b) combining an amount of the lysate from the microbe with a base medium, thereby generating the cell culture medium.33. The method of embodiment 32, wherein generating a lysate from a microbe comprises sonicating the microbe.34. The method of embodiment 33, wherein generating a lysate from a microbe further comprises micro filtering the sonicated microbes to generate a filtered lysate.35. The method of any one of embodiments 32-34. wherein the microorganism is selected from the group consisting of Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21(BL21), Bacillus subtilis, Lactobacillus lactis, Latilactobacillus sakei, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, and Lactobacillus gasseri or wherein the microorganism is Vibrio natriegens. Lactobacillus plantarum, Saccharomyces cerevisiae. Escherichia coli Nissle 1917 (EcN), ox Escherichia coli BL21 (BL21).36. The method of any one of embodiments 32-35, wherein the microorganism is Vibrio natriegens.37. The method of any one of embodiments 32-36, wherein the amount is about 1 |lg / ml to about 100 pg / ml.38. The method of any one of embodiments 32-37, wherein the amount is about 10 |lg / ml to about 100 |ig / ml.39. The method of any one of embodiments 32-37, wherein the amount is about 10 pg / ml to about 50 pg / ml.40. The method of any one of embodiments 32-37, wherein the amount is about 10 |lg / ml to about 40 pg / ml.41. The method of any one of embodiments 32-37, wherein the amount is about 40 |lg / ml.42. The method of any one of embodiments 32-37, wherein the amount is about 20 |ig / ml.43. The method of any one of embodiments 32-37, wherein the microorganism is Vibrio natriegens and an amount is about 20 pg / ml.44. The method of any one of embodiments 32-37, wherein the microorganism is Saccharomyces cerevisiae and an amount is about 10 pg / ml to about 100 g / ml.45. The method of any one of embodiments 32-37, wherein the microorganism is Escherichia coli Nissle 1917 (EcN) and an amount is about 10 pg / ml.46. A method of generating cultivated meat, the method comprising culturing an animal cell in the medium of any one of embodiments 1-20 for a sufficient time to generate cultivated meat.47. The method of embodiment 46, wherein culturing an animal cell comprises culturing the animal cell on a scaffold.48. The method of embodiment 46, wherein the animal cell is a multipotent animal cell.49. The method of embodiment 46, wherein the animal cell is a satellite cell.50. The method of embodiment 46, wherein the animal cell is a bovine satellite cell.51. The method of embodiment 46, wherein the animal cell is a fish cell.52. The method of embodiment 46, wherein the animal cell is a mackerel cell.53. The method of embodiment 46, wherein the animal cell is a mackerel satellite cell.54. The method of any one of embodiments 46-53, wherein the method causes cell doubling at a similar or greater rate than culturing the animal cells in B9 medium.55. The method of any one of embodiments 46-53, wherein the method results in a similar or greater level of Pax7+cells than culturing the animal cells in B9 medium.56. A method of detecting a suitable microorganismal lysate for use as a mammalian serum replacement for animal cell culture, the method comprising:(a) preparing a lysate from a sample of a species of cultured microorganisms;(b) culturing (1) a test plurality of animal cells in a test medium, wherein the test medium comprises the lysate prepared in step (a), and (2) a control plurality of animal cells in a control medium;(c) analyzing the growth of the test plurality of animal cells and the control plurality of animal cells; and(d) detecting the lysate as a suitable microorganismal lysate for use as a mammalian serum replacement for animal cell culture wherein growth of the test plurality of animal cells is about similar or greater than the control plurality of animal cells.57. The method of embodiment 56. wherein the animal cell is a multipotent animal cell.58. The method of embodiment 56, wherein the animal cell is a satellite cell.59. The method of embodiment 56, wherein the animal cell is a bovine satellite cell.60. The method of embodiment 56, wherein the animal cell is a fish cell.61. The method of embodiment 56, wherein the animal cell is a mackerel cell.62. The method of embodiment 56, wherein the animal cell is a mackerel satellite cell.63. The method of embodiment 56, wherein analyzing the growth of the test plurality of animal cells and the control plurality of animal cells comprises analyzing the doubling time of the test plurality of animal cells and the control plurality of animal cells.64. The method of any one of embodiments 56-63. wherein analyzing the growth of the test plurality of animal cells and the control plurality of animal cells comprises analyzing anumber of successful passages of the test plurality of animal cells and the control plurality of animal cells.65. The method of any one of embodiments 56-64, wherein the number of successful passages comprises the greatest passage number x where the doubling time of passage number x-1 is about equal or greater than the doubling time of passage number x.66. A kit, system, or platform comprising a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Bacillus subtilis. Lactobacillus lactis, Latilactobacillus sakei, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, and Lactobacillus gas s er i.67. The kit, system, or platform of embodiment 66, wherein the lysate is packaged as a unit dose wherein the unit dose comprises an amount of the lysate for use as a substitute for serum in animal cell culture.68. The kit, system or platform of embodiment 66 or 67, wherein the unit dose comprises an amount of the lysate for use as a substitute for serum in animal cell culture when dissolved in a predetermined amount of a base medium.69. The kit, system, or platform of any one of embodiments 66-68, further comprising one or more of sodium selenite, albumin, transferrin, transforming growth factor beta, L- ascorbic acid, insulin, neuregulin 1 (NRG1), fibroblast growth factor 2 (FGF-2), DMEM, HEPES, or sodium bicarbonate.EXAMPLES

[0113] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.Example 1 - Microbial lysates as low-cost serum replacements for media for cellular agriculture

[0114] Abstract

[0115] Cultivated meat, the process of generating meat in vitro without the the need to sacrifice animals, is a promising alternative to the traditional practice of livestock agriculture. However, the success of this field depends on finding sustainable and economic replacements for animal-derived and expensive fetal bovine serum (FBS) that is typically used in cell culture processes. Here, the inventors outline an effective screening process to vet the suitability of microbial lysates to support the growth of immortalized bovine satellite cells (iBSCs) and mackerel (Mackl) cells. The inventors show that easily producible, low-cost whole-cell lysates of Vibrio natriegens bacteria can be used to create serum-free media for the long-term growth of iBSCs. The optimized medium, named “VN40'’ (basal B8 media containing Vibrio natriegens lysate proteins at 40 pg / mL), outperforms previously established serum-free media, while maintaining cell phenotype and myogenicity. Overall, this study shows a novel approach to produce serum-free media for cultivated meat production using microbialfy-derived lysates.

[0116] Many attempts have been made to create serum-free media (SFM) for cultivated beef and fish with varying levels of success. Of note is recent work adapting serum-free “B8” media designed for human iPSC culture13to the culture of bovine satellite cells14via the addition of recombinant albumin, leading to the creation of “Beefy 9-’ medium. Still, efforts to replace FBS with recombinant albumin14-15or other mitogenic proteins like fetuin16for cultivated meat production require expensive purified recombinant protein on the g / L scale. Cunent estimates show cultivated meat may require as much as 42 liters of media per kilogram to produce17, making the dependence on such quantities of recombinant proteins economically infeasible. Other methodologies using crude extracts or hydrolysates from plant18or microalgal19-20sources to supplement SFM have the potential to reduce raw material costs considerably. However, these methods can be labor-intensive-18 21, require corrosive chemicals20, or are otherwise still dependent on animal serum19or animal by-products22.

[0117] Microbes such as yeast, bacteria, and algae may provide a promising solution to the longstanding problem of creating serum-free media formulations, as they are animal-free, inexpensive to produce, highly renewable, and rich in proteins and other nutrients. Microbes can also be engineered to express growth factors, which currently represent 99% of the cost of cultured meat media at scale12. Yeast hydrolysates have longstanding use in creating serum-free media23, 24, and recent reports detail using extracts from Saccharomyces cerevisiae yeast25. Enterococcus hirae bacteria26, cyanobacteria27-29, and Chlorella algae30, 20in cultured meat production. However, current research has not demonstrated success in identifying a non-pathogenic, rapidly replicating microbial extract capable of stimulating long-term cellular grow thin serum-free conditions. Microalgae are slow-growing, taking several days to culture20, while cyanobacteria can have toxic effects on cells31. Yeast and bacterial extracts still cannot completely substitute FBS or support long-term growth of animal cells26, 31. For a more comprehensive list of previous attempts to replace serum in cultivated meat media, see Table 2.

[0118] Media for the production of cultivated meat must strive to minimize time, labor, and material costs in order to make cultivated meat economically viable. Such media must also be animal-free and safe to consume and produce. Finally, cultivated meat media must stimulate longterm cell growth with minimal impacts on cell characteristics. While microbial extracts are a promising ingredient for replacing FBS in cell culture media, there have not been any results that satisfy the criteria necessary for commercial use in cultivated meat production. Further, it is known that media requirements vary significantly depending on cell type32.

[0119] In this work, the inventors tested multiple lysates from Gram-positive and Gramnegative bacteria, as well as a yeast, to determine their suitability as replacements for FBS in bovine and fish cell culture media. Based on an initial screen, the inventors found that lysates derived from Vibrio natriegens could serve as an alternative to FBS for iBSCs. A similar screen also identified that lysates from Saccharomyces cerevisiae could support growth of Mackl cells in reduced serum conditions. The inventors show that iBSCs can be adapted to grow' rapidly in Vibrio natriegens lysate-containing media by long-term and short-term serially passaging. Cells cultured in this microbial lysate media (called VN40) demonstrate robust growth, preservation of satellite cell phenotype, and an ability to differentiate into multinucleated myotubes. These results highlight the promise of the whole-cell lysate derived from the fast-growing non-pathogenic marine bacterium, V. natriegens, as an easily producible, low-cost supplement for long-term serum-free growth of iBSCs for cultivated meat. Further, the inventors’ approach may be generalizable to help identity' novel microbial lysates as FBS substituents for different cultivated meat cell lines.

[0120] MATERIAL & METHODS

[0121] Culture and extraction of microbial lysate

[0122] Escherichia coh BL21(DE3) and Nissle 1917, Vibrio natrigens 14048, Lactobacillus plantarum WCF51, Bacillus subtilis 168, and Saccharomyces cerevisiae CEN.PK and BMA-64were streaked out on plates using their respective solid media (see Table 1). Single colonies from the plates were used to inoculate 25 mL of overnight cultures. The next day, cells were harvested by centrifuging at 4000 rpm for 5 min (ThermoScientific Sovrall Legend X1R Centrifuge). Harvested cells were prepared for sonication by washing and resuspending in 1ml sterile phosphate buffer saline, without any additives.

[0123] To generate microbial lysates, cells were sonicated (Branson 150 sonicator and 10 s Pulse, 30 s gap between each pulse and a total 7 min at 45 % amplitude) on ice. After sonication, the soluble lysate was obtained by centrifuging the sonicated solution at 21000 x g for 20 min. The supernatant was then filtered through a 0.2 pm syringe filter (Coming, #431219). The total protein content of the lysate was determined using Bradford Assay (Coomasie Plus™ Protein Assay Reagent. #1856210). (Figure 1A)Microorganisms Medium Growth ConditionsE. coli BL21 (BL21), E. coll Nissle (ECN), Lysogeny Broth (LB) Overnight at Bacillus subtilis 168 (BS) 37 °CVibrio natrigens ATCC 14048 (VN) LB with 5 % NaCl Overnight at 37 °CLactiplantibacillus plantarum WCFS1 (LP), De Man-Rogosa- Overnight at Lactococcus Lactis MG1363 (LL) Sharpe (MRS) 37 °CS. cerevisae CEN.PK (CEN.PK) Yeast peptone adenine Overnight at dextrose (YPAD) 30 °C

[0124] SDS-PAGE

[0125] Denaturing SDS-PAGE was performed following the Invitrogen NuPAGE® protocol. Lysate samples containing 30 pg of protein were adjusted to a final volume of 50 pL with PBS and mixed with 10 pL of 6* SDS loading buffer. The samples were incubated at 95°C for 10minutes. Subsequently, 10 pL of the sample was loaded onto a pre-cast NuPAGE Novex 4-12% Bis-Tris 1.0 mm mini-gel (Invitrogen). For each gel run, 5 pL of pre-stained SDS-PAGE standards (Bio-Rad) were included. Electrophoresis was conducted at a constant voltage (200 V) in lx MES (2-Morpholinoethanesulphonic acid) buffer solution at room temperature for approximately 45 min.

[0126] Lysate fractionation

[0127] Vibrio natrigens lysate was fractionated using a 3 kDa molecular weight cutoff filter (MilliporeSigma™ Amicon™ Ultra- 15 Centrifugal Filter) at 4000 x g. This resulted in two fractions i.e., protein rich fraction greater than 3kDa (HMW fraction) and low molecular weight fraction lower than 3KDa (LMW fraction). The fractions were then tested individually on iBSC cells.

[0128] To test for additive effect of fractions, the inventors used Equation 1 and Equation 2 to determine the sum of means ( / ) and standard deviations ( ), respectively, of fractions’ impact on cell growth. These effects were compared to the impact of whole cell lysates, which are HMW and LMW fractions combined2 H(LMW, HMW) = n(LMW) + p(HMW) (Equation 1)2cr(LMWZ, HMW) = Ja2(LMW) + a2(HMW) (Equation 2)

[0129] iBSC and Mackl culture and maintenance

[0130] iBSCs were previously isolated from the semitendinosus of a 5-week-old Simmental calf at the Tufts Cummings School of Veterinary Medicine14. These cells were engineered to constitutively express bovine telomerase reverse transcriptase (TERT) and cyclin-dependent kinase 4 (CDK4) in prior studies8. iBSCs at passage 40 were thawed from liquid nitrogen stock and passaged in iBSC grow th media (iBSCGM) containing DMEM+Glutamax (ThermoFisher #10566024, Waltham, MA, USA) with 20 % FBS (ThermoFisher #26140079), 1 ng / mL human fibroblast growth factor (FGF)-2 (PeproTech #100-18B, Rocky Hill, NJ, USA), supplemented with 1 % antibiotic / antimycotic (ThermoFisher #1540062) and 2.5 pg / mL puromycin(ThermoFisher #A1113803) as a selection compound. iBSCs were cultured at 37°C and 5 % humidity on tissue culture polystyrene (TCPS) and routinely passaged at 80-90% confluency using 0.25% trypsin-EDTA (ThermoFisher #25200056) to disassociate cells from TCPS. During passaging, cells were dissociated, centrifuged at 300 x g. and seeded at 1,500-2,000 cells / cm2Mackl cells (Kerafast, #ETU008-FP) isolated from Atlantic mackerel were cultured in Mack growth media (MackGM) containing Leiovitz’s L-15 medium (ThermoFisher #11415064) with 20 % FBS, 20 mM HEPES (Sigma Aldrich #H4034, St. Louis, MO, USA) buffer solution (pH 7.4), 1 % antibiotic / antimycotic, 1 ng / mL human FGF-2, and 10 pg / mL gentamicin (Sigma Aldrich #G1397)4, 9Mackl cells were incubated at 27°C without CO2 on TCPS, routinely detached at 70-80 % confluency using 0.05% trypsin-EDTA (ThermoFisher #25300054), centrifuged at 300 x g. and seeded at 3,500-4,000 cells / cm2. Both cell lines (iBSCs and Mackl) were fed every 2-3 days with iBSCGM and MackGM. respectively.

[0131] Short-term iBSC and Mackl cell growth screening with microbial lysates

[0132] For iBSC short-term screening on microbial lysate-based media, iBSCs were harvested and resuspended into serum-free B8 media containing Hi-Def B8 aliquots (Defined Bioscience #LSS-204, San Diego, CA, USA) in DMEM / F12 (ThermoFisher #11320032) supplemented with 1 % antibiotic / antimycotic and 2.5 pg / mL puromycin. Cells were seeded onto 96-well plates at 2.500 cells / cm2with 1.5 pg / cm2of truncated recombinant human vitronectin (rhVTN-N; ThermoFisher #A31804). Cells were allowed to adhere for >3 h, then were washed once with DPBS (ThermoFisher #14190250) and media was changed to B8 + microbial lysates at given protein concentrations. Negative control conditions were fed B8 alone. Positive control conditions were fed Beefy 9, containing B8 supplemented with 0.8 mg / mL of recombinant albumin (Sigma Aldrich #A9731)14. Mackl cells were harvested and resuspended in MacklGM and plated onto 96-well plates at 2,500 cells / cm2. After more than 3 hour-incubation, cells were washed once with DPBS and media was changed to 2.5% FBS-containing L-15 media (other media components, including HEPES, antibiotic / antimycotic, FGF-2, and gentamicin, were added at the same concentrations as MacklGM) + microbial lysates at give protein concentrations. Negative controls were MacklGM without FBS and 2.5% FBS-containing L-15 media. Media formulations without cells were used as blanks to account for media autofluorescence. All samples were allowed to grow' undisturbed for 4 (Mackl) and 5 days (iBSCs), at which point wells were washedonce with DPBS and frozen overnight at -80°C. Samples were then analyzed for short-term grow th using direct DNA quantification with CyQUANT Cell Proliferation Assay (ThermoFisher #C7026) using manufacturer’s protocol. Emission / excitation was read at 480 / 520 nm on a ThermoFisher Varioskan LUX multimode microplate reader. Blank values from media-only samples were subtracted from experimental values, and these values w ere normalized to grow th using either B8 alone (iBSCs) or 2.5% FBS-containing L-15 media (Mackl) to compare the growth between conditions. Experiments were repeated using a narrower range of lysate concentration and using serum-free adapted iBSCs passaged 4 times consecutively in Beefy-9.

[0133] Adaptation and long-term growth in Vibrio natriegens-based media

[0134] After choosing V. natrigens 10 mg / ml serum-free media as a starting point for adaptation based on short-term screens, iBSCs, now at passage 60, were resuspended and seeded at 2,000 cells / cm2in B8 media with 1.5 pg / cm2vitronectin in a 6-well plate. After adhering for >3 hours, cells were fed 2 ml B8 media supplemented with 10 mg / ml V. natrigens lysate. Cells were fed V. natriegens lysate-based media every 2-3 days until reaching 80-90% confluence. At this point, cells were passaged using TrypLE Express enzy me (ThermoFisher #12604013) and counted using a NucleoCounter NC-200 (Chemometec, Allerod, Denmark). Doubling time (DT) was calculated using Equation 3.At

[0135] DT = (Equation 3)l°g2

[0136] with At being time between passages, Cf being cell count at passage, and Ctbeing total cells seeded. Cells were then centrifuged at 300 x g and seeded in a new well. This process was repeated continuously. When doubling time reached a minimum or plateaued, V. natriegens concentration was doubled, until doubling time was comparable to that of iBSCGM, which was when V. natrigens 40 mg / rnL (VN40) was used. At this point, VN40-adapted cells were seeded in triplicate in a 6-well plate and fed VN40. Cell doublings were measured at each passage for each replicate. This same process was repeated simultaneously for Beefy 9-adapted cells fed with Beefy 9. Cells were periodically imaged using phase contrast microscopy (KEYENCE, BZ-X700, Osaka, Japan; Olympus, CKX53, Tokyo, Japan)

[0137] Characterization and differentiation of VN40 adapted cells

[0138] After 20 passages in VN40 media, iBSCs were allowed to adhere in triplicate in 48-well plates at 6000 cells / cm2and their satellite cell identity was characterized via staining for Paired-box 7 (Pax7). Cells were fixed with 4 % paraformaldehyde in PBS (ThermoFisher #AAJ61899AK) for 20 min, washed with DPBS containing 0.1 % Tween-20 (ThermoFisher, BP337), permeabilized for 10 min with DPBS containing 0.1 % Triton X-100 (ThermoFisher, BP151), washed again with DPBS+Tween-20, and blocked for 30 min using a blocking solution containing DPBS with 5 % goat serum (ThermoFisher #16210064) and 0.05 % sodium azide (Sigma #S2002). A primary antibody solution containing 1:500 Pax7 antibody (ThermoFisher #PA5-68506) and 1:1000 actin stain Phalloidin 488 (ThermoFisher A12379) in blocking solution was added to cells and incubated overnight at 4°C. Cells were then washed with DPBS+Tween-20 and blocked for 30 min once again. A solution containing 1:500 Pax7 secondary antibody (ThermoFisher #A11072) and 1:1000 nuclear stain DAPI (ThermoFisher #D1306) in blocking solution was added to cells and incubated for 1 h at room temperature. Cells were then washed with DPBS+Tween-20 and kept in DPBS for imaging. Imaging was carried out using fluorescence microscopy (KEYENCE, BZ-X700, Osaka, Japan). Sternness was quantified using a Celigo automated image cytometer (Revvity, Waltham, MA) to determine cells positive for Pax7. This was repeated for cells maintained in iBSCGM without VN40 adaptation to determine if adaptation altered iBSC satellite cell phenotype. Cells were differentiated by seeding at 5,000 cells / cm2in 48-well plates, feeding with VN40 media, and initiating differentiation when cells appeared confluent ~4 days after initial seeding. Differentiation media contained DMEM+Glutamax supplemented with 2 % horse serum (ThermoFisher #16050130). 0.5 mg / mL recombinant human albumin, lx ITS-X (ThermoFisher #51400056), 0.5mMLDN193189 (Sigma #SML0559), 1 % anti bi otic / antimycotic, and 2.5 pg / mL puromycin. Differentiation continued for 18 days with cells fed fresh differentiation media every 2-3 days. After mature myotubes were observed, cells were fixed, permeabilized, and blocked as described. Cells were then stained using myosin heavy chain (MHC; Developmental studies hybridoma bank #MF-20, Iowa City, IA, USA) and 1:200 desmin (Abeam #abl5200, Cambridge, UK) primary antibodies diluted in blocking solution. Secondary' antibodies for MHC (ThermoFisher #A32723; 1:200) and desmin (ThermoFisher #A11072; 1:500) along with 1:1.000 DAPI, diluted in blocking buffer, were then applied as described. Imaging was carried out using fluorescence microscopy (KEYENCE, BZ-X700, Osaka, Japan).

[0139] Sample preparation for metabolomics analysis

[0140] A 100 % methanol extraction was performed on the lysate. Methanol and VN lysate were mixed in a 3:1 proportion and vortexed. The mixture was centrifuged at 15,000 * g at 4 °C for 15 min. Then, 250 pL supernatant were transferred to each Eppendorf tubes and samples were dried by speed vacuuming (Eppendorf Vacufuge™) and stored at -20°C. Prior to the LC-MS run, samples were reconstituted by adding methanol and water at 1: 1 proportion to dried pellet, mixed by vortexing and then centrifuged at 15,000 x g at 4 °C for 15 min (ThermoScientific Sovrall Legend X1R Centrifuge). Finally, 100 uL of the supernatant was transferred to sample tubes for LC-MS.

[0141] Liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0142] Untargeted analysis of metabolites present in extracted lysate samples was preformed using information-dependent acquisition (IDA) experiments on a quadrupole time-of flight (TOF) mass spectrometer (TripleTOF 5600+, AB Sciex, Framingham, MA, USA) with an electrospray ionization (ESI) source. IDA experiments include a single TOF MS scan corresponding to four dependent product ion (MS / MS) scans based on the highest intensity unique mass. Fragmentation was triggered when precursor ion counts rose quickly over several scans to ensure ions were selected near the top of their LC peaks. A total of four methods was applied to each sample using either a hydrophilic interaction chromatography (HILIC) column (Phenomenex Luna NH2, Torrance, CA, USA) or a reverse-phase column (Phenomenex Synergi Hydro-RP, Torrance, CA, USA) in both positive and negative ionization modes. The chromatographic separation was performed with a binary pump HPLC system (1260 Infinity. Agilent, Santa Clara, CA. USA). Chromatographic gradient methods and mobile phases described in supplementary information.

[0143] Feature annotation

[0144] LC-MS features were annotated using the Biologically Consistent Annotation of Metabolomics Data computational tool (BioCAn)33. Briefly, BioCAn assess potential annotations of mz values and MS / MS spectra using the following annotation tools: CFM-ID34, NIST17 (NIST / EPA / NIH. 2017), MONA (https: / / massbank.us / ), andHMDB35Features could be assigned multiple annotations either by a single annotation tool or by disagreement between assignments designated by different tools to determine the most likely annotation for a feature, the feature wasmapped to a metabolite in the V. natrigens metabolic network and assigned an annotation score by BioCAn. Annotation scores were based on agreement of the annotation assignment by the integrated databases as well as confidence in annotation of neighboring metabolites within the metabolic network.

[0145] Data analysis

[0146] The peak area corresponding to the integrated area under the curve (AUC) of each ion chromatograph was blank subtracted to reduce noise. Each peak area was then normalized to the total ion chromatogram (TIC) for each replicate (n =3). Unannotated features were subsequently removed. In addition, features showing annotation scores less than 1 were removed due to the low likelihood of the annotation. Finally, annotated features showing negative peak areas were removed as their concentration is likely lower than the limit of detection for the applied method. The remaining metabolites were then sorted by peak area.

[0147] Cost analysis

[0148] The inventors compared the cost of VN lysate containing media with BSCGM and B9 media. The details of the cost comparison are in supplementary’ information. [[Inventors: please provide or provide a summary of the comparison]]Please find in the attached file - also in sec.0139.

[0149] RESULTS

[0150] Mechanical lysis of gram-negative bacteria yields extracts with high protein content.

[0151] The overall workflow of lysate production and testing with mammalian cells is described in Figure 1A. The Gram-negative bacteria strains ECN, BL21, and VN, along with the yeast strain CEN.PK, demonstrate relatively higher protein yields per liter of culture compared to Gram-positive bacteria (Figure IB). This discrepancy can be attributed to the structural complexity of Gram-positive bacteria, which possess multiple layers of peptidoglycan in their cell walls, making them more resistant to lysis36. Inefficient lysis of Gram-positive bacteria likely resulted in their lower protein yield.

[0152] The inventors analyzed the protein profiles of lysates obtained from various microbial strains via SDS-PAGE to assess their composition and potential for supporting cell growth. Figure 1C illustrates the SDS-PAGE results for the lysates from V. natriegens, both E. coli strains, B. subtilis, L. plantarum, L. lactis, and S. cerevisiae. Each lane shows distinct protein band patterns, highlighting the differences in protein composition across species.

[0153] Screening helps identify compatibility between lysates and cells lines.

[0154] The iBSCs used in this study were extensively characterized in the inventors’ previous studies8-37. As an initial screen for iBSC growth in serum-free lysate-based media, cells were tested directly after passaging in serum-containing growth medium (iBSCGM). Performance (cell growth) was compared to previously established Beefy-9 serum-free mediuml4 , which contains similar ingredients as the inventors’ tested formulations, except the recombinant human albumin present in Beefy-9 was replaced with the microbial lysates. The results (Figure 2A) show that many of the lysates were inhibitory to cell growth after 5 days of exposure. All lysates elicited decreased cell growth at higher concentration (up to 500 pg / mL). Lysates from V. natriegens (10 pg / mL) and L. plantarum (10 and 100 pg / mL) exhibited short-term cell growth similar to that of the Beefy-9 control.

[0155] The inventors also screened the short-term growth-promoting effects of lysate-based media on Mackl cells (Figure 2B). Given that all lysates at 500 pg / mL showed detrimental effects on iBSC growth, the inventors tested a narrower range of lysate protein concentrations for Mackl cells. Unlike iBSC, V. natriegens and L. plantarum inhibited short-term Mackl cell growth compared to the other microbes. However, S. cerevisiae and E. coli (BL21(DE2) andNissle 1917) significantly increased the growth of the cells after 4 days of exposure. Specifically, Mackl cells performed better in the presence of S. cerevisiae (up to 100 pg / mL) and E. coli Nissle 1917 (10 pg / mL) lysates.

[0156] Despite the inventors’ success with Mackl cells, the inventors found greater success in adaptation of iBSCs to serum-free media using lysates and proceeded with subsequent studies using only iBSCs. Since serum-free adaptation significantly alters cellular phenotype and gene expression38, transferring cells directly from serum-containing media to lysates may not be an appropriate strategy to assess the long-term potential of lysates. So, next the inventors re-screenedthe lysates using iBSCs grown in Beefy-9 (serum-free) medium (4 passages) with select lysates (Figure 2C). The bovine cells were grown in lysate concentrations low er than those of the first screen, since concentrations above 10 pg / mL were shown to be cytotoxic. The results showed that cells cultured in serum-free conditions responded differently to lysates than those cultured only in serum-containing media. Cells cultured in V. natriegens media at 10 pg / mL out-performed those grown at this concentration of B. subtilis and L. plantarum. Cell growth improved with increased V. natriegens lysate concentration between 2.5 and 10 pg / mL. while the opposite effect was seen in cells grown in L. plantarum and B. subtilis. While initial screens found that nonadapted cells proliferated in L. plantarum media, these results did not translate to cells adapted to serum-free media. Given that V. natriegens performed well with serum adapted and un-adapted cells, the inventors chose it for further investigations.

[0157] iBSCs can adapt to long-term growth on V. natriegens lysate containing media.

[0158] iBSCs were cultured directly from BSCGM into serum-free media containing B8 supplemented with 10 pg / mL Vibrio natriegens lysate. Growth was monitored throughout several passages and lysate concentration in feeding media was increased when cell growth slowed. This strategy7led to immediate increases in cell growth rates and was used to slowly adapt cells to media with V. natriegens lysate protein concentration of 40 pg / mL (Figure 3 A), dubbed “VN40”. At this concentration, cell growth rate plateaued, and doubling time reached 24.9 h, similar to those observed using serum-containing BSCGM (21.9 ± 3.1 h) during the course of the study. Cells showed a significant increase in viability between 10 pg / mL V. natriegens feeding (avg.71.5 % viability) versus 40 pg / mL feeding (avg. 93.9 % viability ), though this may have been a factor of initial population enrichment during adaptation to lysate-based media. In all, the process of adapting cells to VN40 took approximately 2.5 months (passage 1 to passage 14), in line with previous durations of adapting mammalian cells to serum-free media39-41 . Adaptation of iBSCs to VN40 was expedited by passaging cells directly into 20 pg / mL VN media (Figure 3B), reducing total adaptation time to 4 weeks. After adaptation of cells in VN40 media, cells w ere grown in triplicate to compare to growth in serum-free Beefy-9 media. Both cell populations were passaged continuously over 24 days with collective cell doublings recorded at each passage (Figure 3C). Cells grown in VN40 significantly outgrew cells grown in Beefy-9, with higher collective cell doublings and lower average doubling times (31.2 ± 4.8 h for VN40; 43.1 ± 8.7 hfor Beefy-9). An increase of V. natriegens lysate content in the media beyond 40 pg / mL hindered cell growth compared to VN40 (Figure 7). Cells had higher confluence after 3 days of grow th in VN40 compared to Beefy-9 (Figure 3D-E). VN40 also resulted in less observable lipid accumulation compared to Beefy-9 (Figure 3F-G). Beefy-9 has been shown to cause aberrant lipid accumulation 14, potentially due to induced insulin resistance from the high concentration of insulin in B8, or via adipogenic signaling. Components of VN40 may be preventing such insulin resistance and / or adipogenic signaling from taking place, which could be beneficial in long-term maintenance of myogenicty and proliferative capacity. Cells in VN40 have maintained proliferation over 108 passages and can survive long-term storage and thawing from liquid nitrogen.

[0159] High and low molecular weight components of V. natriegens lysate are additive in promoting iBSC proliferation.

[0160] To determine if a specific molecular weight fraction contributed to the mitogenic effect of Vibrio natriegens lysate-based media, the inventors fractionated the lysate by ultrafiltration using a 3 kDa molecular weight cutoff membrane. iBSC growth was determined via the same protocols as earlier, with short term growth compared to B8 (negative control) and Beefy-9 (positive control). Formulations using the protein-rich high molecular w eight (HMW) >3 kDa fraction, low molecular weight (LMW) <3 kDa fraction, and whole-cell V. natriegens lysate, were prepared at various concentrations in B8 media. For total lysate and HMW fractions, protein concentrations were determined using Bradford assay, as described previously. Since the protein component of the LMW fraction w as below7the detection limit for Bradford assay, the inventors used the same composition as would have been required for the whole cell lysate. The results (Figure 4A) showed that for concentrations of V. natriegens protein equal to or above 40 pg / mL. whole cell lysates out-performed HMW or LMW fractions alone. It can be concluded from this data that no individual fraction of V. natriegens lysate was cytotoxic compared to whole-cell lysates, and that larger proteins act together with small molecule metabolites to have an overall pro-mitogenic effect on iBSCs. This has been shown in prior work in which metabolomics and proteomics showed the indispensability of both amino acid and protein fractions in Auxenochlorella pyrenoidosa extracts for goldfish muscle cell growth30. Whole-cell lysates of VN40 outperformed individual fractions, suggesting that the proteins and metabolite fractionshave an additive impact (Figure 4B). Ultimately, the inventors’ data shows the importance of using unfractionated VN40 in stimulating iBSC growth.

[0161] VN40 cultured iBSC maintain sternness and can be differentiated.

[0162] The inventors validated continued satellite cell phenotype via Pax7 expression42 of iBSCs after 22 passages in VN40. showing that expression was maintained throughout serum-free adaptation (Figure 6A-B). According to quantitative imaging cytometry', cells were 99.7% positive for Pax7 after adaptation (Figure 6C), indicating maintenance of a pure satellite cell population. After validation of phenotype, the inventors successfully differentiated VN-40 adapted cells into mature multi-nucleated myotubes expressing both Desmin and Myosin Heavy Chain proteins (Figures 6D-K), indicative of early-stage and late-stage differentiation respectively43, 44. This implies that VN40-adapted cells maintain their myogenicity after extensive serum-free passaging, essential for creating functional cultivated meat products45. Cells differentiated after slow, gradual adaptation (10 pg / mL 20 pg / mL — ► 40 pg / mL VN lysate) (Figure 6D-G) differentiated more slowly’ and less robustly than cells differentiated after a quicker adaptation (20 pg / mL — > 40 pg / mL) (Figure 6H-K). This is likely due to the impact of higher passage number on myogenicity46. Differentiation media was optimized to contain ITS-X47 and LDN- 19318948 , which were necessary' for differentiating these overall high passage (>p70) cells. Ultimately, the inventors have arrived at a bacterial lysate-based serum-free formulation which allows for successful proliferation and differentiation of iBSCs.

[0163] LC-MS / MS identifies major metabolites in V. natri egens lysate.

[0164] V. natriegens lysate was analyzed for metabolite composition using a series of untargeted LC-MS / MS assays. The untargeted analysis detected a total of 11,306 features in four total methods. Biologically Consistent Annotation (BioCAn) computation tool successfully- annotated a total of 403 metabolites from the 11.306 total features. Although BioCAn uses biological network data to reduce misannotation, errors may still occur. Thus, only annotations with a score greater than 1.0 were further considered. An annotation score greater than 1.0 indicates strong agreement across integrated databases, as well as similar agreement in the annotation of metabolites w ithin tw o reactions of the annotation in the metabolic network. A total of 155 metabolites were identified across the 4 methods with annotation scores equal to. or above.1.0 (Table 1), which included 80 unique metabolites. Interestingly, purine and pyrimidine derivates, and aromatic amino acids show- the greatest area under the curve, suggesting potentially elevated concentrations of these metabolites in V. natriegens lysate extracts.Table 1. Annotated metabolites showing greatest AUC detected in each LC-MS / MS method. Each metabolite's area is normalized to that w ith the highest AUC for each LC-MS method (n=3).Annotation Normalized Method Metabolite M / Z RT Score Average AUC HILIC (-) Uracil 111.020 251.478 1.2 1.00 HILIC (-) Hypoxanthine 135.031 531.488 6.4 0.26 HILIC (-) Xanthine 151.026 791.072 6.3 0.17 HILIC (-) L-Phenylalanine 164.072 535.862 1.6 0.14 HILIC (-) Guanine 150.042 532.663 4.8 0.05 HILIC (-) L-Arogenate 226.075 510.862 1.3 0.05 HILIC (-) L-Glutamate 146.046 980.112 5 0.04 HILIC (-) L-Citrulline 174.088 645.793 2.2 0.03D-Xylulose 5- HILIC (-) phosphate 229.012 1584.138 2.4 0.02 HILIC (-) L-Methionine 148.044 547.405 1.7 0.02 HILIC (+) 5-Aminopentanoate 118.082 504.533 1 1.00 HILIC (+) Uracil 113.030 249.740 1.2 0.04 HILIC (+) Cytosine 112.048 297.273 1.2 0.003 HILIC (+) L-Proline 116.066 991.431 2.1 0.002 HILIC (+) Inosine 269.090 3112.661 3.3 0.002 HILIC (+) L- Serine 106.046 659.801 3.6 0.001 HILIC (+) Benzonitrile 104.050 546.203 1.1 0.001 HILIC (+) Portulacaxanthin II 375.117 257.946 1.3 0.0003 RP (-) Xanthine 151.026 709.452 6.3 1.00RP (-) L-Phenylalanine 164.072 1220.405 1.6 0.87RP (-) Inosine 267.074 1253.153 3.3 0.55RP (-) Uracil 111.020 299.736 1.2 0.44RP (-) Hypoxanthine 135.031 498.233 6.4 0.31RP (-) L-Tyrosine 180.066 736.295 1.5 0.26RP (-) L-Tryptophan 203.083 1607.048 3.2 0.26RP (-) Guanosine 282.084 1257.624 2.8 0.25RP (-) Xanthosine 283.069 1298.537 3.7 0.19RP (-) L-Citrulline 174.088 187.141 2.2 0.14RP (+) L-Phenylalanine 166.086 1218.830 1.6 1.00 RP (+) L-Tyrosine 182.080 721.470 1.5 0.56 RP (+) Guanine 152.056 310.407 4.8 0.55 RP (+) Hypoxanthine 137.045 560.229 6.4 0.37 RP (+) Uracil 113.034 321.318 1.2 0.30 RP (+) Guanosine 284.097 1265.559 2.8 0.29 RP (+) L-Methionine 150.057 286.392 1.7 0.28 RP (+) Xanthine 153.040 776.350 6.3 0.17 RP (+) Guanine 152.056 1288.269 4.8 0.15RP (+) Inosine 269.087 1248.965 3.3 0.11

[0165] VN40 component costs are competitive relative with Beefy 9.

[0166] Overall production cost of cultivated meat includes at least three main factors -bioreactors, labor, and cell culture medium. For this study the inventors have focused on the economics of FBS, a significant factor in cell culture medium content and costs. Understanding the cost of VN lysate compared to FBS at a large scale is necessary to predict the sustainability of cultivated meat made using lysates as a key media component. VN lysate at 40ug / mL used as a replacement of FBS is sustainable and cost effective at $2 per liter compared to $264 of FBS per liter, a 99.2 % price reduction (Table 4). Thus, based on the calculations, VN40 reduces media cost from $323.30 to $158.60, a 51% reduction (Figure 7). This accounts for raw materials involved in the production of V. natri egens lysate.

[0167] DISCUSSION

[0168] Issues of increasing global food insecurity and climate crisis2'4, coupled with the growing demand for meat1, create an urgent need for more sustainable approaches to meat production. Cultivated meat, the process of growing meat in vitro without the need to raise livestock, is a viable alternative approach6, 7. However, production of cultivated meat needs to be inexpensive, reliable, and relatively simple to compete with the low costs of traditional meat products produced by industrial farming methods. Moreover, the reliance on FBS for cell culture is a significant roadblock to scaling and commercializing cultivated meat due to FBS's animal origin, high cost, lot-to-lot variability, and potential as a source of contamination10, n. Attempts to use recombinant proteins8, 15, 16, 49or crude extracts or hydrolysates from organisms18, 20, 22, 50are costly, labor intensive, and at times, still reliant on animal products or serum. Microbial extracts can be a protein-rich, inexpensive, and scalable alternative to generating FBS-replacing media supplements. Microbial extracts have been utilized for cultivated meat production25, 26, 28, but still depended on serum25, toxic bacteria28, or otherwise animal-derived microbes26to stimulate growth. Further, manufacturing these microbe-based serum-free media (SFM) formulations is limited by relatively slow' growth rates of the chosen microbes.

[0169] In screening microbial lysates for cell line growth, the inventors anticipated positive outcomes in either Gram positive bacteria (lacking lipopolysaccharides), or food-associated / probiotic microbes such as S. cerevisiae and E. coli Nissle 1917. While S. cerevisiae proved beneficial for Mackl cell growth, the beneficial impact of V. natriegens lysate on iBSCs was a surprise, highlighting the importance of individualized screens for separate cell lines, potentially due to unique nutritional and signaling requirements32. After screening cell growth using lysates of seven microbial strains, V. natriegens (a fast-growing marine bacterium) was chosen for use in long-term adaptation of iBSCs to SFM. By sequentially increasing V. natriegens lysate concentration in SFM, the inventors were able to achieve maximal doubling rates of iBSCs using the VN40 formulation. This led to cell growth rates exceeding previously established Beefy -9 mediuml4, while preserving iBSC phenotype and myogenicity. VN40 was able to stimulate long-term growth in iBSCs at up to 24.9 h doubling times, rivaling that of serum-containing media. The lysates were simple to produce and highly potent, stimulating cell growth at a mere 40 pg / mL. Further results indicated the importance of both low- and high-molecular weight components in the microbial extract, suggesting there is no need for process-intensive ultrafiltration of lysate fractions to achieve optimal performance as in other studies18, 21-50. LC-MS analysis of VN lysate revealed the presence of purine derivatives, nucleoside intermediates, and aromatic amino acids, which have also found to be present in FBS51. Aromatic amino acids are synthesized in cow's rumen by fermentation52, and can be found in FBS and basal media used for mammalian cell culture53. These aromatic amino acids found in VN lysate could sen e as a nutritional requirement of iBSCs by aiding protein synthesis, cellular signaling and oxidative stress response53. Purine, pyrimidine and their derivatives in VN lysate could contribute to the growth of iBSC in absence of any serum. Supplementation of nucleosides like uracil, in CHO cell cultures have shown improved growth rate54.

[0170] V. natriegens is a promising ingredient in cultivated meat media, as it is the fastest growing non-pathogenic bacteria known, with a generation time of < 10 min55. Further, its nutritional needs are both minimal and flexible56. The inventors’ work shows its potential to supply the cultivated meat industry with an inexpensive on-demand serum-free supplement in a potent form. Previous environmental impact assessments using microbial lysates assumed a need for microbial protein on the scale of milligrams per milliliter of media27. The inventors' media, only requiring 40 pg / mL protein, would reduce such needs by a factor of one hundred. This would result in significant decreases in water usage, energy usage, and greenhouse gas emissions associated with cultivated meat production, which largely stem from production and processing of media ingredients6. This high potency would also largely impact cost, for which media and additional grow th factors largely account for in cultivated meat production12.Table 2. Previous research exploring cultivated meat media, with advantages of current work indicated.Ref. Active ingredient Low- Minimally Rapidly NonFree ofFBS Long-term for serum cost labor- replicating toxic or other cell replacement intensive source animal growth & production organism ingredients myogenic capacity i Recombinant X X X X X albumin2 Recombinant X X X X X human serumalbuminFetuin + other X X X X X growth factors4 Recombinant X Xheme5 Recombinant X X growth factors,bovine albumin,ethanolamine6 Rapeseed protein X X X X isolates7 4. pyrenoidosa X XExtract8 C. vulgaris extract X X9 C. vulgaris extract X X X10 Ultrafiltered Okara X X Xextract11 Soy and gluten X X X hydrolysates12 Invertebrate X Xhydrolysates13 Growth factorX X Xexpressing Ncerevisiae14 E. hirae - secreted X X X Xpostbiotics15 Anabaena extract X X16 S', maxima extract X X XThis V. natriegens X X X X X Xwork lysateTable 3 is shown in FIG. 16. Cost comparison of itemized media components and total cost of iBSCGM and B9 with VN40 medium.

[0171] The cost calculation for VN lysate was based on LB media (BD Difco, 244620). One gram of LB costs 0.346 USD. The inventors prepare 25 mL of VN culture in LB. yielding approximately 4 mg / mL of lysate (total protein content). For 25 mL of culture, the inventors use 0.625 grams of LB. Therefore, 0.625 grams of LB yields 4 mg / mL of lysate, and 40 pg / mL lysate is produced from 0.006 grams of LB. The cost of 0.006 grams of LB is $0,002, making the cost for 40 pg / rnL VN lysate $0,002. This translates to $0.00005 per 1 pg / mL of VN lysate, and thus, 40,000 pg / mL would cost ~$2.Table 4: VN lysate is 99.2% cheaper than FBS.Unit Cost / Amount / per Cost Cost / L Component Unit L (rxn) Supplier Cat# order (USD) Unit (rxn)ThermoFisherScientifimL 200 mL 26140079 500 660 1.32 264FBS cV.natriegenslysate Pg 40 mg / L NA NA NA NA 0.0005 2References(1) OECD; Food; Nations, A. O. o. t. U. OECD-FAO Agricultural Outlook 2021-2030 2021. 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H.; Ostrov, N.; Wong, B. G.; Gold, M. A.; Khalil, A. S.; Church, G. M. Functional genomics of the rapidly replicating bacteriumby CRISPRi. Nat Microbiol 2019, 4 (7), 1105-1113. DOI: 10.1038 / s41564-019-0423-8. (56) Ellis, G. A.; Tschirhart, T.; Spangler, J.; Walper, S. A.; Medintz, I. L.; Vora, G. J. Exploiting the Feedstock Flexibility of the Emergent Synthetic Biology Chassis for Engineered Natural Product Production. Mar Drugs 2019, 17 (12). DOI: AREN 679 10.3390 / mdl7120679.Supplemental MethodsAdditional details on LC-MS / MS methods.Reverse phase (RP) chromatography method(Phenomenex Synergi Hydro-RP Column). Solvents:A: 0.1% formic acid in waterB: 0.1% formic acid in methanol• Column temperature: 15°C. Flow rate: 0.2 mL / min• Ion source: Turbo spray (ESI). Ion source Gas 1: 35• Ion source Gas 2: 45Curtain Gas: 25Temperature: 450ClonSpray Voltage Floating: ±4500 VGradient:Time (min) %B0-8 38-38 3 ^ 9538-45 9545-47 95 — > 347-55 3Hydrophilic interaction chromatography (HILIC) method(Phenomenex Luna NH2 Column). SolventsA: 95:5 water: acetonitrile± 20mM ammonium acetate, pH to 9.45 using ammonium hydroxideB: 100% acetonitrile• Column temperature: 25°C. Ion source: Turbo spray (ESI). Ion source Gas 1: 35• Ion source Gas 2: 45. Curtain Gas: 25. Temperature: 450C. lonSpray Voltage Floating: ±5500 VGradient:Time (min) %B0-15 85 ^ 015-28 028-30 0 > 8530-60 85References for supplemental methods(1) Stout. A. J.; Mirliani. A. B.; Rittenberg, M. L.; Shub, M.; White, E. C.: Yuen, J. S. K.; Kaplan, D. L. Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat. Commun Biol 2022, 5 (1). DOI: ARTN 466 10.1038 / s42003-022-03423-8.(2) Kolkmann, A. M.; Van Essen, A.; Post. M. J.; Moutsatsou, P. Development of a Chemically Defined Medium for Expansion of Primary Bovine Satellite Cells. Bront BioengBiotech 2022, 10. DOI: ARTN 895289 10.3389 / fbioe.2022.895289.(3) Skrivergaard, S.; Young, J. F.; Sahebekhtiari, N.; Semper, C.; Venkatesan, M.; Savchenko. A.; Stogios, P. J.; Therkildsen, M.; Rasmussen, M. K. A simple and robust serum-free media for the proliferation of muscle cells. Food Res Int 2023, 172. DOI: ARTN 113194 10.1016 / j.foodres.2023.113194.(4) Seo,Y. A.; Cha, M. J.; Park, S.; Lee, S.; Lim, Y. J.; Son, D. W.; Lee, E. J.; Kim, P.; Chang, S. Development of a Normal Porcine Cell Line Growing in a Heme-Supplemented, Serum-Free Condition for Cultured Meat. Int J Mol Sci 2024, 25 (11). DOI: 10.3390 / ijms25115824 From NLM Medline.(5) Dai, W.: Chen, Y; Xiong. W.; Li, S.; Tan, W. S.; Zhou. Y. Development of a serum-free medium for myoblasts long-term expansion and 3D culture for cell-based meat. J Food Sci 2024, 59 (2), 851-865. 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Development of serum-free and grain-derived-nutrient-free medium using microalga-derived nutrients and mammalian cell-secreted grow th factors for sustainable cultured meat production. Sci Rep 2023, 75 (1), 498. DOI: 10.1038 / s41598-023-27629-w From NLM Medline.(10) Teng, T. S.; Lee, J. J. L.; Chen, W. N. Ultrafiltrated Extracts of Fermented Okara as aPossible Serum Alternative for Cell Culturing: Potential in Cultivated Meat Production. Acs FoodSci Technol 2Q23, 3 (4), 699-709. DOI: 10.1021 / acsfoodscitech.2c00401.(11) Radosevic, K.; Dukic, B.; Andlar, M.; Slivac. I.; Gaurina Srcek. V. Adaptation and cultivation of permanent fish cell line CCO in serum-free medium and influence of protein hydrolysates on growth performance. Cytotechnology 2016, 68 (1), 115-121. DOI: 10.1007 / sl0616-014-9760-x FromNLM PubMed-not-MEDLINE.(12) Batish, I.; Zarei, M.; Nitin, N.; Ovissipour, R. Evaluating the Potential of Marine Invertebrate and Insect Protein Hydrolysates to Reduce Fetal Bovine Serum in Cell Culture Media for Cultivated Fish Production. Biomolecules 2022, 12 (11). DOI: 10.3390 / bioml2111697 FromNLM Medline.(13) Lei, Q. Z.; Ma, J.; Du, G. C.; Zhou, J. W.; Guan, X. Efficient expression of a cytokine combination in for cultured meat production. Food Res Int 2023, 170. DOI: ARTN 113017 10.1016 / j.foodres.2023.113017.(14) Celebi-Birand, D.i Gene, K.; Agun, I.; Erikci, E.; Akcali. K. C.; Kiran, F. Microbiota-Derived Postbi otics Enhance the Proliferative Effects of Growth Factors on Satellite Cells in Cultivated Meat Applications. Sustainability-Basel 2023, 75 (23). DOI: ARTN 16164 10.3390 / sul52316164.(15) Ghosh, J.; Haraguchi, Y.; Asahi, T; Nakao, Y; Shimizu, T. Muscle cell proliferation using water-soluble extract from nitrogen-fixing cyanobacteria Anabaenasp. PCC 7120 for sustainable cultured meat production. Biochem Bioph Res Co 2023, 682, 316-324. DOI: 10.1016 / j.bbrc.2023.10.018.(16) Jeong, Y; Choi, W. Y; Park, A.; Lee, Y. J.; Lee. Y.; Park, G. H.; Lee, S. J.; Lee, W. K.; Ryu, Y. K.; Kang, D. H. Marine cyanobacterium as an alternate to the animal cell culture medium supplement. Sci Rep-Uk 2021, 11 (1). DOI: ARTN 4906 10.1038 / s41598-021-84558-2.Example 2 - Screening of bacterial and fungal lysates as serum substitutes

[0172] In screening iBSC grow th using lysates from 2 fungal strains and 4 lactic acid bacteria, we found all lysates to exhibit similar growth-inducing performance to previously established Beefy 9. Some lysates induced growth at specific concentrations, while others showed growth over a range. Most lysates induced growth at protein concentrations of <10 pg / ml, showing these lysates as a promising source of potent, easy to produce, low-cost serum substitutes. Detailed results can be seen in Fig. 9.

[0173] These results are similar to short-term screens conducted previously for Vibrio natriegens lysates which proved successful for long-term iBSC growth, showing these results to be especially promising.Example 3 - Recycling of spent mammalian culture media to grow microbial lysates for generation of serum replacements

[0174] The inventors show successful growth of V. natriegens in spent medium that has been autoclaved in a 40 minute cycle and supplemented with 3% NaCl (Figure 15). While V. natriegens grown in spent medium does not reach the same peak OD600 as V. natriegens growth in fresh medium, we observe peak growth after 8 hours of culture, indicating that we can harvest lysate at this timepoint.

[0175] The inventors show successful expression of bovine FGF2 in V. natriegens (Fig.10), and successful growth of iBSCs using VNFGF lysates without the need for FGF supplementation (Fig.11). iBSCs grown in VN40FGF media without FGF supplementation grew equivalently to those grown in VN40 media with supplemented FGF, showing VN40FGF can replace the need for supplemented FGF (Fig. 11 & 12). iBSCs adapted to growth in VN40FGF media maintained their muscle satellite cell phenotype, preserving their ability to differentiate into myotubes (Fig. 13) and preserve a state of sternness before differentiation (Fig. 14).

[0176] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein wdthout departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use ofsuch terms and expressions of excluding any equivalents of the features show n and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0177] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

[0178] SequencesSEQ ID NO DescriptionBovine FGF-21 AABovine FGF-22 DNA3 Full vector

Claims

CLAIMS1. A cell culture medium comprising an amount of a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Bacillus subtilis, Lactobacillus lactis, Latilactobacillus sakei, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, and Lactobacillus gasseri.

2. The cell culture medium of claim 1 , wherein the microorganism is Vibrio natriegens. Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), or Escherichia coli BL21 (BL21).

3. The cell culture medium of claim 1 , wherein the microorganism is Vibrio natriegens.

4. The cell culture medium of any one of claims 1-3, wherein the amount is about 1 pg / ml to about 100 pg / ml.

5. The cell culture medium of claim 1 , wherein the microorganism is Vibrio natriegens and an amount is about 20 Ltg / ml.

6. The cell culture medium of claim 1, wherein the microorganism is Saccharomyces cerevisiae and the amount is about 10 (Jg / ml to about 100 pg / ml.

7. The cell culture medium of claim 1, wherein the microorganism is Escherichia coli Nissle 1917 (EcN) and the amount is about 10 pg / ml.

8. The cell culture medium of claim 1, wherein the cell culture medium comprises one or more of: sodium selenite, albumin, transferrin, transforming growth factor beta, L-ascorbic acid, insulin, neuregulin 1 (NRG1), or fibroblast grow th factor 2 (FGF-2).

9. The cell culture medium of claim 1, wherein the cell culture medium comprises DMEM, HEPES, and / or sodium bicarbonate.

10. The cell culture medium of claim 1, wherein the cell culture medium supports short or long term grow th or differentiation of cells, wherein the cells are optionally mammalian, fish, and / or insect muscle or adipose cells.

11. The cell culture medium of claim 1 , wherein the cell culture medium supports short or long term grow th or differentiation of bovine satellite cells or mackerel cells (Mackl).

12. The cell culture medium of claim 1, wherein the medium does not contain serum.

13. The cell culture medium of claim 1, wherein the medium comprises one or more of the following compounds: uracil, hypoxanthine, xanthine, L-phenylalanine, guanine, L-arogenate, L-glutamate, L-citrulline, D-xylulose 5-phosphate, L-methionine, 5-aminopentanoate, cytosine, L-proline, inosine, L-serine, benzonitrile, portulacaxanthin II, L-tyrosine, L-tryptophan, guanosine, or xanthosine.

14. The cell culture medium of claim 1, wherein the microorganism expresses FGF-2.

15. The cell culture of claim 3, wherein the Vibrio natriegens expresses FGF-2.

16. The cell culture medium of claim 1, wherein the medium comprises one or more of (1), (2), (3), or (4), wherein:(1) the following compounds present at about the relative concentration to uracil listed below or the absolute concentrations listed belowCone. RangeRelativeCompound (ng / mL)concentration1 - 1,000Uracil 10.26 - 260Hypoxanthine 0.26Xanthine 0.17 0.17 - 170L- 0.14 - 1400.14PhenylalanineGuanine 0.05 0.05 - 500.05 - 50L-Arogenate 0.050.04 - 40L-Glutamate 0.04L-Citrulline 0.03 0.03 - 300.02 - 20D-Xylulose0.025-phosphate0.02 - 20L-Methionine 0.02(2) the following metabolites present at about the relative concentration to 5-aminopentanoate listed belowCone. RangeRelativeCompound (ng / mL)concentration5- 25 - 25,0001Aminopentanoate1 - 1,000Uracil 0.04Cytosine 0.003 0.075 - 750.05 - 50L-Proline 0.002Inosine 0.002 0.05 - 500.025 - 25L-Serine 0.0010.025 - 25Benzonitrile 0.001Portulacaxanthin 0.0075 - 7.50.0003II(3) the following metabolites present at about the relative concentration to xanthine listed belowCone. RangeRelativeCompound (ng / mL)concentration2.25 - 2250Xanthine 1L- 2 - 20000.87PhenylalanineInosine 0.55 1.25 - 12501 - 1000Uracil 0.44Hypoxanthine 0.31 0.7 - 7000.5 - 500L-Tyrosine 0.260.5 - 500L-Tryptophan 0.26Guanosine 0.25 0.5 - 5000.4 - 400Xanthosine 0.190.3-300L-Citrulline 0.14or (4) the following metabolites present at about the relative concentration to L- phenylalanine listed belowCone. RangeRelativeCompound (ng / mL)concentrationL- 3 - 30001Phenylalanine2 - 2000L-Tyrosine 0.56Guanine 0.55 2 - 20001.25 - 1250Hypoxanthine 0.37Uracil 0.3 1 - 10001 - 1000Guanosine 0.291 - 1000L-Methionine 0.28Xanthine 0.17 0.5 - 5000.5 - 500Guanine 0.150.3 - 300Inosine 0.1117. An aqueous solution comprising a serum replacement, wherein the serum replacement comprises a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Lactobacillus lactis, Bacillus subtilis, and Lactobacillus plantarum, and one or more of sodium selenite, albumin, transferrin, transforming growth factor beta, L-ascorbic acid, insulin, neuregulin 1 (NRG1), fibroblast growth factor 2 (FGF-2), DMEM. HEPES, or sodium bicarbonate.

18. The aqueous solution of claim 17, wherein the microorganism is Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), or Escherichia coli BL21 (BL21).

19. The aqueous solution of claim 18, wherein the microorganism is Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), ox Escherichia coli BL21 (BL21).

20. The aqueous solution of claim 19, wherein the microorganism is Vibrio natriegens.

21. The aqueous solution of claim 17, wherein the microorganism is Vibrio natriegens and the lysate comprises FGF-2.

22. A system for generating cultivated meat, the system comprising:(a) the cell culture medium of any one of claims 1-20; and(b) at least one animal cell.

23. The system of claim 25, wherein the animal cell is a multipotent animal cell.

24. The system of claim 25, wherein the animal cell is a satellite cell.

25. A method of making the cell culture medium of claim 1, the method comprising:(a) generating a lysate from a microbe; and(b) combining an amount of the lysate from the microbe with a base medium, thereby generating the cell culture medium.

26. The method of claim 25, wherein generating a lysate from a microbe comprises sonicating the microbe.

27. The method of claim 26, wherein generating a lysate from a microbe further comprises micro filtering the sonicated microbes to generate a filtered lysate.

28. The method of claim 25, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), Escherichia coli BL21 (BL21), Bacillus subtilis, Lactobacillus lactis, Latilactobacillus sakei, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, and Lactobacillus gasseri or wherein the microorganism is Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), or Escherichia coli BL21 (BL21).

29. The method of claim 25, wherein the microorganism is Vibrio natriegens.

30. The method of claim 25, wherein the amount is about 1 pg / ml to about 100 pg / ml.

31. The method of claim 25, wherein the microorganism is Vibrio natriegens and an amount is about 20 pg / ml.

32. The method of claim 25, wherein the microorganism is Saccharomyces cerevisiae and an amount is about 10 Lig / ml to about 100 pg / ml.

33. The method of claim 25, wherein the microorganism is Escherichia coli Nissle 1917 (EcN) and an amount is about 10 pg / ml.

34. A method of generating cultivated meat, the method comprising culturing an animal cell in the medium of claim 1 for a sufficient time to generate cultivated meat.

35. The method of claim 34, wherein culturing an animal cell comprises culturing the animal cell on a scaffold.

36. The method of claim 34, wherein the animal cell is a multipotent animal cell.

37. The method of claim 34, wherein the animal cell is a satellite cell.

38. The method of claim 34, wherein the method causes cell doubling at a similar or greater rate than culturing the animal cells in B9 medium.

39. The method of claim 34, wherein the method results in a similar or greater level of Pax7+cells than culturing the animal cells in B9 medium.

40. A method of detecting a suitable microorganismal lysate for use as a mammalian serum replacement for animal cell culture, the method comprising:(a) preparing a lysate from a sample of a species of cultured microorganisms;(b) culturing (1) a test plurality of animal cells in a test medium, wherein the test medium comprises the lysate prepared in step (a), and (2) a control plurality of animal cells in a control medium;(c) analyzing the growth of the test plurality of animal cells and the control plurality of animal cells; and(d) detecting the lysate as a suitable microorganismal lysate for use as a mammalian serum replacement for animal cell culture wherein growth of the test plurality of animal cells is about similar or greater than the control plurality of animal cells.

41. The method of claim 40, wherein the animal cell is a multipotent animal cell.

42. The method of claim 40, wherein the animal cell is a satellite cell.

43. The method of claim 40, wherein analyzing the growth of the test plurality of animal cells and the control plurality’ of animal cells comprises analyzing the doubling time of the test plurality of animal cells and the control plurality’ of animal cells.

44. The method of claim 40, wherein analyzing the growth of the test plurality of animal cells and the control plurality' of animal cells comprises analyzing a number of successful passages of the test plurality of animal cells and the control plurality of animal cells.

45. The method of claim 44, wherein the number of successful passages comprises the greatest passage number x where the doubling time of passage number x-1 is about equal or greater than the doubling time of passage number x.

46. A kit, system, or platform comprising a lysate from a microorganism, wherein the microorganism is selected from the group consisting of Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN). Escherichia coli BL21 (BL21), Bacillus subtilis. Lactobacillus lactis, Latilactobacillus sakei, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, and Lactobacillus gasseri.

47. The kit. system, or platform of claim 46, wherein the lysate is packaged as a unit dose wherein the unit dose comprises an amount of the lysate for use as a substitute for serum in animal cell culture.

48. The kit, system or platform of claim 46, wherein the unit dose comprises an amount of the lysate for use as a substitute for serum in animal cell culture when dissolved in a predetermined amount of a base medium.

49. The kit, system, or platform of claim 46, further comprising one or more of sodium selenite, albumin, transferrin, transforming growth factor beta, L-ascorbic acid, insulin, neuregulin 1 (NRG1), fibroblast growth factor 2 (FGF-2), DMEM, HEPES. or sodium bicarbonate.

50. A method comprisingculturing a plurality of cells on a cell culture medium;removing the plurality of cells from the cell culture medium to generate a cell-free spent culture medium; andculturing microorganisms on the cell free spent culture medium.

51. The method of claim 50, wherein the cell culture medium comprises or consists of the cell culture medium of claim 1.

52. The method of claim 50, wherein the microorganism is Vibrio natriegens, Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), or Escherichia coli BL21 (BL21).

53. The cell culture medium of claim 52, wherein the microorganism is Vibrio natriegens.

54. The method of claim 50, further comprising generating a lysate from the microorganisms cultured on the cell free spent cultured medium.

55. The method of claim 54, further comprising generating a cell culture medium comprising the lysate.

56. The method of any one of claims 50-54. wherein the method further comprises adding about 0.01 to about 10% Nad, by weight, to the spent medium.

57. The method of claim 56, wherein the NaCl is about 3% by weight.

58. A composition comprising spent cell culture media comprising a lysate from a microorganism.

59. The composition of claim 58. wherein the microorganism is Vibrio natriegens. Lactobacillus plantarum, Saccharomyces cerevisiae, Escherichia coli Nissle 1917 (EcN), or Escherichia coli BL21 (BL21).

60. The composition of claim 58, wherein the spent culture media comprises at least about 0.3 g / ml lactate and / or at least about 0.4 mM ammonium.

61. The composition of any one of claims 58-60, wherein the composition further comprises about 0.01 to about 10% NaCl, by weight.

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