Methods and systems for depleting undesired cell culturing products from cell culture media

Abstract

A method of depleting undesired cell culturing products from a cell culture medium is provided. The method comprises contacting a cell culture medium which comprises lactate and ammonia with a plant tissue under conditions which allow depletion of the lactate and ammonia from the culture medium by the plant tissue, wherein the contacting is effected in an aseptic environment, thereby depleting undesired cell culturing products from the cell culture medium.

Description

[0001] METHODS AND SYSTEMS FOR DEPLETING UNDESIRED CELL CULTURING

[0002] PRODUCTS FROM CELL CULTURE MEDIA

[0003] RELATED APPLICATIONS

[0004] This application claims the benefit of priority from U.S. Provisional Patent Application No. 63 / 535,597 filed August 31, 2023, the contents of which is incorporated herein by reference in its entirety.

[0005] FIELD AND BACKGROUND OF THE INVENTION

[0006] The present invention, in some embodiments thereof, relates to methods and systems for depleting undesired cell culturing products from cell culture media.

[0007] The cultivated meat competitive landscape has grown at an astounding pace, blossoming from just four companies to several dozen in less than two years. However, the field still faces shared challenges around scaleup and cost reduction. One of the major challenges in large scale production oaf cells is the cost of media. Large scale cultivation of cells increases the amount of dissolved species (sugars, minerals, buffers, waste metabolites, CO2, etc.) that contribute to osmolality, increase in pH and decrease in essential nutrients that in turn leads to medium toxicity and culture collapse. Hence the concentrations of these species must be tightly controlled. Waste catabolites like ammonia and lactate are particularly toxic and inhibit cell growth even at relatively low concentrations. Indeed, in cell culture for biopharmaceuticals, accumulation of toxic catabolites is a more frequently encountered limit than any physical limit of the bioreactor itself. The inhibiting concentrations of ammonia and lactate vary with cell line, as do the rates at which cells produce these. A range of 2-10 mmol / L NH3 has been reported for mammalian cells [L. Xie and D. I. C. Wang, “Stoichiometric analysis of animal cell growth and its application in medium design,” Biotechnology and Bioengineering, vol. 43, no. 11, pp. 1164-1174, May 1994], while lactate inhibition levels are generally at least an order of magnitude higher [Pasitka et al. (2023). Nature Food, 4(1), 35-50].

[0008] Hence, there is a need to develop technologies that would allow regeneration or recycling of media, thereby facilitating the transformation of the concept of cultured meat from fiction to reality.

[0009] Many plants are known to date for use in waste water purification and they play a vital role in natural water filtration processes. These include, for example, Water Hyacinth, Phragmites, Cattails, Moringa, Aloe Vera and more. Not all are edible plants and not all absorb nitrate -based compounds, but rather heavy metals, Phosphorous and pesticides. Carrot and tomato have been used in Co(II) absorption from waste water (Changmai et al J. Environmental Chemical Engineering Volume 6, Issue 1, 2018, 246-257). Likewise, vegetable waste has been used in the bioremediation of lead and chromium (Gill et al. J. Bio. & Environ. Sci. Vol. 5, No. 2, p. 306-317, 2014). A vast amount of work has been dedicated to the bioremediation properties of one such plant that has been used in wastewater treatment and belongs to the genus of Duckweeds. Duckweeds are the smallest angiosperms belonging to the family Lemnaceae, normally occupying the surface of lentic habitats. Besides their basic role in aquatic ecosystems, they offer a suitable model for ecologists, physiologists, geneticists, and researchers of many other fields. Moreover, from a practical point of view, they can help in coping with various aspects of the current humanitarian and environmental crisis, serve as indicators or test bioassay for environmental monitoring, help in removing water pollutants, and provide valuable biomass for easing the worldwide protein shortage or the hunger for renewable energy.

[0010] Concerning human food, in Asian countries such as Thailand, the genus Wolffia, especially Wolffia globosa, are easily found in the food markets. The availability of Duckweeds as a food source in several markets led to the investigation of their amino acids contents, and several studies have reported a suitable composition for human ingestion. Cultivation of Wolffia globosa in a greenhouse precision aquaculture results with a composition (dry) of as much as 45 % protein, 7 % minerals, 37 % carbohydrates, 8 % fats, and 3 % water and providing nine essential amino acids; iron; vitamins A, E, and B12; omega 3; zinc; potassium; and folate (Hinoman — Cultivating Natures Wonder. www(dot)hinoman(dot)com).

[0011] On the environmental front, Duckweed, can be used to treat wastewater containing very high total ammonia concentrations as long as certain pH levels are not exceeded. It also contributes to organic matter degradation. The degradation of organic material is enhanced by Duckweed through both additional oxygen supply and additional surface for bacterial growth. The Duckweed mat with attached bacteria and algae is, independent of the loading rates, responsible for three- quarters of the total nitrogen (N) and phosphorus (P) loss in very shallow systems. Its ability to grow as a mat, floating on shallow ponds render it a natural filter, sequestering toxic compounds. Before collapse, the mat is removed and replaced with a fresh inoculate. Naturally, the detoxification of waste and degradation of organic material are done under non-aseptic conditions, facilitated by bacteria.

[0012] Additional related background art:

[0013] Kumla et al. Additional related background art:

[0014] Burapha Science Journal Volume 27 (No.3) September - December 2022; Wang et al. Environmental Research Volume 52, Issue 1, June 1990, Pages 7-22;

[0015] Korner et al. J Environ Qual . 2003 Sep-Oct;32(5): 1583-90;

[0016] Ozengin et al. J Environ Biol 2007 Apr;28(2):307-14;

[0017] Al-Hashimi Journal of King Saud University - Engineering Sciences Volume 22, Issue 1, January 2010, Pages 11-18;

[0018] Suppadit et al. ScienceAsia 34 (2008): 163-168;

[0019] Zhou et al. Plants (Basel) 2023 Jan 29;12(3):589;

[0020] Farid Chemosphere Volume 304, October 2022, 135262;

[0021] Abdul Aziz et al. Appl. Sci. 2020, 70(16), 5397;

[0022] Li et al. Bioresource Technology Volume 318, December 2020, 123858;

[0023] CN114560564

[0024] AU2022425698

[0025] SUMMARY OF THE INVENTION

[0026] According to an aspect of some embodiments of the present invention there is provided a method of depleting undesired cell culturing products from a cell culture medium, the method comprising contacting a cell culture medium which comprises lactate and ammonia with a plant tissue under conditions which allow depletion of the lactate and ammonia from the culture medium by the plant tissue, wherein the contacting is effected in an aseptic environment, thereby depleting undesired cell culturing products from the cell culture medium.

[0027] According to an aspect of some embodiments of the present invention there is provided a cell culturing system comprising a chamber comprising a plant tissue and a cell culture medium which comprises lactate and ammonia, the system being an aseptic environment and allows depletion of the lactate and ammonia by the plant tissue.

[0028] According to an aspect of some embodiments of the present invention there is provided a resin for depleting lactate and ammonia from a cell culture medium comprising an aseptic plant tissue, optionally wherein said resin is immobilized on a solid support.

[0029] According to some embodiments of the invention, the aseptic environment is a sterile environment.

[0030] According to some embodiments of the invention, stiffness of the plant tissue is at least as that of a carrot sample of 50 mm diameter and 7700 mm2volume the minimum forces required for bruising, bending, and shearing of the carrot being 71 Newton (N), 48 N, and 41 N, respectively.

[0031] According to some embodiments of the invention, the plant tissue is hydrous comprising a water content of at least 75 % v / w. According to some embodiments of the invention, the plant tissue is of a root, fruit or a vegetable.

[0032] According to some embodiments of the invention, the plant is carrot.

[0033] According to some embodiments of the invention, the plant is Duckweed.

[0034] According to some embodiments of the invention, the Duckweed is selected from the group consisting of a Wolffia species and Lemna species.

[0035] According to some embodiments of the invention, the Wolffia species is selected from the group consisting of Wolffia angusta, Wolffia arrhiza, Wolffia australiana, Wolffia borealis, Wolffia brasiliensis, Wolffia Columbiana, Wolffia cylindracea, Wolffia elongata, Wolffia globose, Wolffia microscopica and Wolffia neglecta.

[0036] According to some embodiments of the invention, the Wolffia species is Wolffia arrhiza (e.g., var. spotless watermeal).

[0037] According to some embodiments of the invention, the Lemna species is selected from the group consisting of Lemna minor L., Lemna disperma L., Lemna ecuadoriensis L., Lemna gibba L., Lemna gibba L. , Lemna obscura L. and Lemna trisulca L..

[0038] According to some embodiments of the invention, the plant tissue is differentiated.

[0039] According to some embodiments of the invention, the plant tissue is diced, shredded and powder.

[0040] According to some embodiments of the invention, the plant tissue is viable.

[0041] According to some embodiments of the invention, the plant tissue is inactivated.

[0042] According to some embodiments of the invention, the plant tissue is immobilized on a solid support.

[0043] According to some embodiments of the invention, the system is a module in a fed batch or perfusion culturing system of a target cell of interest.

[0044] According to some embodiments of the invention, the system further comprises a target cell of interest.

[0045] According to some embodiments of the invention, the target cell of interest and the plant tissue are in separate chambers.

[0046] According to some embodiments of the invention, the separate chambers are in fluid communication.

[0047] According to some embodiments of the invention, the plant tissue and the target cell of interest are in a single chamber. According to some embodiments of the invention, the system comprises an inlet for introducing the cell culture medium into the chamber and an outlet for collecting the culture medium following assimilation of the lactate and ammonia by the plant tissue.

[0048] According to some embodiments of the invention, the system comprises at least one sensor for detecting at least one of lactate, ammonia, glucose, conductivity, pH, pCCh and pCh in the cell culture medium.

[0049] According to some embodiments of the invention, the method further comprises isolating the cell culture medium from the plant tissue following the contacting.

[0050] According to some embodiments of the invention, the contacting is for a time period which is 1-60 minutes, e.g., 1- 10 min.

[0051] According to some embodiments of the invention, the cell culture medium comprises glucose.

[0052] According to some embodiments of the invention, the cell culture medium comprises a base medium and at least one of a growth factor, a hormone and an antibody.

[0053] According to some embodiments of the invention, the base medium is selected from the group consisting of PBS, Hanks’ BSS, Earle’s salts DPBS, HBSS, EBSS, DMEM, MEM, GMEM , RPMI 1640 Leibovitz L-15, TC 100 Graces insect medium Schneider's Insect medium, Ham F10, Ham F12, DMEM / F12 and Serum-Free Insect Medium 1.

[0054] According to some embodiments of the invention, the method further comprises a target cell of interest.

[0055] According to some embodiments of the invention, the target cell of interest is a cell line, e.g., a mammalian cell line.

[0056] According to some embodiments of the invention, the target cell of interest is a genetically modified cell.

[0057] According to some embodiments of the invention, the genetically modified cell secretes a recombinant polypeptide.

[0058] According to some embodiments of the invention, the plant tissue is generally recognized as safe (GRAS).

[0059] According to some embodiments of the invention, the plant tissue is subjected to a treatment which renders it inactive.

[0060] According to some embodiments of the invention, the treatment is selected from the group consisting of an ethanol fixation, sonication, autoclave, freeze-drying, oven-drying, steam sterilization and radiation (e.g., UV radiation). According to some embodiments of the invention, ethanol for the ethanol fixation is about 70 % v / v.

[0061] According to some embodiments of the invention, the at least 2 times are separated by using the medium resultant of one time of the at least 2 times in cell culturing.

[0062] According to some embodiments of the invention, at least one time of the at least 2 times is effected with fresh plant tissue.

[0063] According to some embodiments of the invention, at least one time of the at least 2 times is effected with plant tissue having been used according to the method.

[0064] According to some embodiments of the invention, the method further comprises adding glucose to the refurbished culture medium resultant of the method.

[0065] According to some embodiments of the invention, the method further comprises analyzing at least one parameter selected from the group consisting of lactate, ammonia, glucose, protein, osmolarity, pH and amino acids.

[0066] According to an aspect of some embodiments of the present invention there is provided a refurbished culture medium obtainable as described herein.

[0067] According to an aspect of some embodiments of the present invention there is provided a method of culturing target cells of interest, the method comprising contacting the cells with the culture medium as described herein, to thereby culturing the cells.

[0068] According to some embodiments of the invention, the cells are a cell line.

[0069] According to some embodiments of the invention, the cells are human cells.

[0070] According to some embodiments of the invention, the cells are non-human cells.

[0071] According to some embodiments of the invention, the cells are non-human cells of an edible species.

[0072] According to some embodiments of the invention, the edible species is selected from the group consisting of a ruminant species, an avian species and an aquatic species.

[0073] According to some embodiments of the invention, the cells are non-genetically modified.

[0074] According to some embodiments of the invention, the cells are genetically modified.

[0075] According to some embodiments of the invention, the cells are genetically modified to express a recombinant secreted polypeptide.

[0076] According to some embodiments of the invention, the cells are genetically modified to express an antibody or a growth factor.

[0077] According to an aspect of some embodiments of the present invention there are provided cells or a culture comprising the same obtainable according to the method as described herein. According to an aspect of some embodiments of the present invention there is provided a processed product comprising the cells as described herein.

[0078] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0079] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0080] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0081] In the drawings:

[0082] Figure 1 shows treatment of H2O with 10 ppt salinity supplemented with 34 mg / L ammonia. Samples were collected after 20, 40, and 60 minutes. A dose dependent and a time dependent absorption of ammonia by the Wolffia arrhiza are evident. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium. 100 % ammonia was absorbed within 60 minutes of treatment.

[0083] Figure 2 shows that removal of ammonia (34 mg / L starting concentration) in the presence of 1000 mg / L lactate seems to be more efficient (80 % removal after 3 hours) than in the presence of 3000 mg / L lactate (70 % removal after 3 hours). The medium is H2O with 10 ppt salinity supplemented with 34 mg / L ammonia. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0084] Figure 3 shows removal of lactate (1000 mg / L) which was found to be 20 % and 30 % after 1 and 3 hours, respectively. A time dependent depletion was evident in 10 ppt saline water. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0085] Figure 4 shows removal of lactate (3000 mg / L starting concentration). Depletion was found to be 10 % and 15 % after 1 and 3 hours, respectively, in 10 ppt saline water. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium. Figure 5 shows that removal of ammonia (10 mg / L) in the presence of 1000 mg / L lactate is similar under different light conditions with 65 % and 80 % removal after 1 and 2 hours, respectively. The medium was DMEM medium with high glucose (4500 mg / L), without L- glutamine, without phenol red (supplied by Sartorius), buffered with HEPES 10 mM spiked by initial concentration of 1000 mg / L Lactate and 10 mg / L ammonia. . The experiment was at a concentration of 15 ml (10 gr) biomass of duckweed in 20 ml medium.

[0086] Figure 6 shows lactate removal, which is light regimen independent. The medium was DMEM medium (Sartorius) with high glucose (4500 mg / L), without L-glutamine, without phenol red, buffered with HEPES 10 mM spiked by initial concentration of 1000 mg / L Lactate and 10 mg / L ammonia. The experiment was at a concentration of 15 ml (10 gr) biomass of duckweed in 20 ml medium.

[0087] Figure 7 shows ammonia removal of 60 % and 85 % after one hour and 3 hours, respectively, again in a time dependent manner. Medium used was serum-free DMEM basal medium supplemented with 3000 mg / L glucose, proteins: including insulin, albumin, growth factor (FGF) buffered with HEPES 15 mM. The medium was spiked with a starting concentration of ammonium 10 mg / L and Lactate 1000 mg / L. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0088] Figure 8 shows lactate removal of 30 % (300 mg / L) after 1 hour. Medium used was serum- free DMEM basal medium supplemented with 3000 mg / L glucose, proteins including insulin, albumin, growth factor (FGF) buffered with HEPES 15 Mm. The medium was spiked with a starting concentration of ammonium 10 mg / L and Lactate 1000 mg / L. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0089] Figure 9 shows ammonia removal of 63 % and 90 % after 1 and 3 hours, respectively. Medium used was serum-free DMEM basal medium with 3000 mg / L glucose, proteins: including insulin, albumin, growth factor (FGF) and buffered with HEPES 15 mM. The medium was spiked with a starting concentration of ammonium 34 mg / L and Lactate 3000 mg / L. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0090] Figure 10 shows lactate removal of 25 % after 1 hour. Medium used was serum-free DMEM basal medium with 3000 mg / L glucose, proteins: including insulin, albumin, growth factor (FGF) and buffered with HEPES 15 mM. The medium was spiked with a starting concentration of ammonium 34 mg / L and Lactate 3000 mg / L. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0091] Figure 11 shows ammonia removal of 100 % after 20 minutes with a starting concentration of 12 mg / L in DMEM F12 medium that was spiked with initial concentration 12 mg / L ammonia and 1000 mg / L lactate. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0092] Figure 12 shows a time-dependent ammonia removal of 100 % after 1 hrin DMEM F12 medium that was spiked with 34 mg / L ammonia and 3000 mg / L lactate. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0093] Figure 13 shows lactate removal of 50 % after 2 hours with a starting of 1,000 mg / L in DMEM F12 medium that was spiked with initial concentration 10 mg / L ammonia and 1000 mg / L lactate. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0094] Figure 14 shows lactate removal of 50 % after from DMEM F12 medium that was spiked with 34 mg / L ammonia and 3000 mg / L lactate. The experiment was at a concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0095] Figure 15 illustrates the results of lactate removal within a time frame of 1 to 2 hours using various treatments of Duckweed. These treatments included a suspension of ethanol for either 2 or 24 hours, as well as an autoclave treatment. The findings indicate that all treated Duckweed demonstrated a reduction in ammonia levels. The medium was DMEM medium with high glucose (4500 mg / L), without L-glutamine, without phenol red (supplier by Sartorius), buffered with HEPES 10 Mm spiked with 3000 mg / L Lactate and 34 mg / L ammonia. The experiment was at concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0096] Figure 16 shows protein content in the medium following various treatments of duckweed as determined by a Bradford assay. The medium was DMEM medium with high glucose (4.5 g / L), without L-glutamine, without phenol red (supplier by Sartorius) spiked by 1000 mg / L Lactate and 10 mg / L ammonia and 200 mg / L of Bovine Albumin Serum (BSA) , buffered with HEPES 10 mM. The experiment was in concentration of 10 ml (7.5 gr) biomass of duckweed in 20 ml medium.

[0097] Figure 17 is a schematic illustration of a process for preparing a live or an inactivated plant (e.g., carrot) tissue that can be used in medium treatment for removal of undesired components such as lactate and ammonia.

[0098] Figures 18 shows lactate removal by carrot tissue inactivated with different ethanol doses. Black- Control (no tissue added), Grey- carrot cubes. 2 g tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius) spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA) and buffered with HEPES 10 mM. Contact time 10 min.

[0099] Figure 19 shows ammonia removal by carrot tissue inactivated with different concentrations of ethanol. Black- Control (no tissue added), Grey- carrot cubes. 2 g tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius) and buffered with HEPES 10 mM spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA). Contact time 10 min.

[0100] Figure 20 shows the effect of inactivated carrot cubes weight on lactate (solid line) and ammonia (dashed) removal. 0-2 g tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius) and buffered with HEPES 10 mM spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA). Contact time 10 min.

[0101] Figure 21 shows lactate removal by the indicated inactivated plant tissues at various time points. 2 g tissue per 5 ml DMEM medium supplemented with glucose (4500 mg / L), without L- glutamine, phenol red (Sartorius) spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA) and buffered with HEPES 10 Mm. Contact time 0, 10, 120 min.

[0102] Figure 22 shows lactate (black) and ammonia (grey) removal by various inactivated plant tissues. 2 g tissue per 5 ml DMEM medium supplemented with glucose (4500 mg / L), without L- glutamine, phenol red (Sartorius) spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA) and buffered with HEPES 10 mM. Contact time 10 min.

[0103] Figure 23 shows the effect of treatment cycles with new (fresh) inactivated carrot cubes subjected to the same media on lactate (black) and ammonia (grey) removal. 2 g tissue per serum- free DMEM basal medium with 3000 mg / L glucose, proteins: includes insulin, albumin and fibroblast growth factor 2 (FGF2) and buffered with HEPES 10 mM. The medium was spiked with starting concentration of lactate 3000 mg / L ammonium 34 mg / L. Contact time 10 min.

[0104] Figure 24 shows the effect of different washing solutions on the removal of lactate by two inactivated carrot types: black- no tissue added, filled - cubed carrot, blank (dots) - shredded carrot. Washing time (Ihr). 2 g tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L- glutamine, phenol red (Sartorius) spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA) and buffered with HEPES 10 mM. Contact time 10 min.

[0105] Figure 25 ion absorption by the carrot cubes from the DMEM solution. 2 g tissue per 5 ml of DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius) , buffered with HEPES 10 mM.

[0106] Figure 26 shows the effect of different washing solutions (water triangles, DMEM-squares ) on lactate (solid line) and ammonia dashed line removal by cycles of new inactivated carrot tissue to the same media. Washing time (Ihr). 2 g tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius) spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA) , buffered with HEPES 10 mM. Contact time 10 min.

[0107] Figure 27 shows the effect of different washing solutions (water triangles, DMEM-squared) on conductivity (solid line) and pH (dashed) by cycles of new inactivated carrot tissue to the same media. Washing time(lhr). 2 g tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius) spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA) , buffered with HEPES 10 mM. Contact time 10 min.

[0108] Figure 28 shows lactate (black) and ammonia (grey) removal by sequentially re-using the same inactivated carrot tissue. 2 g tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius) spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA) , buffered with HEPES 10 mM. Contact time 10 min.

[0109] Figure 29 is a graph showing proliferation of MIA CaPa-2 in the indicated media as determined by a CCK assay.

[0110] Figure 30 shows DF-1 cell line 1 growth kinetics in various media.

[0111] Figure 31 shows DF-1 cell line growth kinetics in various media.

[0112] Figure 32 shows the protein concentrations of bovine serum albumin (BSA), Carbonic anhydrase from bovine erythrocytes (Ert), and insulin (Ins) in media treated with carrots. These proteins were analyzed using a 16% SDS-PAGE gel with tricine running buffer. Lysozyme (Lyz) served as a normalization control. Lanes 1-3 on the right display the calibration curves. Lane 4 represents the control (medium without carrots) at 60 minutes, while lanes 5 and 6 show experimental samples at 60 minutes. Lane 7 is the control at 120 minutes, with lanes 8 and 9 representing experimental samples at 120 minutes. Lane 10 contains a protein ladder (250-5 kDa, Bio-Rad).

[0113] Figures 33A-C show different analytes in media with 3000 mg / L lactate, 34 mg / L ammonium and 500 bovine serum albumins (BSA) having been exposed to the indicated treatments. (A) ammonia concentration in the media with and without treatments. (B) lactate concentration in the media with and without treatments. (C) glucose concentration in the media with and without treatments.

[0114] Figure 34 shows the concentration of bovine serum albumin (BSA) in a culture medium treated with a carrot tissue, as determined by 12 % SDS-PAGE with glycine running buffer. Carbonic anhydrase from bovine erythrocytes served as a normalization control. The gel includes control samples in lanes 1 and 2, medium treated with carrot cubes in lanes 3-5, medium treated with Wolffia in lanes 6-9, and a protein weight ladder (10-250 kDa, Biolab) in lane 10. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0115] The present invention, in some embodiments thereof, relates to methods and systems for depleting undesired cell culturing products from cell culture media.

[0116] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being carried out in various ways.

[0117] The accumulation of excreted metabolites to toxic levels, limits cell proliferation and culture productivity and requires discarding expensive media. Selective removal of such compounds will enable effective recycling of the media for continuous growth. Disposing large quantities of toxic media may also have an environmental effect. Hence provided herein are methods and systems for depleting undesired cell culturing products from cell culture media, thereby allowing recycling at least some components of the media.

[0118] Thus, according to an aspect of the invention there is provided a method of depleting undesired cell culturing products from a cell culture medium. The method comprising contacting a cell culture medium which comprises lactate and ammonia with a plant tissue under conditions which allow depletion of the lactate and ammonia from the culture medium by the plant tissue, wherein the contacting is effected in an aseptic environment, thereby depleting undesired cell culturing products from the cell culture medium.

[0119] As used herein “depleting” refers to removal or reduction in the concentration of a specific substance i.e., a waste material e.g., ammonia and / or lactate from a solution e.g., a culture medium. Removal can be of at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 % or complete removal such that it is no longer detectable using analytic methods which are well known in the art. For example, enzymatic assays for the detection of ammonia and lactate are well known in the art and typically make use of glutamate dehydrogenase (ammonia assay), L-lactate dehydrogenase and glutamate-pyruvate transaminase (lactate assay). Commercial kits can be used such as K-AMIAR and K-LATE kits, available from Megazyme, Neogen (Ireland) that detect ammonia and lactate, respectively.

[0120] According to a specific embodiment, the reduction is by a range of 20-50 %, e.g., 20-40 %, 30-40 % (w / w) of lactate. According to a specific embodiment, the plant tissue to solution ratio to achieve the desired reduction falls within the range of 1:5 to 1:2 (v / w). This means that for every unit of plant tissue weight, between 5 and 2 units of solution volume can be used, according to some embodiments of the invention. According to a specific embodiment, the reduction is by a range of 20-50 %, e.g., 20-40 %, 30-40 % (w / w? 1:5- 1:2 ) of ammonia. . According to a specific embodiment, the plant tissue to solution ratio to achieve the desired reduction falls within the range of 1:5 to 1:2 (v / w). This means that for every unit of plant tissue weight, between 5 and 2 units of solution volume can be used, according to some embodiments of the invention.

[0121] According to a specific embodiment, depleting lactate and / or ammonia is by at least 25 % as compared to their concentration in the medium prior to contacting with the plant tissue.

[0122] As used herein, the term “medium” or “cell culture medium” refers to a medium as known in the art, and compositions comprising ingredients of biological origin. Such media and cultures may contain nutrients, supplements and feeds, amino acids, peptides, proteins and growth factors (such as albumin, e.g., BSA, catalase, transferrin, insulin, erythropoietin, fibroblast growth factor (FGF), TGF beta, IGF, and others), vitamins, polyamines, sugars, carbohydrates, lipids, nucleic acids, hormones, fatty acids, trace materials, certain salts (such as potassium salts, calcium salts, magnesium salts), as well as waste materials such as ammonia, lactate, toxins and sodium salts. The medium is typically an aqueous based solution that promotes the desired cellular activity, such as viability, growth, proliferation, differentiation of the cells cultured in the medium. The pH of a culture medium should be suitable to the cells that will be grown. Most bacteria grow in pH 6.5 - 7.0 while most animal cells thrive in pH 7.2 - 7.4. Hence contemplated herein is growth in pH of 6.5-7.4, e.g., 7, 6.5-7, 6.5-7.2, 6.8-7.2, 6.9-7.1, 6.9-7.4, 6.9-7.3. 6.9-7.2.

[0123] In cell culturing, various types of media are employed to provide the necessary nutrients and environment for cell growth and maintenance. These media can be broadly categorized into natural and synthetic media, each of which is contemplated herewith as a separate embodiment. Natural media include complex substances like serum, plasma, and tissue extracts, which contain a mixture of growth factors, hormones, and other nutrients. Serum, often derived from fetal bovine sources, is particularly rich in growth factors but can vary in composition. Synthetic or defined media, on the other hand, are precisely formulated to include specific concentrations of salts, amino acids, vitamins, glucose, and other essential nutrients. These media may also be supplemented with growth factors, hormones, and antibiotics to promote cell proliferation and prevent contamination. A common synthetic medium is Dulbecco's Modified Eagle Medium (DMEM), often used in conjunction with fetal bovine serum (FBS) and antibiotics. Other specialized media, such as RPMI 1640, Minimal Essential Medium (MEM), and Roswell Park Memorial Institute (RPMI) medium, are tailored to specific cell types and applications, ensuring optimal conditions for diverse cellular processes, each of which is contemplated herewith as a separate embodiment.

[0124] According to a specific embodiment, the medium comprises animal derived components. According to a specific embodiment, the medium is free of animal derived components (i.e., xeno-free).

[0125] When referring to animal derived components it does not mean to encompass the cells which are cultured in the medium, but rather the medium components which are typically soluble in the medium.

[0126] The medium may comprise cells in which case it is referred to herein as “a cell culturing system”.

[0127] The eukaryotic cells may be mammalian cells, e.g., edible cells, e.g., chicken cells, crustacean cells, or fish cells.

[0128] Also included herein are including physiological cell suspensions such as blood or bone marrow aspirates.

[0129] According to a specific embodiment, the cells are a cell line, such as lines which are used in recombinant protein or mRNA production. Several cell lines are commonly used, some of which are described infra without the meaning to limit to those examples: CHO (Chinese Hamster Ovary) cells are one of the most prevalent mammalian cell lines. HEK293 (Human Embryonic Kidney) cells, particularly the HEK293T and HEK293E variants, are typically used for their high transfection efficiency. The NSO cell line, derived from murine myeloma cells, is another mammalian line used in the industry. Insect cells include, for example, the Sf9 and Sf21 cell lines, derived from the fall armyworm (Spodoptera frugiperda) and are typically employed using the baculovirus expression system.

[0130] Saccharomyces cerevisiae is widely used in baking, brewing, winemaking, and biotechnology. Pichia pastoris is utilized in recombinant protein production. Candida utilis, also known as torula yeast, is grown for use as animal feed, a flavor enhancer, and a source of singlecell protein. Kluyveromyces lactis is cultivated for lactose fermentation and enzyme production. Schizosaccharomyces pombe is grown for research purposes.

[0131] The cells are contemplated as genetically modified to express a heterologous product of interest or not.

[0132] According to a specific embodiment the plant tissue is generally recognized as safe (GRAS).

[0133] According to a specific embodiment, and without being bound by theory, the medium is depleted from waste molecules by the action of assimilation by the plant tissue, e.g., duckweed or carrot or absorption or adsorption to the plant tissue, thereby the medium or at least components thereof can be recycled. As used herein, the terms “waste material(s)” and “waste molecule(s)” are interchangeable. There are any materials / molecules that interfere with desired growth and / or desired differentiation of the cells that are cultured in a cell culture medium, e.g., inhibit cell growth and / or differentiation or induce cell death. By way of non-limiting examples, the waste materials / molecules include, but are not limited to, ammonia, lactate, toxins, sodium salts, alanine, glutamic acid, aspartic acid, ammonium, reactive oxygen and nitrogen species. According to a specific embodiment, the waste is lactate and / or ammonia.

[0134] As part of the metabolic processes associated with cell growth, waste products such as, metabolites, alcohol, alkaloids, carbon dioxide, lactate and ammonium ions are generated, and accumulated in the culture media.

[0135] As used herein “lactate” may be interchangeably used with “lactic acid” a metabolism biproduct in cell culturing.

[0136] According to a specific embodiment, the concentration of lactate before contacting is 1000- 3000 mg / L, e.g., 1500-3000 mg / L, 1000-2000 mg / L, 1500-2500 mg / L.

[0137] As used herein “ammonia” also includes “ammonium” a metabolism bi-products in cell culturing.

[0138] According to a specific embodiment, the concentration of ammonia before contacting is 1- 5 mM , e.g., 1-3 mM, e.g., 1.5-2.5 mM, e.g., about 2 mM.

[0139] Other waste materials which may be depleted include, but are not limited to, amphiphilic toxins, sodium salts, alanine, glutamic acid, aspartic acid, reactive oxygen, and nitrogen species. In some embodiments, the waste materials have a molecular weight no greater than 100 Da, e.g., 90 Da, 80 Da, 70 Da, 60 Da, 50 Da, 40 Da, 30 Da, 20 Da, 10 Da, 5 Da.

[0140] In an alternative or an additional embodiment, the waste material has a molecular weight of at least 1 Da, e.g., 1-10 Da, 1-100 Da, 1-50 Da, 1-20 Da.

[0141] By way of non-limiting example, the at least one waste material may comprise ammonia, ammonium, and / or lactate.

[0142] Culturing systems in which the methods and recycling module can be included are described for example in WO2022238867.

[0143] According to a specific embodiment, the treatment with the plant tissue does not affect the protein content in the medium.

[0144] As used herein “protein content” refers to concentration and / or composition of the proteins present in the medium. Hence, the protein is not affected by the plant tissue and remains about the same. As shown in the Examples section which follows, the amount of BSA, erythropoietin, insulin and lysozyme was not affected by the treatment. The proteins are not adsorbed by, incorporated in or degraded by the plant tissue. This attests to the specificity of the contemplated treatment towards waste products such as lactate and ammonia but not to the proteins which can be recycled safely.

[0145] As mentioned, the contacting is performed under conditions which allow depletion of the lactate and ammonia from the medium.

[0146] These conditions refer to any one of or all of: temperature, pH, contact time, shaking. For example, temperature which can be 10-42 °C, e.g., 15-42 °C, 20-42 °C, 10-38 °C, 10-37 °C, 10-36 °C, 10-25 °C, 37-42 °C, 25-38 °C, 25-37 °C, 25-36 °C, 10-20 °C ; pH from 6-8, 6.5-7.5, 6.8-7.2; contact time range from 1 min-24 hr, 1-5 min, 5 min to 24 hr, 1 min to 12 hrs, 1 min to 6 hrs, 1 min to 2 hrs, 1 min to 1 hr, 1 min to 30 min, 1 min to 20 min, 1 - 10 min, 2-20 min, 2-20 min, 2-5 min, 5 - 20 min, 5-15 min, 5-10 min.

[0147] According to a specific embodiment, the plant tissue does not refer to isolated cells (single cells or up to cell clumps of less than 200 cells or less than 20 or 50 cells).

[0148] According to a specific embodiment, the plant tissue does not refer to a whole plant.

[0149] According to a specific embodiment, the plant tissue does not refer to a whole organ.

[0150] According to a specific embodiment, the plant tissue refers to a whole plant.

[0151] According to a specific embodiment, the plant tissue refers to a whole organ.

[0152] The term '"plant" as used herein encompasses tissues from plants or plant parts, including seeds, fruit, root, leaves, shorts, bark, tubers and fronds. The plant tissue can be differentiated. According to other embodiments, the plant tissue is undifferentiated e.g., callus or meristems. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantee, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Cannabaceae, Cannabis indica, Cannabis, Cannabis sativa, Hemp, industrial Hemp, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi / losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, zucchini, cucumber, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash, tea, trees. According to a specific embodiment, the plant tissue is of a fruit or a vegetable.

[0153] According to a specific embodiment, the tissue is of a plant selected from the group consisting of artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, zucchini, cucumber, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato and squash.

[0154] According to a specific embodiment, the fruit is a fleshy fruit (e.g., berry, drupe, pome, hesperidium or cucurbiteae).

[0155] According to a specific embodiment, the vegetable is selected from the group consisting of a leafy vegetable, root vegetable, tuber vegetable, flower vegetable, stem vegetable, bulb vegetable, pod or seed vegetable and sea vegetable. According to a specific embodiment, the plant tissue is hydrous and comprises a water content of at least 75 %, 80 %, 85 %, 90 % v / w.

[0156] According to a specific embodiment, the plant tissue is hydrous and comprises a water content of at least 75 % v / w.

[0157] Methods of determining water content of a plant tissue are well known in the art. For example, measuring the amount of water present in the tissue relative to its dry weight. The most common method used for this purpose is the gravimetric method, which involves drying the tissue and measuring the weight loss.

[0158] According to a specific embodiment, the plant tissue is of a root vegetable e.g., carrot.

[0159] According to a specific embodiment, the plant is a duckweed.

[0160] According to a specific embodiment, the Duckweed is selected from the group consisting of a Wolffia species and Lemna species.

[0161] According to a specific embodiment, the Wolffia species is selected from the group consisting of Wolffia angusta, Wolffia arrhiza, Wolffia australiana, Wolffia borealis, Wolffia brasiliensis, Wolffia Columbiana, Wolffia cylindracea, Wolffia elongata, Wolffia globose, Wolffia microscopica and Wolffia neglecta.

[0162] According to a specific embodiment, the Wolffia species is Wolffia arrhiza (e.g., var. spotless watermeal).

[0163] According to a specific embodiment, the Lemna species is selected from the group consisting of Lemna minor L., Lemna disperma L., Lemna ecuadoriensis L., Lemna gibba L., Lemna gibba L. , Lemna obscura L. and Lemna trisulca L.

[0164] According to a specific embodiment, the stiffness of the plant tissue is at least as that of a carrot sample of 50 mm diameter and 7700 mm2volume the minimum forces required for bruising, bending, and shearing of the carrot fruit being 71 N, 48 N, and 41 N, respectively.

[0165] Once the plant tissue is selected, it can be used viable or subjected to treatment that renders it inactive.

[0166] According to a specific embodiment, the plant tissue is diced, shredded or powder.

[0167] According to a specific embodiment, the plant tissue is diced.

[0168] Figure 17 shows specific embodiments for plant tissue preparation to be used as a filter for removing lactate and / or ammonia and optionally other waste materials.

[0169] Thus, schematically illustrated is a process for the preparation of plant tissue to be used in removal of lactate and ammonia from tissue culture media. The process can use a live (noninactivated) or inactivated tissue. According to a specific embodiment, the plant tissue is viable. Of note, viable and alive are interchangeably used.

[0170] According to another embodiment, the plant tissue is subjected to a treatment which renders it inactive.

[0171] According to a specific embodiment, the treatment is selected from the group consisting of an ethanol fixation, sonication, autoclave, freeze-drying, oven-drying, steam sterilization, radiation (e.g., UV radiation) and detergent treatment.

[0172] According to a specific embodiment, inactivation is in Ethanol such as at a ratio of 1-4 g (tissue) in 10 ml ethanol for 30 min.

[0173] According to a specific embodiment, ethanol for said ethanol fixation is about 50- 100 % v / v, e.g., 70 % (v / v).

[0174] According to some embodiments, the detergent can be ionic, non-ionic or zwitterionic.

[0175] According to a specific embodiment, the detergent is Sodium Dodecyl Sulfate (SDS): A synthetic organic compound used as a detergent and surfactant. According to a specific embodiment SDS is used at a concentration of 0.1-5 %, e.g., about 1 %. For instance, soaking the plant tissue biomass in x 3 volume of 1 % Sodium dodecyl sulfate (SDS), e.g., for 2 hr. and then wash with deionized water. According to some embodiments the following conditions may be used: Triton X-100 a non-ionic detergent with moderate detergent power, used at a concentration of 0.1- 3 %. Sodium Deoxycholate, an ionic detergent, can be used at a concentration of 0.5 % to 2 % (w / v). CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-l -propanesulfonate), a zwitterionic detergent can be used at a concentration of 0.1-0.5% (w / v). Tween-20 (polysorbate) non-ionic detergent can be used at a concentration of 0.05% to 0.5% (v / v).

[0176] According to a specific embodiment, the plant tissue is inactivated.

[0177] As used herein “inactivated” or “inactive” refers to a plant tissue which is non-dividing yet still comprises enzymatic activity. As used herein “viable” or “live” refers to a plant tissue which has not undergone inactivation and therefore its cells are still dividing. Methods of determining plant tissue viability include but are not limited to (Triphenyl) Tetrazolium Chloride (TTC) staining, Evans blue staining, fluorescein diacetate staining, neutral red staining.

[0178] As mentioned, Figure 17 describes specific embodiments for plant tissue treatment as further detailed below.

[0179] As used herein “washing” refers to reduction of osmolality of the tissue, e.g., by washing the plant tissue with a pre-determined volume e.g., x 5, x 10, x 20 x 5-20, x 5-10, e.g., about xlO volumes of deionized water for 1-20 min. Filtration is used to separate the plant tissue from the wash water by any separation methods such as but not limited to, filter press or gravity sedimentation.

[0180] Both the inactivated tissue and the viable tissue maintain selectivity and specificity to removing ammonia and lactate but do not substantially (+ / - 10 % w / v) remove proteinaceous compositions from the medium.

[0181] The selectivity and specificity mean that the plant tissue will preferentially remove ammonia and lactate over proteinaceous compositions from the medium.

[0182] According to a specific embodiment, the system and methods are operated in the dark.

[0183] According to a specific embodiment, the system and methods are operated under aseptic conditions. Sterile implies the absence of all life, desired or undesired. Aseptic implies life in the absence of foreign pathogens. A bioreactor is sterile before a batch starts, and is operated aseptically after inoculation. Piping and connected equipment outside the aseptic bioreactor remain sterile. Sanitary implies that pathogens may be present, but have been reduced to levels where they cannot cause disease. Within these definitions, there exists an important distinction between aseptic industrial fermentation and aseptic animal cell culture: While industrial fermentation of a commodity bioproduct can generally be carried out in the presence of small numbers of foreign organisms — as long as these are not pathogenic to the production cells or users of the end product — any stray microbial life is functionally pathogenic to animal cells in culture. At neutral pH, elevated temperature, and (according to cultured-meat messaging) in the absence of antibiotics, a cellculture bioreactor will readily harbor and be overcome by the contaminating microbe, which proliferates much faster than animal cells. Typically, animal cell cultures can thus be further classified as axenic, i.e., free of any foreign organisms.

[0184] According to a specific embodiment, the aseptic environment is a sterile environment.

[0185] The methods described herein for medium depletion (recycling) can be done in a dedicated system. Thus, according to an aspect of the invention, there is provided a cell culturing system comprising a chamber comprising a plant tissue and a cell culture medium which comprises lactate and ammonia, the system being an aseptic environment and allows depletion of said lactate and ammonia by said plant tissue.

[0186] According to a specific embodiment, the plant tissue used in the method or system described herein is immobilized on a solid support.

[0187] According to another embodiment, the plant tissue is not immobilized to a solid support and in fact remains in a "free" state. Instead of being fixed or attached to a solid support or matrix, the tissue remains dispersed and freely moving within the medium. The plant tissue serves as a resin for depleting ammonia and / or lactate from a tissue culture medium and can be packed as an article of manufacture for that purpose optionally attached to a solid support as described herein and / or packed as a column.

[0188] According to a specific embodiment, the system of chambers is a module in a culturing system of a target cell of interest.

[0189] According to a specific embodiment, the system further comprises a target cell of interest.

[0190] According to a specific embodiment, the target cell of interest and said plant tissue are in the same chamber (though there may be more than one, i.e., a plurality of chambers).

[0191] In some embodiments, the plurality of chambers are fluidically connected in a row or in parallel to each other.

[0192] According to a specific embodiment, the plant tissue and said target cell of interest are in a single chamber.

[0193] According to a specific embodiment, the target cell of interest and said plant tissue are in separate chambers.

[0194] According to a specific embodiment, the separate chambers are in fluid communication.

[0195] According to a specific embodiment, the system comprises an inlet for introducing the cell culture medium into the chamber and an outlet for collecting said culture medium following assimilation / adsorption of the lactate and ammonia by the plant tissue.

[0196] According to a specific embodiment, the culturing system is based on fed batch or perfusion (e.g., ATF).

[0197] Thus, the chambers described above and herein may be used in conjunction with a perfusion based culturing system, that make use of e.g., alternating tangential flow (ATF) system, a Tangential Flow Filtration (TFF) system, or a fed- batch culturing system. Thus, the chamber which comprises the plant tissue may be directly or indirectly connected to at least one liquid flow adaptor, configured to connect the chamber to an alternating tangential flow (ATF) system, to a Tangential Flow Filtration (TFF) system, or to a fed-batch culturing system.

[0198] In some embodiments, the chamber is directly or indirectly connected to a flow adaptor configured to connect the chamber to a hemoperfusion system.

[0199] In some embodiments, the chamber is configured to treat up to 1000 liters of liquid.

[0200] According to some embodiments the system comprises at least one chamber as described above and herein; means for flowing a medium / media through the plurality of chambers which comprise the plant tissue; means for circulating the medium; and a bioreactor.

[0201] In some embodiments, the at least one chamber is disposable.

[0202] In some embodiments, the means for flowing a medium comprises or is a pump. In some embodiments, the system further comprises at least one sensor configured to record values of at least one parameter related to the flow of the medium through the chamber and / or content of the medium.

[0203] According to a specific embodiment, the at least one sensor is for detecting at least one of lactate, ammonia, glucose, conductivity, pH, pCCh and pCh in said cell culture medium.

[0204] In some embodiments, the system may further comprise a controller electrically connected to the pump and the at least one sensor, wherein the controller is configured to activate the pump based on signals received from the at least one sensor.

[0205] In some embodiments, the chamber is configured to have a dead volume of less than 100 ml, e.g., less than 50 ml, less than 20 ml, less than 10 ml, less than 5 ml or any intermediate, smaller or larger volume.

[0206] In some embodiments, a single chamber is configured to recycle up to 1000 liters of liquid, e.g., up to 500 liters, up to 100 liters or any intermediate, smaller or larger volume of liquid.

[0207] In some embodiments, the system comprises a passive flow chamber. In some embodiments, the passive flow chamber is configured to recycle the culture medium by osmosis or diffusion. In some embodiments, the passive flow chamber is an add-on or removable device to a cell culture container, e.g., a cell culture plate, a cell culture flask, or a cell culture bioreactor. In some embodiments, the passive flow chamber is submerged at least partly in a culture medium within a cell culture container such as a bioreactor. In some embodiments, the passive flow chamber is removably attached to a wall of the cell culture container. Alternatively, the passive flow chamber is non-removably attached to and is an integral part of the cell culture container.

[0208] As mentioned the system described above which comprises the plant tissue is in a flow communication with a cell culture container, e.g., a cell culture plate, a cell culture flask or a bioreactor. The culture medium from the container flows through the chamber which comprises the plant tissue. In some embodiments, the culture medium flowing may comprise cells or tissues cultured in suspension. In some embodiments, the culture medium flowing into the chamber which comprises the plant tissue also comprises waste molecules and nutrients that are needed for proper growth and / or differentiation of the cells. By way of non-limiting examples, the nutrients include, but are not limited to, at least one of proteins, hormones, and growth factors. However, as mentioned it is believed that the protein content is not affected by the plant tissue.

[0209] According to a specific embodiment, contacting with the plant tissue is for 1-120 minutes, e.g., 5-120 minutes, 10-120 minutes, 30-120 minutes, 60-120 minutes; 1-90 minutes, e.g., 5-90 minutes, 10-90 minutes, 30-90 minutes, 60-90 minutes; 1-60 minutes, e.g., 5-60 minutes, 10-60 minutes, 30-60; 1-30 minutes, e.g., 5-30 minutes, 10-30 minutes, 20-30; 1-20 minutes, e.g., 5-20 minutes, 10-20 minutes.

[0210] According to a specific embodiment, contacting is for a time period which is 1-60 minutes, e.g., 1- 10 min.

[0211] Following the treatment, the medium may reach a lactate level of 500-1000 mg / L, e.g., 500- 800 mg / L, 500-700 mg / L and an ammonia level of 10-28 mg / L (0.55-1.55 mM). Sometimes more than one cycle of treatment (i.e., at least 2 times) with the plant tissue may be needed to reach the desired level of ammonia and / or lactate. In such a case fresh plant tissue (e.g., inactivated as described herein) may be used though this is not obligatory. Reduction of ammonia levels to undetectable may require the use of viable tissue, e.g., in the case of Duckweed.

[0212] According to a specific embodiment, the at least 2 times of treatment are separated by using the medium resultant of one time of the at least 2 times in cell culturing.

[0213] According to a specific embodiment, the at least one time of the at least 2 times is effected with fresh plant tissue.

[0214] The use of “fresh” means a plant tissue not having been used in the methods described herein. Such a tissue is naive to the treated medium (wasn’t exposed to media before).

[0215] According to a specific embodiment, at least one time of the at least 2 times is effected with plant tissue having been used according to the method.

[0216] Thus, improved removal of lactate and / or ammonia may be achieved by subjecting the medium more than once to treatment (more than one cycle) and / or by adding a new batch of plant tissue.

[0217] As mentioned above and throughout, the methods and systems described herein encompass the step of isolating the cell culture medium from the plant tissue following the contacting.

[0218] According to a specific embodiment, the method further comprises adding glucose to the refurbished culture medium resultant of the method. Thus, the method may further comprise standardize the glucose concentration to 4.5 g / L with D-glucose.

[0219] According to a specific embodiment, the method further comprises analyzing at least one parameter in the refurbished medium selected from the group consisting of lactate, ammonia, glucose, protein, osmolarity, pH and amino acids and optionally amending as needed (e.g., glucose) or exposing it to an additional cycle(s) of treatment, e.g., when lactate or ammonia levels haven’t reached the desired level.

[0220] Thus, according to a specific embodiment, there is provided a refurbished culture medium obtainable by the method of depletion as described herein. Such a refurbished medium can then be directly flowed or added to the target cells or stored (e.g., -20 °C to room temperature, e.g., 4 °C or frozen).

[0221] According to a specific embodiment, the refurbished culture medium further comprises fresh powder of medium (typically the same base medium) optionally at a concentration of 20-50 %.

[0222] It will be appreciated that the addition of fresh medium can aid in achieving the right nutritional values (as detailed above) as well as correcting the osmolarity e.g., to 250-500 mOsm / 1, e.g., 250-350 mOsm / 1, 280-320 mOsm / 1.

[0223] The refurbished medium may be packed in an article of manufacture such as a bottle or a tube.

[0224] The refurbished medium can be used in a variety of commercial and research applications. Typically, it is used in applications which require the growth of large amounts of cells.

[0225] Such applications include production of cultured meat or production of recombinant polypeptides / mRNA e.g., antibodies.

[0226] Thus, according to an aspect of the invention, there is provided a method of culturing target cells of interest, the method comprising contacting the cells with the refurbished medium to thereby culture the cells.

[0227] According to some embodiments, the target cells are a cell line.

[0228] According to some embodiments, the target cells are human cells.

[0229] According to some embodiments, the target cells are non-human cells.

[0230] According to some embodiments, the target cells are non-human cells of an edible species.

[0231] According to some embodiments, the target edible species is selected from the group consisting of a ruminant species, an avian species and an aquatic species.

[0232] According to some embodiments, the target cells are non-genetically modified.

[0233] According to some embodiments, the target cells are genetically modified.

[0234] According to some embodiments, the target cells are genetically modified to express a recombinant secreted polypeptide.

[0235] According to some embodiments, the target cells are genetically modified to express an antibody or a growth factor.

[0236] Also provided herein are cells or a culture comprising same obtainable according to the method as described herein.

[0237] These may be the final product or further processed such as in the cultured meat industry, molded, printed, extruded alone or in combination with other edible ingredients to provided a processed product which comprising the cells having been cultured as described herein. As used herein the term “about” refers to ± 10 %.

[0238] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0239] The term “consisting of’ means “including and limited to”.

[0240] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0241] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0242] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0243] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0244] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0245] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0246] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0247] EXAMPLES

[0248] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0249] EXAMPLE 1

[0250] EFFICIENCY TESTING BY REMOVAL RATE ANALYSIS IN DIFFERENT MEDIA MATERIALS AND METHODS (RELEVANT TO ALL WOLFFIA

[0251] EXPERIMENTS)

[0252] Wolffia arrhiza was purchased from GreenOnyx .LTD. The duckweed, Wolffia arrhiza was cultivated as described in Appenroth, Klaus-J., et al. 2018 doi: 10.3389 / fchem.2018.00483. Medium was changed every 2 to 3 days to insure minimal microbial growth and healthy W. arrhiza biomass.

[0253] To determine W. arrhiza volume for experiments, the plant was put in a graduated cylinder, washed with distilled water and excessive water was removed from biomass prior to experiment and then weighted. Experiments were performed in petri plates under 11,500 LUX light intensity.

[0254] Samples for ammonia and lactate measurements were taken in 100 ul volumes and stored under 4 °C conditions until processed. Assays for ammonia and lactate were performed with K- AMIAR and K-LATE kits, respectively, by Megazyme, NEOGEN (Ireland). The assay of protein content was performed in Bradford reagent from Sigma.

[0255] RESULTS

[0256] The duckweed, Wolffia arrhiza was tested in different conditions such as - time, concentrations, medium types. It was also exposed to different ammonia and lactate concentrations. The ammonia and lactate tests measured the specific removal rate SRR (mg removed / gr sorbent / time) by enzymatic methodology assays (K-AMIAR and K_LATE kits Megazyme, Ireland).

[0257] Over 200 experiments were performed that tested the ability Wolffia arrhiza to remove ammonia and lactate from 4 different types of media. A. Saline water - H2O with 10 ppt salinity (NaCl)

[0258] B. Medium DMEM high glucose (4500mg / L), w / o L-glutamine, w / o phenol (HEPES 10 Mm)

[0259] C. Medium based on serum-free DMEM basal medium supplemented with 3000 mg / L glucose, proteins: in the range of mg / L selected from the group consisting of insulin, albumin and growth factors (e.g., FGF), buffered by HEPES 15 mM.

[0260] D. DMEM-F12 medium for cells proliferation, (Gibco™ DMEM / F-12 Catalog number: 11320033)

[0261] A. H2O with IQppt salinity

[0262] Two biomass concentrations were tested: 5 ml (3.35 gram) and 10 ml (7.7 gram) 1 in 10 ml (50% and 100%) volume H2O with 10 ppt salinity supplemented with 34 ppm ammonia (v / v). Samples were collected after 20, 40, and 60 minutes. As shown in Figure 1, a dose dependent and time dependent absorption of ammonia by the Wolffia arrhiza were evident. At a concentration of 10 ml (7.7 gram) in 10 ml volume all the 34ppm ammonia were absorbed within 60 minutes of treatment.

[0263] Thereafter the remediation capacity of a biomass of 15 ml in 20 ml volume (7 5%) supplemented with 34 mg / L Ammonia together with 1,000 mg / L and 3,000 mg / L lactate was tested. Samples were retrieved after 1 and 3 hours.

[0264] Figure 2 shows removal of ammonia (34 mg / L) in the presence of 1000 mg / L lactate seems to be more efficient (80% removal after 3 hours) than in the presence of 3000 mg / L lactate (70 % removal after 3 hours).

[0265] Figure 3 shows removal of lactate with initial concentration 1000 ppm which was found to be 20 % and 30% after 1 and 3 hours, respectively, again in a time dependent manner. A lactate reduction of 300 mg / L demonstrating when normalized that 0.8 mg lactate is absorbed by 1 gram duckweed .

[0266] Figure 4 shows removal of lactate (3000 mg / L) which was found to be 10 % and 15 % after 1 and 3 hours, respectively. The normalized value is 1.3 mg lactate per 1 gram duckweed.

[0267] B. DMEM medium with high glucose (4500mg / L), without L-glutamine, without phenol red and buffered by HEPES 10 mM:

[0268] A biomass of 15 ml (10 gram) was combined with 20 ml volume medium supplemented with initial concentration of 10 mg / L ammonia together with 1,000 mg / L lactate under different light conditions: high (11,500 LUX), low (4,000 LUX) and darkness. Samples were retrieved (t-0 about 1-5 min following contact with the Duckweed) after 1 (t-1) and 2 (t-2) hours. Figure 5 shows that removal of ammonia (10 mg / L) in the presence of 1000 mg / L lactate is similar under different light conditions with 65 % and 80 % removal after 1 and 2 hours, respectively.

[0269] Figure 6 shows lactate removal, which is time dependent with high variation, no significantly different results between light regimen. The lactate reduction is around 700 mg / L, the normalize number 1.4 mg lactate per 1 gram duckweed.

[0270] The light regime is important in two parameters:

[0271] • Some of the medium components like vitamin C are sensitive to light.

[0272] • Live Wolffia arrhiza is Mixotrophic, for which light is important.

[0273] Hence, showing that there is no change in removal rate of lactate under the indicated light regimen is important. Due to photosensitivity of some components, it may therefore be desirable to work on the dark.

[0274] C. Medium used is serum-free DMEM basal medium supplemented with 3000 mg / L glucose and proteins selected from the group consisting of insulin, albumin and growth factors (e.g., FGF) and buffered by HEPES 15 mM.

[0275] A wet biomass of 15 ml (10 gram) after filtration as describe in Figure 17 was combined with 20 ml medium . supplemented with 10 mg / L ammonia together with 1,000 mg / L lactate. Samples were retrieved after 1 and 3 hours.

[0276] Figure 7 shows ammonia removal of 60 % and 85 % after one hour and 3 hours, respectively, again in a time dependent manner.

[0277] Figure 8 shows lactate removal of 30 % after 1 hour from a concentration of 1000 mg / L. A lactate reduction of 314 mg / L. The normalized value is 0.624 mg lactate per 1 gram duckweed.

[0278] Thereafter, a concentration of 15 ml (10 gram) biomass in 20 ml with 34 ppm ammonia together with 3,000 mg / L lactate was tested. Samples were retrieved after 1 and 3 hours.

[0279] Figure 9 shows ammonia removal of 63 % and 90 % after one hour and 3 hours, respectively.

[0280] Figure 10 shows lactate removal of 20 % after 1 hour from a concentration of 3000 mg / L. A lactate reduction of 770 mg / L. The normalized value is 1.54 mg lactate per 1 gram duckweed.

[0281] D. DMEM F12 medium for cells proliferation, (Gibco™ DMEM / F-12 Catalog number: 11320033)

[0282] A biomass of 15 ml (10 gram) was combined with 20 ml medium supplemented with 10 mg / L ammonia together with 1,000 mg / L lactate and medium supplemented with 34 mg / L ammonia together with 1,000 mg / L lactate. Samples were retrieved after 20, 40, 60, and 120 minutes kinetics demonstration. Figure 11 shows ammonia removal of 100 % after 20 minutes, again in a time dependent manner.

[0283] Figure 12 shows ammonia removal of 100% after one hour, and figure 13 shows lactate removal of 50 % after 1 hour from concentration of 3000 mg / L.

[0284] Figure 13 shows lactate removal of 50 % after 2 hours with a starting of 1,000 mg / L in DMEM F12 medium that was spiked with initial concentration 10 mg / L ammonia and 1000 mg / L lactate. A lactate reduction of 610 mg / L. The normalized value is 1.22 mg lactate per 1 gram duckweed.

[0285] Figure 14 shows lactate removal of 50 % after 1 hour from DMEM F12 medium that was spiked with 34 mg / L ammonia and 3000 mg / L lactate. A lactate reduction of 1200 mg / L. The normalized value is 2.4 mg lactate per 1 gram duckweed.

[0286] EXAMPLE 2

[0287] Effect of pretreatment of duckweed like ethanol exposure and autoclave on lactate and ammonia removal by carrot cubes

[0288] The rational; was to compare the kinetics of lactate removal after different pretreatments.

[0289] Wolffia arrhiza biomass was exposed to different pretreatments which inactivated the duckweed before it was mixed with the medium.

[0290] The pretreatments were:

[0291] • Autoclave for 3 (hr) in temperature of 121 °C.

[0292] • Soaking the duckweed biomass in x3 volume of 100 % ethanol for 2 or 24 hr.( Ethanol 2 hr. or Ethanol 24 hr.)

[0293] • Non treated duckweed will be noted as ’’live”.

[0294] 7.5 gram of pretreated biomass was combined with 20 ml medium. DMEM medium with high glucose (4500mg / L), without L-glutamine, without phenol red. The biomass was initially mixed and maintained without mixing for 2 hr, sampling at t=0, t=l hr and t=2 hr.

[0295] Figure 15 illustrates the results of lactate removal within a time frame of 1 to 2 hours using various treatments of Duckweed. The findings indicate that all treated Duckweed demonstrated a reduction in lactate but under ethanol pretreatment the removal of lactate occurred faster than with autoclave. The normalized value was 2.2-2.6 mg lactate per 1 gram duckweed.

[0296] . Figure 16 presents the protein content in the medium following different treatments of Duckweed as measured using a Bradford assay. The untreated medium exhibited a 10 % increase in protein content from t=0 to t=2 hours. In contrast, all other treatments showed a 10% reduction in protein content compared to the untreated medium, with no significant changes observed between t=0 and t=2 hours. However, the medium treated with the plant after autoclaving showed a notable exception, with a more pronounced reduction in protein content during this period.

[0297] EXAMPLE 3

[0298] A process for the preparation of plant tissue to be used in removal of lactate and ammonia from tissue culture media

[0299] Figure 17 describes schematically a process for the preparation of plant tissue to be used in removal of lactate and ammonia from tissue culture media. The process can use a live (non-inactivated) or inactivated tissue.

[0300] EXAMPLE 4

[0301] Effect of ethanol concentrations on lactate and ammonia removal by carrot cubes

[0302] The rational was to test the most effective concentration for ethanol inactivation of the plant tissue.

[0303] Control: No carrot tissue. Treatment: Carrot tissue.

[0304] Experiment: 2 gr per 5 ml DMEM spiked with ammonia 34 mg / L, lactate 3000 mg / L, Bovine Serum Albumin (BSA) 200 mg / L and buffer by HEPES 10 mM. Contact time: 10 min.

[0305] Inactivation: 1 g per 10 ml ethanol. Contact time: 30 min.

[0306] Results

[0307] Selectivity: Protein: Not effected. Glucose: Not tested. Both are not shown.

[0308] Figures 18-19 show that 70 % ethanol inactivation is the most effective in lactate removal. Ammonia was not affected. A linear trend in lactate removal is displayed until 70 %.

[0309] EXAMPLE 5

[0310] Effect of the weight of plant tissue on lactate and ammonia removal

[0311] The rational was to test the correlation between tissue weight and lactate / ammonia removal. Experiment: 0-2 gr of inactivated tissue per 5 ml DMEM spiked with ammonia 34 mg / L, lactate 3000 mg / L, Bovine Serum Albumin (BSA) 200 mg / L and buffer by HEPES 10 mM. Contact time: 10 min.

[0312] Results

[0313] Figure 20 shows a linear correlation between tissue weight and lactate and ammonia removal. Specifically, removal of 1 gr / L (30%) of lactate and 0.01 g / L ammonia by 2 g of shredded carrot.

[0314] Selectivity: Protein: Not effected. Glucose: Decrease by 30 %. Both are not shown. EXAMPLE 6

[0315] Kinetics of lactate removal by inactivated plant tissues

[0316] The rational; was to compare the kinetics of lactate removal by different plants: Duckweed (DW), Lamella (LAM), Alphalpha sprout, Radish sprout, Carrots and Callus (Crocus).

[0317] Experiment: 2 gr of inactivated tissue per 5 ml DMEM spiked with ammonia 34 mg / L, lactate 3000 mg / L, Bovine Serum Albumin (BS A) 200 mg / L and buffer by HEPES 10 mM. Contact time: 10 min or 120 min.

[0318] Results

[0319] Most plants exhibit similar removal capability after 10 min.

[0320] As shown in Figure 21, most tissues removed 750 mg / L (25 %) lactate after 10 minute. Minor removal of lactate by callus.

[0321] No further removal of lactate after 120 min is shown. Longer time (20 hours) were tested on D and L samples with no further removal.

[0322] Selectivity: Protein: Not effected. Glucose: Not tested.

[0323] EXAMPLE 7

[0324] Lactate and ammonia removal by various types of inactivated plants tissues

[0325] The rational was to compare different plant tissues: taproot (carrot), fruit (cucumber, zucchini), leaf (cabbage), stem (celery), tuber (potato), seeds (sesame, chickpea)

[0326] Experiment: 2 gram of inactivated tissue per 5 ml DMEM spiked with ammonia 34 mg / L, lactate 3000 mg / L, Bovine Serum Albumin (BS A) 200 mg / L and buffer by HEPES 10 mM. Contact time: 10 min.

[0327] Results

[0328] Figures 22 shows that most hydrous tissue types have similar capabilities of lactate and ammonia removal. Approx. 750 mg / L (25 %) lactate and 15 mg / L (45 %) ammonia. Normalized value of the lactate is 1.88 mg lactate per gram hydrous tissue.

[0329] Selectivity: Protein: No decrease. Glucose: Carrot and cabbage - No removal. Cucumber and Zucchini - 10 % removal. Celery - 15 % removal.

[0330] EXAMPLE 8

[0331] Removal of lactate and ammonia by replenishing with new carrot cubes

[0332] The rational was to reach maximal removal of lactate and ammonia from a tissue culture medium by replenishing with new carrot cubes.

[0333] Experiment: Effect of cycles (supplementating) of new inactivated carrot cubes to the same media on lactate and ammonia removal. 2 gram tissue per DMEM basal medium serum free with 3000 mg / L glucose, proteins: includes insulin, albumin and growth factor (FGF) and buffer by HEPES 15 mM. The medium was spiked with starting concentration of lactate 3000 mg / L ammonium 34 mg / L. Contact time 10 min. in each cycle the old cube was removed and new tissue cube was added.

[0334] Results

[0335] Figure 23 shows that repetitive exposure of new carrot to the same media enabled greater removal of lactate and ammonia /

[0336] Removal of 2100 mg / L (70 %) lactate and 28 mg / L (85 %) ammonia was achieved after 5 cycles.

[0337] Selectivity: Protein: No major effect was observed. Glucose : Removal of 200 ppm (10 %) was observed.

[0338] Conductivity: a reduction of 64 % was reached at cycle 5. pH: 0.1 decrease in pH for each cycle, value remained between 7-8 (not shown).

[0339] EXAMPLE 9

[0340] Effect of Pre- washing with Lactate on Inactivated Carrot Tissues

[0341] Rational to test the effect of washing the carrot tissue with DMEM with or without lactate on removal efficacy.

[0342] Experiment: Effect of different washing solutions on the removal of lactate by two inactivated carrot types: no tissue added, cubed carrot, shredded carrot. Washing time (Ihr). 2 g tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius), buffered with HEPES 10 mM spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA). Contact time 10 min.

[0343] Loading: 1 g per 3 ml loading solution. Contact time: Ihr.

[0344] Results

[0345] Figure 24 shows the effect of different washing solutions on the removal of lactate by two inactivated carrot types: black- no tissue added, filled - cubed carrot, blank (dots) - shredded carrot. Washing time (Ihr). 2 g tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L- glutamine, phenol red (Sartorius) spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA) and buffered with HEPES 10 mM. Contact time 10. The experiment investigated the impact of washing tissue with lactate solution on its ability to remove lactate. Washing the tissue with DMEM that contains lactate appeared to disrupt its capacity for lactate removal. Interestingly, no significant difference was observed in lactate reduction (750 mg / L, representing a 25% decrease) between carrot tissues washed with deionized water and those washed with DMEM medium. This suggests that the type of washing solution (deionized water or DMEM) did not significantly affect lactate removal in this case.

[0346] Selectivity: Glucose removal was affected by the washing solution. Samples treated with plant tissue washed in deionized water showed a 25 % decrease in glucose concentration. In contrast, samples treated with plant tissue washed in DMEM exhibited no change in glucose concentration. This suggests that the washing solution may influence the tissue's ability to remove glucose, with deionized water potentially facilitating glucose removal.

[0347] EXAMPLE 10

[0348] Effect of washing the tissue with DMEM in time on conductivity of the washing solution

[0349] The rational was to reach time of equilibrium for the correct time duration for washing the carrot in DMEM solution. The time frame was 10-140 min .

[0350] Experiment: ion absorbed by the carrot cubes from the DMEM solution. 2 gram of inactivated tissue per DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius), buffered with HEPES 10 mM.

[0351] Results

[0352] Results

[0353] Figure 25 shows saturation of conductivity of DMEM (washing solution) is achieved by a 30 min incubation with carrot cubes.

[0354] High (20 ml) and low (2 ml) volume to tissue ratio were tested with a similar trend (only 20 ml shown).

[0355] EXAMPLE 11

[0356] Effect of washing solution on lactate and ammonia removal by additional cycles with new carrot cubes

[0357] The rational was to reach maximal removal of lactate and ammonia in DMEM with minimal change of components.

[0358] Experiment: Effect of different washing solution (water, DMEM) on lactate and ammonia removal by cycles of new inactivated carrot tissue to the same media. Washing time (Ihr). 2 gr of inactivated tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius), buffered with HEPES 10 mM spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA). Contact time 10 min. Results

[0359] Figure 26 shows that lactate and ammonia removal profiles were not effected by DMEM washing. Removal of 1.8 g / L (70 %) lactate and 0.028 g / L (75 %) ammonia was achieved in 5 cycles. Selectivity: Protein: Not effected. Glucose: Water - not effected, DMEM - slight increase in cycle 5 of -700 ppm. Not shown.

[0360] EXAMPLE 12

[0361] Effect of washing on Chemical Components by Repetition Cycles with New Carrot Cubes £111

[0362] The rational; was to compare washing with deionized water and DMEM aiming for minimal change of in medium components: checked by conductivity.

[0363] Experiment: 2 gram of inactived tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius), buffered with HEPES 10 mM spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA). Contact time 10 min.

[0364] Results

[0365] As shown in Figure 27 washing with DMEM has maintained conductivity after two cycles. Reduction of 30 % in conductivity is shown by DMEM in 5th cycle. Reduction of 70 % in conductivity is shown by in the medium treated by carrots that were washed in water in 5th cycle.

[0366] EXAMPLE 13

[0367] The effect of reusing the same carrot cubes after washing on lactate and ammonia removal

[0368] Rational - Test recycling capability meaning multiple use of the same tissue in media spiked with lactate.

[0369] Experiment: Lactate and ammonia removal by sequentially re-using the same inactivated carrot tissue. 2 g tissue per 5 ml DMEM medium with glucose (4500 mg / L), without L-glutamine, phenol red (Sartorius), buffered with HEPES 10 mM spiked by 3000 mg / L lactate, 34 mg / L ammonia and 200 mg / L Bovine Serum Albumin (BSA). Contact time 10 min.

[0370] Results

[0371] Re-usage of the same carrot cubes is highly significant. Long term re-usage (1 and 2 weeks) achieved similar results (not shown).

[0372] The results show that removal of 0.85 g / L (-30%) lactate and 0.005 g / L (15%) ammonia was achieved using the same carrot tissue for 8 rounds. See Figure 28. Selectivity: Protein: Not effected in all 8 iterations. Glucose : Not effected in all 8 rounds. EXAMPLE 14

[0373] The effect of recycled medium compared to new medium on cell growth

[0374] Rational - This study compares the growth of cell cultures in medium treated with either carrots or Wolffia arrhiza and those cultured in fresh or spent media. The experiment included two control groups: a negative control consisting only of spent medium, and a positive control where spent medium was supplemented with the same amount of DMEM powder as the spent medium that was treated with Wolffia or carrot tissue.

[0375] Goal: To assess the efficacy of different medium treatments, including plant-based remediation and basal was done by Co lab, Israel, an external lab with blind test.

[0376] Materials and Experimental Procedures

[0377] Cell cultures:

[0378] Mia Capa2 cells are derived from human pancreatic cancer tissue. Characterized as an epithelial-like cell line, they serve as a valuable model for cancer research, drug discovery, and investigating the intricacies of pancreatic cancer biology.

[0379] Procedure:

[0380] MiaCapa2 Cell Culture and Media Preparation.

[0381] On day 0, a vial of MiaCapa2 cells were thawed and inoculated into a 75 cm2tissue culture flask with DMEM high glucose produced by Bio-west with 10 % bovine serum. The culture medium was replaced on day 1. The cells were transferred to a 175 cm2flask on day 4, and subsequently split into four 175 cm2flasks on day 7. Cells were cultured until reaching confluence, at that point the medium was replaced. Medium was monitored for a color change to yellow, indicating exhaustion.

[0382] Media Collection: Spent medium was collected on day 5 in 50 ml tubes and centrifuged at 125 x g for 5 minutes to remove cellular debris. The supernatant was filtered through a 0.22 pm filter, stored at 4 °C.

[0383] Medium treatments: 20 grams of carrots or Wolffia arrhiza inactivated by ethanol was combined with 50 ml spent medium for 10 min in room temperature in 50 ml flask.

[0384] The spent media, both those treated by the plant tissue and those left untreated, were supplemented with 30 % (w / v) of DMEM fresh basal media components in powder form (D5030, Sigma- Aldrich). Furthermore, the glucose concentration in both media types was adjusted to a standard level of 4.5 g / L using D-glucose (D9559, Sigma- Aldrich). Table 1: Measured Parameters for Growth Experiments for the Mia Capa2 cell line.

[0385] All the samples of the medium were sent back to Co LAB for growing cell experiments.

[0386] Cell Culture and Experimental Setup

[0387] MIA CaPa-2 cells were trypsinized and seeded at densities of 2500 and 5000 cells per well, respectively, in 96-well plates containing 100 pL of DMEM high glucose (Biowest) supplemented with 10 % bovine serum. On day 1, the culture medium was replaced with the test medium outlined in Table 1. Cell morphology was monitored daily, and calibration curves were generated on days 2 and 3 as follows: known amounts of cells were sown in a plate of 96 wells in a range of 5,000 to 60,000 cells per well. Cell viability and proliferation were assessed using a CCK-8 assay on days 3 and 4. The CCK-8 assay is a colorimetric method that quantifies metabolically active cells by measuring the absorbance of a formazan dye product.

[0388] Results

[0389] Figure 29 illustrates the growth kinetics of MIA CaPa-2 cells cultured in various media. The positive control (fresh medium) exhibited the highest cell density, reaching 40,000 cells / mL after four days. Spent medium treated with Wolffia achieved a cell density of 31,000 cells / mL. Carrot- treated medium showed intermediate growth (21,000 cells / mL), while both corrected and uncorrected spent media exhibited significantly lower cell densities (12,000 and 9,000 cells / mL, respectively). EXAMPLE 15

[0390] Growth of a cultivated meat cell line on recycled medium in comparison to new medium

[0391] Rational To evaluate the potential of recycled spent culture medium, this study compares the growth potential of a cell culture in medium treated with Wolffia arrhiza to fresh or untreated spent medium. Additionally, a control group includes utilizing spent medium supplemented with the same basal medium, i.e., spent corrected.

[0392] Materials and Experimental Procedures

[0393] Cell cultures:

[0394] DF-1 is a cell line derived from chicken embryonic fibroblasts. These cells exhibit a fibroblast-like morphology and robust growth rate. Primarily utilized in virology, cellular and molecular biology research, and recombinant protein production, DF-1 cells have become a staple in avian-related studies. Meat companies use DF-1 for producing cultured meat.

[0395] Experiment 1

[0396] Cell culture media composition

[0397] Media consisted of DMEM High Glucose w / L-Glutamine w / o Sodium Pyruvate -L0102 with added 10 % (BV) Fetal bovine serum (S1400) and 1 % (BV) and antibiotics: penicillinstreptomycin.

[0398] Media collection, Treatment and Correction

[0399] Cells were obtained from frozen stocks at 40,000 cells / cm2 density in culture T flasks (SPL Life Sciences), T75 with 15 ml media. After reaching 90-100 % confluence, cells were dislocated and transferred into 3 T175 with 33 ml media. Cells were cultured for 5 days or until lactate levels reached an approx, concentration of 3000 mg / L. The liquid spent was filtered through a 0.22 pm filter and stored at 4 °C or at -18 °C and thawed overnight before usage.

[0400] Medium treatments 20 grams of ethanol inactivated Wolffia arrhiza was combined with 50 ml spent medium for 10 min in room temperature in 50 ml flask.

[0401] The treated and untreated spent media were supplemented with a different concentration of DMEM basal medium components as powder (D5030, Sigma- Aldrich) and sodium bicarbonate (S5761 Sigma- Aldrich). Additionally, the glucose concentration in each medium was standardized to 4.5 g / L using D-glucose (D9559, Sigma- Aldrich). The goal is to accomplish a final medium osmolarity in optimal the range of 280-320 mOsm / 1. Table 2 shows DMEM percentage of correction from concentration of new medium and the measured parameters.

[0402] Cell culture in 96 wells experiments

[0403] Cells for seeding were sourced from frozen stocks at 40,000 cells / cm2ratio in culture T flasks (SPL Life Sciences), T75 with 15 ml media. After reaching 90-100 % confluence (approx. 2-3 days in a flask, media color is changed and cells were visually assessed with a Nikon Eclipse TS100 microscope) the cells were harvested for seeding, 100 pl of media at concentration of 6000 cells / well in 96 well tissue plates.

[0404] Harvesting was done by using standard protocol (Masters and Stacey, “Changing medium and passaging cell lines,” Nat. Protoc. 2007 29, vol. 2, no. 9, pp. 2276-2284) for disassociating culture cells with trypsin and PBS which were then centrifuged with ROTOFIX 32 at 1000 RPM for 5 min. The pellet was suspended with new medium DMEM. Counting live cells from flasks was tested by taking a 10 pl sample mixed with 10 pl Trypan blue and measuring it through DeNovix CellDrop Brightfield Cell Counter (Hylabs) on the Trypan blue setting.

[0405] Experimental Setup

[0406] On day 0 DF-1 cells were seeded at a concentration of 6000 cells / well in 96 well tissue plates in 100 pl of fresh medium. On day 1, the culture medium was replaced with the test medium outlined in Table 2. Cell morphology was monitored daily, and calibration curves were generated on days 2 and 3. Cell viability and proliferation were assessed using a CCK-8 assay on days 2, 3 and 4.

[0407] Cell count was performed using the cell counting kit -8 (CCK-8 96992) - Sigma- Aldrich) for estimating the number of living cells in the cultures after the indicated treatments. A calibration curve both for spectra reading timing and for initial number of seeding cells was generated. All readings were done after 4 hr incubation in 37 C° from the time of introducing the CCK-8 reagent to the culture. Each 96 plate (Thermo Fisher Scientific CAT No. 167008) was accompanied with control seeding curve (concentration ranging 0-48000 cells / well) and was assessed by TECAN program (2 sec with amplitude of 1 mm (orbital), 432 RPM) before absorbance reading.

[0408] Table 2: Measured parameters for growth Experiments for the DF-1 cell line.

[0409] Results:

[0410] Figure 30 shows the growth kinetics of DF-1 cells cultured in the indicated media. The positive control (fresh medium) exhibited the highest cell density, reaching 23,000 cells / well after four days. Spent medium treated with Wolffia demonstrated the second-best growth performance, achieving a cell density of 12,000 cells / well. While both corrected and uncorrected spent media exhibited significantly lower cell densities (-1000 cells / well).

[0411] To conclude, these results show that the improved performance of the treated media is attributed to reduced levels of inhibitory metabolites, such as lactate and ammonia (Table 2), and the restoration of optimal osmolarity (320-280 mOsm / kg) compared to the hyperosmotic untreated media.

[0412] Experiment 2

[0413] Rational

[0414] A second experiment was conducted using the DF-1 cell line to evaluate cell growth rates under two specific conditions:

[0415] 1. Equal amounts of basal DMEM medium (30% of the original DMEM concentration) were added to both Wolffia-treated and untreated spent media (Cl in Table 3). 2. The osmolarity of the Wolffia- treated spent medium (C2 in Table 3) was adjusted to match that of the untreated spent medium corrected (line 3 in Table 3) by adding 50% of the original DMEM concentration.

[0416] The rational of condition 2 is to mimic a hyper osmotic environment to DF-1 cell growth that is common to the bioreactor growth used in cultivated meat production.

[0417] The collection of spent media, experimental setup, and Wolffia treatment procedures remained consistent with the initial DF-1 experiment. The sole modification was the altered percentage of medium correction, as detailed in Table 3, to achieve the desired experimental conditions of equal basal DMEM content and osmolarity. Table 3: Measured parameters for growth experiments for the DF-1 cell line at the start of the experiments. Second set experiment.

[0418] Results

[0419] Figure 31 shows the growth kinetics of DF-1 cells cultured under various medium conditions. The positive control, which used fresh medium, achieved the highest cell density of 52,000 cells per well. Spent medium treated with Wolffia arrhiza (C2) and 50 % DMEM correction, demonstrated intermediate performance, yielding a cell density of 42,000 cells per well. Medium treated with Wolffia arrhiza (Cl) and 30% DMEM correction, as well as untreated spent medium with 30% DMEM correction, both resulted in similar cell densities of 31,000 cells per well. The negative control, consisting of spent medium without any correction, exhibited significantly lower cell densities, approximately 4,000 cells per well.

[0420] The results indicate that both Wolffia arrhiza-treated spent medium (Cl) and untreated spent medium corrected with 30 % DMEM achieved similar cell densities, suggesting that higher concentrations of DMEM can enhance cell growth. Notably, the Wolffia arrhiza-treated medium (C2) with 50 % DMEM correction achieved a higher cell density despite having a similar osmolarity (-430 mOsm / L) compared to the 30 % DMEM corrected spent medium. These findings suggest that Wolffia arrhiza treatment effectively reduces inhibitory metabolites, such as lactate and ammonia, thereby allowing for a greater degree of basal medium correction without surpassing the osmolarity threshold of 500 mOsm / L that can inhibit cell growth. Consequently, the Wolffia arrhiza-treated spent medium corrected with 50 % DMEM (C2) supports superior cell growth while utilizing the same volume of spent medium for cell culture.

[0421] EXAMPLE 16

[0422] Determining the effect of carrot treatment on protein content in the media

[0423] This experiment investigated the effect of carrot cube treatment on proteins in the media, while focusing on protein concentration in the medium. The experiment was conducted at pH 7 to mimic the physiological pH of growing cells. The study evaluated the effects of both standard treatment durations (5-20 minutes) and extended incubation times (1-2 hours) to emphasize the impact of slower processes.

[0424] Experimental Procedures:

[0425] The carrots underwent an inactivation process using 50% ethanol for 30 minutes. Following this treatment, they were rinsed with deionized water. The carrots were then cut into cubes with average dimensions of 3 mm x 3 mm x 6 mm for use in the experiment. The medium described is a new high-glucose DMEM (Dulbecco's Modified Eagle Medium) containing 15 mM HEPES buffer, supplied by Sartorius. The pH of this medium was adjusted to 7.0 using 0.1 N HC1 After pH adjustment, the following proteins were added to the medium:

[0426] • Bovine Serum Albumin (BSA) - 500 mg / 1 (Sigma A8022)

[0427] • Carbonic Anhydrase from bovine erythrocytes - 500 mg / 1 (Sigma C7025)

[0428] • Insulin - 500 mg / 1 (Sigma 10516)

[0429] • Lysozyme - added as a normalizing protein for SDS-PAGE analysis (Sigma L7651) These proteins are commonly added to serum-free cell culture media. They represent a significant portion of the protein content typically found in fetal bovine serum (FBS), which is traditionally added to serum-containing medium.

[0430] Table 4. Data on investigated proteins

[0431] Duplicate experiments were initiated by combining four grams of ethanol-treated carrots with ten milliliters of protein-containing medium in 50 ml containers, maintaining a 1:2.5 carrot- to-medium ratio (w / v). The mixture was incubated at 50 rpm for two hours at room temperature. Samples were collected at the start, one hour, and two hours into the incubation period. A control containing only the protein-containing medium was analyzed in the same experiment. Following incubation, carrots were separated from the medium through gravity sedimentation. Collected medium samples underwent preparation for protein quantification via SDS-PAGE gel electrophoresis.

[0432] Results

[0433] SDS-PAGE analysis was conducted on samples obtained throughout the treatment processes. The electrophoresis was performed using a 16% gel with Tricine running buffer. To achieve an optimal protein load of 8 pg per lane, protein samples were diluted to a concentration of 80 mg / L. A volume of 10 pL of this diluted sample was then loaded into each well of the gel. Lysozyme was used as a normalization control (the third band from the top), and a protein ladder (250-5 kDa, Bio-Rad) served as a molecular weight marker on the left side of the SDS-PAGE.

[0434] Figure 32 present calibration curves (100 %, 70 %, 35 %) for each protein that were included on the left side of SDS-PAGE (lanes 1-3). Lanes 4-9 present simulated physiological pH conditions (pH 7) during the experiments, on those lanes the concentrations of BSA (Top band), Carbonic anhydrase from bovine erythrocytes (send band form the top ) and insulin (lower band), remained consistent throughout the 120-minute experimental period, as evidenced by the absence of changes in protein band intensities. These findings indicate that the proteins analyzed: Insulin, Bovine serum albumin (BSA) and Carbonic anhydrase from bovine erythrocytes in this study were not absorbed or degraded during the carrot treatment process.

[0435] EXAMPLE 17

[0436] Effect of pretreatment of duckweed and carrot cubes with Sodium dodecyl sulfate (SDS) on lactate and ammonia removal from cell culture medium

[0437] Wolffia arrhiza and carrot cube biomass was exposed to different pretreatments which inactivated the duckweed plant tissues before it was mixed with the medium.

[0438] The pretreatments were:

[0439] Soaking the duckweed biomass in x 3 volume of 1 % Sodium dodecyl sulfate SDS for 2 hr. and then wash by 10 volumes of deionized water.

[0440] Soaking the carrot cubes biomass in x 3 volume of 1 % Sodium dodecyl sulfate SDS for 2 hr. and then wash by 10 volumes of deionized water.

[0441] In this experiment, 7.5 grams of pretreated biomass were combined with 20 ml of medium. The medium used was DMEM with high glucose (4500mg / L), lacking L-glutamine and phenol red, but supplemented with 3000 mg / L lactate, 34 mg / L ammonium, and 500 mg / L bovine serum albumin (BSA). The biomass was initially mixed for 1 hour, with samples taken at the start (t=0) and after one hour (t=l hr).

[0442] The results are shown in Figures 33A-C. Figure 33B shows the results of lactate removal over a 1-hour period. The data indicates that all SDS-treated Duckweed and carrot samples achieved the same lactate reduction of 33% Figure 33A illustrates the reduction in medium ammonia concentration, with Wolffia and carrots demonstrating 30 % and 35 % reductions, respectively. Figure 33C shows a 30 % reduction in medium glucose concentration for both Wolffia and carrots.

[0443] A Bradford assay was conducted to assess total protein content, which showed no change. Additionally, an SDS-PAGE analysis is presented in Figure 34. This gel image reveals no alterations in the BSA protein band. The samples in the SDS-PAGE, from left to right, are arranged as follows: lanes 1-2 contain control samples, lanes 3-5 show medium treated with carrot cubes, lanes 6-9 display medium treated with Wolffia and lane 10 ladder 10-250 kDa purchase from Biolab.

[0444] The SDS-PAGE intensities were analyzed by ImageJ program (version 1.54g, National Institutes of Health, USA). Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety

Claims

WHAT IS CLAIMED IS:

1. A method of depleting undesired cell culturing products from a cell culture medium, the method comprising contacting a cell culture medium which comprises lactate and ammonia with a plant tissue under conditions which allow depletion of said lactate and ammonia from said culture medium by said plant tissue, wherein said contacting is effected in an aseptic environment, thereby depleting undesired cell culturing products from the cell culture medium.

2. A cell culturing system comprising a chamber comprising a plant tissue and a cell culture medium which comprises lactate and ammonia, the system being an aseptic environment and allows depletion of said lactate and ammonia by said plant tissue.

3. A resin for depleting lactate and ammonia from a cell culture medium comprising an aseptic plant tissue, optionally wherein said resin is immobilized on a solid support.

4. The method or cell culture system or resin of any one of claims 1-3, wherein said aseptic environment is a sterile environment.

5. The method or cell culture system or resin of any one of claims 1-4, wherein depleting lactate and / or ammonia is by at least 25 % as compared to their concentration in said medium prior to said contacting.

6. The method or cell culture system or resin of any one of claims 1-4, wherein stiffness of said plant tissue is at least as that of a carrot sample of 50 mm diameter and 7700 mm2volume the minimum forces required for bruising, bending, and shearing of the carrot fruit being 71 N, 48 N, and 41 N, respectively.

7. The method or cell culture system or resin of any one of claims 1-4, wherein said plant tissue is hydrous comprising a water content of at least 75 % v / w.

8. The method or cell culture system or resin of any one of claims 1-4, wherein said plant tissue is of a fruit or a vegetable.

9. The method or cell culture system or resin of any one of claims 1-6, wherein said plant is carrot.

10. The method or cell culture system or resin of any one of claims 1-4, wherein said plant is Duckweed.

11. The method or system of claim 8, wherein said Duckweed is selected from the group consisting of a Wolffia species and Lemna species.

12. The method or system of claim 11, wherein said Wolffia species is selected from the group consisting of Wolffia angusta, Wolffia arrhiza, Wolffia australiana, Wolffia borealis, Wolffia brasiliensis, Wolffia Columbiana, Wolffia cylindracea, Wolffia elongata, Wolffia globose, Wolffia microscopica and Wolffia neglecta.

13. The method or system of claim 11, wherein said Wolffia species is Wolffia arrhiza (e.g., var. spotless watermeal).

14. The method or system of claim 11, wherein said Lemna species is selected from the group consisting of Lemna minor L., Lemna disperma L., Lemna ecuadoriensis L., Lemna gibba L., Lemna gibba L. , Lemna obscura L. and Lemna trisulca L.

15. The method or cell culture system or resin of any one of claims 1-14, wherein said plant tissue is differentiated.

16. The method or cell culture system or resin of any one of claims 1-15, wherein said plant tissue is diced, shredded and powder.

17. The method or cell culture system or resin of any one of claims 1-15, wherein said plant tissue is viable.

18. The method or cell culture system or resin of any one of claims 1-15, wherein said plant tissue is inactivated.

19. The method or cell culture system or resin of any one of claims 1-15, wherein said plant tissue is immobilized on a solid support.

20. The system of claim 2, being a module in a fed batch or perfusion culturing system of a target cell of interest.

21. The system of claim 2 or 20, wherein further comprising a target cell of interest.

22. The system of claim 21, wherein said target cell of interest and said plant tissue are in separate chambers.

23. The system of claim 22, wherein said separate chambers are in fluid communication.

24. The system of any one of claims 21-23, wherein said plant tissue and said target cell of interest are in a single chamber.

25. The system of any one of claims 2-24, comprising an inlet for introducing said cell culture medium into the chamber and an outlet for collecting said culture medium following assimilation of said lactate and ammonia by said plant tissue.

26. The system or resin of any one of claims 2-25, comprising at least one sensor for detecting at least one of lactate, ammonia, glucose, conductivity, pH, pCCh and pCh in said cell culture medium.

27. The method of any one of claims 1-26 further comprising isolating said cell culture medium from said plant tissue following said contacting.

28. The method of any one of claims 1-27, wherein said contacting is for a time period which is 1-60 minutes, e.g., 1- 10 min.

29. The method, system or resin of any one of claims 1-28, wherein said cell culture medium comprises glucose.

30. The method, system or resin of any one of claims 1-29, wherein said cell culture medium comprises a base medium and at least one of a growth factor, a hormone and an antibody.

31. The method, system or resin of claim 30, wherein said base medium is selected from the group consisting of PBS, Hanks’ BSS, Earle’s salts DPBS, HBSS, EBSS, DMEM, MEM, GMEM , RPMI 1640 Leibovitz L-15, TC 100 Graces insect medium Schneider's Insect medium, Ham F10, Ham F12, DMEM / F12 and Serum-Free Insect Medium 1.

32. The method, system or resin of claim 30, wherein said cell culture medium comprises a base medium and at least one of BS A, insulin, lysozyme and erythropoietin.

33. The method or system of any one of claims 1-31, further comprising a target cell of interest.

34. The method or system of any one of claims 1-33, wherein said target cell of interest is a cell line, e.g., a mammalian cell line.

35. The method or system of any one of claims 1-34, wherein said target cell of interest is a genetically modified cell.

36. The method or system of claim 35, wherein said genetically modified cell secretes a recombinant polypeptide.

37. The method, system or resin of any one of claims 1-36, wherein said plant tissue is GRAS.

38. The method, system or resin of any one of claims 1-37, wherein said plant tissue is subjected to a treatment which renders it inactive.

39. The method, system or resin of claim 38, wherein said treatment is selected from the group consisting of an ethanol fixation, sonication, autoclave, freeze-drying, oven-drying, steam sterilization, radiation (e.g., UV radiation) and detergent treatment.

40. The method, system or resin of claim 39, wherein ethanol for said ethanol fixation is about 70 % v / v.

41. The method, system or resin of claim 39, wherein said detergent is sodium dodecyl sulfate (SDS).

42. The method of any one of claims 1-41, comprising at least 2 times of contacting that are separated by using the medium resultant of one time of said at least 2 times in cell culturing.

43. The method of claim 40, wherein at least one time of said at least 2 times is effected with fresh plant tissue.

44. The method of claim 40, wherein at least one time of said at least 2 times is effected with plant tissue having been used according to the method.

45. The method of any one of claims 1-44, wherein further comprising adding glucose to the refurbished culture medium resultant of the method.

46. The method of any one of claims 1, 4-19 and 27-44, wherein further comprising analyzing at least one parameter selected from the group consisting of lactate, ammonia, glucose, protein, osmolarity, pH and amino acids.

47. A refurbished culture medium obtainable by the method of any one of claims 1 and 27-46.

48. A method of culturing target cells of interest, the method comprising contacting the cells with the culture medium of claim 47, thereby culturing the cells.

49. The refurbished culture medium of claim 47 or the method of claim 48 further comprising fresh medium optionally at a concentration of 20-50 % (w / v).

50. The method of claim 48, wherein said cells are a cell line.

51. The method or system of any one of claims 48-50, wherein said cells are nonhuman cells.

52. The method or system of any one of claims 48-50, wherein said cells are nonhuman cells of an edible species.

53. The method or system of claim 52, wherein said edible species is selected from the group consisting of a ruminant species, an avian species and an aquatic species.

54. The method or system of any one of claims 48-52, wherein said cells are non- genetically modified.

55. The method or system of any one of claims 48-52, wherein said cells are genetically modified.

56. The method or system of any one of claims 48-52, wherein said cells are genetically modified to express a recombinant secreted polypeptide.

57. The method or system of claim 56, wherein said cells are genetically modified to express an antibody or a growth factor.

58. Cells or a culture comprising same obtainable according to the method of any one of claims 48-57.

59. A processed product comprising the cells of claim 58.

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