Systems and methods of use for 3D culture of plant cells

Abstract

The present invention relates to a method of three-dimensionally culturing proliferative plant cells in a packed bed bioreactor, the bioreactor comprising at least one packed bed chamber configured to confine the proliferative plant cells therein such that the proliferative plant cells become a three-dimensionally stationary biomass phase; and at least one container comprising a fluid medium configured to flow through the proliferating plant cell stationary biomass phase, where flow of the fluid medium through the proliferating plant cell stationary biomass phase allows a compound to be transferred from the fluid medium into the cells with low shear forces within the at least one packed bed chamber, and vice versa, whereby a natural growth environment for proliferating plant cells is simulated within the 3D bioreactor.

Description

Technical Field

[0001] The present invention is generally in the field of plant cell cultivation and, in particular, relates to plant cell cultivation in a packed bed bioreactor. Background Art

[0002] Climate change and an increasing world population raise real concerns and fears that conventional farming methods will not be able to meet the expected growth in food demand as the amount of arable land per person available for food production decreases due to factors such as urbanization, erosion, soil salinization and desertification. In addition, the sustainability of food production and the threat of crop losses due to climate change and plant diseases play an increasingly important role and need to be considered. Therefore, various alternative agricultural strategies are constantly being considered.

[0003] Plants are essential components for producing carbohydrates, lipids (fatty acids), proteins (amino acids) and vitamins, and have attracted the attention of the biotechnology industry for producing secondary metabolites and recombinant proteins. Secondary metabolites are generally smaller but complex molecules, which cannot be chemically synthesized or are expensive in many cases. Based on their metabolic pathways and their biogenetic precursors, they can be classified into three groups: terpenes (e.g., paclitaxel, ginsenosides), alkaloids (e.g., morphine) and phenols (e.g., shikonin, rosmarinic acid). Extracting secondary metabolites from plants traditionally grown in the field remains the main production method for these substances. However, as mentioned above, there are many shortcomings related to the excessive changes in traditional farming and environmental conditions over time and regional changes, which lead to unpredictable differences in the quality and quantity of raw materials. Many secondary metabolites are produced in plants that are not suitable for agricultural production or are difficult to grow outside their local ecosystems. As a result, it is not surprising that about one-fifth of the 50,000 medicinal plants used today are listed on the list of endangered species. Therefore, plant cells and tissue cultures grown in bioreactors provide ecologically sustainable alternatives. In addition, it is believed that plant cells and tissue cultures represent the major drawbacks of solving traditional herbal medicine cultivation and avoid the appropriate method for problems associated with extracting products from protected wild plants. A common example of a medicinal secondary metabolite derived from plant cell culture is paclitaxel, which is an anticancer drug. Previously, it was produced by harvesting bark from Taxus sp. trees, and the large-scale production of paclitaxel is carried out in stainless steel bioreactors today. In addition, completely eliminating environmental changes leads to improved batch consistency, which is essential for obtaining official approval. Labor-intensive greenhouse or field production of whole plants is avoided, not only in upstream processing, but also in downstream processing, particularly in the case of secretion to the product in the culture medium, to reduce costs. Obviously, when operating in a closed bioreactor system, the process is greatly improved in terms of product contamination by endotoxins and mycotoxins and equally important in terms of the safety of the environment by artificial, genetically modified plant contamination.

[0004] Therefore, the present invention aims to provide a possible cost and time effective solution by cultivating plant cells in a packed bed bioreactor in a proprietary method that simulates the natural environment of the plant. The systems and methods disclosed herein can be applied to all and / or most plant species and can be used for cultivation, and can present possible solutions because they allow for the controlled, closed and sustainable manufacturing of plant-based products for various industries such as food, pharmaceuticals, cosmetics, etc. Summary of the invention

[0005] In one main aspect, the present invention relates to a three-dimensional (3D) bioreactor for culturing proliferating plant cells, comprising: at least one packed bed chamber, the packed bed chamber being configured to confine the proliferating plant cells therein so that the proliferating plant cells become a three-dimensional stationary biomass phase; and at least one container containing a fluid culture medium, the fluid culture medium being configured to flow through the proliferating plant cell stationary biomass phase; wherein the flow of the fluid culture medium through the proliferating plant cell stationary biomass phase allows compounds to be transferred from the fluid culture medium to the cells at low shear forces within at least one packed bed chamber, and vice versa, thereby simulating the natural growth environment of the proliferating plant cells within the 3D bioreactor.

[0006] For purposes of clarity, the term "culturing" as used herein means maintaining plant cells under conditions suitable for growth / metabolite production.

[0007] The term "harvesting" as used herein includes extracting cells from a bioreactor or separating cellular contents from cells during or after a culture period.

[0008] As used herein, the term "compound" includes primary and secondary metabolites.

[0009] As used herein, the term "plant-based metabolite compound" includes primary or secondary metabolites. Some non-limiting examples of plant-based metabolites include amino acids, sugars, organic acids, phenols, alkaloids, terpenes, and flavonoids.

[0010] The terms "plant cell," "plant cell biomass," "plant cell callus," "callus," "aggregate," "plant cell aggregate," "plant cell culture," "plant culture," and "plant cell callus" are used interchangeably and are all intended to describe plant cells.

[0011] The term "suspension culture" refers to a culture in which plant cells are exposed to conditions that are compatible with cell proliferation and growth and that allow the cells to grow in suspension.

[0012] The terms "three-dimensional culture" and "3D culture" refer to a culture in which plant cells are exposed to conditions that are compatible with cell growth and allow the cells to grow in a 3D orientation relative to each other. Such conditions will typically utilize a 3D growth surface (also referred to as a "scaffold," "carrier," or "3D matrix"), including in some embodiments an adherent material present in a 3D culture vessel. Preferably, the 3D culture is performed in conjunction with the modular device.

[0013] In some optional embodiments, the bioreactor may further include at least one monitoring and / or control unit for monitoring and / or controlling at least one parameter in the culture conditions and culture medium parameters. Non-limiting examples of parameters that can be monitored and / or controlled are pH, temperature, stirring speed, flow rate, gas concentration, amino acid levels, vitamin levels, mineral levels, growth factor levels, dissolved oxygen levels, glucose levels, lactic acid (lactate) levels, lactate dehydrogenase levels, NH3 levels, glutamate (glutamate) levels or combinations thereof.

[0014] Optionally, but not necessarily, the fluid culture medium can be a growth medium that provides nutrients to the proliferating plant cells and absorbs plant cell metabolites and secreted compounds from the plant cells into the growth medium. Other fluid culture media may also be used depending on the plant cell type and desired application.

[0015] In some embodiments, the fluid culture medium can be exchanged for any one of differentiation medium, maturation medium and induction medium and combinations thereof in any order, thereby allowing continuous development of plant cells and simulating the natural growth process of plant cells within the 3D bioreactor.

[0016] In some embodiments, the bioreactor may further include at least one inlet port to allow for the addition of gas and / or liquid and / or solid materials into the 3D bioreactor during the culturing of the proliferating plant cells.

[0017] In yet further embodiments, the bioreactor may further include at least one outlet port to allow removal of fluid culture medium from the 3D bioreactor during cultivation of the proliferating plant cells, for collection of metabolites secreted by the quiescent biomass phase of the proliferating plant cells during the cultivation process, and / or for removal of plant-secreted compounds / metabolites, and / or for balancing fluid culture medium parameters.

[0018] Optionally, the bioreactor may further comprise a vibrating arm functionally connected to the packed bed chamber, thereby allowing vibration of the proliferating plant cells, for minimizing channel effects and improving contact between the fluid culture medium and the proliferating plant cell stationary biomass phase, and / or for harvesting the proliferating plant cell stationary biomass phase for collecting cells or for lysing the cells to obtain their contents. The harvesting of plant cells may be obtained by combining enzyme reactions and applying a vibrating force to the plant cells, thereby lysing the cells and collecting their contents from the culture medium.

[0019] The packed bed chamber may be located within the vessel with the flowing fluid medium or it may be located outside the vessel with the fluid medium and connected to the vessel by a tube that flows the fluid medium through the quiescent biomass phase of the proliferating plant cells.

[0020] In some embodiments, the packed bed chamber is divided into one or more subchambers by at least one perforated disk, such that each subchamber contains proliferating plant cell biomass cells in similar size or gradient size according to the hole size of the at least one perforated disk.

[0021] Optionally, at least one packed bed chamber comprises a carrier configured to allow growth of proliferating plant cells within and / or above the carrier.

[0022] In some optional embodiments of the present invention, the bioreactor further comprises an agitation mechanism functionally connected to the packed bed chamber and configured to compound the proliferating plant cell quiescent biomass phase within the basket, thereby rearranging the plant cell biomass and allowing culture medium to flow smoothly through the plant cells.

[0023] The agitation mechanism may include at least a large gear attached to an agitator located within the packed bed chamber and a small gear attached to the handle.

[0024] However, in some embodiments, the packed bed chamber is a separate chamber from the fluid culture medium container, and the plant cell resting biomass phase is used in agriculture to inoculate the resting cell biomass to obtain a plant or part thereof.

[0025] Optionally, the plant cell resting biomass phase is harvested for collecting compounds produced by the plant cell resting biomass phase. The compounds can be used for industrial applications. Some non-limiting examples of industrial applications are food manufacturing, pharmaceuticals, industrial applications and cosmetic compositions.

[0026] In yet further embodiments of the present invention, the bioreactor may further comprise a perfusion chamber comprising a 3D packed bed matrix comprising one or more macrocarriers, microcarriers, or a combination thereof.

[0027] The present invention further relates to methods for culturing and propagating plant cells using the bioreactor disclosed above for use of the plant cells and / or compounds produced by said plant cells in medical, cosmetic, food technology, agricultural and industrial applications.

[0028] In a specific embodiment, the present invention relates to a method for culturing proliferating plant cells in a 3D bioreactor, the method comprising the following steps: a) confining the proliferating plant cells in at least one packed bed chamber so that the proliferating plant cells become a stationary biomass phase; and b) allowing culture medium from at least one container containing fluid culture medium to flow through the proliferating plant cell stationary biomass phase; wherein the flow of fluid culture medium through the proliferating plant cell stationary biomass phase allows compounds to be transferred from the fluid culture medium to the cells at low shear force in at least one packed bed chamber, and vice versa, thereby simulating the natural growth environment of the proliferating plant cells in the 3D bioreactor.

[0029] Optionally, the above method for culturing and proliferating plant cells may further comprise the step of c) monitoring and / or controlling at least one of the culture conditions and culture medium parameters by at least one monitoring and / or control unit.

[0030] Also provided herein is a method for culturing proliferating plant cells using the bioreactor of the present invention as described above to use the plant cells and / or compounds produced by the plant cells for medical, cosmetic, food technology, agricultural and industrial applications, the method comprising: a) inputting the proliferating plant cells into at least one packed bed chamber in the bioreactor, wherein the packed bed chamber confines the proliferating plant cells within the packed bed chamber so that the proliferating plant cells produce a stationary biomass phase; and b) flowing at least one fluid culture medium from at least one container containing a fluid culture medium through the stationary biomass phase in a low shear environment, allowing efficient transfer of compounds between the fluid and the cells, wherein the fluid culture medium contains one or more of nutrients, hormones, plant growth regulators (PGRs), vitamins, amino acids, trace elements, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain aspects of the subject matter disclosed herein and, together with the description, help explain some principles associated with the disclosed embodiments.

[0032] Figure 1A is a schematic cross-sectional view of an exemplary bioreactor that can be used to culture plant cells.

[0033] Figure 1B Schematic isometric top view of an example of a growth and vibration basket for plant cells.

[0034] Figure 2 is a schematic diagram of an exemplary embodiment of a modular bioreactor for growing proliferative plant cells.

[0035] Figure 3A is a schematic isometric cross-sectional elevation view of an exemplary plant cell apparatus.

[0036] Figure 3B for Figure 3A Schematic close-up isometric bottom view of the upper perforated disk of the plant cell apparatus.

[0037] Figure 4A is a schematic diagram of an exemplary agitation mechanism for agitating plant cell aggregates within a plant growth basket.

[0038] Figure 4B For assembling on a cell growth basket of a packed bed bioreactor according to an embodiment of the present invention Figure 4A Schematic diagram of the stirring mechanism.

[0039] Figures 5A-5E Schematic diagrams and photographs of various plant cell aggregates at different stages of growth grown in a packed bed bioreactor with or without carriers. Figure 5A Schematic partial view of a 3D micro packed bed bioreactor showing the growth of plant cell aggregates on top of a scaffold within a growth basket. The plant cell aggregates are filling the growth basket and are retained within the basket due to upper and bottom perforated disks that mechanically restrict their ability to exit the basket into the culture medium above or below the basket. Figure 5B-Figure 5C This is a photograph showing a cannabis cell embedded in a 3D carrier. The photograph was made using blue Hoechst staining. Figure 5D Schematic illustration of plant cell seeding and growth in a 3D microbial system without a carrier. Figure 5E Close-up of cannabis cell culture aggregates adhered to a 3D support.

[0040] Fig. 6A Graph showing cannabinoid production ratios in packed bed bioreactors relative to suspension cultures.

[0041] Figure 6B Schematic diagram showing the amount of cannabinoids obtained in a packed bed bioreactor compared to the amount of cannabinoids obtained when plant cells are cultured in suspension culture shake flasks. DETAILED DESCRIPTION

[0042] Before explaining at least one embodiment of the present invention in detail, it should be understood that the present invention is not limited to the details set forth in the following description or the details exemplified by the embodiments in its application. The present invention can have other embodiments or be implemented or carried out in various ways. In addition, it should be understood that the wording and terminology used herein are for the purpose of description and should not be considered as limiting.

[0043] In one main aspect, the present invention relates to a three-dimensional (3D) bioreactor for culturing proliferating plant cells, comprising at least one packed bed chamber configured to confine the proliferating plant cells therein so that the proliferating plant cells become a three-dimensional stationary biomass phase; and at least one container containing a fluid culture medium, the fluid culture medium configured to flow through the proliferating plant cell stationary biomass phase; wherein the flow of the fluid culture medium through the proliferating plant cell stationary biomass phase allows compounds to be transferred from the fluid culture medium to the cells at low shear forces within at least one packed bed chamber, and vice versa, thereby simulating the natural growth environment of the proliferating plant cells within the 3D bioreactor.

[0044] The present invention further relates to a method for culturing proliferating plant cells in a 3D bioreactor, the method comprising the steps of: a) confining the proliferating plant cells within at least one packed bed chamber so that the proliferating plant cells become a stationary biomass phase; and b) allowing culture medium from at least one container containing a fluid culture medium to flow through the proliferating plant cell stationary biomass phase; wherein the flow of the fluid culture medium through the proliferating plant cell stationary biomass phase allows compounds to be transferred from the fluid culture medium to the cells at low shear forces within at least one packed bed chamber, and vice versa, thereby simulating the natural growth environment of the proliferating plant cells within the 3D bioreactor.

[0045] In yet a further aspect, the present invention relates to a method for culturing proliferating plant cells using the bioreactor described herein for use of the plant cells and / or compounds produced by the plant cells for medical, cosmetic, food technology, agricultural and industrial applications, the method comprising: a) inputting the proliferating plant cells into at least one packed bed chamber in the bioreactor, wherein the packed bed chamber confines the proliferating plant cells within the packed bed chamber so that the proliferating plant cells produce a stationary biomass phase; and b) flowing at least one fluid culture medium from at least one container containing a fluid culture medium through the stationary biomass phase in a low shear environment, allowing efficient transfer of compounds between the fluid and the cells, wherein the fluid culture medium comprises one or more of nutrients, hormones, plant growth regulators (PGRs), vitamins, amino acids, trace elements, or combinations thereof.

[0046] The main aspects of the present invention and the optional modes of implementing the present invention will be better understood through the following detailed description of various exemplary non-limiting drawings and examples described below. Referring now to the drawings:

[0047] Figure 1ASchematic cross-sectional view of an example of a bioreactor 400 that can be used to culture plant cells. The terms "plant cell", "plant cell culture", "plant cell aggregate", "plant cell callus", "callus" and "proliferating plant cell" are all intended to describe cells derived from plants and are used interchangeably below. Callus is a multipotent plant cell mass produced by wound signals or hormone treatment. Callus can be produced by a single differentiated plant cell, and multiple callus cells (callus) are capable of regenerating the entire plant body. As known in the art, callus produces a three-dimensional structure in the following three stages: induction, division and differentiation. These developmental stages can be characterized by changes in average cell size and the structure and overall metabolic status of plant tissues. In the division stage, which is performed throughout the outer region (periphery), the callus cell size continues to decrease, while a small core of undivided cells remains inside. Then, these outer regions differentiate and begin to mature. Proliferating cells produce a three-dimensional structure in which each cell grows in a 3D manner relative to adjacent cells, thereby forming a 3D cell aggregate. The 3D cell aggregates themselves produce independent biomass, which, unlike animal cells, can grow independently without a carrier (for further details, see: Feher A (2019) Callus, Dedifferentiation, Totipotency, Somatic Embryogenesis: What These Terms Mean in the Era of Molecular Plant Biology? Front. Plant Sci. 10:536).

[0048] In the depicted embodiment, the growth and vibration basket 416 (hereinafter referred to as "basket") is preferably, but not necessarily, loaded with a carrier, such as, but not limited to, a polyester tray 10. The terms "carrier" and "scaffold" can be used interchangeably and both refer to an element configured to be added to the basket 416 and used as a platform on or within which plant cells are based and grown. The basket wall 4161 is preferably separate from the bioreactor inner wall 40 and can be moved up and down. In some embodiments, the bioreactor 400 is filled with deionized water or an isotonic buffer via an inlet tube 420, which is configured and operable to deliver various culture media to the bioreactor, and then optionally autoclaved. In other embodiments, after sterilization, the liquid is replaced with a growth medium that saturates the basket 416 and its contents. Basket 416 actually divides the fluid within bioreactor 400 into three main parts: an upper portion 422 containing mainly fresh culture medium inserted via inlet tube 420, a middle portion 424 containing culture medium surrounding plant cells and / or carriers within basket 416, and a lower portion 426 containing mainly culture medium flowing through plant cells, and compared to the fresh culture medium at upper portion 422, lower portion 426 is generally lacking in nutrients consumed by plant cells in the middle and rich in compounds and debris secreted by plant cells. In the embodiments described herein, the culture medium at upper portion 422 is manipulated by impeller 419 that produces fluid motion as shown by arrow 409. In still further embodiments, temperature, pH, dissolved oxygen concentration, etc. are set before inoculation and are continuously adapted to culture conditions as needed. In still further embodiments, a slow initial agitation rate of the culture medium is used to promote cell adhesion, and then the agitation rate is increased. If desired, during the growth of plant cells, metabolites can be harvested from the cell-free culture medium below the basket. In some embodiments, the rotation of the impeller 419 creates a negative pressure in the draft tube 450, which draws the cell-free culture medium out of the lower portion 426 through the draft tube 450 and then into the upper portion 422 through the impeller 419 port, thereby circulating the culture medium uniformly in a continuous loop in the direction according to arrow 409. In still further embodiments, the culture medium can be adjusted by monitoring various parameters via electrodes 406 to control various parameters of the fluid. In some optional embodiments, an annular sparger (not visible) is located inside the impeller aeration chamber 11 for oxygenating the culture medium flowing through the impeller 419 port via gas added from external port 403 (which can be maintained inside the housing 5) and sparger line 7. In some other optional embodiments, gas can be added through inlet 420. Alternatively, the sparged gas can be restricted to a distant chamber and absorbed by the nutrient medium, which flushes the fixed plant cells. In some optional embodiments, a water jacket 417 covers the culture medium area within the bioreactor 400, with ports 13 and 14 for moving jacket water in and out.A removal tube 410 is placed along the bioreactor and has an opening in the lower medium portion 426 to allow, if desired, the harvesting of metabolites secreted by the plant cells from the cell-free medium below the basket 416. The removal tube 410 can also be used to collect harvested cells, remove debris, and to refresh the medium when the spent medium portion is removed and fresh medium is added.

[0049] In some embodiments, a continuous stirred tank bioreactor can be used, wherein the culture medium is continuously fed into the bioreactor and the product is continuously extracted to maintain a steady state with constant time in the bioreactor. A stirred tank bioreactor with a fiber bed basket can be obtained, for example, from New Brunswick Scientific Co., Edison, NJ). Other bioreactors can be used, such as but not limited to fixed bed bioreactors, perfusion bioreactors with multi-active foams, radial flow perfusion bioreactors comprising tubular poly-L-lactic acid (PLLA) porous scaffolds, and any other bioreactors known in the art suitable for the purposes of the present invention. "Fixed bed bioreactor" refers to a bioreactor in which the cell growth matrix is ​​usually not raised from the bottom of the incubation apparatus in the presence of a growth medium. For example, the matrix can have enough density to prevent it from being raised and / or it can be filled by mechanical pressure to prevent it from being raised. The matrix can be a single body or multiple bodies. Typically, the matrix remains substantially in place during the standard stirring rate of the bioreactor. In some embodiments, a plurality of carriers are loosely filled, for example, forming a loose packed bed, which is immersed in a nutrient medium.

[0050] In certain embodiments, a perfusion bioreactor is used, wherein the perfusion chamber comprises a 3D matrix. In certain embodiments, the 3D matrix is ​​in the form of a carrier 10. The carrier can be, for example, a macrocarrier, a microcarrier, or a mixture thereof. Non-limiting examples of commercially available microcarriers include alginate-based microcarriers (GEM, Global Cell Solutions), dextran-based microcarriers ( GE Healthcare), collagen-based microcarriers ( Percell Biolytica) and polystyrene-based microcarriers (SoloHill Engineering). In certain embodiments, the microcarriers are packed inside a perfusion bioreactor.

[0051] In some embodiments, the carrier in the perfusion bioreactor is filled, for example, to form a packed bed, which is immersed in a nutrient medium. Alternatively or additionally, the carrier may include an adhesive material. In other embodiments, the surface of the carrier includes an adhesive material, or the surface of the carrier is sticky. In yet other embodiments, the material exhibits a chemical structure that allows cell adhesion, such as a charged surface exposed group. Optionally, the carrier may include a fibrous material, optionally a sticky fibrous material, which may be a woven fiber matrix, a non-woven fiber matrix, or both in a more specific embodiment. The carrier may be composed of naturally occurring polymers, such as, but not limited to, proteins, alginates, cellulose, carbohydrates, silk, wool, natural rubber (latex), etc. Alternatively, a fiber carrier available on the market may also be used according to an optional embodiment of the present invention. In some embodiments, the culture medium in the bioreactor does not include any carrier, and the plant cells can grow in a growth basket due to their genetically acquired ability to grow as aggregates, as will be referred to below Figure 5D Described in detail.

[0052] In a further embodiment, cells are seeded onto the support by suspending the cells in the culture medium and introducing the culture medium into the equipment. In some further embodiments, stirring speed is gradually increased, for example, by starting with 40RPM for 4 hours, then speed is gradually increased to 400RPM. In certain embodiments, the glucose or sucrose level of the culture medium can be regularly tested, and the perfusion speed of adjustment maintains an acceptable glucose / sucrose concentration, in certain embodiments, between 5-30,000mg / l, between 5-1,000mg / l, between 1,000-5,000mg / l, between 1000-10,000mg / l, between 5,000-10,000mg / l, between 10,000-20,000mg / l, between 15,000-25,000mg / l. In yet other embodiments, at the end of the culturing process, the carriers are removed from the packed bed and, in some embodiments, washed with an isotonic buffer, and the cells are treated by agitation and / or enzymatic digestion or removed from the carriers.

[0053] In certain embodiments, the bioreactor is inoculated with a weight range of proliferating plant cells between 0.05-1.0 mg / ml, between 0.25-0.5 mg / ml, or between 0.5-1.0 mg / ml.

[0054] In still other embodiments, 0.5-140 grams of cells / gram (gr) of carrier (matrix) is inoculated, or in other embodiments 0.5-2 grams of cells / gram of carrier, or in other embodiments 2-4 grams of cells / gram of carrier, or in other embodiments 4-8 grams of cells / gram of carrier, or in other embodiments 8-16 grams of cells / gram of carrier, or in other embodiments 16-32 grams of cells / gram of carrier, or in other embodiments 16-32 grams of cells / gram of carrier, or in other embodiments 32-48 grams of cells / gram of carrier, or in other embodiments 48-64 grams of cells / gram of carrier, or in other embodiments 64-80 grams of cells / gram of carrier, or in other embodiments 80-92 grams of cells / gram of carrier, or in other embodiments 92-104 grams of cells / gram of carrier, or in other embodiments 104-116 grams of cells / gram of carrier, or in other embodiments 116-128 grams of cells / gram of carrier, or in other embodiments 128-140 grams of cells / gram of carrier.

[0055] Figure 1B Schematic isometric top view of an optional example of a growing and vibrating basket 416 for propagating plant cell culture in a packed bed bioreactor. The top and bottom borders of the basket 416 are formed by perforated discs 432 each having a plurality of holes 4321 of a predetermined diameter. The perforated discs 432 are also denoted hereinafter as "discs", "grids", "upper grids", "lower grids", "middle grids", all of which are used interchangeably and refer to the upper, middle or lower wall of the basket 416. In some optional embodiments, when carriers are used, the carriers "sit" on the bottom grid and block the ability of aggregates to come out the other side, so that the propagating plant cells do not leave the basket from that direction. From the top side where they enter the basket, the flow in the basket is established so that it always flows inwards, so that plant cell aggregates entering the basket 416 cannot move against the flow and will remain within the basket 416 (the cells are trapped within the basket and remain there).

[0056] In other optional embodiments, at the beginning of the culturing process of the proliferating plant cells in the bioreactor, the plant cells can pass through the holes of the disk, as long as the proliferating plant cell aggregates have a size smaller than the holes of the disk. As the proliferating plant cell aggregates grow in the bioreactor, at a certain point in time, the plant cell aggregates remain "trapped" in the basket 416 and only the culture medium keeps flowing between them, flushing the plant cell aggregates. This is a unique plant-specific feature compared to animal cells that must adhere to a carrier to remain in the growth basket and do not circulate with the flowing culture medium. The inventors of the present invention have discovered a method for culturing plant cells in a packed bed chamber with or without the use of a carrier, as long as a static biomass of proliferating plant cells is obtained in the packed bed chamber.

[0057] In the center of the basket 416, an impeller flow guide 450 is shown, and a removal tube 410 is also shown, passing through both the upper and lower discs 432 to reach the lower culture medium portion (not shown), and allowing the collection of culture medium (if necessary) to harvest metabolites secreted by plant cells from the lower culture medium portion 426. The basket 416 is preferably connected to one or more vibration rods 436 configured to allow vertical vibration of the basket 416. When performed at low intensity, the vertical vibration of the basket 416 can be used to avoid channel effects, thereby enabling the maintenance of uniform distribution of culture medium within the basket, thereby maintaining uniform growth for a longer period of time, and further releasing blockage. When the vibration is performed at a moderate intensity, it can be used to release the proliferating plant cell resting biomass from the basket 416, and when the vertical vibration is maintained at a high intensity, it can be used to separate cell aggregates to collect cells, and for cell lysis and collection of metabolites from the harvested cultured proliferating plant cell resting biomass. For this purpose, a combination of enzyme reactions and strong vibration forces can be applied.

[0058] Also shown in this figure is a gasket 433 configured to prevent plant cells outside and around the culture basket 416 from migrating into the lower portion 426 of the bioreactor 400 during the growth phase. Cell aggregates may be collected directly from the basket 416.

[0059] Figure 2Schematic diagram of an exemplary embodiment of a modular bioreactor 500 for propagating plant cell culture according to some optional embodiments of the present invention. In the example shown herein, the modular bioreactor 500 includes: a central culture medium container (reservoir) 502 containing a cell culture medium 5021, wherein the central culture medium container 502 does not contain plant cells; and one or more 3D propagation plant cell growth apparatuses 501. Each of the propagation plant cell growth apparatuses 501 may include microcarriers consisting of a 3D matrix 509 (such as but not limited to a fiber matrix) that can be made of a synthetic matrix or a natural matrix. The central culture medium container 502 is operably connected to each propagation plant cell growth apparatus 501 via a pipe (tubing) 550 so that the culture medium from the culture medium container 502 flows through the apparatus 501 in parallel or sequentially, and in some embodiments flows through the carrier or microcarrier (if used). In certain embodiments, the flow can be against gravity. More specifically, the apparatus can be placed in a vertical orientation, wherein the flow is in an upward direction. In other embodiments, a plurality of plant cell carriers (not depicted) consisting of 3D matrices 509 are configured in each proliferating plant cell apparatus 501. Optionally, the modular bioreactor 500 is aseptically sealed. In other embodiments, the described bioreactor is a closed system. The reason for using one or more proliferating plant cell culture apparatuses 501 simultaneously is to improve the growth production capacity of the modular bioreactor 500, thereby increasing the size of the batch. By using several separated subunits to grow the proliferating plant cells, the scale expansion of the growth process is achieved without facing the challenges that usually occur due to scale expansion and may affect the growth of the proliferating plant cells. Cultivating proliferating plant cells in a bioreactor may have certain limitations associated with scale expansion, which may damage the cultivation process. By dividing the plant cell culture apparatus into a plurality of apparatuses that are all connected to each other, the inventors have expanded the process scale without suffering the damage that may occur when the process scale is expanded in a large container.

[0060] Optionally, the modular bioreactor 500 further comprises one or more circulation pumps 503 or other means for actively transporting culture medium through the proliferating plant cell apparatus 501. In certain embodiments, each proliferating plant cell apparatus 501 is connected to a dedicated pump 503.

[0061] In yet other embodiments, each of the plurality of apparatuses 501 is temperature-insulated. Additionally or alternatively, the central medium container 502 is temperature-insulated. Non-limiting examples of temperature insulation are the medium container water jacket 518 and the apparatus water jacket 517, which can independently be various types of water jackets known in the art. In yet other embodiments, both the central medium container 502 and the plant cell growth apparatus 501 are temperature controlled or, in other embodiments, operably connected to a thermostat and / or a thermometer and / or other device for monitoring and controlling the temperature of the fluid contents thereof.

[0062] The term "3D proliferating plant cell growth apparatus", as used herein, refers to an apparatus (e.g., as depicted in 501) configured to contain a proliferating plant cell quiescent biomass with a fluid culture medium flowing through the proliferating plant cell quiescent biomass, wherein the proliferating plant cells can grow on a carrier 509 or in the absence of a carrier. Thus, in certain embodiments, the proliferating plant cells in the apparatus grow as callus that itself produces quiescent biomass, and in certain other embodiments, the plant cells adhere to the carrier during culture, such as Figures 5A-5D In more specific embodiments, carrier can be selected from macrocarrier, microcarrier or both. Non-limiting examples of commercially available microcarrier include microcarrier based on alginate (GEM, Global Cell Solutions), microcarrier based on dextran (Cytodex, GE Healthcare), microcarrier based on collagen (Cultispher, Percell) and microcarrier based on polystyrene (SoloHill Engineering). In certain embodiments, microcarrier is packaged in utensil. In other embodiments, carrier is fibrous 3D carrier comprising adhesive material.

[0063] The culture medium container 502 may include a stirring element such as an impeller 504, which is driven by a motor 505 to mix the culture medium 5021 within the culture medium container 502. Those skilled in the art will recognize in light of this disclosure that other suitable mixing devices may be used, such as, but not limited to, marine-blade impellers, pitched-bladed impellers, hydrofoil impellers, Rushton impellers, pitched-bladed impellers, Cell lift impeller, A320 impeller (SPX Flow), HE3 impeller (Chemineer), etc.

[0064] Optionally, the culture medium container 502 can be connected with one or more control elements 506 for monitoring and controlling various parameters, including pH rate, dissolved oxygen concentration and temperature. Other parameters (subsequent cell proliferation, culture absorption rate and state and culture medium osmotic pressure) can also be monitored. In some optional embodiments, the concentration of minerals and compounds (such as phosphate, ammonia, glucose and sucrose) that affect cell growth can be monitored. Optionally, the optical density (OD) of the culture medium can also be monitored, which indicates the cell adhesion to the carrier in the growth apparatus 501. The feed line 520 for introducing fresh culture medium into the culture medium container 502 and the waste line 510 for removing the culture medium circulating through the propagation plant cell from the culture medium container 502. Preferably, perfusion relates to the function of the feed line 520 and the waste line 510. The control element 506 can include a pH regulating solution line (not depicted) for adding alkaline or acidic solution to regulate pH as required. Perforated disk 532 defines top and bottom boundaries of grow basket 516 and is configured to restrict growth of proliferating plant cell aggregates within grow basket 516 and prevent them from circulating with culture medium via tube 550 .

[0065] Figure 3A is a schematic isometric cross-sectional elevation view of another exemplary plant cell bioreactor 600, the exemplary plant cell bioreactor 600 being Figure 1A The bioreactor 400 shown in FIG. 4 is similar but has a cell basket 416 that is divided into at least one subchamber 439 by an additional perforated disk 432 disposed along the longitudinal axis of the basket 416. In the specific example shown in this figure, the cell plant basket 416 is divided into a total of three subchambers by adding two more perforated disks 432 between the upper and lower perforated disks that define the upper and lower boundaries of the cell basket. The size of the holes in each of the perforated disks 432 can be the same, or can be different from each other, for example, the diameter of the holes can decrease from the bottom to the top, thereby creating a gradient of callus size within the sub-growth chamber 439, so that the upper cell chamber will contain the smallest proliferating plant cells, while the lower cell chamber will contain the largest. Other optional variations can be applied. As described above, the culture plant cell basket 416 can accommodate a support also denoted as a "scaffold" below, both terms having the same meaning according to the present invention and can be used interchangeably. The scaffold is intended to provide a growth platform for the proliferating plant cells. In some other embodiments, the culture plant cell basket 416 does not contain any carrier, thereby allowing the proliferating plant cells to grow independently. The use of a carrier can vary depending on the plant cell type, the compound of interest, and the specific growth protocol. When a carrier is used, the culture cell basket 416 can be completely filled with the carrier or partially filled with the carrier.

[0066] The plant cell bioreactor 600 may further include a basket locating pin 436 that may mate with a hollow longitudinal center axis 435 (hereinafter referred to as “longitudinal axis”) of the basket 416 .

[0067] The growing basket 416 can have a variety of sizes and volumes. At certain sizes, dividing the basket into several subchambers allows for more efficient vibration of the cells within the basket because the perforated disk 432 more efficiently transmits the vibration force to each adjacent proliferating cell in a vertical manner, upward and downward (as shown by arrows 490 and 490', respectively). As described above, vibrating the basket at a low intensity effectively avoids channeling and improves the flow of the growth medium (or any other medium that may be used) through the stationary biomass of the proliferating plant cells being cultured within the growing basket 416, and ensures maximum contact between the proliferating plant cells and the flowing medium. In addition, when the vibration is strong, the vibration power can be used to break the cells and harvest their contents. For harvesting, the basket 416 can be oscillated upward 490 and downward 490' along the longitudinal axis 435 within (and relative to) the bioreactor 600.

[0068] In some embodiments, plant cells can be propagated using a suitable growth medium / culture medium known in the art, for example, before and in combination with a disclosed modular bioreactor, respectively, by using a combination of a suspension and a 3D matrix. Unless otherwise indicated, the term "culture medium" refers to a liquid composition designed for in vitro replication of proliferating plant cells. In addition, non-limiting examples of suitable culture media are mentioned herein. The term "growth" and / or "cultivation" of a proliferating plant cell population is intended to be synonymous with the amplification of a proliferating plant cell population. In various embodiments, the described amplification can be performed in a suspension and then in a 3D matrix.

[0069] In other embodiments, a method for culturing a proliferating plant cell is provided, comprising amplifying cells in the described device. In certain embodiments, culturing in the described device is performed by suspension culture. Any of the described device embodiments can be applied to the culturing method.

[0070] In certain embodiments, the bioreactor system for plant cell culture described herein is a closed system. Alternatively or additionally, the described process is a process of at least partial automation. Those skilled in the art will appreciate that the closed system is sealed with the external environment in a manner that enables aseptic maintenance in view of the present disclosure. In a further embodiment, the closed system is sealed in a manner to prevent the unintentional contamination caused by the material outside the system. In yet other embodiments, the closed system is sealed in an airtight manner. The technician will further appreciate that the closed system enables the manipulation of its contents without the need to manipulate in a sterile hood or sterile room.

[0071] In some optional embodiments, any of the methods further comprises determining the concentration of plant cell aggregates in the growing apparatus / basket. Thus, the described growing apparatus / plant cell basket is optionally further operably connected to a sensor for determining cell concentration.

[0072] In yet other embodiments, any of the methods further comprises measuring the viability of the cells in the growing apparatus / basket. In other embodiments, the described apparatus further comprises a probe or other device for measuring the viability of the cells in the growing apparatus.

[0073] In yet other embodiments, any of the methods further comprises monitoring and / or controlling the pH of the culture medium in the central culture medium container. Those skilled in the art will appreciate in light of the present disclosure that the pH of the liquid formulation can be adjusted in a variety of ways known in the art, non-limiting examples of which are adding carbon dioxide (CO2), alkaline solutions, acid solutions, and / or pH buffers to the formulation. Non-limiting examples of devices for adjusting pH include for adding CO2 to the formulation, 2、 Input channels and pumps for base solutions, acid solutions and / or pH buffers. In certain embodiments, the described systems include adjustable controls for the pH of the formulation.

[0074] In other embodiments, any of the methods further includes monitoring and / or controlling the dissolved oxygen concentration (pO2) inside the culture medium container, or in other embodiments, any of the methods further includes monitoring and / or controlling the dissolved oxygen concentration (pO2) inside the growth apparatus, or in other embodiments, any of the methods further includes monitoring and / or controlling the dissolved oxygen concentration (pO2) inside both the culture medium container and the apparatus. In other embodiments, the equipment may further include an instrument or other device for monitoring and / or controlling the dissolved oxygen concentration inside the culture medium container. (As a non-limiting example) pO2 can be adjusted by adding O2 to the preparation, using a pump in some embodiments. In certain embodiments, the described system includes an adjustable controller for the pO2 of the culture medium inside the culture medium container. In yet other embodiments, pO2 is measured for estimating the number of viable cells in the apparatus.

[0075] In other embodiments, any of the methods further include monitoring and / or controlling the temperature of the culture medium inside the culture medium container, or in other embodiments, any of the methods further include monitoring and / or controlling the temperature of the culture medium inside the apparatus, or in other embodiments, any of the methods further include monitoring and / or controlling the temperature of the culture medium inside both the culture medium container and the apparatus. Therefore, the equipment can further include a thermometer, a thermostat or other devices for monitoring and / or controlling the temperature of the culture medium inside the culture medium container and / or the growth apparatus, and its non-limiting examples are thermometers, insulation materials (insulation) and external containers for fluids (for example, liquids or gases) that can manipulate their temperature. Methods for determining and regulating the temperature of the culture medium are well known in the art. In certain embodiments, the system described includes an adjustable controller for the temperature of the culture medium.

[0076] In yet other embodiments, any of the methods further comprises collecting and / or storing data about the conditions inside the culture medium container, which can be, for example, glucose concentration, temperature, pH, dissolved oxygen concentration, etc. In other embodiments, the apparatus optionally further comprises a meter (connected to an external computer) and / or other means for collecting and / or storing data about the conditions inside the culture medium container. In certain embodiments, the data is used to generate a report.

[0077] In some other embodiments, any one in the method further includes collecting and / or storing data about fluid transfer to the culture medium container and / or transfer out from the culture medium container, or in other embodiments, any one in the method further includes collecting and / or storing data about fluid transfer to the utensil and / or transfer out from the utensil, or in some other embodiments, any one in the method further includes collecting and / or storing data about fluid transfer to the culture medium container and the utensil and / or transfer out from the culture medium container and the utensil. In other embodiments, equipment optionally further includes an instrument (being connected to an external computer) and / or other devices for collecting and / or storing data about fluid transfer to the culture medium container and / or the container and / or transfer out from the culture medium container and / or the utensil. In certain embodiments, data are used to generate a report.

[0078] In yet other embodiments, any of the methods further comprises controlling the flow rate of the culture medium transferred into and / or out of the central culture medium container, or in other embodiments, any of the methods further comprises controlling the flow rate of the culture medium transferred into and / or out of the plant culture vessel, or in yet other embodiments, any of the methods further comprises controlling the flow rate of the culture medium transferred into and / or out of both the culture medium container and the vessel. In other embodiments, the apparatus optionally further comprises an instrument (connected to an external computer) and / or other means for controlling the flow rate of the culture medium transferred into and / or out of each culture vessel.

[0079] In yet other embodiments, any of the methods further comprises promoting uniform mixing of the liquid contents of the described culture medium container when the agitator / stirring device is activated (e.g., rotated). Therefore, the culture medium container optionally further comprises one or more baffles protruding inwardly from the inner surface of the container.

[0080] In yet other embodiments, the described culture medium container is optionally further operably connected to an automatic calibrator and / or other device for calibrating other components and / or sensors described herein and / or monitoring failure of one, some or all of these components, which represent separate embodiments.

[0081] Each of the optional method steps and optional components represents a separate embodiment, and they can be freely combined in various embodiments.

[0082] Those skilled in the art will appreciate that while three growth subchambers 439 are depicted in this figure, the use of different numbers and configurations of growth subchambers is consistent with the present disclosure.Each growth subchamber may have a similar or different volume compared to the other subchambers.

[0083] Figure 3B for Figure 3ASchematic close-up cross-sectional isometric bottom view of the upper perforated disk 432 of the growth basket 416. Shown in this view are holes 4321, all of a fixed diameter, at predetermined locations on the top of the disk so that they functionally create a physical barrier that allows culture medium to flow through them but keeps the plant cell aggregates within the basket. When a carrier is used, when the cell aggregates are small, they physically block the passage of cells through the lower perforated disk, and then the cell aggregates grow to a size larger than the diameter of the holes 4321 and cannot pass through them. The size and distribution of the holes on the disk allow the turbulence from the impeller to be evenly and laminarly distributed at the entrance into the basket 416. In addition, the more evenly distributed the holes, the more even the material transfer to all parts of the basket, allowing the diameter of the basket to expand. Also shown in this view is a hollow longitudinal center axis 435.

[0084] Figure 4A-4B Schematic isometric view of an exemplary agitation mechanism 700 for agitating plant cell aggregates within a plant growth basket in a packed bed bioreactor and a schematic elevation view of agitation mechanism 700 embedded within a growth basket 716 of a packed bed bioreactor 770, respectively.

[0085] The stirring mechanism 700 includes a rotating cylinder 750, which is generally arranged along the central axis of the packed bed bioreactor 770. Spokes 760 extend radially from the rotating cylinder 750 inside the growing basket area 716, causing the basket contents to move when the rotating cylinder 750 rotates. The rotating cylinder 750 can be operably connected to an external component capable of transmitting an applied torque, which can optionally be rotated manually or connected to a motor (not shown) via a handle 735. In the example shown in these figures, the stirring mechanism 700 includes a gear mechanism, which includes a small gear 730 attached to the handle 735 that extends to the outside of the growing basket 716 and can be rotated manually or with the help of a motor, and a large gear 740 attached to the impeller 704. The large gear 740 is connected to the rotating cylinder 750 by a ring 742 mounted on the top of the rotating cylinder 750 and rotates with it. The rotating cylinder 750 causes the spokes 760 to rotate to thereby stir the contents of the growing basket 716, minimizing the channeling effect and increasing the contact area between the growth medium (or any other medium used) and the plant cells within the basket 716. In addition, the stirring mechanism 700 can also be used to improve the harvesting of cells. The rotating cylinder 750 can rotate in a continuous rotational motion, or in other embodiments, the rotating cylinder 750 can rotate in a partial rotational motion, such as an oscillating partial rotational motion. It can rotate clockwise and counterclockwise, all depending on the operator's needs and design.

[0086] In still other embodiments, the rotating cylinder 750 is operably connected to the growth basket 716 in a manner that enables the basket itself to rotate relative to the outer chamber wall of the bioreactor 770. The impeller 704 is also Figure 4B 704. According to some optional embodiments of the present invention, the impeller 704 can be a cell-lift impeller that generates a vacuum pull from below, thereby causing a downward fluid flow within the cell basket 716. The cell-lift impeller 704 preferably rotates about the same axis as the rotating cylinder 750, but is not operably connected to the rotating cylinder 750. As used herein, the term "cell-lift" impeller refers to an impeller having a vertical tube, whose rotational motion generates a low pressure differential at the bottom of the tube. It should be clear that other types of impellers can also be used to implement the present invention.

[0087] Additional components such as removal tube 710 (outlet port) are provided along the bioreactor and, if desired, allow for the collection of metabolites secreted by the plant cells from the cell-free culture medium below the basket 716, electrodes 706 that allow for monitoring and control of various parameters of the culture process and culture medium, and upper and lower perforated trays 732 that define the upper and lower boundaries of the plant cell culture basket 716 and allow for the plant cells to be harvested and retained from flowing with the culture medium and into a stationary biomass phase.

[0088] Figures 5A-5E Schematic diagrams and photographs of various plant cell aggregates at different stages of growth grown in a packed bed bioreactor with or without carriers.

[0089] Figure 5A 8 is a schematic partial view of a 3D micro packed bed bioreactor 800 showing the growth of plant cell aggregates 822 on top of scaffolds 824 within a growth basket 816. The plant cell aggregates fill the growth basket and are retained within the basket due to upper and bottom perforated disks 832 that mechanically restrict their ability to exit the basket into the culture medium on top of or below the basket 816.

[0090] Figure 5B and 5C For 3D Fibracell TM Illustration of the expansion of Cannabis Indica plant cells within a carrier. The plant cells were cultured in a packed bed bioreactor for a period of 21 days. This resulted in the expansion of cell aggregates, as shown in these figures. The 3D carrier was stained with the specific DNA marker Hoechst. Cell aggregates were observed to be inserted into the fibers of the carrier. Scale bar 100 μm.

[0091] Figure 5DSchematic diagram of plant cell inoculation and growth in a 3D microbiosystem without a carrier. Cannabis cells were then cultured in a 3D microbioreactor. Although no carrier was used in the culture process, the average cell aggregate diameter increased by about 10 times after three weeks of culture, indicating cell culture proliferation.

[0092] Figure 5E Adhesion of Cannabis sativa plant cell aggregates to Fibracell TM Schematic representation of the carrier. Cultivation of plant cells in a packed bed bioreactor for a period of 21 days resulted in cell aggregates adhering to the 3D carrier. Proliferation of cell aggregates is shown, since the cell culture was filtered to reach 300 μm at the time of inoculation and the final aggregate size was measured to be above 500 μm. Scale bar 530 μm.

[0093] Fig. 6A Graph showing cannabinoid production ratios in packed bed bioreactors relative to suspension cultures. As shown in this graph, cannabinoid concentrations in 3D bioreactors increased significantly relative to suspension cultures. This increase reached over a 1000-fold increase in the 3D bioreactor system.

[0094] Figure 6B Schematic diagram showing the amount of cannabinoids obtained when culturing plant cells in a packed bed bioreactor of the present invention compared to the amount of cannabinoids obtained when culturing plant cells in suspension culture shake flasks (where all other conditions were similar). These results demonstrate the ability of the 3D system to induce and significantly increase the cannabinoid production potential of cell cultures. This potential is reflected in the amount of cannabinoids produced under both the suspension and 3D systems, showing a maximum 8-fold increase in cannabinoids in the 3D system. Biomass yield, metabolite content and profile results indicate that biomass and cannabinoid yields are higher in the packed bed bioreactor in both total amount and profile.

[0095] Example

[0096] General plant cell culture process workflow in a 3D packed bed bioreactor

[0097] After growing cell cultures in 2D and suspension methods, the cultures are processed to reach final product specifications and to increase production yields via 3D systems.

[0098] The following steps are a general description of the process and are applicable to a variety of plants and product lines.

[0099] Step 1.3D Pre-seeding Procedure

[0100] After cell separation and cultivation of cells derived from specific tissues, cell cultures for 3D inoculation in 3D systems are prepared. The purpose is to achieve the required aggregate size to adapt to the microbiological system. This is done to achieve cell culture uniformity and enable the physical process of inoculation through small diameter pipes. During this process, the inventors investigated aggregate size (0-700 μm) and inoculation procedure technical details such as cell biomass concentration, flow rate, time and RPM in the entire process.

[0101] Certain parameters were looked at to ensure that the vaccination procedure went as planned.

[0102] Step 2. Cell Growth for Biomass Accumulation

[0103] In this phase, specific culture media are used to increase biomass accumulation and initiate differentiation of the cell culture so that the desired secondary metabolites and / or active compounds can be produced. For example, when the goal is to produce cannabinoids, the culture media provided to the cell culture shell contains specific cannabinoid precursors, such as oleic acid. The duration of this phase can be between 10-28 days, depending on the desired action and plant strain. The action can be to produce a complete or large spectrum of cannabinoids, or it can be a specific cannabinoid such as one or more minor cannabinoids.

[0104] In this step, the aim is to expand the cells to reach the highest biomass of the cell culture. During this phase, the medium uptake is monitored and, if necessary, refilled by perfusion. Once biomass accumulation is reached, the next step of cell differentiation and / or metabolite production can be started.

[0105] Step 3. Cell culture manipulation

[0106] During this stage, the inventors "drive" the cell culture to produce and / or reach the product manufacturing process. The characteristics of this stage are highly dependent on the final product, and it can include cell culture maturation, differentiation and metabolite production via elicitation. For cannabis, for example, elicitation is used to activate cell culture to produce the desired product for a period of 1-16 days. Since secondary metabolites in plants act as protective compounds, stress conditions (biological / abiotic) are generated to induce a stress response for activating cell culture to produce its "defense" metabolites. Manipulation of the culture medium contents via activation of cell differentiation and maturation can be adapted to specific plant species and metabolites of interest.

[0107] Step 4. Final product manufacturing

[0108] During this phase, further production of certain metabolites may be induced, and the growth medium may be altered to allow for long-term stability of the desired product or to alter specific properties in the final product. For example, when accumulation of metabolite production is observed in the cell culture and growth medium, the cell culture is washed with a control feed medium. Optionally, during steps 3 and 4, the culture medium may be collected, as the culture medium is composed of specific desired metabolites, such as cannabinoids, caffeine, and vanillin, etc., depending on the plant cells being cultured and the culture conditions.

[0109] The above general workflow will be better understood through the following detailed examples.

[0110] Example 1: Inoculation of Cannabis Plant Cells into a Packed Bed Bioreactor

[0111] Hemp callus cell cultures derived from sterile internode tissue were maintained in liquid MS medium (Duchefa cat. M0221) containing essential vitamins (Gamborg B5), sugars and phytohormones. After sufficient biomass accumulation, a 1 mm filter was used to select the culture aggregate size for inoculation. The cell culture was poured into a 1 mm filter set on a beaker and washed thoroughly. The beaker thus contained cells with a diameter of less than 1 mm. The cells were collected and used as the initial inoculum for bioreactor inoculation. In addition, the bioreactor head plate was modified to increase the inner diameter of the pipes to prevent clogging of the lines. The minimum inner diameter of the pipe used was 6 mm long. A wet weight of 8.5 g of the hemp cell culture was inoculated at 200 rpm for a period of five (5) hours, or until the optical density (OD) of the growth medium was similar to that of the blank medium measurement (see Table 1 below). As can be seen in Table 1, the hemp cell culture was inoculated at 200 rpm for a period of five (5) hours. Figure 5A OD measurements supported visual inspection of medium turbidity and cell content as observed in the literature. Cell cultures were grown in a 350 ml packed bed modified AppliKon TM The OD measurement results are listed in Table 1 below.

[0112] Table 1: Optical density measurements during cell culture inoculation of packed bed bioreactors.

[0113]

[0114]

[0115] As shown in the above data, the OD measurement five (5) hours after inoculation, which represents the cell content of the medium, was lower than the blank medium measurement. This indicates that all cells were adhered / trapped within the packed bed area. Therefore, efficient inoculation of cell culture was demonstrated in the packed bed bioreactor inoculation procedure.

[0116] Example 2: Monitoring Cannabis Cell Culture Proliferation in a Packed Bed Bioreactor (InProcess Control (IPC) Follow-up)

[0117] The inventors of the present invention have developed a method for monitoring the growth of plant cells within a packed bed bioreactor during operation of the bioreactor.An optional method is described herein.

[0118] 8.5 g wet weight of Cannabis indica cell culture was inoculated as described above in Example 1. Inoculations were performed in biological triplicates in both suspension culture and bioreactor systems (systems 1, 2 and 3 in Table 2 below represent triplicates).

[0119] After inoculation, the bioreactor was grown for a period of twenty-eight (28) days with an average perfusion rate of 190 ml / day. The total packed bed volume comprised 64% Fibra-Cel TM Carrier (a lower amount relative to the amount of carrier used in the growth of animal cells in a packed bed bioreactor). The carrier is applied to allow the plant cell culture to expand in volume throughout the culture period. The pH set point of the culture medium is 5.8, the dissolved oxygen (DO) set point is 30%, and the temperature set point is 25°C. These parameters are maintained by the system controller during the cell culture period. In order to characterize cell growth and viability, several parameters were followed up. These include culture medium absorption measurements and biomass. The growth rate is monitored using the daily glucose consumption rate (GCR / day). The GCR of the cell culture increased from 46 (GCR / day) to 129 (GCR / day) during the cell proliferation phase. The data supports the accumulation of biomass from 8.5g to 18g, which is a 2.1-fold increase.

[0120] The biomass accumulation data listed in Table 2 below demonstrate that the packed bed bioreactor system is more efficient in terms of growth and medium usage of plant cells relative to traditional parallel suspension cultures of plant cells in shake flasks.

[0121] Table 2: Comparison of biomass accumulation per volume (g / L)

[0122]

[0123]

[0124] Example 3: Plant somatic embryo development in a packed bed bioreactor.

[0125] For plant somatic embryo differentiation and development, the selected plant cell culture derived from germinating seeds is first inoculated and cultured as described in the above examples. In the next step, the plant hormone cytokinin TDZ (Duchefa cat.T0916) at a concentration of 2 mg / l and Auxin2,4D (Duchefa cat.D0911) at a concentration of 1 mg / l are optionally applied to the cultivated plant cell culture with growth factors such as 1 mg / l gibberellic acid (Duchefa cat.G0907). Cell culture is carried out at 25°C for 2 to 4 weeks until the spherical stage is observed. High percentage carbon source content (high percentage carbonsource content) such as 0.15 mole glucose and 0.25 mole sucrose are applied to the culture medium to start or improve the efficiency of somatic embryo development and maturation (H.Lou, S.Kako, 1995). In order to eliminate the inhibitory effect of secreted plant cell metabolites on differentiation, a high perfusion rate (> 1200 ml / day) is used. Geovanny I. Nic-Can et al., 2015 ).

[0126] Example 4: Production of carnosic acid from rosemary (Rosmarinus officinalis) cells

[0127] Carnosic acid is a natural benzyl abietane diterpene found in Rosmarinus officinalis (rosemary) and Salvia officinalis (common sage). It is commonly used as an antioxidant preservative in food and non-food products.

[0128] To produce carnosic acid in a packed bed bioreactor, rosemary cells were first inoculated and cultured as previously described in Examples 1 and 2 above. To maintain high cell culture proliferation and metabolite production potential, plant hormones 0.5 mg / l kinetin and 1 mg / l TDZ were applied. The cell culture was cultured until a stationary phase was reached, which was manifested by a consistent carbon source consumption rate. After the stationary phase, the cells were harvested, lysed, and carnosic acid was collected.

[0129] Example 5: Vanillin production using specific metabolite precursors.

[0130] Vanillin is the main component of vanilla bean extract. Vanillin is widely used as a flavoring agent in foods, beverages, cosmetics and pharmaceuticals. To produce vanillin in a packed bed bioreactor, Vanilla Planifolia cells are cultured as described in Example 2 above. To enhance the natural biosynthetic pathway, flavonoid precursors such as tyrosine and phenylalanine are applied at concentrations of 100-500 μM. Specific precursors of vanillin such as 50-500 μM p-coumaric acid are added to the growth medium. Once vanillin accumulates in the cell culture, the cells can be lysed or more precisely harvested and the desired compound extracted from the growth medium. Alternatively, conventional extraction methods such as ethanol extraction can be used.

[0131] Example 6: Cannabis plant cell-based cannabinoid production in a packed bed bioreactor

[0132] The cannabis callus cell culture derived from sterile internode tissue was treated as described in Examples 1-2 above. After treatment, the cell culture medium was supplemented with stress-inducing factors in a process called induction. In addition, in order to activate the production of general secondary metabolites in plant cells, the growth medium contained 100 μM methyl jasmonate (MeJA, Duchefa cat. M0918) for an activation period of three (3) days. After three (3) days, perfusion was increased to remove MeJA from the culture medium and 15 mg / l chitosan (Sigma, cat. 448869) and 100 mg / l olivetol were added to the culture medium as precursors (Sigma cat. 152633). In order to keep the concentrations of chitosan and olivetol constant, perfusion was increased to 2 L / day.

[0133] Induction was performed for periods of nine (9) and fourteen (14) days. Post-induction cultures were washed with control MS medium for a period of two (2) days. Cells were harvested from the bioreactor using mechanical force and cell aggregates were separated from the carrier. Chemical analysis of the cannabinoid content of the cell cultures was performed using LC-MS / MS. Figure 6A-6B As shown, the biomass yield, metabolite content and profile results indicate higher biomass and cannabinoid production in the packed bed bioreactor, both in terms of total amount and profile.

[0134] The above results indicate the beneficial effects on plant cell biomass accumulation and secondary metabolite production in packed bed bioreactors compared to suspension cultures.

[0135] It should be clear that the embodiments and descriptions of the drawings set forth in this specification are only for a better understanding of the present invention and do not limit its scope. It should also be clear that those skilled in the art may make adjustments or modifications to the drawings and the above-mentioned embodiments after reading this specification, and the adjustments or modifications will still be covered by the current subject matter.

Claims

1. A three-dimensional (3D) bioreactor for culturing and proliferating plant cells, comprising: a. at least one packed bed chamber, the packed bed chamber being configured to confine the proliferating plant cells therein so that the proliferating plant cells become a three-dimensional stationary biomass phase; and b. at least one container comprising a fluid culture medium configured to flow through the resting biomass phase of the proliferating plant cells; Wherein, the flow of the fluid culture medium through the proliferating plant cell static biomass phase allows compounds to be transferred from the fluid culture medium to the cells within the at least one packed bed chamber with low shear force, and vice versa, thereby simulating the natural growth environment of the proliferating plant cell static biomass within the 3D bioreactor.

2. The bioreactor according to claim 1, further comprising at least one monitoring and / or control unit for monitoring and / or controlling at least one of the culture conditions and culture medium parameters.

3. A bioreactor according to claim 2, wherein the at least one parameter monitored and / or controlled is selected from the group consisting of: pH, temperature, agitation speed, flow rate, gas concentration, amino acid level, vitamin level, mineral level, growth factor level, dissolved oxygen level, glucose level, lactate level, lactate dehydrogenase level, NH3 level, glutamate level, and combinations thereof.

4. The bioreactor of claims 1-3, wherein the fluid culture medium is a growth medium that provides nutrients to the proliferating plant cells and absorbs plant cell metabolites and secreted compounds from the plant cells into the growth medium.

5. The bioreactor according to any of the preceding claims, further comprising at least one inlet port to allow the addition of gas and / or liquid and / or solid material into the 3D bioreactor during the cultivation of the proliferating plant cells.

6. A bioreactor according to any of the preceding claims, further comprising at least one outlet port to allow removal of fluid culture medium from the 3D bioreactor during cultivation of the proliferating plant cells, for collecting metabolites secreted by the resting biomass phase of the proliferating plant cells during the cultivation process, and / or for removing plant-secreted compounds / metabolites, and / or for balancing fluid culture medium parameters, and / or for collecting cells after harvesting.

7. A bioreactor according to any of the preceding claims, further comprising a vibrating arm functionally connected to the packed bed chamber, thereby allowing vibration of the proliferating plant cells, for minimizing channel effects and improving contact between the fluid culture medium and the proliferating plant cell stationary biomass phase, and / or for harvesting the proliferating plant cell stationary biomass phase for collecting cells or for lysing the cells to obtain their contents.

8. The bioreactor according to claim 7, wherein the harvest of plant cells is obtained by combining enzymatic reactions and applying vibrational forces to the plant cells, thereby lysing the cells and collecting their contents from the culture medium and / or lysing plant cells from stationary biomass.

9. A bioreactor according to any of the preceding claims, wherein the packed bed chamber is located within a container with a flowing fluid culture medium or outside a container with a fluid culture medium and is connected to the container by a tube that allows the fluid culture medium to flow through the stationary biomass phase of the proliferating plant cells.

10. The bioreactor according to any of the preceding claims, wherein the packed bed chamber is divided into one or more subchambers by at least one perforated disk, so that each subchamber contains proliferating plant cell biomass cells in similar size or gradient size according to the hole size of the at least one perforated disk.

11. A bioreactor according to any of the preceding claims, wherein the fluid culture medium can be exchanged for any one of a differentiation medium, a maturation medium and an induction medium and combinations thereof in any order, thereby allowing continuous development of plant cells and simulating the natural growth process of plant cells within the 3D bioreactor.

12. The bioreactor according to any one of the preceding claims, wherein the at least one packed bed chamber comprises carriers configured to allow growth of the proliferating plant cells within and / or above the carriers.

13. The bioreactor of any one of the preceding claims, further comprising an agitation mechanism functionally connected to the packed bed chamber and configured to compound the proliferating plant cell quiescent biomass phase within the basket, thereby rearranging the plant cell biomass and allowing culture medium to flow smoothly through the plant cells.

14. The bioreactor of claim 13, wherein the stirring mechanism comprises at least a large gear attached to an agitator and a small gear attached to a handle, the agitator being located within the packed bed chamber.

15. The bioreactor according to any one of the preceding claims, wherein the packed bed chamber is a separate chamber from the fluid culture medium container and the plant cell resting biomass phase is used in agriculture to inoculate the resting cell biomass to obtain plants or parts thereof.

16. The bioreactor according to any one of the preceding claims, wherein the plant cell quiescent biomass phase is harvested for the collection of compounds produced by the plant cell quiescent biomass phase and / or for the collection of the plant cells.

17. The bioreactor of claim 16, wherein the compound or cell is for industrial application.

18. The bioreactor according to claims 16-17, wherein the industrial application is selected from the group consisting of: food manufacturing, pharmaceuticals, industrial applications and cosmetic compositions.

19. The bioreactor of any one of claims 1-18, further comprising a perfusion chamber comprising a 3D packed bed matrix containing one or more macrocarriers, microcarriers, or a combination thereof.

20. A method of culturing and proliferating plant cells using a bioreactor according to any one of claims 1 to 19 for using the plant cells and / or compounds produced by the plant cells for medical, cosmetic, food technology, agricultural and industrial applications.

21. A method for culturing and proliferating plant cells in a 3D bioreactor, comprising: a. confining the proliferating plant cells in at least one packed bed chamber so that the proliferating plant cells become a stationary biomass phase; and b. flowing medium from at least one container containing a fluid culture medium through the resting biomass phase of the proliferating plant cells; Wherein, the flow of the fluid culture medium through the proliferating plant cell static biomass phase allows compounds to be transferred from the fluid culture medium to the cells and vice versa within the at least one packed bed chamber at low shear force, thereby simulating the natural growth environment of the proliferating plant cell static biomass within the 3D bioreactor.

22. The method for culturing and proliferating plant cells according to claim 21, further comprising: At least one of the culture conditions and the culture medium parameters is monitored and / or controlled by at least one monitoring and / or control unit.

23. A method for culturing and proliferating plant cells using a bioreactor according to any one of claims 1 to 19 to use the plant cells and / or compounds produced by the plant cells for medical, cosmetic, food technology, agricultural and industrial applications, the method comprising: a) inputting the proliferating plant cells into at least one packed bed chamber in a bioreactor, wherein the packed bed chamber confines the proliferating plant cells within the packed bed chamber so that the proliferating plant cells produce a quiescent biomass phase; and b) flowing at least one fluid culture medium from at least one container containing a fluid culture medium through the stationary biomass phase in a low shear environment, allowing efficient transfer of compounds between the fluid and the cells, wherein the fluid culture medium comprises one or more of nutrients, hormones, plant growth regulators (PGRs), vitamins, amino acids, trace elements, or combinations thereof.

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