US-12624328-B2 - System for 3D cultivation of plant cells and methods of use

US12624328B2US 12624328 B2US12624328 B2US 12624328B2US-12624328-B2

Abstract

This invention is directed to a method for three-dimensional cultivation of proliferating plant cells in a packed bed bioreactor, the bioreactor comprising at least one packed bed chamber configured to confine said proliferating plant cells within it such that the proliferating plant cells become a three-dimensional stationary biomass phase; and At least one container comprising a fluid media, the fluid media is configured to flow through said proliferating plant cells stationary biomass phase, wherein, the flow of the fluid media through the proliferating plant cells stationary biomass phase allows transfer of compounds from the fluid media into the cells and vice versa in low shear forces within the at least one packed bed chamber thereby imitating natural growth environment of the proliferating plant cells within the 3D bioreactor.

Inventors

Lior Raviv
Eran Ben Eliezer

Assignees

PLURI BIOTECH LTD.

Dates

Publication Date: 20260512
Application Date: 20240318
Priority Date: 20230608

Claims (19)

1 . A three-dimensional (3D) bioreactor for cultivating proliferating plant cells comprising: at least one packed bed chamber configured to confine proliferating plant cells within it such that the proliferating plant cells remain as a stationary three-dimensional biomass phase during cultivation; and at least one container comprising a fluid media, the fluid media is configured to flow through proliferating plant cells stationary three-dimensional biomass phase; vibrating arms functionally connected to the packed bed chamber, so as to allow vibrating of the proliferating plant cells for minimizing channeling effect and improving contact between the fluid media and the proliferating plant cells stationary biomass phase, and/or for harvesting said proliferating plant cells stationary three-dimensional biomass phase for either collecting the cells or for lysing the cells to obtain their content; wherein the packed bed chamber is configured so that, after initial seeding, the plant cells are physically retained and do not circulate with the fluid media, thereby enabling in-situ development of large, stationary plant cell aggregates; wherein, the flow of the fluid media through the proliferating plant cells stationary three-dimensional biomass phase allows transfer of compounds from the fluid media into the cells and from the cells into the fluid media in a low shear force environment within the at least one packed bed chamber thereby replicating the natural growth environment of the proliferating plant cells stationary three-dimensional biomass within the 3D bioreactor.
2 . The bioreactor of claim 1 , further comprising a monitoring unit for monitoring at least one parameter of the culturing conditions and media parameters.
3 . The bioreactor of claim 2 , wherein said at least one parameter monitored and/or controlled is selected from the group consisting of: pH, temperature, stirring velocity, flow rate, gases concentration, amino acids levels, vitamins levels, minerals levels, growth factors levels, dissolved oxygen levels, glucose levels, lactate levels, lactate dehydrogenase levels, NH.sub.3 levels, glutamate levels, and combinations thereof.
4 . The bioreactor of claim 1 , wherein said fluid media is a growth media that provides nutrients to the proliferating plant cells, and uptakes plant cells metabolites and secreted compounds from the plant cells into the growth media.
5 . The bioreactor of claim 1 , further comprising at least one inlet port to allow addition of gases and/or liquids and/or solid material into the 3D bioreactor during the cultivation of the proliferating plant cells.
6 . The bioreactor of claim 1 , further comprising at least one outlet port to allow removal of fluid media out from the 3D bioreactor during the cultivation of the proliferating plant cells for collecting metabolites secreted by the proliferating plant cells stationary biomass phase during the cultivation process and/or for removing plant's secreted compounds/metabolites and/or for balancing the fluid media parameters and/or for collecting cells after harvesting.
7 . The bioreactor of claim 1 , wherein said harvesting of the plant cells is obtained by combining enzymatic reaction and applying vibration force on the plant cells so as to lyse the cells and collect their content from the media and/or to lyse the plant cells from the stationary three-dimensional biomass.
8 . The bioreactor of claim 1 , wherein said packed bed chamber is either positioned within the container having the flowing fluid media or positioned outside the container having the fluid media and connected thereto by tubes that flow the fluid media through said proliferating plant cells stationary three-dimensional biomass phase.
9 . The bioreactor of claim 1 , wherein said packed bed chamber is divided into one or more sub-chambers by at least one perforated disk, such that each sub-chamber contains proliferating plant cells biomass cells either in a similar size or in gradient sizes according to the hole dimensions of the at least one perforated disk.
10 . The bioreactor of claim 1 , wherein said fluid media may be exchanged to either one of a differentiating media, a maturation media, and an elicitation media, and combinations thereof in any order, so as to allow a consecutive development of the plant cells and imitate the natural growth process of plant cells within the 3D bioreactor.
11 . The bioreactor of claim 1 , wherein said at least one packed bed chamber comprises carriers configured to allow said proliferating plant cells to grow within them and/or on top of them.
12 . The bioreactor of claim 1 , wherein said packed bed chamber is a separated chamber from said fluid media container and said plant cells stationary three-dimensional biomass phase is used in agriculture for seeding said stationary three-dimensional biomass phase to obtain plants or parts thereof.
13 . The bioreactor of claim 1 , wherein said plant cells stationary three-dimensional biomass phase is harvested for collecting compounds produced by said plant cells stationary three-dimensional biomass phase and/or for collecting the plant cells.
14 . The bioreactor of claim 13 , wherein said compounds or cells are used for industrial applications.
15 . The bioreactor of claim 13 , wherein said industrial applications are selected from: food manufacturing, medicaments, industrial applications, and cosmetic compositions.
16 . The bioreactor of claim 1 , further comprising a perfusion chamber containing a 3D packed bed substrate containing one or more macro carriers, microcarriers, or combinations thereof.
17 . A method for cultivating proliferating plant cells for usage of the plant cells and/or compounds produced by said plant cells for medical, cosmetic, food tech, agriculture, and industrial applications using the bioreactor according to claim 1 .
18 . The bioreactor of claim 1 , further comprising a controlling unit for controlling at least one parameter of the culturing conditions and media parameters.
19 . A three-dimensional (3D) bioreactor for cultivating proliferating plant cells comprising: at least one packed bed chamber configured to confine proliferating plant cells within it such that the proliferating plant cells remain as a stationary three-dimensional biomass phase during cultivation; and at least one container comprising a fluid media, the fluid media is configured to flow through proliferating plant cells stationary three-dimensional biomass phase; a stirring mechanism functionally connected to said packed bed chamber and configured to blend the proliferating plant cells stationary biomass phase within said chamber, so as to rearrange the plant cells biomass and allow fluent flow of the fluid media through the plant cells, wherein said stirring mechanism comprises at least a large gear attached to a stirrer, said stirrer is positioned within said packed bed chamber, and a small gear attached to a handle; wherein the packed bed chamber is configured so that, after initial seeding, the plant cells are physically retained and do not circulate with the fluid media, thereby enabling in-situ development of large, stationary plant cell aggregates; wherein, the flow of the fluid media through the proliferating plant cells stationary three-dimensional biomass phase allows transfer of compounds from the fluid media into the cells and from the cells into the fluid media in a low shear force environment within the at least one packed bed chamber thereby replicating the natural growth environment of the proliferating plant cells stationary three-dimensional biomass within the 3D bioreactor.

Description

FIELD OF THE INVENTION This invention is in the field of plant cells culturing in general and directed to plant cells culturing in a packed bed bioreactor in particular. BACKGROUND The climate change and the constantly increasing world population leads to a real concern and fear that traditional farming methods will not be able to meet the anticipated growth in food demands, with amount of arable land per capita available for food production decreasing due to factors such as urbanization, erosion, soil salinization, and desertification. Furthermore, sustainability in food production and the threat of crop losses due to climate change and plant diseases are playing an increasingly important role and need to be taken into account. Therefore, various alternative agricultural strategies are constantly being considered. Plants are an essential component to produce carbohydrates, lipids (fatty acids), proteins (amino acids), and vitamins and have been drawing attention for the biotechnology industry with the production of secondary metabolites and recombinant proteins. Secondary metabolites are usually small but complex molecules, which are in many cases impossible or expensive to synthesize chemically. Based on their metabolic pathways and their biogenetic precursors, they can be classified into three groups: terpenoids (e.g., paclitaxel, ginsenosides), alkaloids (e.g., morphine) and phenolics (e.g., shikonin, rosmarinic acid). The extraction of secondary metabolites from plants, which were traditionally grown in fields, is still the main production method for these substances. However, as per the above, there are a number of disadvantages associated with traditional farming and the excessive variation of environmental conditions over time and region, which leads to unpredictable differences in the quality and quantity of the raw materials. Many secondary metabolites are produced in plants that are not suited to agricultural production or can be hard to grow outside their local ecosystems. As a result, it is not surprising that around one fifth of the 50,000 medical plants that are used today are on the list of threatened species. Therefore, plant cell and tissue cultures grown in bioreactors offer an eco-sustainable alternative. Furthermore, plant cell and tissue cultures are believed to represent an appropriate method that addresses the main drawbacks of traditional farming of herbs and avoids the problems associated with extracting products from protected wild plants. One popular example of a pharmaceutically used secondary metabolite derived from plant cell cultures is paclitaxel, an anti-cancer drug. Previously produced by harvesting the bark from Taxus sp. trees, today, the large-scale production of paclitaxel is performed in stainless steel bioreactors. Furthermore, the complete elimination of environmental variations leads to improved consistency between batches, which is crucial for gaining official acceptance. The avoidance of labor-intensive greenhouse or field production of whole plants reduces costs, not only in upstream processing, but also in downstream processing, in particular the case of products which are secreted into the medium. Obviously, the safety of the process with regards to product contamination with endotoxins and mycotoxins and, of no less importance, with regards to environmental contamination with artificial, genetically modified plants is tremendously enhanced when operating in a closed bioreactor system. Thus, the present invention is aimed to provide a possible cost and time effective solution by cultivating plant cells in a packed bed bioreactors in a proprietary method that mimics the natural environment of the plant. The system and methods disclosed herein may be applicable to all and/or most plant species and may be used for cultures may present a possible solution, as they allow for controlled, closed and sustainable manufacturing of plant based products that are used in various industries such as food, pharmaceuticals, cosmetics and else. SUMMARY OF THE INVENTION In one main aspect, the present invention is directed to a three-dimensional (3D) bioreactor for cultivating proliferating plant cells comprising at least one packed bed chamber configured to confine said proliferating plant cells within it such that the proliferating plant cells become a three-dimensional stationary biomass phase; and at least one container comprising a fluid media, the fluid media is configured to flow through said proliferating plant cells stationary biomass phase; wherein, the flow of the fluid media through the proliferating plant cells stationary biomass phase allows transfer of compounds from the fluid media into the cells and vice versa in low shear forces within the at least one packed bed chamber thereby imitating natural growth environment of the proliferating plant cells within the 3D bioreactor. For clarification purposes the term “culturing” as used herein means maintaining the plan