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WO2018178434A1 - Bioréacteur de type colonne à bulles pour culture de cellules végétales en suspension - Google Patents

Bioréacteur de type colonne à bulles pour culture de cellules végétales en suspension Download PDF

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Publication number
WO2018178434A1
WO2018178434A1 PCT/ES2018/070152 ES2018070152W WO2018178434A1 WO 2018178434 A1 WO2018178434 A1 WO 2018178434A1 ES 2018070152 W ES2018070152 W ES 2018070152W WO 2018178434 A1 WO2018178434 A1 WO 2018178434A1
Authority
WO
WIPO (PCT)
Prior art keywords
bioreactor
bubble column
suspension
plant cells
culture
Prior art date
Application number
PCT/ES2018/070152
Other languages
English (en)
Spanish (es)
Inventor
Roque Bru Martinez
Juan Carlos VERA URBINA
Original Assignee
Universidad De Alicante
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad De Alicante filed Critical Universidad De Alicante
Publication of WO2018178434A1 publication Critical patent/WO2018178434A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a product consisting of a bioreactor for carrying out, under aseptic conditions, the cultivation of plant cells in suspension, whose design features also allow the culture medium to be recovered, replaced by another culture medium and reused. the remaining biomass in it for a next cultivation operation.
  • bioreactors are widely used for the cultivation of microorganisms (bacteria, filamentous fungi, etc.) both on a laboratory scale and on an industrial scale.
  • microorganisms bacteria, filamentous fungi, etc.
  • the characteristics of its design typically of a tank agitated by mechanical devices, are not always suitable or optimal for the cultivation of plant cells. This is especially due to the fact that they show a lot of sensitivity to shear stress, have low oxygen demand (about ⁇ ⁇ 0 2 to 10 "6 cells), low growth rate (doubling time 25 to 11 Oh) and often occur as large aggregates, 2-4 mm in diameter (Rao, RS and Ravishankar, GA (2002). Plant cell cultures: Chemical faetones of secondary metabolites. Biotechnol Adv. 20: 101-153).
  • bioreactor such as the concentration of dissolved oxygen, pH, temperature, mixing and the nutrient supplement in order to favor the desired functions both for the maintenance of the cells and for the production of the metabolite
  • concentration of dissolved oxygen, pH, temperature, mixing and the nutrient supplement in order to favor the desired functions both for the maintenance of the cells and for the production of the metabolite
  • Different bioreactor designs have been shown to be suitable for the cultivation of plant cells and tissues, with pneumatic agitation of the bubble column and airlift type being the most appropriate for the culture of cells in suspension.
  • a problem they present is the need for a homogeneous and constant aeration.
  • Plant cells have been grown to produce biomass with various applications and also natural compounds excreted in the culture medium. In both cases, the process may require changing the chemical composition of the medium, which becomes effective by replacing the medium, even more than once, which is a major problem because this may require manipulation of the culture even outside the bioreactor. greatly increasing the risk of contamination and crop loss.
  • the invention relates to the design of a new bioreactor for the growth of plant cells under aseptic conditions in suspension in a liquid medium whose composition satisfies the nutritional and physiological requirements of the cells and is capable of maintaining appropriate physicochemical conditions.
  • the prototype has been made for laboratory-scale tests, so its size has been limited by the restriction imposed by the dimensions of standard laboratory sterilization equipment.
  • the bioreactor is 7 liters with cylindrical geometry and pneumatic agitation by bubbling suitable for carrying out the cultivation of plant cell suspensions, suitable for the recovery and replacement of the culture medium and suitable for the reuse of biomass, all in an environment permanent aseptic
  • Reusable materials such as glass and metal
  • pneumatic agitation designs are the most appropriate for plant cell culture.
  • the bioreactor is composed of three main parts assembled and sterilized which are the following:
  • Body container of circular cross-section with straight walls comprising at least two parts, a cylindrical part and a spherical part.
  • Cover cylindrical piece that closes the open upper part of the body.
  • Support piece that allows the support of the body and the cover.
  • the body comprises a porous base that separates the two parts thereof, the cylindrical and the spherical part, allowing the flow of fluids in a constant and uniform way between both parts (but not of particles larger than the pore) and comprises also at least two piped holes, one of them connected to an oil-free sterile air line with adjustable flow and the other to circulate liquids or gases; in order to supply air to the cell culture and perform pneumatic agitation of the same while maintaining its homogeneity and an effective mass transfer.
  • the lid comprises at least holes for filling, transfer of the culture and entry / exit of gases which allows the change of medium in the bioreactor under aseptic conditions.
  • the body comprises a plurality of cased holes for the connection of flexible tubes and the inlet and outlet of fluids.
  • the body in its cylindrical part, comprises a double wall that is topped with a horizontal flange that allows the support of the body in the support and of the cover on the body for the hermetic closing of the bioreactor.
  • the body in the double wall of the cylindrical part, comprises at least one inlet and one outlet orifice for a tempered liquid to circulate and thus exchange heat with the culture and keep its temperature constant. In any case, the temperature can be kept ambient by other means.
  • the body is transparent or opaque. In a particular embodiment, the body is made of glass or steel.
  • the lid has a plurality of holes to be able to fix it to the support and attach accessories, such as stopper, sampling and filling tubes.
  • the lid is made of stainless steel.
  • the support is a piece comprising at least four tubes, in equidistant parallel arrangement, welded at the end of support to the ground, to a tube in the form of incomplete circumference and on the other, to a solid ring with holes that allow the coupling with the lid.
  • the support is made of stainless steel.
  • the bioreactor is connected to reservoirs for the exchange of liquid. These reservoirs are connected by autoclavable silicone flexible tubes with the inputs and outputs of the bioreactor. By derivation of the tubes and valves, the flow of air can enter through the base of the bioreactor body to aerate the crop, or through the lid, producing in this case the emptying of the liquid into an empty reservoir. Reconfiguring the line again with the valves, the medium contained in a full reservoir it can be pushed by pressure towards the bioreactor and, entering through the base, lead to filling it. They are essential to carry out the operations of extraction and / or replacement of culture medium for which this design has been specifically devised.
  • the bioreactor is connected to an aseptic sampling device. This device can be homemade or commercial and only needs to be connected to the sampling tube assembled in the lid.
  • FIGURE 1 Bioreactor body design: exterior view and section showing the interior design.
  • FIGURE 2 Bioreactor lid design.
  • FIGURE 3 Design of the bioreactor support.
  • FIGURE 4 Assembled bioreactor parts.
  • FIGURE 5 Reservoir for fluid exchange with the bioreactor.
  • the bioreactor 4 comprises the following parts:
  • Body 1 single-piece hollow vessel of circular section with straight walls and curved bottom, ie hollow cylindrical shape like a hollow spherical cap.
  • the body comprises the cylindrical part 6 and the spherical 7, internally separated by the porous base 5, of glass or steel, thus delimiting the two aforementioned parts, whose porosity allows the flow of fluids between them, but not of particles of larger size to the pore.
  • the cylindrical part 6 has a double wall to delimit the compartment 8 with cased holes of inlet 9 and outlet 10 through which a tempered liquid can circulate to regulate the temperature of the cylindrical part 6.
  • the upper part of the body is finished off with a horizontal flange 11 which allows the support of the body in the support and the lid on it for hermetic closing once the parts are assembled.
  • the spherical part 7 of the body has at least two tubing holes 12 and 13 for the connection of flexible tubes and the inlet and outlet of fluids.
  • Cover 2 solid cylindrical metal piece of a few millimeters thick and a diameter somewhat larger than that of the upper edge of the body.
  • This lid has several round holes 14 to be able to screw it to the support and others to attach accessories, such as cap 15, sampling tubes 16, filling 17 and transfer of the crop 18.
  • the lid On the outer face, the lid has welded short hatches of the same material to connect flexible tubes for the addition of fluids 19 and gas inlet / outlet 20.
  • the cover On the inner side, has a peripheral groove for the coupling of a rubber or silicone O-ring to enable a tight seal with the body.
  • Support 3 metallic piece of four tubes of a few millimeters of section 21, in parallel arrangement, welded at one end (the ground support) to a tube that forms an incomplete circumference 22 and on the other, to a solid ring 23 of the same external diameter as the lid and internal diameter slightly larger than the body.
  • This ring 23 has several holes that coincide with those of the cover 14 so that they can be coupled by screws that pass through both parts.
  • the ring also has welded handles 24 to facilitate the transport of the piece and three screwed pieces 25 finished off in flexible material, such as Teflon, where it supports the body to avoid breaking the glass due to stress.
  • Preparatory sterilization operations are carried out separately from the bioreactor assembled with all accessory pipes and screws and all closed holes except the 20 where an air filter is attached.
  • the closure of the reservoir has two piped holes, one for air inlet / outlet 28, to which the air filter is coupled, and another for liquid inlet / outlet 27, to which a flexible tube of sufficient length to reach internally at the bottom of the bioreactor.
  • the three vessels, assembled bioreactor and the two reservoirs are disconnected from the air and water lines.
  • the bioreactor is connected by means of a bifurcated sterile silicone tube with a T between the holes 17 and 27 with the two reservoirs 26 filled with sterile medium.
  • the liquid from one reservoir 26 is transferred to the bioreactor by clamping the tube that goes to the other reservoir and connecting the air line to the air inlet / outlet 28 in the reservoir that has the free passage. To transfer the liquid from the other reservoir, it would be operated in the same way.
  • the oil-free sterile air line is connected to the tubed hole 13 allowing air to enter the spherical part 7, the passage of air through the porous base 5, which has a diffusing effect, and the passage of air diffused in the form of small bubbles to the cylindrical part 6 where they rise freely through the liquid producing the effect of aerated and agitated pneumatic.
  • the air that passes through the liquid leaves the cylindrical part 6 through the gas inlet / outlet 20, preventing overpressure in the container.
  • the remaining holes in the bioreactor are closed.
  • the sterile air line must provide a flow of at least 5 liters per minute for effective aeration and agitation.
  • the bioreactor is inoculated.
  • the air inlet 13 is temporarily disconnected and closed and the bioreactor is placed in an aseptic environment, such as a laminar flow hood.
  • the plug of the hole 15 of larger diameter than the rest is removed and a certain amount of water is poured through the bioreactor suspension of cells to be cultured.
  • the plant cells come from a cell suspension obtained by dispersion in liquid medium with moderate orbital agitation (100-150 rpm) of a callus of differentiated cells from in vitro cultured plant tissue (Vera-Urbina et al., 2013). The operation is facilitated with the help of a previously sterilized funnel.
  • hole 15 is closed again with its cap.
  • plant cells are grown in "batch" mode from a cell suspension inoculum.
  • the bioreactor is removed from the aseptic environment and the sterile air line is reconnected at 13.
  • the culture in the bioreactor is maintained with a continuous supply of air to favor the homogeneity and growth of the crop for the necessary time, until that it reaches the stationary phase of growth or a desired amount of biomass.
  • the culture is finally recovered by disassembling the lid and transferring it to another container.
  • the air line 13 can be saturated with water by passing it through a sterile water container, which would prevent losses of bioreactor volume during long periods of cultivation.
  • plant cells are grown in "fed batch” mode from a previous in situ culture.
  • the depleted nutrient medium is displaced from the bioreactor to an empty reservoir 26 by the emptying operation described previously.
  • the biomass is retained in the bioreactor on the porous base since the size of the cell aggregates is larger than the pores.
  • the bioreactor is filled with new nutrient-rich medium contained in another reservoir 26 by means of the filling operation described previously. Once filled, continue according to the "batch” crop description again. This cycle of emptying of depleted medium, supply of rich medium and technically growing can be repeated as many times as desired. After the last cycle, the crop is recovered as described for "batch" mode.
  • EXAMPLE 1 Cultivation of a Vitis vinifera cell suspension in batch mode.
  • the culture was done in duplicate. A quantity of drained fresh cells that had been cultured in shake flasks up to a stationary phase equivalent to 2.2% weight / volume was inoculated in the bioreactor containing 6675mL of culture medium whose composition is described in the literature (Bru, R., Selles, S., Casado-Vela, J., Belchi-Navarro, S., Pedre ⁇ o, M. A. (2006). Modified cyclodextrins are chemically defined glucan inducers of defense responses in grapevine cell cultures. J Agr Food Chem. 54 (1): 65-67). The temperature of the culture was kept constant at 24 ° C by flow of thermostated water through the double wall of the body.
  • EXAMPLE 2 Extracellular production of the trans-resveratrol compound in batch mode and batch mode fed by a Vitis vinifera cell suspension.
  • the culture temperature was kept constant at 24 ° C and the aeration also at a flow of 0.43 ⁇ . 1 .min "1.
  • a sample of the culture was periodically taken to measure the concentration of extracellular trans-resveratrol, in addition to variables related to the metabolic activity of the culture (sucrose, glucose, pH, conductivity) by methods described in the literature (Vera -Urbina et al., 2013)
  • the concentration of trans-resveratrol exceeded 3g / L, the nutrient-depleted and product-rich medium was replaced by fresh elicitation medium, by means of the "fed batch" embodiment In this way, 3 production cycles were performed, the results of which are summarized in Table 2.
  • trans-resveratrol obtained in elicitation medium (Lijavetzky et al. 2008) by batch-fed by direct inoculation (25% -m / v-) with vine cell suspensions (Vitis vinifera L. cvGamay) in the bioreactor.
  • EXAMPLE 3 Extracellular production of the trans-resveratrol compound in fed batch mode coupled to a batch mode culture of a Vitis vinifera cell suspension.
  • the two processes necessary for the production of the extracellular compound by plant cell culture are integrated into a single process operating only in a fed batch mode.
  • a stronger inoculum was used to shorten the biomass growth period, an amount of 580g of fresh cells drained that has been cultivated in shake flasks until stationary phase that was inoculated in the bioreactor containing 5300ml_ of culture medium giving a culture volume of 5827ml_ (inoculum equivalent to 10% weight / volume).
  • the culture temperature was kept constant at 24 ° C and the air flow was adjusted from initial 0.47 to 0.54 ⁇ . 1 .min "1 final. Growth and variables related to the metabolic activity of the culture were measured as in Example 1.
  • Table 3 Values of trans-resveratrol production obtained with vine cell suspensions in the middle of elicitation (Lijavetzky et al. 2008) in the bioreactor implementing a batch-fed production system from the biomass production coupling followed by elicitation .

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Abstract

La présente invention concerne un bioréacteur de type colonne à bulles pour mettre en oeuvre, dans des conditions aseptiques, la culture de cellules végétales en suspension, dont les caractéristiques de conception permettent en outre de récupérer le milieu de culture, de le remplacer par un autre milieu de culture et de réutiliser la biomasse restant dans ledit bioréacteur pour une opération de culture suivante. Le bioréacteur comprend trois parties principales, un corps, un couvercle et un support, qui doivent être assemblés, stérilisés et reliés à une conduite d'air stérile exempt d'huile par minute pour son fonctionnement.
PCT/ES2018/070152 2017-03-30 2018-03-01 Bioréacteur de type colonne à bulles pour culture de cellules végétales en suspension WO2018178434A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES201730479A ES2611958B2 (es) 2017-03-30 2017-03-30 Biorreactor tipo columna de burbujeo para cultivo de células vegetales en suspensión
ESP201730479 2017-03-30

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WO2018178434A1 true WO2018178434A1 (fr) 2018-10-04

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11299700B1 (en) 2021-02-19 2022-04-12 Acequia Biotechnology, Llc Bioreactor containers and methods of growing hairy roots using the same

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109810898A (zh) * 2019-03-27 2019-05-28 厦门鹭港兆康生物科技有限公司 一种细胞悬浮培养生物反应器及细胞悬浮培养的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08163981A (ja) * 1994-12-15 1996-06-25 Shiseido Co Ltd 植物の器官または細胞の培養方法およびその装置
WO2008135991A2 (fr) * 2007-05-07 2008-11-13 Protalix Ltd. Bioréacteur jetable à grande échelle

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08163981A (ja) * 1994-12-15 1996-06-25 Shiseido Co Ltd 植物の器官または細胞の培養方法およびその装置
WO2008135991A2 (fr) * 2007-05-07 2008-11-13 Protalix Ltd. Bioréacteur jetable à grande échelle

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAMACHANDRA RAO S ET AL.: "Plant cell cultures: Chemical factories of secondary metabolites", BIOTECHNOLOGY ADVANCES, vol. 20, no. 2, 1 May 2002 (2002-05-01), pages 101 - 153, XP004357338 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11299700B1 (en) 2021-02-19 2022-04-12 Acequia Biotechnology, Llc Bioreactor containers and methods of growing hairy roots using the same

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ES2611958B2 (es) 2017-10-18

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