WO2018178434A1

WO2018178434A1 - Bubble column-type bioreactor for the cultivation of vegetable cells in a suspension

Info

Publication number: WO2018178434A1
Application number: PCT/ES2018/070152
Authority: WO
Inventors: Roque Bru Martinez; Juan Carlos VERA URBINA
Original assignee: Universidad De Alicante
Priority date: 2017-03-30
Filing date: 2018-03-01
Publication date: 2018-10-04
Also published as: ES2611958B2; ES2611958A1

Abstract

The invention relates to a bubble column-type bioreactor for carrying out the cultivation of vegetable cells in a suspension in aseptic conditions, the design characteristics of which also permit the recovery of the culture medium, the substitution thereof for another culture medium, and the reusing of the remaining biomass therein for a subsequent cultivation process. The bioreactor comprises three main parts - a body, a cover and a support - which are to be assembled, sterilised and connected to an oil-free, sterile air line every minute for the functioning thereof.

Description

BIORREACTOR TYPE BUBBLE COLUMN FOR CULTURE CELLS

VEGETABLES IN SUSPENSION

DESCRIPTION

Bubble column bioreactor for plant cell culture in suspension. FIELD OF THE INVENTION

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. STATE OF THE PREVIOUS TECHNIQUE

The use of bioreactors is widely used for the cultivation of microorganisms (bacteria, filamentous fungi, etc.) both on a laboratory scale and on an industrial scale. However, 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 Ι μηιοΙ 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).

To design an appropriate bioreactor for a particular bioprocess, it is necessary to know the biological system, both its cell growth pattern and its metabolism and the factors that influence them to understand the requirement of the cells on their physical and chemical environment.

In addition, it is necessary to optimize and control the operating parameters of the 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 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. However, a problem they present is the need for a homogeneous and constant aeration.

As for construction materials, there has been a tendency in recent years to use special plastics for the manufacture of single-use fermentation vessels, however, high consumable costs are only justified if the product obtained is of a high value, therefore, has only been generalized for the production of biopharmaceuticals (antibodies, vaccines, therapeutic proteins) by culturing mammalian and insect cells.

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.

In order to address this problem, the basic requirements for the cultivation of vine cells and for the production of the frans-resveratrol bioactive compound in a commercial bioreactor of mechanical agitation were studied. Based on this, a prototype of a low-cost pneumatic agitation by inverted cone-shaped bubble agitation bioreactor was designed and constructed and a comparative study with a commercial one was carried out (Vera-Urbina, JC; Sellés-Marchart, S .; Martínez-Esteso, MJ; Pedreño, MA and Bru-Martinez, R. (2013) Production of grapevine cell biomass (Vitis vinifera L. Gamay) and resveratrol in custom and commercial bioreactors using cyclodextrins and methyl jasmonate elicitors. In. Resveratrol: Sources , Production, and Health Benefits Publisher: Dominique Delmas pp19-39 Nova Science Publishers, Inc.). The results obtained demonstrate the feasibility of scaling flasks to low-cost bioreactors, but above all they show clear limitations in the production of biomass and bioactive compounds, due to the design shortcomings. In particular, this design could not meet the oxygen demand of the culture at high cell densities that occur in advanced stages of the growth cycle as well as under conditions of elicitation metabolite production. For all the above, it is necessary to carry out a new bioreactor design that presents the optimal characteristics for plant cell culture and that overcome the limitations in production of biomass and bioactive compounds, for which the change of culture medium is a requirement.

EXPLANATION OF THE INVENTION

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, with 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.

Characterized in that 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. In addition, 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. In a particular embodiment, the body comprises a plurality of cased holes for the connection of flexible tubes and the inlet and outlet of fluids. In a particular embodiment, 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.

In a particular embodiment, 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. In a particular embodiment, the body is transparent or opaque. In a particular embodiment, the body is made of glass or steel.

In a particular embodiment, 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.

In a particular embodiment, the lid is made of stainless steel.

In a particular embodiment, 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.

In a particular embodiment, the support is made of stainless steel.

In a particular embodiment, 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. In a particular embodiment, 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.

The invention provides a number of advantages:

· Design adapted to the needs of plant cell culture in suspension.

• Structural cost reduction compared to commercial models of agitated tank or airlift of similar size: any non-essential device / sensor is eliminated (eg pH stato).

• Reduction of operational cost compared to single-use models, especially suitable for obtaining products (biomass and / or metabolites) of low or moderate commercial value.

• Possibility of changing medium and reusing biomass aseptically without the need for an aseptic environment (eg laminar flow cabinet). BRIEF DESCRIPTION OF THE FIGURES

To complete the description that is being made and in order to help a better understanding of the features of the invention, the following figures are attached as an integral part of said description, which are illustrative and not limiting, which represent the following:

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.

DETAILED EXHIBITION OF REALIZATION MODES

For a better understanding of the invention, a detailed description of the parts that make up the different configurations that are presented has been developed. As seen in Figure 1, 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. 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. On the inner side, the cover 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.

The operation of the bioreactor is detailed below:

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. There are also two reservoirs 26 of hermetic closure and sufficient capacity to contain the entire operating volume of the bioreactor, both containing culture medium. 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.

Next, filling or emptying of the sterilized assembled bioreactor occurs. To do this, 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. These operations can be reversed at any time for the emptying of the bioreactor by changing the air line from 28 to 20, closing the hole 17 and using a sterile bifurcated silicone tube with a T connecting the bioreactor with an empty reservoir 26 between the holes 12 and 27. The emptying and filling cycle can be repeated as many times as desired. This embodiment is what makes it possible to change the medium in the bioreactor under aseptic conditions.

In the next step, the aerated and agitated tire of the full bioreactor is produced. Thus, 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. Considering the dimensions of the bioreactor, the sterile air line must provide a flow of at least 5 liters per minute for effective aeration and agitation.

Next, the bioreactor is inoculated. To do this, the air inlet 13 is temporarily disconnected and closed and the bioreactor is placed in an aseptic environment, such as a laminar flow hood. In that environment, 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. Finally, hole 15 is closed again with its cap.

Then, plant cells are grown in "batch" mode from a cell suspension inoculum. After inoculation 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.

In addition, plant cells are grown in "fed batch" mode from a previous in situ culture. After a batch 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. Once the medium has been emptied, 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.

Here are some examples of cultivation:

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., Pedreño, 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. Initially air was blown at a flow of 0.3 liters per liter of culture and minute (Ι. ¹ .min ^"1 ) and was adjusted to avoid cell sedimentation throughout the culture to a maximum value of 0.6 Ι . ¹ .min ^"1 . Periodically, the medium was transferred to an empty container (see embodiments) to measure the volume of cellular packing (VEC) as a measure of biomass by means of a scale attached to the body. Likewise, a sample of the culture was periodically taken to measure 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 result of the two replicas and the resulting parameters are shown in Table 1. On average, the culture took 28.5 days to reach the stationary phase, increasing the biomass by 7, 1 times. Table 1. Production values and kinetic parameters of the growth of vine cell biomass in the bioreactor. TC: cultivation time, VO: operating volume, PF: fresh weight, FM: Multiplication factor, td: Duplication time.

EXAMPLE 2: Extracellular production of the trans-resveratrol compound in batch mode and batch mode fed by a Vitis vinifera cell suspension.

A quantity of 1395g of drained fresh cells that had been grown in shake flasks until stationary phase was inoculated in the bioreactor containing 4500ml_ of elicitation medium whose composition is described in the literature (Lijavetzky, D., Almagro, L, Belchi-Navarro, S., Matínez-Zapater, JM, Bru, R., Pedreño, MA (2008). Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures. BMC Research Notes. 1: 132), giving a final volume of 5500ml_ of culture (inoculum equivalent to 25.3% weight / volume). The culture temperature was kept constant at 24 ° C and the aeration also at a flow of 0.43 Ι. 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) When 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. The average duration of the cycles was 4.2 days with an average production of 15.2 g / cycle. 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.

Using the advantageous bioreactor design, 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 Ι. ¹ .min ^"1 final. Growth and variables related to the metabolic activity of the culture were measured as in Example 1. After 8 days of growth, 26.6% VEC (equivalent to 18.4% weight / volume) was reached. At that time, the growth medium is replaced by fresh elicitation medium, by means of the batch-fed embodiment, proceeding from here in the same way as in Example 2, so that 4 cycles were performed of production whose results are summarized in Table 3. The fourth cycle was very unproductive, so the yields were calculated on cycles 1 to 3. The average duration of the cycles was 5.6 days with an average production of 1 1, 9 g / cycle.

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 .

Production Productivity Specific production

Time of

Cycle

elicitation

production

(days)

(mg.mL ¹ ) (mg) (mg.ml days ¹ ) (mg. g biomass ^"1 ) (mg. g substrate ^{" 1} )

Cycle 1 6 3.57 ± 0.04 14 867.8 ± 169.3 0.60 ± 0.01 14.0 ± 0.2 165.2 ± 1.9

Cycle 2 5 2.48 ± 0.13 10 679, 1 ± 539.7 0.50 ± 0.03 10.0 ± 0.5 118.7 ± 6.0

Cycle 3 6 2.35 ± 0.03 10 018.6 ± 144.4 0.39 ± 0.01 9.4 ± 0.1 111.3 ± 1.6

Cycle 4 4 0.23 ± 0.01 1 018.4 ± 18.5 0.06 ± 0.00 0.96 ± 0.02 11.32 ± 0.2

Average

5.6 ± 0.6 2.80 ± 0.67 11 856.7 ± 2,630 0.49 ± 0.10 11.1 ± 2.5 131.7 ± 29.2 (cycles 1-3)

Claims

Bubble column bioreactor for suspension plant cell culture comprising three main parts assembled and sterilized:

• Cover: cylindrical piece that closes the open upper part of the body.

• Support: piece that allows the support of the body and the cover.

Characterized in that the body comprises a porous base that separates the two parts thereof, the cylindrical and the spherical part; and also comprises at least two piped holes, one of them connected to a sterile oil-free air line with adjustable flow and the other for the circulation of liquids or gases. In addition, the lid comprises at least holes for filling, culture transfer and gas inlet / outlet.

Bubble column bioreactor for the cultivation of plant cells in suspension according to claim 1, wherein the body comprises a plurality of tubed holes for the connection of flexible tubes and the entry and exit of fluids.

Bubble column bioreactor for growing plant cells in suspension according to claim 1, wherein the body, in its cylindrical part, comprises a double wall that is topped with a horizontal flange.

Bubble column bioreactor for cultivation of plant cells in suspension according to claim 1, wherein the body, in the double wall of the cylindrical part, comprises at least one inlet orifice and one outlet for circulating a tempered liquid.

Bubble column bioreactor for suspension plant cell culture according to claim 1, wherein the body is transparent or opaque.

Bubble column bioreactor for growing plant cells in suspension according to claim 1, wherein the body is made of glass or steel.

Bubble column bioreactor for growing plant cells in suspension according to claim 1, wherein 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.

8. Bubble column bioreactor for growing plant cells in suspension according to claim 1, wherein the lid is made of stainless steel.

9. Bubble column bioreactor for cultivation of plant cells in suspension according to claim 1, wherein the support is a piece comprising at least four tubes, in equidistant parallel arrangement, welded at the end of ground support, to a tube in Incomplete circumference shape and on the other, to a solid ring with holes that allow the coupling with the lid. 10. Bubble column bioreactor for suspension plant cell culture according to claim 1, wherein the support is made of stainless steel.

1 1. Bubble column bioreactor for culture of suspended plant cells according to claim 1, wherein the bioreactor is connected to reservoirs for the exchange of liquid by means of autoclavable silicone flexible tubes with the bioreactor inlets and outlets.

12. Bubble column bioreactor for plant cell culture in suspension according to claim 1, wherein the bioreactor is connected to an aseptic sampling device through the sampling tube of the lid.