WO2011051523A2

WO2011051523A2 - Cell-culture flow bioreactor

Info

Publication number: WO2011051523A2
Application number: PCT/ES2010/070651
Authority: WO
Inventors: Pedro Moreo Calvo; Victor ALASTRUÉ VERA; Carlos Marzo Mainar
Original assignee: Ebers Medical Technology; Universidad De Zaragoza
Priority date: 2009-10-26
Filing date: 2010-10-08
Publication date: 2011-05-05
Also published as: WO2011051523A3; ES2372108B1; ES2372108A1

Abstract

Cell-culture flow bioreactor that comprises an incubator (1), the receptacle (14) of which, to which access is gained via a hermetically sealed door (13), is defined by thermally insulating walls, and which is provided with a system for pumping fluid that circulates via the interior of a plurality of culture circuits where the cell-seeded scaffolds are housed, which comprises: - at least one motor (6, 7) for actuating at least one impulsion means (8, 20) arranged in the exterior of the incubator (1); - at least one fluid-impulsion means (8, 20) arranged in the interior of the incubator; - and a plurality of hermetic motor/impulsion-unit connection means that traverse a plurality of through-holes in at least one of the walls of the incubator (1).

Description

FLOW BIORREACTOR FOR CELL CULTURE

DESCRIPTION

OBJECT OF THE INVENTION

The invention relates to a bioreactor for tissue engineering, which, formed by a fluid pumping system integrated in an incubator, allows the controlled application of fluid flow over or through cell cultures housed in a culture circuit, guaranteeing conditions suitable for cell development, of temperature, composition of the atmosphere inside the incubator, diffusion of nutrients in the culture and mechanical stimulation of the cells, while, thanks to the placement of the pumping system drive out from! Incubator receptacle, the introduction of motors inside is avoided, reducing the risk of contamination, eliminating the possibility of overheating the crop and increasing the useful space in the receptacle.

FIELD OF APPLICATION OF THE INVENTION

The present invention is included in the technical field of apparatus for cell culture, in particular, equipment for the realization of cell cultures in tissue engineering.

BACKGROUND OF THE INVENTION

Tissue engineering is fitted within bioengineering discipline whose main purpose is the in vitro regeneration of biological substitutes that can be implanted in the human body so ^'as to improve or restore function of organs and tissues. Tissue regeneration by this technique usually requires the cultivation of cells on three-dimensional substrates, using scaffolds that support cell proliferation. The realization of this type of three-dimensional cultures presents particular problems in comparison with the cell culture on flat surfaces, some of the most important being the difficulty to maintain an adequate distribution of nutrients in a constant way throughout the crop and the need to apply on the mechanical stimulus cells that reproduce the natural conditions of cell development in vivo, for which devices known as tissue engineering bioreactors are used whose main purpose is to provide the conditions that enable and optimize tissue regeneration including physicochemical conditions, of temperature, pH, availability of nutrients and mechanical stimulation.

Regarding the latter, there are several methods to mechanically stimulate cell cultures, and bioreactors can be classified for tissue engineering according to the type of technology used for this purpose. One of the types is known as flow bioreactors, which use the application of a flow of culture medium through the scaffolding in which the crop is developed - perfusion - or around the surface of the themselves - periphery—, to mechanically stimulate cells, which also leads to an improvement in the supply of nutrients and the elimination of waste substances resulting from cellular metabolism.

Therefore, tissue engineering flow bioreactors are constituted by a culture chamber in which the scaffolds that support the cell culture are housed, a system of tubes or conduits, reservoirs or auxiliary elements, which serve for the storage, renovation and transport of the culture medium to and from the culture chamber. In addition, in order to circulate the medium through the interior of the culture circuit and control the flow that is passed through the scaffolding, a pumping system is used, comprising, for example, a peristaltic, syringe or other pump, driven by a drive system, consisting of an electric motor. In this sense, you can mention as relevant examples documents US20080311650-A1 and US0064 6995-B1.

However, currently existing flow bioreactors are not able to provide the rest of the necessary conditions that allow cell culture, specifically, they are not able to maintain constant temperature conditions or control the atmosphere in which the cultures are carried out, so that it is essential to introduce the bioreactor in a CO2 incubator that guarantees conditions of temperature and composition of atmospheric gases suitable for cultivation. Said incubator has a receptacle inside which the bioreactor is arranged, in which the composition of the atmosphere and the temperature are permanently controlled in the ranges that allow the survival of the cell cultures. To obtain this temperature, the incubators have a control system that only allows the inside of the receptacle to be heated, since this is of a value normally higher than the temperature of the laboratories where the incubators are arranged (approximately 37 ° C), of so that the desired temperature for the crop is reached and maintained, being also equipped with thermal insulation systems that minimize heat losses to the outside. Therefore, it is clear that incubators are only capable of obtaining a certain temperature in their receptacle when it is necessary to heat their interior.

Currently, and that are known by the applicant, all commercial flow bioreactors intended for cell culture in tissue engineering are based on the introduction of culture circuits together with pumping systems inside incubators, which raises several drawbacks Significant factors associated with maintaining the appropriate environmental conditions for the crop described below:

First, the continuous operation of the pumping system for periods of several weeks or even months of typical duration of tissue engineering cultures causes overheating in the incubator receptacle, since it is unable to dissipate the heat generated by the drive motor of the pumping system, with the immediate consequence of the overheating of the incubator receptacle and the consequent death of the culture cells, since they are unable to withstand high temperatures.

- Secondly, it is necessary to note that the power supply of the bioreactor drive systems that are introduced inside the incubator is carried out by means of connection to the power grid, given the length of the cultivation periods. However, commercial - incubators are not prepared for the passage of cables from the outside to the inside of their receptacle, so the introduction of electrical cables, necessary for feeding the motors of the pumping system, has to be done through the incubator door, preventing its good closing, losing the tightness of the system, generating leaks and increasing the risk of contamination.

- Third, the atmosphere typically corrosive existing in the receptacle from the incubator, due to the usual presence of CO ₂ and water vapor, can seriously damage to engines used in operating the pumping system, unless choose models specifically designed for use in corrosive environments, high cost.

- Finally, the introduction of the drive system inside the incubator presents the added drawback of the decrease in useful space that could be used for other purposes.

These drawbacks seriously compromise the viability of the crops made with this type of systems and imply the presence of limitations of a practical nature, so it would be desirable to have a tissue engineering flow bioreactor that, controlling the environmental parameters necessary to develop the cell cultures and allowing to apply fluid flow in a controlled way on scaffolding, solved the inconveniences associated with the need to introduce motors inside an incubator. EXPLANATION OF THE INVENTION

The present invention relates to a flow bioreactor that allows the controlled application of a fluid flow on cell cultures through the use of pumping systems integrated in an incubator, guaranteeing the maintenance of conditions suitable for cell development during periods of prolonged cultivation solving the aforementioned drawbacks, the distinguishing details being properly set out in the independent and dependent claims that accompany the present specification.

The bioreactor of the invention comprises:

- an incubator, whose receptacle, which is accessed through a hermetically sealed door, is limited by thermal insulating walls,

- a fluid pumping system, comprising:

or at least one drive motor of at least one drive means, disposed outside the incubator, or at least one fluid drive means disposed inside the incubator,

or a plurality of hermetic motor-impeller connection means that pass through a plurality of through holes located in at least one of the walls of the incubator.

Specifically, the bioreactor of the invention comprises an incubator provided with a heating system or a cooling system that allows to regulate the temperature inside. The incubator preferably further comprises a system for regulating the presence of gases in the atmosphere inside such as C0 ₂ and water vapor. This incubator has one or more through holes, as many as desired, in one or several of its walls, through each of which airtight connection means is passed as an axis that completely crosses the wall of the incubator, sealing the hole existing between each axis and the edge of the hole through which It passes through a gasket, airtight seal, in such a way that the loss of heat is minimized and the passage of gases through the holes is prevented, while allowing the rotation of the shafts. Said shafts are preferably made of a corrosion resistant material.

The drive of each axis is carried out by means of a motor, preferably electric, which is arranged in. the outside of the incubator and connects to the outer end of the shaft by means of a coupling. In turn, the inner end of each axis is designed so that an actuator or impeller means that allows the fluid flowing through each of the culture circuits that are introduced into the incubator receptacle to be coupled. This actuator or drive means may consist, for example, of a peristaltic pump head into which a pipe section of! cultivation circuit Preferably, the connection between the actuator or drive means and the inner end of the shaft should be non-permanent, so that the actuator or drive means can be removed for cleaning, sterilization and replacement.

Each of the assemblies formed by a driving means, a shaft and drive system constitutes a pumping subsystem, which can be dependent or independent depending on whether they share the same drive system or motor or not, respectively. Importantly, the bioreactor can have several pumping subsystems, which allows the use of several culture circuits simultaneously. In this case, the subsystems must be positioned on the walls of the incubator in such a way that each drive means can be accessed without being disturbed by the excessively close presence of a drive means of another adjacent pumping subsystem.

The fundamental advantages provided by the equipment of the invention are given by the placement of the drive motors outside the incubator. Thanks to this arrangement, the problem of excessive heat generation in the receptacle of the same due to the continuous operation of the motors is solved and the need to introduce electric cables through the closure of the door is also eliminated, thus ensuring the tightness of the system. Likewise, it With this invention it is possible to increase the useful space in the incubator receptacle and avoid the need to use specially designed motors to withstand the presence of corrosive environments.

In addition, as indicated above, it should be remembered that the purpose of the use of the pumping system is due, among other reasons, to the need to apply mechanical stimulation on the cells of the culture, which is achieved by applying fluid flows to Through or around the scaffolds on which the cells are grown, it is necessary to precisely regulate the flow rate of said flow depending on the type of culture that is performed. In this aspect, the invention included here has an electronic control system that, by independently regulating the rotation speed of each of the drive motors of the pumping system, allows the application of flow rates , continuous or variable over time, in the culture circuits, as indicated by the user. Specifically, a microcontroller or a programmable logic controller is preferably used to perform this electronic control using as an interface a data input medium and a data display medium, such as a touch screen integrated in the incubator itself or, alternatively, a computer. In any case, the equipment may be equipped with any other control system that allows to regulate the flow driven by the pumping system.

Therefore, it is clear that the proposed system clearly implies an improvement over the other products on the market, since, allowing to apply flow in a controlled manner on cell cultures housed in circuits located inside an incubator, avoids the associated problems to the introduction of bioreactors driven by motors inside it, which opens a wide range of possibilities.

Thus, the proposed equipment provides a wide range of applications within the cell culture and can be used, for example, for the realization of tissue regeneration cultures, provided that it is used together with a culture circuit adapted to the specific needs of the specific tissue that you want to regenerate.

The bioreactor described for the realization of cell cultures under flow conditions represents, therefore, an innovative team of structural characteristics unknown until now for this purpose, reasons that together with its practical utility, provide it with sufficient foundation to obtain the privilege of exclusivity that It is requested.

DESCRIPTION OF THE DRAWINGS

To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, this descriptive report is attached, as an integral part thereof, of a set of drawings, in which with character Illustrative and not limiting, the following has been represented:

Figure 1 shows a schematic front view of the object of the invention, particularized in the case of having two pumping subsystems, in which the driving means of! pumping system so that the arrangement of the axes of said system in the incubator receptacle can be appreciated, in an example in which the control system of the pumping system is integrated in the incubator.

Figure 2.- Shows a schematic view of the rear part of the object of the invention, particularized in the case of having two motors and two driving means, where the arrangement of the motors that drive each of them is appreciated. axes that cross the wall of the incubator.

Figure 3.- Shows a view of the arrangement of two culture circuits inside the system object of the invention, particularized in the case of having two motors and two driving means, specifically, two peristaltic pump heads. Figure 4.- Shows a sectional view of! system object of the invention particularized in the case of having two motors and two driving means, in which the main parts and elements of the pumping system are appreciated, as well as the presence of two culture circuits in the incubator receptacle.

PREFERRED EMBODIMENT OF THE INVENTION

In view of the aforementioned figures, and according to the numbering adopted, two examples of preferred embodiment of the invention described below can be observed, which are not intended to be limiting of their scope.

Preferred Embodiment 1

In this preferred embodiment, a version of the equipment is shown in which the pumping system comprises two motors and two driving means. Thus, as can be seen in Figures 1 and 2, the equipment comprises an incubator (1), into whose receptacle (14) the culture circuit (s) will be inserted. The walls of the incubator (1) are thermal insulators and incorporate a heating system that allows heat to be transmitted to the receptacle (14). Access to the receptacle (14) is carried out by means of a door (13). In the rear wall of the incubator (1) there are two axes (4 and 5) that cross the wall of the same being connected to two motors (6 and 7), preferably electric, located outside the receptacle, responsible for printing movement to axes.

The movement is transmitted to the interior receptacle (14) by the shafts (4 and 5), preferably made of a corrosion-resistant material, such as stainless steel, which pass through sealed holes with airtight gaskets (15 and 16), which They minimize thermal losses and at the same time prevent the motors (6 and 7) from being damaged due to the corrosive atmosphere inside the receptacle (14). The inner end of the axes (4 and 5) It is equipped with the appropriate form so that the impeller means that is desired to be used to drive the fluid in the culture circuits can be coupled thereto.

Therefore, it is remarkable to note that the fact of arranging the motors (6 and 7) outside the receptacle (14) where the cultivation circuits are to be located, solves the problem caused by the generation of heat due to the operation of the drive motors Likewise, the arrangement of the motors (6 and 7) on the outside of the receptacle (14) prevents them from being damaged due to the corrosive atmosphere that can occur inside it and having to introduce electric power cables that damage the door seal (13).

The control of. engines (6 and 7) are performed electronically, the control system hardware being located in a compartment (2) arranged in the upper area of the equipment, where a touch screen (3) is also provided on its front face, through of which the operating parameters of the pumping system can be entered. In the upper compartment (2) the electronics of the temperature control system of the receptacle (14) are also provided and, in the event that the equipment incorporates it, the control system of the composition of the atmosphere of the receptacle (14) ,,

Preferred Embodiment 2

A second preferred embodiment of the present invention is presented in Figures 3 and 4, in which the equipment of the particularized preferred embodiment 1 is shown so that as pumping means (8 and 20) pump heads are used peristaltic, as well as culture circuits composed each of them by tube (10 and 18), culture chamber (12 and 17) and reservoir (11 and 19). In addition, a tray (9) has been used that allows the volume of the receptacle (14) to be separated into two parts, upper and lower, so that in each of them a culture circuit can be placed. Independent. A section of the tube (10 and 18) of each culture circuit is introduced into the corresponding drive means (8 and 20). In this way, when the motors (6 and 7) drive the driving means (8 and 20), the culture medium is circulated that circulates inside the tubes (10 and 18) from the reservoirs (11 and 19) towards the culture chambers (12 and 17), where the scaffolds sown with cells are housed, and from there back to the reservoirs (11 and 19). Using the touch screen (3) you can choose the application of time-varying flow rates, so that the mechanical stimulation that is exerted on the cells when passing flow of culture medium through or around the scaffolds is more intense .

Describing sufficiently the nature of the present invention, as well as the way of putting it into practice, it is not considered necessary to make its explanation more extensive so that any person skilled in the art understands its scope and the advantages that derive from it, stating that , within its essentiality, may be implemented in other embodiments that differ in detail from those indicated by way of example, and to which it will also achieve the protection that is sought as long as it does not alter, change or modify its principle fundamental.

Claims

1. - Flow bioreactor for cell culture, comprising an incubator (1), whose receptacle (14), which is accessed through a hermetically sealed door (13), is limited by thermal insulating walls, and which is equipped with a fluid pumping system that circulates inside a plurality of culture circuits, where the cell-scaffolds are housed, characterized in that the pumping system comprises:

- at least one drive motor (6.7) of at least one drive means (8, 20), arranged outside the incubator (1),

- at least one fluid driving means (8, 20) disposed inside the incubator,

- a plurality of hermetic motor-impeller connection means that pass through a plurality of through holes located in at least one of the walls of the incubator (1),

2. - _; Flow bioreactor for cell culture, according to claim 1, characterized in that the pumping system comprises at least one independent pumping subsystem comprising a drive motor (6, 7) connected by motor-impeller connection means to a driving means ( 8, 20).

3. - Flow bioreactor for cell culture according to claim 1, characterized in that the pumping system comprises at least one dependent pumping subsystem comprising a drive motor connected by motor-impeller connection means to a plurality of driving means .

4- Flow bioreactor for cell culture, according to claims 1 and 2, characterized in that the motor-impeller connection means comprise an axis (4, 5) that completely crosses the wall of the incubator and is connected at one end to the motor and on the other to the driving means, and a gasket of <sealing sealing of the gap between each axis and the edge of the hole through which it passes.

5- Flow bioreactor for cell culture, according to claim 4, characterized in that the motor-impeller connection means comprise a coupling means from the outer end of the shaft to the motor (6, 7) and a coupling means from the inner end of the shaft to the corresponding drive means (8, 20).

6. - Flow bioreactor for cell culture, according to previous claims, characterized in that the drive motors (6,7) are electric motors.

7. - Flow bioreactor for cell culture, according to claims 1-6, characterized in that the driving means of the pumping system (8,20) is a peristaltic pump head.

8. - Flow bioreactor for cell culture, according to claims 1-6, characterized in that the driving means of the pumping system (8,20) is a syringe pump.

9. - Flow bioreactor for cell culture, according to previous claims, characterized in that the rotation of the motors is controlled by an electronic control system regulating independently of the flow rate provided by each pumping subsystem.

10. - Flow bioreactor for cell culture, according to claim. 9, characterized in that the control system of the motors of the pumping system is fully integrated in the incubator, the electronic components thereof being placed in a compartment inside the incubator (2), and outside of which means are provided data input (3) for the introduction, by the user, of the operating parameters of the pumping system.

11. - Flow bioreactor for cell culture, according to claim 10, characterized in that the data input means is a touch screen (3).

12. - Flow bioreactor for cell culture, according to claims 9 and 10, characterized in that the control system of the motors of the pumping system is a unit selected from a microcontroller, a programmable logic controller or a computer.

13. - Flow bioreactor for cell culture, according to previous claims, characterized in that the incubator comprises a system for regulating the atmosphere of the receptacle, controller of, at least, the concentration of C0 ₂ .

14-- Flow bioreactor for cell culture, according to claims 1-13, characterized in that the incubator comprises a heating system inside.

15.- Flow bioreactor for cell culture, according to claims 1-13, characterized in that the incubator comprises a cooling system inside. ^'