Patent Grant
7790444
The present invention relates to a bioreactor for studying the effects of stimuli of physical, chemical, mechanical and electromagnetic nature on cellular activity, for applications in many fields among which: in tissue engineering for development of biological constructs; in the industrial field for pharmacological “testing”, and in the cosmetic field for studying allergologic reactions to the developed products.
It is well known that each biological tissue during its evolution and its normal activity is subject to physical and chemical stimuli that both determine its pathological and physiological status and affect its normal function. For this purpose, systems capable of reproducing physical or chemical stimuli have been sought in order to study its influence on the normal cellular activity.
Presently, real systems are known that reproduce a pressure stimulus, for studying the influence on the gangliar or endothelial cells. Other known systems reproduce a laminar flow, or turbulent flow, for simulating the permeation of nutrients through cellular membrane, as normally occurs in any biological tissue owing to blood flow.
Concerning isobar cell culture two systems are known:
So called flow bioreactors also exist, which provide a chamber for cell culture that is arranged in series with a nutrient flow system. The applications of such bioreactors are various, such as the study of pathologies, the regeneration of cardio-muscular tissues, the development of hepatic functional substitutes, the regeneration and the testing of cartilage.
Flow bioreactors have been studied for high density cultures. In fact, the flow of nutrients that passes through a bioreactor allows a much easier perfusion of the same and a most effective removal of the cellular catabolites. These systems increase the growth speed of cellular mono-layers up to a confluence from 100% up to 200% and optimize the function, the morphology and the differentiation of the cells.
On the market, the many bioreactors differ from one another essentially for a variety of types of the culture chambers:
The main limit of most of these systems is that they are not autonomous, since they require an incubator in order ensure required values of pH and temperature in the chamber. The presence of the incubator does not allow, in particular, the use of a computer for following in real time the progressive change of the parameters in order to adjust them during the experiment.
Bioreactors also exist where the presence of an incubator is not necessary; however, the structure of the chamber for cell culture does do not allow to follow the experiment in real time, by means of optical and/or fluorescence microscope, and then to determine the development of the cellular processes.
In conclusion, autonomous bioreactors do not presently exist that are at the same time capable of keeping the pressure the pH and the temperature in a culture chamber and to change it quickly in a controlled manner, as well as capable of generating in the cells a fixed flow of culture medium, with possibility of looking in real time at what is happening inside.
It is an object of the present invention to provide a device with the functions of a bioreactor that uses culture chambers that are easily modellable and conformable, and that allow the use of transducers and regulators, for monitoring in real time what happens in a culture chamber and for adjusting the parameters and the physical-chemical stimuli that are simulating physiological and/or pathological conditions.
Another object of the invention is to provide a device with the function of bioreactor where a flow of culture medium is present that allows a reduction of the amount of culture medium used for each experiment that is from 10 to 30 times less with respect to other devices present on the market, with considerable savings concerning both the amount of culture medium and the analysis of the substances in it contained.
A further feature of the present invention is to provide a bioreactor having means for conveying the culture medium in the culture chambers that do not damage the cells and cellular aggregates, do not have means for stirring, gas bubbling or other mechanical moving parts in the culture chamber.
These and other objects are achieved by a bioreactor for monitoring cellular activity in the presence of physical, chemical and mechanical stimuli, whose characteristic is of providing:
Advantageously, said means for controlling comprise a specially developed software, which by a graphic interface easy to operate by each user allows both setting the parameters of the experiment and looking in real time at what happens to the cells.
This way an analysis in real time is allowed of a culture chamber without having the need to use an incubator surrounding the culture chamber.
In particular, said culture chamber is made of silicone rubber and is shaped in such a way that a desired laminar flow of the culture medium is created that can be outlined by a computer aided design program.
Preferably said cell is made of at least two parts that can overlap, where at least one has a recess in such a way that once overlapped to the other a passage for the culture medium is provided.
Advantageously, along said passage for the culture medium at least one of said parts that can overlap provides a glass slide for laboratories, in order to allow a microscope observation of the implanted cells. Said two parts that can overlap can be pressed on each other by two stiff plates kept together by releasable coupling means.
Preferably, said means for conveying in a controlled manner said culture medium through said culture chamber comprises:
In particular, said premixing chamber comprises:
Preferably, said container of inert material is shaped as a glass flask.
Advantageously the means for operatively measuring the physiological parameters of the culture medium can comprise:
Advantageously, in and at the bottom of the premixing chamber, a conical frustum concave structure is present where said pH sensor is arranged, in order to preserve it from a direct contact with possible bubbles of gas, which is introduced in said premixing chamber for adjusting the flow of culture medium and keeping it at a predetermined pH.
Preferably, means are provided for operatively adjusting the physiological parameters of the culture medium comprising:
Preferably, the means for monitoring and controlling the physical-chemical stimuli applied to the cells in the culture chamber are selected from the group comprised of:
Advantageously, several culture chambers can be connected by means of ducts of predetermined length for simulating the behaviour of biological organs even complex, so that the cells contained in the chambers that are arranged upstream produce metabolites that, transported by the culture medium, feed the cells contained in the chambers that are arranged downstream.
Advantageously more chambers connected to each other are integrated on a single miniaturized support, in particular of stiff material, creating a circuit for the flow of culture medium that feeds, in a predetermined way and in succession, all the culture chambers.
Advantageously, said support of stiff material is made through a process of microforming, in particular, by photolithography and/or electroerosion.
Advantageously said support blocks the cells and the ducts with at least one glass slide of transparent material, allowing the microscope observation of the development of the cells contained in the chambers.
Advantageously, said inlet and outlet ducts of gas into/away from said premixing chamber can be operated by electrovalves driven by an electrical control unit.
Preferably, the bioreactor uses an electronic control unit for amplifying and filtering the electrical signals coming from the sensors, for measuring said physiological parameters of the culture medium, and located separately from said electrical drive control unit for the electrovalves, to avoid electromagnetic interferences.
The invention will now shown with the following description of an exemplary embodiment thereof, exemplifying but not limitative, with reference to the attached drawings wherein:
In
In particular, the device uses a sensorized premixing chamber 1 that has the task of preparing a culture medium that is used for feeding the cells, which are arranged in a culture chamber 2, for eventually observing the development of the cells by a microscope 40. The signals at the outlet of the sensors are transmitted to a signal amplifying and filtering control unit 50, which transmits the treated signals to a computer 52 that saves them by an I/O data acquisition board.
Said computer is connected to a control unit 51 that operates electrovalves 20, 21 and 22, which adjust the introduction of air and carbon dioxide in the premixing chamber. Said control unit 51 is connected to control unit 50 by an electrical cable 53, in order to eliminate the interference of the electric supply network with the signal amplification and filtering system.
The culture medium is drawn from the premixing chamber 1 by duct 4 and its flow is adjusted by a peristaltic pump 30. The culture medium crosses then culture chamber 2 and continues its path through duct 3, returning again in the premixing chamber 1. At the outlet of culture chamber 2 the duct has a pick up point 23 for spilling out a sample of culture medium to analyse. Immediately before, along the duct, a temperature sensor is provided 24 that transmits a signal to the control unit 50 by electrical cable 9.
Premixing chamber 1 comprises a pH sensor 2, which transmits a signal to the control unit 50 by electrical cable 10. Anther parameter determined in the premixing chamber is pressure, through a pressure sensor 1 that transmits a signal to the control unit 50 by electrical cable 10.
The premixing chamber contains a controlled environment by a controlled introduction of air through duct 7 and of CO2 through duct 8, which flow then in a duct 6. Such introductions are controlled respectively by electrovalves 21 and 22, operated by control unit 51. Premixing chamber 1 has also a gas outlet duct 5, which is also controlled by a servomechanism 20 operated by the same control unit 51, which allows keeping the pressure fixed in the bioreactor as imposed by the software.
The control of the physical-chemical and physiological parameters is carried out in order to follow the data imposed by the software through an algorithm of PID type, so that the system is steady, and this would not have happened if an ON/OFF control had been used, and corrections are made only when the system alone cannot turn back to the starting equilibrium, in order to reduce the effects coming from the outer environment and to simulate as far as possible an homeostasis of the cellular system.
The premixing chamber is described in more detail in
On the bottom of container 60, a base 62 is made of silicone rubber that has a frustum conical recess for reducing the space about pH sensor 64, to obtain a more accurate measure, avoiding that the added gas changes the pH.
The temperature of the culture medium 59 present in the container 60, is adjusted by a flow of liquid at a chosen temperature flowing in a duct 61 surrounding the container 60.
In particular, in
As shown in
Downstream from each cell a pick up point 152 and 153 can be provided for spilling out an amount of culture to analyse.
In
In the example described a culture medium inlet channel is used 102 to feed the hepatic cells in chamber 220, from which, through ducts 109 and 110 the culture medium reaches respectively the adipocytes in chamber 223 and the cells endothelial in chamber 222, connected in parallel. From these chambers cells, through the ducts 107, 108 and 105, the culture medium reaches hepatic cells in chamber 220 through duct 106 and through duct 104 running through the pancreatic cells in chamber 221.
The foregoing description of a specific embodiment will so fully reveal the invention according to the conceptual point of view, so that others, by applying current knowledge, will be able to modify and/or adapt for various applications such an embodiment without further research and without parting from the invention, and it is therefore to be understood that such adaptations and modifications will have to be considered as equivalent to the specific embodiment. The means and the materials to realise the different functions described herein could have a different nature without, for this reason, departing from the field of the invention. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.
| Number | Date | Country | Kind |
|---|---|---|---|
| PI2004A0046 | Jun 2004 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2005/001691 | 6/16/2005 | WO | 00 | 1/11/2007 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2005/123258 | 12/29/2005 | WO | A |
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