System for studying biological material also by positron annihilation spectroscopy

Abstract

A system for studying biological material by microscopic and spectroscopic techniques comprising a bioreactor (10) having a culture chamber; said bioreactor (10) comprises a lower body (11 ) and an upper body (12) that can be joined to each other; a seal (27) interposed between said lower body (11 ) and said upper body (12); said lower body (11 ) comprises a through hole (17) closed by a membrane (19); said upper body (12) comprises a through hole (23) closed by an element transparent to visible radiation (28); said seal (27) delimits said culture chamber; said culture chamber being connected to two fluidic channels (18) which extend externally to said lower and upper bodies (11 and 12) via the through holes (25).

Description

[0001] SYSTEM FOR STUDYING BIOLOGICAL MATERIAL ALSO BY POS ITRON ANN IH ILATION SPECTROSCOPY

[0002] DESCRIPTION

[0003] The present invention refers to a system for studying biological material also by positron annihilation spectroscopy.

[0004] Inflammation of the vascular tissue is often one of the first alarm signals for various pathologies, including diabetes, myocardial ischem ia, Alzheimer’s disease and chronic diseases of the veins and arteries. Early detection is therefore essential for timely diagnosis and effective treatment, before the disease progresses and has serious consequences for the patient. Accordingly, the development of models that allow real-time study of the inflammatory phenomena is an objective in the search for more effective diagnostic methods than those currently available.

[0005] For this reason, in vitro cell cultures, chicken embryo models and bioreactors are used, for example, the latter including m icrofluidic systems for maintaining cell vitality, or appropriate biological tissue, providing a continuous, pulsed or variable flow of culture medium or other appropriate solution.

[0006] These chambers are often optically accessible and allow inspection of the cell culture or tissue morphology or other important properties via optical inspection techniques such as optical microscopy.

[0007] The object of the present invention is to provide a system that allows, at the same time or at different times, the study of wet biological material, such as live cells, fixed cells, or appropriate tissue or biological matrix, both via techniques based on particle-matter interaction and techniques based on light-matter interaction.

[0008] The object of the present invention is therefore the use of techniques such as positron annihilation or transm ission / scanning electron m icroscopy to allow the study of cells or other biological matrices.

[0009] A further object is to provide a system that allows the study of cells, or other biological matrix, via optical techniques, such as optical m icroscopy.

[0010] In accordance with the present invention, said objects and others are achieved by a system for the study of biological material by means of m icroscopic and spectroscopic techniques comprising a bioreactor having a culture chamber; said bioreactor comprises a lower body and an upper body that can be joined to each other; a seal interposed between said lower body and said upper body; said lower body comprises a through hole closed by a membrane; said upper hole comprises a through hole closed by an element transparent to visible radiation; said seal delim its said culture chamber; said culture chamber being connected to two fluidic channels, said bioreactor comprising through holes for the external connection of said fluidic channels.

[0011] Further characteristics of the invention are described in the dependent claims.

[0012] This solution offers various advantages with respect to the solutions of the known art.

[0013] The system allows study of the cellular and vascular processes or other relevant biological processes via the use of positron annihilation spectroscopy or other spectroscopy technique, transm ission / scanning electron m icroscopy and optical m icroscopy in vitro and in vivo.

[0014] In particular, the solution allows, via the use of a particle beam such as a positron particle beam , the exam ination of a layer of cells, biological tissues or appropriate biological matrices. By using the Positron Annihilation Spectroscopy (PAS) technique and evaluating the lifetime of the particles (Positron Lifetime Annihilation Spectroscopy - PLAS) or the quantity of motion of the electron-positron pair at the time of the annihilation phenomenon (Doppler Broadening Spectroscopy - DBS) it is possible to obtain information on the m icroscopic structure and on the electronic or physical environment of cells or tissues. In particular, the positron beams are able to modulate the im plantation energy of the particles within the sample, and consequently the depth at which the annihilation phenomenon occurs. In this way, with implantation energy in the range 1 -20 keV, for example, the majority of the annihilation events take place at the level of the biological material within the bioreactor.

[0015] The solution would allow various information to be obtained, for example on the state of health of a biological tissue, evaluating its integrity, and providing an indirect measurement of the presence of an inflammatory or pathological condition. The use of the above-mentioned techniques, appropriately integrated with the device, therefore allow non-destructive m ini-invasive intravital exam ination using methodologies which, at present, cannot be used together. The device consists of a main body made of plastic or appropriate biocompatible material, divided into two parts which are joined to each other, form ing a fluidic culture chamber able to maintain sterile conditions. In the lower part is a thin housing for a membrane permeable to the particles such as, for example, positrons / electrons, resistant to mechanical and chem ical / physical stress, and able, for example, to isolate the biological material from the vacuum system necessary for the propagation of the monoenergetic particle beam used for the positron annihilation spectroscopy analysis. For use with electron m icroscopy, said membrane positioned in the above housing (window) is pierced and covered with appropriate material, for example graphene. The material constituting the membrane can be functionalized so as to facilitate growth and adhesion of the cells or other biological material thereon. Survival of the biological material within the bioreactor culture chamber is guaranteed by the dynam ic circulation, for example, of a suitable culture medium and by maintenance of the optimal temperature (37°C) due to loading of the two body portions (upper and lower) with metal alloys. In the upper portion of the device the presence of a material transparent to visible radiation, for example glass, allows exam ination of the sample by means of optical m icroscopy simultaneously with the spectroscopy measurements.

[0016] The characteristics and advantages of the present invention will be evident from the following detailed description of a practical embodiment thereof, illustrated by way of non-lim iting example in the attached drawings, in which: figure 1 shows a device for the study of biological material, for example cells, seen in an exploded view, according to the present invention; figure 2 shows a lower component of a device for the study of biological material, for example cells, seen from above, according to the present invention; figure 3 shows a lower component of a device for the study of biological material, for example cells, seen from below, according to the present invention; figure 4 shows an upper component of a device for the study of biological material, for example cells, seen from above, according to the present invention; figure 5 shows an upper component of a device for the study of biological material, for example cells, seen from below, according to the present invention; figure 6 shows a flanged device for the study of biological material, for example cells, seen in an exploded view, according to the present invention.

[0017] Referring to the attached figures, a system for the study of biological material, such as cells maintained vital or other suitable biological matrix, according to the present invention, comprises a device form ing the culture chamber, namely the bioreactor 10.

[0018] The bioreactor 10 is composed of two main components, a lower body 1 1 and an upper body 12 which can be joined to each other.

[0019] The shape of the bioreactor is circular but other shapes are possible.

[0020] The lower body 1 1 has on its upper surface, which will be the surface inside the bioreactor 10, a circular containment edge 13 having an opening 14, an inner circular ring 15 coaxial with the edge 13, sunken in the lower body 1 1 , therefore two circle arcs 16 opposite each other, protruding from the lower body 1 1 and inside the ring 15. It also has a central through hole 17 which is connected to two or more fluidic channels 18, in this case two, opposite each other, sunken in the lower body 1 1 , which extend passing between the openings between the two circle arcs 16.

[0021] The lower body 1 1 comprises on the lower surface a housing which circumscribes the hole 17, for fixing, for example by means of glue or other material or appropriate fastening technique, a membrane 19 which is resistant to mechanical and physical / chem ical stress, impermeable to liquids and is an electrical insulator.

[0022] The upper body 12, which will have a dimension such as to be contained inside the edge 13, comprises a tab 20 which will be positioned in the opening 14 of the lower body, for correct alignment of the two bodies 1 1 and 12 of the bioreactor 10.

[0023] The upper body 12 has on its lower surface which will be the surface inside the bioreactor 10, a circular ring 21 , sunken in the upper body 12 positioned at the ring 15, two circle arcs 22, sunken in the upper body 12, opposite each other, inside the ring 21 , positioned at and adapted to cooperate with the circle arcs 16. It also has a central through hole 23 which is connected to two fluidic channels 18 opposite each other, sunken in the upper body 12, which extend passing between the openings located between the two circle arcs 22, and positioned at and adapted to cooperate with the two fluidic channels 18.

[0024] The bioreactor 10 comprises through holes 25 for the external connection of the fluidic channels 18. In particular, the through holes 25 are configured to fluid-dynam ically connect the fluidic channels 18 to the outside of the bioreactor 10, namely the bodies 1 1 , 12. To summarise, therefore, the two fluidic channels 18 term inate in two through holes 25 connected to two tubes 26 positioned on the upper surface of the upper body 12. Around the central hole 23 and the two fluidic channels 18 is a seal 27. The seal 27 has a shape that follows and surrounds the conformation of the central hole 23 and the two fluidic channels 18, and therefore delim its the culture chamber. In particular, it is circular centrally at the hole 23 and extends with two channels along the same axis in the two opposite directions until reaching the two through holes 25.

[0025] The upper body 12 comprises on its upper surface a housing that circumscribes the hole 23, for fixing, by means of glue, an optically transparent element, in this case a transparent slide 28. The lower body 1 1 and the upper body 12 can comprise various through holes, in this case eight 29. To join them , an appropriate clamping method has been chosen, in this case by means of bolts. Alternatively, glue or heat welding can be used to fix the two bodies 1 1 and 12.

[0026] In the ring 15, circular and sunken in the lower body 1 1 and in the corresponding ring 21 circular and sunken in the upper body 12 a seal 30 is positioned to guarantee a good hydraulic seal of the bioreactor 1 0.

[0027] In addition to the device 10, the system can comprise a flange 35 where the device can be mounted.

[0028] The flange 35 preferably has a circular shape and on one of its surfaces comprises a raised edge 36, with diameter smaller than that of the flange 35 and coaxial therewith, having a dimension such that the device 10 can be housed and remain fixed by interference. Inside the edge 36 a through hole 37 is positioned in the area of the membrane 19 or the hole 17.

[0029] The surface of the flange 35 where the device 10 is located will be at atmospheric pressure, whereas the opposite surface will be in a vacuum (with pressure lower than the surrounding environment).

[0030] The flange is made of conductive material and is purposely designed so that it can act as an interface between the vacuum system for directing and focusing the particle beam . Furthermore, the flange 35 allows interaction by heat exchange with the bioreactor (namely heat conduction in order to maintain the biological culture at a temperature of 37°C) and by electrostatic interaction. The latter is functional to the use of a monoenergetic beam as it allows the bioreactor to be earthed, to effectively direct the electron / positron beam . The flange has a seal around the opening to maintain the vacuum once the bioreactor is fixed to a vacuum pump.

[0031] In one embodiment example, the bioreactor 10 has a diameter of 55 mm , and an overall height of 5 mm . The holes 17 and 23 have a diameter of 1 1 mm .

[0032] The tubes 26 have an external diameter of 1 mm and an internal diameter of 0.66 mm .

[0033] The two fluidic channels 18 have a length of 1 1 mm.

[0034] The membrane 19 has a thickness of 100 nm and occupies an area between 1 and 16 mm2.

[0035] The bioreactor 10 is made of a biocompatible polymer material, for example polycarbonate, epoxy resin, etc.

[0036] The membrane 19 must be resistant to mechanical and physical / chem ical stress, impermeable to liquids, permeable to the charged particles and must be an electrical insulator, for example it can be made of appropriately modified silicon, as in the case of silicon nitride (SiNx). This membrane has the function of separating the vacuum chamber necessary for the production of a monoenergetic positron beam from the environment inside the device. The side of this membrane that forms the base of the culture chamber can be appropriately functionalized, for example with fibronectin, to improve the adhesion of the cell or other biological matrix.

[0037] Both the membrane 19 and the slide 28 must be appropriately fixed in their housings, for example by means of glue, or other material or other suitable fixing technique, in order to withstand the various pressure stresses to which they are subject and to ensure the hydraulic seal and sterility of the device.

[0038] The seal 27 is a biocompatible seal, preferably made of silicone, or other suitable material such as, for example, polydimethylsiloxane (PDMS) or nitrile, or other material, and delim its the central culture chamber with the two fluidic channels.

[0039] The membrane 19 is fixed on the lower body 1 1 and the slide 28 is fixed on the upper body 12.

[0040] The three components formed of the lower body 1 1 , the upper body 12 and the seal 27 must therefore be assembled so as to guarantee the leak tightness of the device, the sterility of the biological sample and at the same time the hydraulic seal in the case of flushing of the culture medium in said chamber.

[0041] The tab 20 is positioned in the opening 14 to guarantee correct assembly of the bioreactor 10. The circular seal 30 is inserted in the recesses 15 and 22.

[0042] Furthermore, to avoid movements between the two bodies 1 1 and 12, the two circle arcs 16, protruding from the lower body 1 1 , are inserted in the two circle arcs 22, sunken in the upper body 12.

[0043] The components are preferably assembled by means of bolts in the through holes 29 or by gluing or welding techniques.

[0044] The assembled closed bioreactor is connected to a circuit for circulating the fluid to sustain the biological material for the duration of the experiment. Both assembly and closure and, lastly, connection of the circuit are carried out in sterile conditions. The bioreactor thus connected can be moved and positioned in an appropriate mounting, located in the term inal part of the path of a monoenergetic positron beam .

[0045] For use of the device with the positron annihilation spectroscopy (PAS) technique, a positron beam is used.

[0046] For this purpose, a positron beam can be obtained from a radioactive source that em its positrons in a continuous energy spectrum such as, for example, the isotope of sodium22Na which has a broad energy spectrum , from 0 to 542 keV.

[0047] The positrons resulting from the decay [3+ are moderated, namely slowed down to obtain particles with energy in the order of a few eV. These particles are guided, accelerated and focused by means of electrostatic or magnetic optics until reaching energy values in the order of tens of keV. The kinetic energy reached by the particles is equivalent to the difference in potential between the moderator and the sample studied, and is provided by a high voltage power supply. A setting typically used with these beams is able to vary the implantation energy of the particles em itted, for example between 1 and 20 keV, consequently varying the implantation depth of the particles in the sample, controlling where the annihilation phenomenon occurs.

[0048] Considering a setting that produces a monoenergetic positron beam , the positron annihilation spectroscopy (PAS) technique is used. The annihilation event of these particles produces rays y which are recorded by a detector system . Two of the possible techniques that can be used are positron annihilation lifetime spectroscopy (PALS) and doppler broadening spectroscopy (DBS).

[0049] For example, for a configuration that uses the PALS measurement system , there is the radioactive source and the attached system for the production of the particle beam towards the bioreactor; therefore, a scintillator with a photomultiplier, a constant fraction discrim inator (CFD) and a delay line. The signal com ing out of the delay line and the particle beam start signal are sent to a time-amplitude converter and then to a multi-channel analyser (MCA).

Claims

CLAIMS1 . A system for studying biological material via m icroscopic and spectroscopic techniques comprising a bioreactor (10) having a culture cham ber; said bioreactor (10) comprises a lower body (1 1 ) and an upper body (12) that can be joined to each other; a seal (27) interposed between said lower body (1 1 ) and said upper body (12); said lower body (1 1 ) comprises a through hole (17) closed by a membrane (19); said upper body (12) comprises a through hole (23) closed by an element transparent to visible radiation (28); said seal (27) delim its said culture chamber; said culture chamber being connected to two fluidic channels (18), said bioreactor (10) comprising through holes (25) for the external connection of said fluidic channels (18).

2. The system according to claim 1 characterised in that it comprises a flange (35) housing said bioreactor (10); said flange (35) comprises a through hole (37) at said hole (17).

3. The system according to claim 2 characterised in that it comprises a chamber to which a vacuum pump is applied, to exert a negative pressure on the membrane (19) via the coupling with the hole (37) of the flange (35).

4. The system according to claim 1 characterised in that said bioreactor (10) is couplable with a radioactivesource or an electron beam for using the positron annihilation spectroscopy (PAS) technique or the scanning electron m icroscope or transm ission electron m icroscope (SEM, TEM) techniques.

5. The system according to one of the preceding claims characterized in that said bioreactor (10) is couplable with a light source to be used for optical m icroscope techniques.

6. The system according to claim 1 characterised in that said membrane (19) is permeable to the passage of charged particles, such as positrons or electrons.

7. The system according to claim 1 characterised in that said membrane (19) is treated to facilitate adhesion of the biological material.

8. The system according to claim 1 characterised in that said seal (27) is a biocompatible seal.

9. The system according to claim 1 characterised in that said seal (27) has a circular shape centrally at said hole (23) and extends with two channels along the same axis in the two opposite directions until reaching said two through holes (25).

10. The system according to claim 1 characterised in that said bioreactor (10) is made comprising a suitable material, with the appropriate addition of or superficially covered with particles made of metal alloy or other suitable alloy.

Images