Process and system for the treatment of the surfaces of supports or containers for two- dimensional neuronal cultures by means of chitosan as a cell adhesion factor

TEXT OF THE DESCRIPTION

Object of the present invention is a process for the treatment of the surfaces of supports or containers for two-dimensional neuronal cultures by means of chitosan as a cell adhesion factor.

The standard methods used traditionally for the functionalisation of supports used for obtaining and maintaining a mono-layered neuronal culture provide the adhesion surface of the biological material is previously treated with the deposition of adhesion proteins (factors) .

The purpose of such pre-treatment is to promote the adhesion process mediated by specific proteins of the cell membrane .

A cell culture is a homogeneous group of eukaryotic cells of tissue origin, able to remain vital in vitro . The cell cultures are maintained in liquid medium inside sterile containers . In particular the cells derived from solid tissues must adhere to the bottom of the container to be able to perform properly their vital functions. The adhesion process is mediated by specific biomolecules which, in vivo, mediate the adhesion between cell and extracellular environment . In vitro such biomolecules , or derivatives thereof , are used to modify the surface of the container or support so to reproduce a favorable environment to the adhesion and cell development .

Traditionally, the protocols for the modification of the surface of containers/supports are based on using natural biopolyrners , as the proteins fibronectin, laminin, collagen or synthetic biopolymers, such as the Poly-DL-ornithine , Poly-DL- Lysine and Polyethylene imine . Such biopolyrners have high costs and also medium-long treatment times.

For what concerns the primary cultures of both hippocampal and cortical neurone, standard protocols for the modification of the surface of containers/supports for the culture are based on the use of laminin, polyornithine or polylysine . Laminin is stored at -20 ºC and for being used it must be slowly thawed to 4 º C to avoid its spelling with consequent impossibility to be used. Then the laminin solution has to be diluted in saline or medium (0.1 mg/ml) and brought into contact with the surface to be treated at 37" C for 8 hours, at the end of which we proceed with washes to eliminate non-adsorbed excess laminin. Also, polyornithine and polylysine must be thawed and diluted, generally, at a concentration of 0.15 mg/ml, brought into contact with the surface to be treated at 37"C for 12 hours, at the end of which we proceed with washes to eliminate excess not adsorbed.

The main disadvantage in using such factors is related to their high cost and to the preparation time of the functionalised supports.

(Letoumeau, Paul C. "Cell-to-substratum adhesion and guidance of axonal elongation . " Developmental biology 44.1 (1975): 92-101; Letoumeau, Paul C. "Possible roles for cell-to- substratum adhesion in neuronal morphogenesis . " Developmental biology 44.1 (1975): 77-91).

For example, polyornithine is a synthetic amino acidic chain the cost of which is high especially for the laboratories making massive use of cell cultures. It is generally used at a concentration between 0.15 and 80 pg/ml in aqueous solution, deposited on the surface of interest in a range between 6 and 12 h at 37ºC.

Mow it has been found a cell adhesion factor, the polysaccharide chitosan, alternatively to using standard factors such as those described above, which allows having low prototyping costs, by reducing the preparation time of the functionalized supports, and which allows having a lower cost still maintaining the same adhesion features of the standard factors.

Chitosan is a linear and cationic polysaccharide derived from the deacetylation of chitin. Chitin is the main component of the exoskeleton of crustaceans, some insects and the mushroom walls.

Chitosan is a natural biopolymer with the ability to support the adhesion and the differentiation of neuronal cultures.

The process we have proposed is based on recognizing the typical features of "adhesion factor" of the deacetylated polysaccharide chitosan.

This particular material which we have identified alternatively to the standard adhesion factors also has the virtue of relatively more affordable costs with respect to the products marketed the main life science comjpsniee .

For example, 1 mg laminin sold by Sigma-Aldrich has a cost of 168 euros, 50 ml, 0.01% w/v polyornithine sold by Sigma-Aldrich has a cost of 106 euros and 50 ml, 0.01% w/v poly-D-lyaine sold by Sigma-Aldrich has a cost of 106 euros, 5 mg 80 euros. 50 g low molecular weight chitosan sold by Sigma-

Aldrich has a cost of 92 euros.

Object of the present invention is a process for the treatment of the surfaces of supports or containers for the in-vitro preparation of a two- dimensional neuronal culture by means of cell adhesion factors, wherein the cell adhesion factor is chitosan .

Chitosan is used in solution preferably at a concentration between 0.01 and 1% w/v.

The stock solution of chitosan is preferably in 0.1 M acetic acid.

Preferably , the container or support is directly brought into contact with the chitosan solution by means of deposition by adsorption or else is treated by means of nebulisation of the chitosan solution by the use of an airbrush.

The deposition by adsorption is preferably carried out for a time between 1.5 and 5 hours, more preferably between 2 and 4 hours, at a temperature between ambient temperature and 40 ºC, more preferably about 37º C.

Preferably, the excess of chitosan solution deposited by adsorption is removed and subsequently subjected to washing either with sterile water or directly with culture medium,

The functionalised substrates by the adsorption process do not provide for a subsequent sterilisation because the polymeric material is previously filtered and made sterile.

The nebulisation is preferably carried out at a nitrogen pressure between 2 and 3 atmospheres, more preferably the value of 2.5 atmospheres , and at ambient temperature .

The airbrush can preferably have a nossle with 0.2 mm diameter.

Preferably, after deposition by airbrush pen, the material is left to dry under chemical hood and the substrates are sterilised afterwards in 70% ethanol over a time between 20 min and 1 hour, finally washing the surface with sterile water or culture medium. Subsequently they can be used for the cell culture.

The process with the airbrush has the advantage of nebulising the material by the use of stencils so to generate adhesion surfaces with patterns generating non-random neuronal networks.

Object of the present invention is the use of chitosan as adhesion factor for the treatment of surfaces of supports or containers for the in-vitro preparation of a two-dimensional neuronal culture.

According to a further embodiment the process for the treatment of the surfaces of supports or containers for the in vitro preparation of a two- dimensional neuronal culture by means of cell adhesion factors, wherein the cell adhesion factor is chitosan, provides the chitosan to be applied with types of techniques other than deposition by means of airbrush .

As an alternative to the airbrush, it is possible to provide the use of coating techniques derived from other technical fields, such as painting in the field of finishing.

Such techniques end the corresponding plants ere configured to treet e high number of pieces end cen be used for the mess production of lerge numbers of perts or suitebly reconfigured elso for the smell series production.

Among the different teclxni^pies used in tlie field of costing, for example, the use of spraying nossles which ere mounted on supports having different degrees of freedom of orientation end displacement of said nossles, is known.

A further variant can provide for example the use of deposition techniques by means of printers of the ink-jet type.

In this case, the control of the deposition and distribution patterns of chitosan can be easily modified depending on the need, simply by modifying the control software of the printer.

By using a monochrome printer and by using the solution containing chitosan instead of the ink it is possible to define the distribution patterns, for exsmple in the form of graphic elements positioned in pre-established positions with reference to an image generation area and having the shape and sise provided for the area on which the chitosan solution must be deposited. The graphic elements can be generated on a virtual drawing sheet by means of software of graphic design and can be transmitted to the printer for being printed in the form of image file to be printed.

According to an optional embodiment , the inkjet printer can be programmed to distribute a pre- established quantity of solution over a pre- established area both uniformly on the entire area and in order to generate parts of said area on which a greater or lesser amount of solution was delivered.

According to an embodiment variant, in this case the distribution function can be related to a gray scale in which the greater or lesser shade of gray represent a greater or lesser amount of solution which must be distributed over said area. This allows making distribution patterns of the solution in a simple way by using graphic software which is graphically representing the areas on which to distribute the solution with corresponding geometrical shapes and corresponding sisee or dimensional ratios, whereas the different quantities of solution for different parts of said areas or else a pre-established quantity of solution to be uniformly distributed on said areas are defined by means of the coloration of said area parts or the entire area with a pre-established gradation of gray related to a pre-established quantity of solution.

Thus, it is possible to generate command file of the deposition member of the chitosan solution by using drawing programs or graphics such as for example Microsoft PowerPoint, Microsoft Word or others. The control unit of the deposition member is in this case provided with drivers controlling the deposition member, which comprise the correlation instructions between the graphic file and the command parameters of the functions of the deposition member related to the displacement of the latter for the delivery of the solution in the pre-established areas of a support and/or related to the quantity per area unit to be deposited on those areas. The ink-jet printer can be further used also for feeding the washing fluid in the step following the deposition for the elimination of the excess solution .

While by using nozzle painting systems it is generally needed to provide for quite large series of substrates having identical and repetitive shape and size, the use of the ink-jet printer allows to economically generate a lower number of substrate pieces , by offering at the same time a remarkable flexibility in adapting to different needs mainly relative to the different shapes and sizes of the substrates on which the chitosan solution must be deposited.

In addition to the flexibility the ink-jet printers are of simple construction, space-saving and relatively inexpensive if compared to other types of coating deposition devices.

In relation to the composition specifications of the chitosan solution, in combination with the two above mentioned additional embodiments, these can be identical to what previously described in combination with the embodiment providing the deposition lay means of airbrush.

The composition of the solution, as well as the ratios of the various components with respect to the chitosan, can vary depending on the deposition technique used for example according to one of the two above mentioned variants . The specific choices are dictated by the configurations of the deposition system used and their determination is within the basic technical knowledge of the expert in the art and within the normal design activities. According to a further aspect, object of the invention is also a system for the mass production of supports or containers for two-dimensional neuronal cultures provided with chitosan coating as cell adhesion factor, which system comprises

one feed station to feed at least one substrate intended to form a support or container;

one deposition station of a chitosan solution for creating a cell adhesion factor wherein the cell adhesion factor is constituted by chitosan ;

one discharging station to discharge the substrate on which a chitosan layer has been laid; one carrier of said at least one substrate from said feed station to said deposition station of the chitosan solution and subsequently to said discharging station,

and wherein the deposition station comprises a sprayer/nebuliser member of the chitosan solution on at least one part of the substrate surface

a control unit of said sprayer/nebuliser member being provided relative to the ejection direction of the jet of chitosan solution and/or to the movement of said member in accordance with a pre-established else and shape of said part of surface of the substrate on which the deposition of the chitosan solution is provided and/or relative to the amount of chitosan solution per unit eise of said surface area.

According to a further feature , the sprayer/nebuliser member can be selected among at least one spraying/nebulisation nosale, or at least one print head of the ink-jet type or a combination of said nossles and said heads, said at least one nossle or said at least one head or the combination of these two elements being supported on an orientating and/or displacing mechanism according to at least one or according to two or more axes.

According to an optional feature , the deposition station and/or the feed station are provided with members detecting the presence and/or position and/or shape and/or sise of the substrate, which are connected to the control unit, whereas said control unit is configured to process the signals of said detecting members and to generate command signals of the deposition members corresponding to the detected position, shape and sires of the substrates .

Still according to an optional feature which can be provided in any combination with the previous features, the system comprises a production unit of the chitoaan solution corresponding to the deposition requirements , which unit comprises at least one tank for chitosan, at least one tank for at least one solvent, one mixer and dosing means, feeders to feed the chitosan and said at least one solvent to said mixer, at least one tank for the chitosan solution prepared for the deposition, which tank is connected to and feeds said deposition members.

Still according to a further feature, said system can optionally comprise a washing station off the deposited and excess chitosan solution.

Such washing station can be made in various ways and depends in part on the shape of the substrates .

In a generic arrangement, the washing station can have supply nossles of jets of washing fluid on the surfaces of the substrates on which the chitosan solution has been deposited and removal members of the washing fluid and/or members for drying the substrates ,

The removal of the washing fluid may require moving the substrates if for example they are in the form of containers , such as for example their complete or partial reversal .

In combination with or alternatively, the drying can occur by means of jets of cold and/or hot air depending on the specific conditions.

Still according to a further feature, between the deposition station and the washing station, a drying station of the chitosan solution deposited on the substrate in the deposition station can be provided. In this station alternatively or in combination drying members can be provided, such as for example jets of cold or hot air, heat treatments or others.

According to a possible embodiment variant, the drying station is provided with a chemical hood under which the drying process off the substrates occurs.

Still an embodiment provides a sterilisation station of the substrates directly upstream of the discharge station.

Alternatively, the sterilisation station or else one or more further sterilisation stations can be provided upstream or downstream of any stations described above with reference to the basic embodiment and the different optional embodiments.

The sterilisation station can be provided with one or more sterilisation members each working according to a technique and a process among those known and which are used depending on the specific case, the technician in the art toeing atole to select the best sterilization technique among those known and available in the state of the art to him.

A sterilization technique particularly suitable in combination with the embodiments described above and related to a highly automated production line can consist of irradiation of substrates with the chitosan deposition layer by means of gamma rays.

Such technique is known per se and thus provides a sterilization station comprising members generating and irradiating substrates entering the same by means of gamma rays .

As it appears evident from what set forth above, the process according to the present invention has the advantage of being able to be configured for exploiting it in the context of mass production. The plant and system for implementing the process are simple and easily achievable . Furthermore depending on the cases , it is possible to provide different configurations without requiring a new design of the system itself.

Depending on the shape and size of the supports or containers, it is possible to treat in parallel a certain number of said supports or containers . In this case the transporter can feed a constant flow of several columns parallel to each other of supports or containers , which columns are arranged next to each other. Therefore, the system allows producing a high number of supports or containers , helping to further reduce costs and making serial the production of supports or containers for the in vitro preparation of a two-dimensional neuronal culture by means of cell adhesion factors. The features of the invention , in particular relative to the deposition process and the structure of an automated plant for the mass production of supports or containers for two-dimensional neuronal cultures provided with a chitosan coating as a call adhesion factor, will better result from the following description of some non-limiting exemplary embodiments depicted in the attached drawings , wherein :

Figure 1 depicts a functional block diagram of a first embodiment of a plant for the mass production of supports or containers for two-dimensional neuronal cultures provided with a chitosan coating as a cell adhesion factor .

Figure 2 schematically shows a further embodiment of a plant according to Figure 1 , in its minimal configuration .

Figure 3 shows an example of a combined support on which surfaces chitosan solution is intended to be applied by means of deposition with a plant according to the present invention.

With reference to Figure 1, as already previously described, among the possible alternatives for the deposition of the chitosan solution on the surface of a substrate intended to act as a support or container of a two-dimensional neuronal culture , an automated plant automatically and serially carrying out the operations described above in any of the modes and/or combinations and subcombinations of features described above is provided.

In particular in Figure 1 the plant is made as an assembly of operational stations distributed along a path for transferring the substrate (s) between different stations , each intended to carry out at least one of the operations widely described above.

The substrates are collected and fed in succession and/or at least in part in parallel to a transport line depicted by the arrows in a feed station 100. Depending on the mode with which the substrates are fed, the feed station 100 can provide different configurations and different picking members from a warehouse at an inlet end of the transport line. Such configurations and such members can be selected by the technician in the art from a wide selection of known solutions , among which for example also the use of robotic arms equipped with gripping members carried by the arm.

The substrates are then fed to a deposition station, in which a deposition member 101 is provided which can be constituted by one or more nossles or by one or more heads of the ink-jet type and by displacing and orientating means of said nossle(s) or said head(s) ,

These are fed with a chitosan solution from a feed tank not depicted or alternatively from an optional preparation and feed station of the chitosan solution denoted by 106 and shown with a dash and dot line .

The preparation of the solution and in particular the composition and ratios of the single components can be for exaiqple provided according to one or more of the modes described above. The dilution ratio and also the composition of the solution can anyway vary depending on the features required by the nossles and/or by the ink-jet heads.

The solution and/or the chitosan and/or the aingle components , separately from each ether, of said solution can be sterilised prior to feed them to the deposition station as denoted by 107, with dash and dot lines to highlight the fact that such step and such station can be optional.

After the deposition on the substrates in the deposition station 101, these are transferred to a discharging and packaging station 105. This station is itself also made according to the contingent features of the substrates and furthermore cam provide, in addition to manipulators and packaging members, also drying mesibere of the layer of material deposited on the substrate as highlighted in the previous description.

By 102, 103 and 104 the optional treatment stations referring to the process steps described above are depicted and in particular by 102 a washing station, by 103 a drying station and by 104 a sterillsation station are depicted.

The washing station 102 can be provided to carry out the step described above of eliminating the excess chitosan solution. This operation can be carried out according to any mode previously described and the washing station 102 is thus provided with washing members which are compatible with the selected mode and which the technician in the art selects from a range of known solutions depending on said selected mode.

The above applies also to the drying station 103. In fact the selection of the drying members which alternately or in combination can be air conditioning flow generators (heating or cooling) and thermal energy generators to which the substrates exiting the washing station 102 are exposed, depends on the type of substrate and/or the type of substances used for the washing, or else by their chemical/physical features .

A sterilisation station 104 can be provided before the discharge of the substrates treated by the production line. Also for the sterilisation station, one or more of the variants currently on the market and known to the technician in the art are possible alternatively or in combination to each other.

In particular an embodiment can provide sterilisation by means of gamma rays . This sterilisation process , which can be used in an automated production line wherein the human staff must not be present in the proximity of the sterilisation station, allows optimally integrating the sterilisation step with the transfer motion of the substrate from one station to the other, without requiring additional handling of the substrates to modify their space orientation and/or positions.

Concerning the substrates, these can be in the form of single pieces separated from each other and each of which is a unit intended to act as a support or container for the culture.

An alternative can provide two or more supports or containers as integrated in an assembly in a side by side position and juxtaposed to one another according to a pre-established order, whereby the individual substrates of the assembly are loaded and subjected to the process all together.

When discharging, the separation step of the single substrates from the assembly can be provided or else the substrate assembly is retained and subjected to packaging in ita complete form comprising several supports or containers.

Figure 3 shows an example in which the assembly of supports 301 in the form of disks is provided in the form of a rectangular plate 300. The individual disks 301 are retained to the remaining material of the plate by means of pre-established weakened lines thanks to which they can be separated at the end of the process prior to their packaging.

As already set forth, it is possible to provide the packaging of the entire plate 300, only part of the or else to separate the disks 301 and packaging them separately from each other or in a group of a pre-established number of disks.

Figure 2 shows a block diagram of a plant operating on a flat support in the form of a single supporting unit or in the form of plate-like element integrating several units of product, such as for example depicted in Figure 3.

The individual supports 250 are fed from a warehouse to a feed station 200. One after the other the supports are positioned on a conveyor belt 240, forming a succession of supports .

These are transferred to a deposition station 210. In the depicted example, the deposition station

210 has a structure of the ink-jet printer type with one or more heads 213 mounted on a displacing mechanism which can operate according to different displacing directions whereas it is also possible to provide further orientating members of the head (s) (not depicted in detail) .

Only by way of example, the head is mounted on a first eliding block 212 sliding on a first guide oriented transversally to the transport direction off the substrate 250. The first guide can be mounted on a second sliding block 211 sliding in a direction parallel to the direction of the conveyor belt 240 on a second guide 214. The latter could not be needed as the displacement of the substrate 250 relative to the head and in a direction parallel to the feed direction of the belt is obtained already by the displacement of the substrate 250 by means of the belt 240. However in case we wanted to make more deposition runs or we wanted to vary the deposition speed per unit of surface area to be treated, the independent and additional displacement of the head(a) 213 in the transport direction of the belt could be convenient .

By 220 is depicted a feed source of the chitosan solution, which can be made according to one or more of the variants described above, in particular also with reference to the example of Figure 1.

By 230 a diaeharg· station is depicted . This has members handling the single substrates for the withdrawal of the same from the outlet end of the belt 240 and depicted by 231, The handling members 231 can serve one packaging unit or collection warehouse of the substrates 250 which is depicted by 232 and in which the substrates treated can be sent to the following treatment steps, not depicted, such as for example one or more of the washing 102 , drying 103, sterilising 104 and final packaging steps described with reference to the previous example ,

The control of the stations and of the members provided in the same is carried out by means of a control unit 260. This can be in the form of a computer, a PC or other system with micro-processor able to load and execute a program codifying the instructions to configure said unit and the peripherals of the same, so to carry out the functions needed to control said stations and said operational members.

In addition to the memories for the control programs and the command drivers of the stations and of the members provided in the same, the control unit is provided in combination with one or more input/output interfaces for the communication with the user denoted globally by 262 and which can comprise one or more of the known interfaces currently in the market.

According to yet a further feature, the control unit 260 can be provided with a communication unit 263 thanks to which it can connect to remote units for exchanging data, such as for example for uploading or downloading data and/or programs and/or also for the connection to a portable unit of a user, which constitutes itself the user interface.

As it is evident, the deposition station 210 can provide also an ink-jet type printer as deposition member, which can be constituted also by a retail type printer to which the chitoaan solution is fed instead of the ink.

As previously described, this solution is particularly cheap. In fact the control of the printer can occur thanks to a text file or image file in which the template (a) of the support or of the assembly of supports is depicted and in which the gray scale represents the quantity of solution per surface unit to be deposited on the areas corresponding to the substrates .

Thanks to this, the preparation of the printer only requires the preparation of a video image by using programs of low difficulty and universal use, without the need, of complex operations for generating the commands of the deposition members.