Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/14—Scaffolds; Matrices
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
  • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli

Definitions

• the present invention relates to a reactor for cell culture.
• these devices also commonly called bioreactors, include a culture chamber where the cells are grown in a liquid solution on a substrate scaffold; the chamber is also maintained in controlled temperature and atmosphere to ensure the correct cell growth.
• This brief introduction aims to better introduce the invention that in its more specific aspect relates to a bioreactor for electromechanical stimulation of stem cells.
• these cells have the ability to acquire the phenotype of a specific cell lineage undergoing transdifferentiation.
• stem cells subjected to a periodic cycle of relaxation and contraction, reflecting that of the heart rate can transdifferentiate towards the cardiac muscle phenotype.
• the cells are seeded on a scaffold of flexible biocompatible material in culture which is stretched and released with cycles of set frequency, so that the cells lying thereon undergo periodic strain cycles accordingly .
• the bioreactor described in this document meets the general criteria outlined above on the state of the art and includes therefore a culture chamber with controlled atmosphere, whereas for the cyclic stretch of the substrate a device is provided with two parallel rotating shafts, with wings between which an elastic scaffold is hold.
• the shafts rotate simultaneously in opposite directions with angles 10°-20° wide following alternate cycles (i.e. clockwise-counter clockwise). Following these alternating rotations, wings diverge and come closer together in succession, causing the elongation and return of the membrane.
• the motion of lifting and lowering requires a deeper pan, to be able to keep it immersed in the liquid.
• bioreactor makes use of two counter-rotating shafts. with an inertia related not only to the use of two shafts per se, but also to the mechanism for their operation.
• This device consists of a cam. also housed in the same culture chamber, making it a factor which inevitably interferes with cell reproduction.
• the technical problem underlying the present invention is to provide a bioreactor for mechanical stimulation of cells, particularly stem cells, with featured structure and operation that could overcome the limits outlined above with reference to known bioreactors.
• the idea of the invention consists in subjecting the scaffold bearing the cells to a linear deformation, preferably directly obtained with a mechanical mobile organ, i.e. without intermediate mechanical transmissions between the driving means and the mobile organ.
• the driving means are arranged outside the culture chamber, so that they do not interfere with the controlled atmosphere within it.
• the biorcactor aiso includes means for measuring strain and stress of the scaffold.
• the drive of the mobile device can be controlled based on the feedback of information (usually in the form of electrical signals) provided by means of measuring the strain or stress applied.
• FIG. 1 is a perspective view of a biorcactor in accordance with the invention
• FIG. 2 is a perspective view of the bioreactor in the previous figure, without the part concerning the culture chamber:
• cuituie chamber is a top view of the cuituie chamber:
• - Fig. 4 is a view from the bottom of the culture chamber:
• FIG. 5 is a perspective view of the culture chamber, showing in detail its components:
• Fig. 6 is an exploded view showing the internal components of the culture chamber:
• - Fig. 7 is a longitudinal section of the bioreactor of the previous figures.
• 1 refers to a bioreactor in accordance with the invention, including a base 2 for a support structure 3 of the culture chamber 4,
• the base 2 hosts a linear electric motor 5 mounted on a wall 6 fixed to the base itself (by welding or another appropriate system).
• the electric motor 5 is preferably of the synchronous so-called "brushless” type, with integrated position sensors that provide output signals for the control of the bioreactor 1 which will be discussed in more detail later, concerning the position of the shaft 53 of the motor.
• linear motors suitable for use in the biorcactor according to the invention arc those marketed undci the brand LinMot * by Swiss company NTI ⁇ O (Spreitcnbachj or its
• the base 2 holds the support structure 3 of the culture chamber, which is also used for heating in order to maintain its temperature controlled, To this end the structure 3 contains a thermostat 30 which is in heat exchange with a fluid flowing in the inlet 3 ] and outlet 32 lines, below it.
• the floor thermostat can be made with a metal plate or a glass, attached to the frame 3 by screws.
• Channels 31 , 32 convey a thermal fluid (liquid or gas) conveniently heated electrically or otherwise placed inside a container in the base 2. and not shown in the drawings for simplicity.
• the upper surface of the thermostat 30 exchanges heat with the bottom 40 of the culture chamber 4; as shown (sec Fig. 4 the bottom 40 is substantially square and consists of a plastic plate, made in polyethylene or similar, which in this example has two handles 41 so that it can be more easily hold.
• a rectangular cavity 42 is formed intended to serve as a container for the culture medium, along the edges of which arc a temperature sensor 43, a pair of electrodes 44 for stimulation and an electrode 45 of detection of acidity (pH) of the liquid cell culture.
• the culture chamber also includes a side wall 46 extending along the four sides 46a, 46b,
• the entry seat 49 for a rod 50 driven by the motor 5 (see Fig. 5 where a portion of the sheath 55 was specifically removed), which serves for the application of mechanical strain to the scaffold hosting the stem cells.
• the seat 49 contains a recirculation ball guide (not shown) that supports the end 50a of the rod protruding from the chamber 4, with a very low friction: the seal around the protruding end 50a is ensured by a clamp 51 tightened by a screw 52. while for the connection of the end of the rod 50a with the shaft 53 of the linear motor 5, a coupling flange 54 is provided.
• two sheaths bellows 55a. 55b arc used fixed in a watertight way to the opposing faces 46b. 46c ot the culture chamber 4, through holders 56a, 56b (of bayonet or similar type).
• the presence of the two sheaths bellows 55a. 55a ensures that during the motion of the rod the interna] volume of the culture chamber 4 stays constant: if it were not so, since the chamber is sealed, the motion of the stem would lead to considerable pressure oscillations.
• an arm 58 having one end fixed on the rod itself and sealed around the sheaths 55a. 55b: the arm 58 engages with a bracket 60. whose function will be better described later, by a pair of sliding at a site
• a similar bracket 64 is engaged as just explained, i.e. through a coupling with a site 65 with guides 66. along with a plaie 67 fixed to a load cell indicated on the drawings with reference number 68.
• This cell comprises a base 69 attached to one side of the side wall 40 of the culture chamber 4. which houses the electronic components for signal detection, and from which extends a cantilevered shelf 70.
• the shelf 70 serves as an amplifier of deformations measured with strain gauges applied to it: the strain gauges 7) can be applied in positions (top. bottom or centre of the shelf 70) and number 1. 2 or more) appropriate for the accuracy of the measurements to be obtained.
• the brackets 60 and 64 serve to support the terminals 72, 73 configured in "I" to stretch the scaffold 75 bearing the cultured stem cells.
• the scaffold 75 must have high properties of bio/cyto-compatibilhy and an elastic behaviour that accompany the deformation to which it is submitted.
• the scaffold 75 can be made in the form of membrane or tissue, preferably based on a biopojymer that has the properties specified above; materials based on polylaetic acid. polyglycolic acid, ⁇ -capro lactone ( PLA. PLGA. etc..) such as those marketed by the company Purac Biomaterials, were successfully tested to this aim.
• the tension clamps 72. 73 have a part fixed to the respective bracket 60, 64 (in this example by screws which engage in respective holes that can be glimpsed in the figures) so that they tighten the flap end of the scaffold 75 and block it in the brackets 60, 64.
• the part of the clamps 72, 73 cantilevered over the latter allows to distance the scaffold 75 and keep it immersed in the tank 42 provided on the bottom 40 oi ⁇ culture chamber 4; the tank is filled with a medium whose formulation is specific for the cultured cells.
• the culture chamber 4 is maintained a controlled atmosphere: for this purpose it is topped by a Hd 80, preferably transparent to allow observation inside, locked tight on the side wall 40 of the chamber 4 using four knobs 81 located at its vertices.
• the bioreactor 1 also includes a computerized control 84 (PC or similar type ) operationally connected to the motor 5 and to the different devices, sensors, detectors, meters, etc... which are part of the reactor 1 but not described as a self-evident, thai serve to govern its operation as follows.
• a computerized control 84 PC or similar type
• the bioreactor 1 is of a type designed for the cultivation of stem cells. to be addressed particularly (but not exclusively) to the muscle cell phenotype. preferentially the cardiac muscle tissue.
• the alternating axial movement (i.e. forward and backward) of the rod 50 are transmitted to the bracket 60 through the arm 58 integral to both: since the bracket 60 is in turn attached to the ends of the elastic scaffold 75 bearing the stem cells, alternating movements of the former generate similar movements of the second.
• the other end of the scaffold 75, fixed to the bracket 64. stands still and is integral with the shelf 70 of the load cell 68.
• the end of it fixed to the bracket 60 will follow the movement solid with the arm 58 and stem 50. while the other end remains stationary joined to the bracket 64 fixed through the plate 67 to the shelf 70: as a result the scaffold 75 extends and shortens clastically in accordance with the movements of the rod 50 and shaft 53 of the linear motor 5.
• This provision makes it possible to detect the state of stress imparted to the scaffold 75. i.e. the traction (e.g. in the order of some Newton) to which it is subjected during the elongation in each cycle.
• the traction e.g. in the order of some Newton
• the load cell 68 is attached to the end of the scaffold 75 through the shelf 70 and the plate 67. so that it can detect directly the tensile stress applied: in other words, the load cell 68 actually measure the effort undergone by the scaffold 75. not an indirect parameter under which this effort is then calculated.
• the stress imparted to the scaffold is controlled upon detecting the electric current drawn by the engine: the stress applied to the elastic scaffold is substantially proportional to the torque transmitted from the engine and it is therefore understandable that the measure may not be very accurate, as it derives from an indirect parameter such as current consumption which does not take into account all friction and inertia that are downstream of the motor.
• the electric motor 5 can therefore be used advantageously to determine precisely (including the order of hundrcdths of a millimetre) the displacement of the shaft 53, by virtue of a direct detection, i.e. without errors resulting from the conversion of angles of rotation shifts into straight movements.
• the electrical signals of sensors placed along the stator of the linear motor 5 arc transmitted electronically to the computer 84 (possibly via an amplifier or servo-controller not shown in the drawings), able to determine at any instant the position of the shaft.
• the computer 84 processes the signals received on the basis of an appropriate program stored in it and provides feedback to the linear motor 5.
• the use of the linear motor 5 in addition to being accurate in itself, can also allow the transmission of the movement to the scaffold 75 bearing the stem cells just as precise, so that the elongation applied to it complies with the commands given by the computer 84 to the linear motor 5.
• the bioreactor 1 can be operated with a control on one or both of them.
• the computer 84 stores programs that allow to adjust the operation of the linear motor 5, working on its electrical supply so as to achieve a taiget stretch of the scaffold 75. or by applying a defined stress on it.
• the electrical signals ⁇ hat identify the movement of the shaft 53, in order to process any changes that must be provided to the elongation of the scaffold 75, while in the second case there will be used as a feedback the signals provided b> the load cell 68 which, as mentioned above, gives an immediate indication of the stress applied to the scaffold 75.
• the computer 84 also controls all othei parameters needed for the culture of stem cells, and this applies for example to the temperature of the medium in the compartment 42. or its acidity, measured respectively by the temperature sensor 43 and the pH sensor 45. both opcratively connected to the computer 84.
• i hese sensors arc known in themselves and are therefore not considered further here. The same is true if the cell culture takes place in the presence of electro stimulation via electrodes
• the current applied by the latter is controlled by the computer 84 to which the electrodes are connected operationally.
• bioreactor I is further equipped with additional sensors (not described because they also are known) for the control of environmental conditions inside the culture chamber 4.
• CCb carbon dioxide
• it can contain thermometers to measure temperature and a pressure gauge to detect the pressure in the culture chamber 4.
• the tissues obtained from ceil culture to whom this invention is intended increase their size over time, as the process develops; it follows that weight. thickness, length and also the consistency of tissue cell grow up in time, and change the mechanical properties thereof, including those of the scaffold 75 bearing it.
• the latter plays the role of a matrix in which cells grow and multiply, so that the fibres of biocompatible material which is composed interact with the cellular tissue, thereby changing their clastic behaviour.
• the scaffold 75 and the cells on it form a whole: it follows therefore that as the tissue grows, changes in mechanical properties of the scaffold 75 are to be expected; the elasticity of the substrate will vary over time and with it the elongation obtained by applying a given effort.
• the bioreactor 1 presented here allows for an optimal control of the process of culture, as it is able to provide precise adjustment in feedback both in terms of stress and strain.
• the linear motor 5 allows to eliminate reduction and / or transmission mechanism interposed between it and the scaffold, so that the elongation of the latter coincide with the translation of the rod 50 and shaft 53.
• the detection of the stress applied to the substrate 75 through the load cell 68, which is connected through the bracket 7Q and the plate 67, is precisely obtained because it is a parameter indicating a direct effort actually applied to the scaffold with the stem cell on it. Thus, it is not a measure derived from the electricity consumed by the motor or from other indirect parameters.
• the biorcactor 1 includes deformation and supporting means of the scaffold 75: the first is obtained essentially with the linear motor 5 with the shaft 53 and the rod 50, which 'deliver the cyclic deformation of the scaffold 75. while the latter depends on the brackets 60 and 64, the clamps 72 and 73. the shelf 70 and the plate 67. which hold the scaffold 75 in the culture chamber 4 in conditions suitable for cell culture (i.e. soaked into the medium contained in the tank 42.
• the scaffold deformation means and supporting means so roughly defined, are connected by the arm 58 which transmits the movements of the rod 50 to the movable bracket 60. and consequently to the end of the scaffold 75 solid therewith.
• the rod 50 and the shaft 53 of the motor 5 as a unique piece, instead of two separate pieces coupled by the flange 54.
• the material of the stem will preferably be the same of the linear motor shaft.
• linear electric motor 5 is certainly preferable because it is more advantageous for the reasons explained above, it is possible to use an alternative motor or other driving systems, provided that they obtain an axial movement of the stem 50.
• a hydraulic or pneumatic actuator could be provided, i.e. devices in which a piston (corresponding to the shaft 53) is controlled by oil, compressed air or another fluid.
• Tlic translation of the rod 50 controlled by the actuator may be detected with as appropriate optical, electromagnetic, or other systems.
• optical sensors that detect the position of the rod 50 could be used, or permanent magnets applied on it and sensing coils excited by these permanent magnets might be arranged within the structure of chamber 4.
• the magnets would induce in the coils a current that can be detected by the control system similar to what happens in the linear electric motor, thereby providing precise data on the movements of the rod 50.
• Optical or electromagnetic sensors of the position of the rod 50 may also be used in combination with the linear motor 5 (i.e. not only with the mechanisms of oscillating glyph or rod-crank).
• brackets 60. 64 or the terminals 72, 73 may be configured differently and, more generally, the solutions to block the ends of the scaffold 75 may be the most diverse. consistent with the functionality of the bioreactor.
• the force applied to the scaffold 75 must be detected; such a detection can be made as appropriate, with the load cell and strain gauges described in the example, or with other equivalent means: therefore, the anchoring systems of the ends of the scaffold 75 must be compatible with the way devised to measure the stress applied to the scaffold.

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• 2009
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