SYSTEM AND METHOD FOR DETERMINING A FORCE APPLIED TO OR GENERATED BY A CELL OR TISSUE CULTURE

Abstract

The invention relates to a system (10) for determining a force applied to a cell or tissue culture (14) arranged in a culture chamber (12), comprising an elastic element (30) mounted in or suitable for mounting in said culture chamber (12), said elastic element (30) being adapted to be coupled with said cell or tissue culture (14) such that a force applied to said cell or tissue culture (14) leads to a deflection of said elastic element (30) against a restoring force thereof, and said elastic element comprising a magnetic element (34) arranged such as to be moved upon deflection of said elastic element (30), a magnetic field sensor (42) mounted or suitable for mounting outside said culture chamber (12), said magnetic field sensor (42), when mounted outside said culture chamber (12), being adapted to detect a change of magnetic field attributable to a corresponding movement of said magnetic element (34) upon deflection of said elastic element (30).

Claims

1-28. (canceled)

29. A system for maintaining a cell or tissue culture, the system comprising: a culture chamber in which said cell or tissue culture is to be arranged; an elastic element adapted to be coupled with said cell or tissue culture such that a force applied to or generated by said cell or tissue culture leads to a deflection of said elastic element against a restoring force thereof; a sensor unit adapted to determine a signal representing at least one of an amount of said deflection of said elastic element and a force corresponding to said deflection; a drive mechanism adapted for applying a force to said cell or tissue culture; and a control unit operatively connected with the drive mechanism.

30. The system of claim 29, wherein said control unit is adapted to control the force applied by the drive mechanism to said cell or tissue culture at least in part in response to said signal determined by said sensor unit.

31. The system of claim 29, wherein said force applied by said drive mechanism is a tensile force.

32. The system of claim 29, wherein said system further comprises one or more electrodes adapted for applying electrical stimuli to said cell or tissue culture.

33. The system of claim 32, wherein said control unit is adapted to control said electrical stimuli.

34. The system of claim 33, wherein said control unit is further adapted to control the force applied by the drive mechanism to said cell or tissue culture at least in part in response to said signal determined by said sensor unit, thus coordinating the mechanical and electrical stimulation.

35. The system of claim 34, wherein said coordinating the mechanical and electrical stimulation comprises simulating a force-length relationship which muscle tissue would generate in its natural physiological environment.

36. The system of claim 29, wherein the sensor unit comprises at least one magnetic field sensor.

37. The system of claim 36, wherein the sensor unit is adapted to determine the amount of said deflection of said elastic element by measuring a change in a magnetic field, said change being caused by a deflection or displacement of said elastic element.

38. The system of claim 29, wherein said drive mechanism is a linear drive mechanism.

39. The system of claim 29, wherein said drive mechanism is adapted to generate the force applied to said cell or tissue culture by linearly moving an attachment structure for the cell or tissue culture.

40. The system of claim 29, further comprising a first holding element, said first holding element comprising a first holding section for holding said cell or tissue culture and a first coupling section for coupling with said drive mechanism.

41. The system of claim 40, wherein said control unit is adapted to determine a position of said first holding element.

42. The system of claim 40, further comprising a second holding element, wherein said holding element comprises a second holding section for holding said cell or tissue culture and a second coupling section for coupling with said elastic element.

43. The system of claim 40, wherein said first holding element and said second holding element are each triangular in shape.

44. A method for applying a controlled force to a cell or tissue culture arranged in a culture chamber, comprising: providing said culture chamber; coupling an elastic element mounted in said culture chamber with said cell or tissue culture such that a force applied to or generated by said cell or tissue culture leads to a deflection of said elastic element against a restoring force thereof; determining an amount of deflection of said elastic element; applying, by a drive mechanism, a force to said cell or tissue culture; and controlling the force applied by the drive mechanism to said cell or tissue culture at least in part in response to said determined amount of deflection of said elastic element.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 is a schematic view of a system according to an embodiment of the invention.

[0047] FIG. 2 is a close up view of the magnetic element and the magnetic field sensor of the system of FIG. 1, further showing the magnetic field generated by the magnetic element.

[0048] FIG. 3 shows the magnetic flux density in two directions measured by the magnetic field sensor, as a function of the deflection of the elastic rod of the system of FIG. 1.

[0049] FIG. 4 shows similar flux densities as FIG. 3, but with a smaller distance between the magnetic field sensor and the magnetic element.

[0050] FIG. 5 shows a combined parameter derived from the flux densities of FIG. 4, as a function of the deflection of the elastic rod of the system of FIG. 1.

[0051] FIG. 6 shows an elastic element with an adjustable spring constant.

[0052] FIG. 7 shows the magnetic field distribution in an embodiment in which the elastic element itself is magnetic.

[0053] FIG. 8 shows the magnetic field component parallel to the direction of motion of the tip of the elastic element of FIG. 7 upon deflection.

[0054] FIG. 9 shows the magnetic field distribution for an embodiment in which the magnetic element is formed by two anti-parallel permanent magnets.

[0055] FIG. 10 shows the magnetic field component perpendicular to the direction of motion of the tip of the elastic element.

[0056] FIG. 11 is a diagram showing the relative twitch force versus cultivation time for samples of adult human myocardium cultivated in a system according to FIG. 1.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0057] For the purposes of promoting an understanding of the principles of the invention, reference will now be made to a preferred embodiment illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated apparatus and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur now or in the future to one skilled in the art to which the invention relates.

[0058] FIG. 1 shows a schematic representation of a system 10 according to a preferred embodiment of the present invention. As seen therein, the system 10 comprises a culture chamber 12 in which a cell or tissue culture schematically shown at 14 is arranged. The cell or tissue culture 14 could for example be a piece of functional bioartificial cardiac tissue that could serve as an artificial patch for transplantation. While not shown in FIG. 1, the culture chamber 12 further includes a nutrition solution which is kept at physiologic temperatures, such that warm and humid conditions are present inside the culture chamber 12.

[0059] As is explained for example in Kensah, G. et al., an optimal tissue replacement should be able to exert contractile forces comparable to that of native cardiac tissue. By mechanically stretching the cell or tissue culture, the tissue formation and contractility of the bioartificial cardiac graft can be improved. For this purpose, a linear drive 16 is provided for applying a force, more precisely a tensile force to the cell or tissue culture 14. More precisely, the cell or tissue culture 14 is held by a first and a second holding element 18, 24, respectively. Herein, the second holding element 24 has a holding section 26 for holding said cell or tissue culture 14, and a coupling section 28 for coupling with the linear drive 16. The first holding element 18 likewise has a holding section 24 for holding the opposite end of the cell or tissue culture 14, and a coupling section 22 for coupling with a rod 30, which rod 30 resembles an example of the elastic element referred to in the introductory portion of the description.

[0060] While not shown in detail in FIG. 1, the holding sections 20 and 26 of said first and second holding element 18, 24 could each be a rod that is integrally cast with a cell/matrix mixture in a similar way as disclosed in Kensah, G. et al. Alternatively, the holding sections 20 and 26 of said first and second holding elements 18, 24 could include a clamping device for clamping the cell or tissue culture 14. Instead of using a clamping device, native or bioartificial tissue, or matrix prepared for cell seeding can be attached to the holding elements 18, 24 by cyanoacrylate or protein-based adhesives, as described by Brandenburger, M et al., Cardiovasc Res. 2012; 93(1): 50-9.

[0061] With further reference to FIG. 1, the rod 30 is made from surgical grade steel. In some embodiments, the rod 30 may additionally be coated with a biocompatible material, such as Teflon or Parylene. The rod 30 has a fixed end 30a that is attached to a wall of the culture chamber 12 using a fastener 32. Opposite to the fixed end 30a, the rod 30 has a free end 30b at which a magnetic element 34 is disposed. In the embodiment shown, the magnetic element 34 is a permanent magnet that is coated with a biocompatible material. More precisely, the magnetic element 34 is a permanent magnet made from an AlNiCo alloy that is goldplated and that is further coated with Parylene or Teflon. When a tensile force is applied to the cell or tissue culture 14 by means of the linear drive 16, this force leads to a deflection of the rod 30, and consequently to a movement of the magnetic element 34, wherein the direction of the movement of the magnetic element 34 is indicated by the arrow 36 in FIG. 1. Moreover, the magnetic element 34 is arranged such that its magnetic dipole moment is parallel to this direction of motion, or at most tilted with regard to this direction of motion by 30?, preferably by less than 20? and most preferably by less than 12?.

[0062] Finally, stimulating electrodes 38 are provided inside the culture chamber 12, which are devised for applying electrical stimuli to the cell or tissue culture 14. Each of the cell or tissue culture 14, the first and second holding elements 18, 24, the rod 30 and the stimulating electrodes 38 are arranged inside the culture chamber 12, and are hence exposed to the humid atmosphere kept at physiological conditions therein.

[0063] Outside the culture chamber 12, a sensor module 40 is provided, which includes a magnetic field sensor 42. The sensor module 40 is removably attached to the culture chamber 12 by means of a receptacle 44 schematically shown in FIG. 1. The receptacle 44 is an example of the adapter referred to in the introductory portion of the specification. The magnetic field sensor 42 is capable of measuring the magnetic field strength, more precisely the magnetic flux density in three dimensions X, Y and Z, wherein the dimensions X and Y are indicated by the coordinate system shown in FIG. 1, and wherein the dimension Z is perpendicular to the paper plane.

[0064] Finally, the system 10 comprises a control unit 46, which is operatively connected with the linear drive 16 and the stimulation electrodes 38 for controlling the same, and which is connected to receive signals representing magnetic field information obtained with said magnetic field sensor 42 from the sensor module 40.

[0065] Next, the function of the system 10 shown in FIG. 1 is explained with reference to FIGS. 2 to 5.

[0066] FIG. 2 is a close up view of the magnetic field sensor 42, the magnetic element 34, the wall of the culture chamber 12 separating the magnetic field sensor 42 from the magnetic element 34 as well as the magnetic field generated by the magnetic element 34. When the magnetic element 34 is moved downwards in FIG. 2, upon deflection of the rod 30 in response to a tensile force applied by said linear drive 16, the magnetic flux density both for the X and the Y component measured by the magnetic field sensor 42 will change, as demonstrated in FIGS. 3 and 4. FIG. 3 shows the magnetic flux densities B.sub.X and B.sub.Y measured by the magnetic field sensor 42 in X and Y direction as a function of deflection of the free end 30b of the rod 30, or in other words, as a function of the movement of the magnetic element 34, provided that the magnetic element 34 and the magnetic field sensor 42 are arranged at a distance of 2.5 mm apart.

[0067] FIG. 4 shows the same parameters in a situation where the magnetic element 34 and the magnetic field sensor 42 are arranged at a distance of only 1.5 mm. Both of these distances are sufficient to accommodate a wall of a culture chamber 12 in between, or in other words, to arrange the magnetic element 34 inside and the magnetic field sensor 42 outside the culture chamber 12.

[0068] As can be discerned from FIG. 3, at a distance of 2.5 mm, the magnetic flux density in X direction is an approximately linear function of the dislocation of the magnetic element 34 within a range of ?0.9 mm to +0.9 mm with respect to a rest position. It was found that from the measured flux density in X direction alone, the dislocation of the magnetic element 34 due to deflection of the rod 30 could be measured with a resolution of 3 ?m.

[0069] When the distance between the magnetic element 34 and the magnetic field sensor 42 is decreased to 1.5 mm, the relative resolution of the magnetic flux density becomes expectedly higher, but the linear region becomes more narrow, as indicated by the shaded area in FIG. 4, and in fact decreases to a range from ?0.6 mm to +0.6 mm.

[0070] FIG. 5 shows an example, where a combined parameter is calculated from B.sub.X and B.sub.Y according to the formula B.sub.X/(B.sub.Y+2?B.sub.Ymin), where B.sub.Ymin corresponds to the minimum value of By in FIG. 5. As seen from FIG. 5, this combined parameter, even when measured at a distance of only 1.5 mm between the magnetic element 34 and the magnetic field sensor 42, shows a linear behavior in a much larger range of dislocations from about ?2.0 mm to +2.0 mm.

[0071] In the embodiment shown, the magnetic field components B.sub.X and B.sub.Y are digitized within the sensor module 40 and transferred to the control unit 46, where the combined parameter of FIG. is calculated therefrom. Accordingly, in this embodiment the control unit 46 resembles an example of the aforementioned logic element suitable for translating the measurement result of the magnetic field sensor 42 into a parameter representing the deflection of the elastic element. The control unit 46 can then, based on a known force-deflection-curve of the rod 30 likewise calculate the force applied to the cell or tissue culture 14.

[0072] Based on the thus determined force applied to the cell or tissue culture 14, the control unit 46 can then provide for a desired mechanical stimulation, which can be accompanied by a suitable electrical stimulation using the stimulating electrodes 38.

[0073] As is seen from FIGS. 3 to 5, the detection of the deflection by means of the magnetic field sensor 42 works well within a certain range of deflections. In order to provide for a wide range of forces that can be measured, in one embodiment of the invention, a set of elastic rods 30 can be provided with different spring constants to choose from, so that depending on the desired force to be applied to the cell or tissue culture 14, a rod 30 can be chosen that will largely exhaust, but not exceed the range of deflection corresponding to the shaded area in FIG. 5.

[0074] Instead of providing a set of rods with different spring constants, it is also possible to provide a rod 30 with an adjustable spring constant. For example, the rod 30 may be provided with a rigid sleeve 48 that can be slipped on the rod 30 to locally stiffen the same and hence increase the spring constant of the rod 30, as shown in FIG. 6. The spring constant can be further adjusted by the position of the sleeve 48 on the rod 30 by means of a threaded connection between a threaded element 50 and the sleeve 48. By turning a knob 52 connected with said threaded element 50, the position of the sleeve 48 can be adjusted along a direction indicated by arrows 54.

[0075] In the embodiment shown, the culture chamber 12 with the rod 30 pre-mounted therein is a disposable product, that is not to be reused. However, using the fastener 32, it is easy for the manufacturer to pre-assemble the culture chamber 12 with rods 30 of different strengths and correspondingly different spring constants. The sensor module 40, on the other hand, is reused and can be easily combined with any fresh culture chamber 12, by simply inserting the same into the receptacle 44 shown in FIG. 1. Importantly, the receptacle 44 is arranged such that when the sensor module 40 is received therein, the magnetic field sensor 42 included in the sensor module 40 will be appropriately arranged with regard to the magnetic element 34 provided at the free end 30b of the rod 30.

[0076] As mentioned in the introductory portion of the specification, in some embodiments the elastic element 30 itself may be magnetic, such that no additional magnetic element needs to be provided. Instead, in such an embodiment, the elastic element itself forms the magnetic element referred to herein.

[0077] FIG. 7 shows the magnetic field distribution of a magnetic elastic rod 30 and a magnetic field sensor 42 arranged in said magnetic field. FIG. 8 shows the magnetic field component parallel to the direction of motion of the tip 30b of the elastic element 30 upon deflection of the elastic element as measured by the magnetic field sensor 42. From the geometry of the magnetic field shown in FIG. 7, it will be appreciated that without deflection, i.e. for vanishing magnet displacement, the magnetic field strength is zero. As further shown in FIG. 8, if the tip 30b of the magnetic elastic element 30 is displaced within a range of ?0.9 mm, the magnetic field strength varies approximately proportionally with the displacement of the tip 30b (referred to as magnet displacement in FIG. 8).

[0078] FIG. 9 shows the magnetic field distribution for a further embodiment, in which the magnetic element 34 is formed by two anti-parallel permanent magnets 56, and the magnetic field sensor 42 is arranged in the corresponding magnetic field.

[0079] FIG. 10 shows the magnetic field component perpendicular to the direction of motion of the magnetic element 34 as measured by the magnetic field sensor 42 upon deflection of the corresponding elastic element 30 (not shown). In FIG. 9, this direction of motion corresponds to a vertical movement. From the geometry of the magnetic field shown in FIG. 9, it will be appreciated that in the rest position shown in the figure (i.e. without deflection), the magnetic field strength perpendicular to the direction of motion is zero. As is further seen from FIG. 10, in the vicinity of the rest position shown in FIG. 9, the magnetic field strength varies approximately proportionately with the displacement of the magnetic element 34 in the vertical direction.

[0080] The ability of the described tissue culture system 10 to maintain vitality and functionality of myocardium in vitro was tested with a typical application. Samples of adult human myocardium 14 were obtained from explanted failing hearts of consenting patients receiving a heart transplant.

[0081] The tissue was cut into thin slices according to an established procedure as described in Brandenburger M, Wenzel J, Bogdan R, Richardt D, Nguemo F, Reppel M, Hescheler J, Terlau H, Dendorfer A. Organotypic slice culture from human adult ventricular myocardium. Cardiovasc Res, 93(1), 50-59, 2012. Vital tissue specimen were glued to triangle tissue holders 18, 24, and were mounted in culture chambers 12, as exemplified in FIG. 1. By means of the described technique, a passive strain could be applied that resembled physiological diastolic stress of the cardiac wall. Under continuous electrical stimulation (0.2 Hz, 1 ms pulse duration, bipolar), active contractions were recorded over a culture period of up to 10 days. In comparison to the force development of hypertrophic human myocardium subjected to a previously established technique of tissue cultivation in a quiescent state (square symbols in FIG. 11), contractile performance was better preserved under biomimetic conditions (round symbols in FIG. 11). It is hence seen that the system 10 of FIG. 1 allows for: [0082] well controlled biomechanic conditions and relevant readouts, [0083] long-term functional preservation, thus enabling chronic manipulation and the investigation of protracted alterations, and [0084] continuous measurement of contractility, thus e.g. enabling assessment of drug effects in a single tissue specimen.

[0085] In general, it is expected that the culture technique described for adult myocardial tissues will also improve the maturation of artificial tissues engineered from differentiated stem cells and a biocompatible matrix. Furthermore, its application to any kind of mechanosensitive tissue (e.g. skeletal muscle, smooth muscle from vessel, gut, bronchi, etc., connective tissue, tendon, cartilage, bone etc.) can be envisaged.

[0086] As was explained above, an important advantage of the system and method of the invention is that the spring constant of the elastic element 30 allows for simulating in vitro the elasticity of the surrounding tissues with the cell or tissue culture would experience in its natural physiological environment, i.e. in vivo.

[0087] To give a specific example, it shall be assumed that for the culture of a heart muscle, the Young's modulus of the heart muscle in systole shall be simulated. As can be taken from Hassaballah A I, Hassan M A, Mardi A N, Hamdi M. An inverse finite element method for determining the tissue compressibility of human left ventricular wall during the cardiac cycle. PLoS One. 2013; 8(12):e82703, this Young's modulus may amount to about 500 kPa. It follows that for a sample size of 5?5?0.3 mm.sup.3 (length?width?thickness), and for a contraction of 0.5 mm, a force of 500 mN/mm.sup.2?5 mm?0.3 mm?0.5 mm/5 mm=75 mN is generated. In order to simulate the elasticity of the surrounding tissue, an elastic element 30 with a spring constant of 150 mN/mm is therefore needed. This can be for example achieved in the setup shown above with a rod 30 formed by steel wire having an effective length of 10 mm and a diameter of 0.28 mm. Namely, the spring constant of a bending rod is defined as 3?E?Iy/I.sup.3 with E (elastic modulus)=180 GPa for V2A steel and Iy (second moment of inertia)=r.sup.4??/4 for a cylindrical rod. With these values, a spring constant 3?E?r.sup.4??/4/1.sup.3 of 150 mN/mm is obtained.

[0088] Although a preferred exemplary embodiment is shown and specified in detail in the drawings and the preceding specification, these should be viewed as purely exemplary and not as limiting the invention. It is noted in this regard that only the preferred exemplary embodiment is shown and specified, and all variations and modifications should be protected that presently or in the future lie within the scope of protection of the invention as defined in the claims.

REFERENCE SIGN LIST

[0089] 10 system [0090] 12 culture chamber [0091] 14 cell or tissue culture [0092] 16 linear drive [0093] 18 first holding element [0094] 20 holding section [0095] 22 coupling section [0096] 24 second holding element [0097] 26 holding section [0098] 28 coupling section [0099] 30 rod [0100] 30a fixed end [0101] 30b free end [0102] 32 fastener [0103] 34 magnetic element [0104] 36 arrows [0105] 38 stimulating electrodes [0106] 40 sensor module [0107] 42 magnetic field sensor [0108] 44 receptacle [0109] 46 control unit [0110] 48 sleeve [0111] 50 threaded element [0112] 52 knob [0113] 54 arrows [0114] 56 permanent magnet