JP4129285B2 - Quality control method for cultured tissue - Google Patents

Description

    The present invention relates to a quality control method for cultured tissue cultured in vitro and a method for producing a cultured tissue having transplantation suitability.

  In recent years, in vitro (in vitro), cells are seeded and cultured on a tissue regeneration substrate (carrier) that serves as a scaffold, and the tissue regeneration is performed to reconstruct human tissue, and the reconstructed cultured tissue is returned to the living body. Regenerative medicine and tissue engineering, which treats by applying, are attracting attention. It is necessary to examine whether or not such a cultured tissue for transplantation is in a state suitable for transplantation when applied to a living body. Alternatively, when such a cultured tissue for transplantation is shipped from a manufacturer, it is necessary to inspect whether or not the tissue is suitable for shipment.

  For example, as a method for confirming the structure of the cultured tissue, a method is known in which a part of the cultured tissue is cut to produce a tissue section, the production substrate is stained with a staining agent, and then the stained image is observed with a microscope. This method can be used as an inspection method for transplantation aptitude, an inspection method over time of the culture state, or an inspection method at the time of shipment (offer).

  However, it takes time to produce a tissue section for examination, and observation with a microscope is troublesome and burdensome for the examiner. Furthermore, in order to obtain a stained image at each observation, a part of the cultured tissue is cut out and used, or another cultured tissue piece cultured under the same conditions (cultured tissue piece of the same lot) is prepared. Many pieces of test tissue are required.

  In addition, it is attempted to process the cultured tissue with a cork borer and investigate the relationship between the amount of the production substrate and the hardness of the cultured tissue by a destructive test such as a compression test. It is necessary to inspect other cultured tissues of the lot as inspection tissue pieces, and many inspection tissue pieces are required as in the case of the stained image observation.

  In addition, cell growth is influenced by the age of the cell provider, or shows different cell growth depending on the individual cell. Therefore, the degree of growth is slightly different for each cultured tissue, and the transplantability of the cultured tissue is appropriate. It was difficult to predict the culture such as when it was in a state. In addition, because it is difficult to predict the culture, it is difficult to accurately control the culture to bring the cultured tissue to a desired state. As a result, the quality of providing the cultured tissue is stabilized, and the culture has transplantability. It was difficult to reliably and easily manufacture the tissue.

  In view of the above prior art, the present invention easily determines the appropriateness of transplantation of a cultured tissue without destroying the cultured tissue, and a quality control method capable of easily and appropriately managing the quality of the cultured tissue, It is an object of the present invention to provide a manufacturing method that can be easily manufactured.

The present invention for solving the technical problem is as follows: ( 1) Measuring the hardness information of a cultured tissue having a three-dimensional structure that is cultured in vitro and changes in hardness or elasticity as the culture period elapses; Based on the measurement results, grasp the state of cell growth and the amount of the production substrate, and based on the state, the quality control method for the cultured tissue for determining the transplantability of the cultured tissue ,
( 2) Calculation of hardness information based on a detection unit including a contact unit, a vibrator connected to the contact unit, a vibration detection unit for detecting vibration of the vibrator, and a detection result from the vibration detection unit A quality control method of the cultured tissue according to (1), wherein a hardness measuring device having a calculation means is used, and the hardness of the cultured tissue is measured by bringing the contact portion into contact with the cultured tissue,
(3) The detection unit further includes a load detection unit that detects a load applied to the contact unit, and a relationship between the vibration of the vibrator detected by the vibration detection unit and the load detected by the load detection unit. The quality control method of the cultured tissue according to (2), wherein the hardness of the cultured tissue is measured based on
(4) The detection unit further includes a displacement amount detection unit that detects a displacement amount of the contact portion from a reference position, and is detected by the vibration of the vibrator detected by the vibration detection unit and the displacement amount detection unit. The quality control method of the cultured tissue according to (2), wherein the hardness of the cultured tissue is measured based on the relationship with the amount of displacement,
(5) The cultured tissue according to any one of (1) to (4), wherein the cultured tissue is produced by seeding and culturing cells on a tissue regeneration substrate having a three-dimensional structure. Quality control method,
(6) The tissue regeneration substrate is collagen, gelatin, hyaluronic acid, fibronectin, fibrin, chitin, chitosan, laminin, dermatan sulfate, heparan sulfate, chondroitin sulfate, calcium alginate, calcium phosphate, calcium carbonate, polyglycolic acid, polylactic acid And a quality control method for a cultured tissue according to (5), comprising any one of polyrotaxane and
(7) As a source cell of the cultured tissue, at least one of chondrocytes, osteoblasts, fibroblasts, endothelial cells, epithelial cells, myoblasts, adipocytes, hepatocytes, nerve cells, or progenitor cells thereof. The quality control method of the cultured tissue according to any one of (1) to (6), and
(8) A method for producing a cultured tissue including the quality control method according to any one of (1) to (7) .
The method for measuring the hardness of the cultured tissue in the quality control method of (2) of the present invention includes a contact part, a vibrator connected to the contact part, and a vibrator for determining the transplantability of the cultured tissue cultured in vitro. Using the hardness measurement apparatus having a detection unit including a vibration detection unit for detecting vibration of the vibrator and calculation means for calculating hardness information based on a detection result from the vibration detection unit, the culture The contact portion is brought into contact with the tissue, and the hardness of the cultured tissue is measured.

  According to this hardness measurement method, when the contact portion comes into contact with the cultured tissue and the vibration frequency of the vibrator changes, the hardness information of the cultured tissue can be obtained, and the hardness of the cultured tissue can be determined from the hardness information. It can be measured. Based on the hardness of the cultured tissue thus obtained, the measurement of the mass of the production base of the cultured tissue or the suitability for transplantation based on this can be determined.

The hardness measurement method in the quality control method of (3) of the present invention is the hardness measurement method of (2) , wherein the detection unit further comprises a load detection unit that detects a load applied to the contact unit, The hardness of the cultured tissue is measured based on the relationship between the vibration of the vibrator detected by the vibration detector and the load detected by the load detector.

  According to this hardness measurement method, the load information applied to the contact portion is obtained, and the hardness of the cultured tissue can be measured based on the relationship between the load information and the vibration of the vibrator. Based on the hardness of the obtained cultured tissue, the transplantability of the cultured tissue can be determined more appropriately.

The hardness measurement method in the quality control method of (4) of the present invention is the hardness measurement method of (2) , wherein the detection unit includes a displacement amount detection unit that detects a displacement amount from a reference position of the contact unit. In addition, the hardness of the cultured tissue is measured based on the relationship between the vibration of the vibrator detected by the vibration detection unit and the displacement detected by the displacement detection unit.

  According to this hardness measurement method, a displacement is obtained by displacing the vibrator from the reference position, and the hardness of the cultured tissue is measured based on the relationship between the displacement and the vibration of the vibrator. Based on the hardness of the obtained cultured tissue, the transplantability of the cultured tissue can be determined more appropriately.

In the hardness measuring method of the quality control method, that the cultured tissue is having a one production substrate produced from seed-cultured cells and the cells in the tissue regeneration substrate having a three-dimensional structure .

  The tissue regeneration substrate in the quality control method for cultured tissue is collagen, gelatin, hyaluronic acid, fibronectin, fibrin, chitin, chitosan, laminin, dermatan sulfate, heparan sulfate, chondroitin sulfate, calcium alginate, calcium phosphate, calcium carbonate, It may contain any one of polyglycolic acid, polylactic acid and polyrotaxane.

  Further, the cells in the quality control method of the above cultured tissue are chondrocytes, osteoblasts, fibroblasts, endothelial cells, epithelial cells, myoblasts, adipocytes, hepatocytes, nerve cells, or precursor cells thereof. At least one of the following.

In the quality control method of the present invention, by using any one of the above (2) to (4), it is possible to objectively determine the transplantability of a cultured tissue based on the hardness measured without destroying the cultured tissue. And appropriately.

The present invention includes a method for producing a cultured tissue including the quality control method according to any one of (1) to (7).
According to this method for producing a cultured tissue, since the culture is performed while measuring the hardness, the cultured tissue can be cultured while determining the suitability for transplantation using the hardness of the cultured tissue as an index. Thereby, a culture tissue suitable for transplantation can be reliably produced.

  As described above, according to the present invention, the hardness of the cultured tissue can be measured without destroying the cultured tissue, and the quality of the cultured tissue can be easily and appropriately managed, and has transplantability. A cultured tissue can be produced reliably and easily.

  With reference to FIG. 1, the hardness measuring apparatus 10 which concerns on this invention is demonstrated. The hardness measurement apparatus 10 includes a probe 20 (corresponding to the detection unit of the present invention) fixedly held on the gantry 12 and a control unit 40 that controls driving of each part of the probe 20 and detection signals from each sensor in the probe 20. (Corresponding to the calculation means of the present invention).

  In the probe 20, a measuring element 22 including a tactile sensor unit 24 and a pressure sensor unit 26, and a motor 30 that drives the measuring element 22 in the vertical direction are arranged. A displacement sensor unit 28 is integrally fixed to the motor 30 and detects the amount of movement of the probe 22 based on the number of rotations of the drive shaft of the motor 30. The motor 30 is fixed to the probe 20, and moves up and down a measuring element 22 that can be moved up and down inside the probe 20.

  The tactile sensor unit 24 is configured by integrally connecting a contact 32, a vibrator 34, and a pickup 36. The contact 32 has a hemispherical shape and is provided at the tip of the probe 22. The diameter of the contactor 32 can be appropriately changed according to the size and type of the cultured tissue (object) to be measured, the measurement method, and the like. The vibrator 34 is composed of a piezoelectric element or the like, and the pickup 36 is composed of a vibration sensing piezoelectric element or the like.

  The pressure sensor unit 26 includes a pressure sensor including a pressure sensitive element. This pressure sensor is fixed to the pickup 36 of the tactile sensor unit 24 and detects a load applied to the contact 32. The pressure sensor is fixed in such a manner that its natural frequency does not affect the frequency detection by the pickup 36.

  The displacement sensor unit 28 includes a displacement sensor including an encoder, a potentiometer, and the like. The displacement sensor detects the displacement of the position based on the amount of movement when the probe 22 moves in the vertical direction in FIG. 1 by driving the motor 30 disposed on the probe 20.

  The probe 20 is attached to the gantry 12 via the arm 14 and the support shaft 16 so as to be able to approach and separate. The stage 12 is provided with a stage 18 so as to face a contact 32 disposed at the lower end of the probe 20. A cultured tissue to be measured is placed on the stage 18.

The control unit 40 includes a control unit 42 and a self-excited oscillation circuit 44, and is connected to a computer not shown.
The self-excited oscillation circuit 44 is composed of an amplification circuit and a band filter not shown, and is connected to the vibrator 34 and the pickup 36. The self-excited oscillation circuit 44 has a circuit configuration in which an output signal from the pickup 36 is amplified by an amplifier circuit and then forcedly fed back to the vibrator 34 through a band-pass filter. As a result, by adjusting the amplification degree of the amplifier circuit and the characteristics of the band filter, the input / output signal phase difference of the self-excited oscillation circuit 44 is adjusted to zero, and the vibrator 34 is vibrated to perform self-excited oscillation.

The control unit 42 is connected to the self-excited oscillation circuit 44, and adjusts the frequency characteristics of the band-pass filter to be applied to the frequency characteristics of the transducer 34 for the vibration of the transducer 34 in the tactile sensor unit 24. The hardness information is calculated.
The control unit 42 is connected to the pressure sensor unit 26 and the displacement sensor unit 28, respectively. The load information and the displacement are respectively determined from the load detected by the pressure sensor unit 26 and the displacement detected by the displacement sensor unit 28. Calculate information.

  The displacement information is calculated based on the position information from the displacement sensor unit 28. In the present embodiment, an arbitrary position is set as an initial position, and this initial position is set as a reference position in the position information. Further, the position information obtained when this reference position is set to zero and the amount of movement of the probe 22 moved downward by the motor 30 is positive is used as displacement information.

  In addition, the section from the initial position of the probe 22 to the contact with the cultured tissue can be set as a free running distance, and the movement amount after the contact with the cultured tissue can be used as the displacement information. In this case, based on the pressure information from the pressure sensor unit 26, the position where the probe 22 contacts the surface of the cultured tissue may be used as the reference position, and the displacement information may be based on the difference from the amount of movement obtained from the displacement sensor unit 28. . Further, the contact position may be determined based on the tactile information from the tactile sensor unit 24 instead of the pressure information from the pressure sensor unit 26.

  When the natural frequency of the pressure sensor of the pressure sensor unit 26 affects the frequency detection in the pickup 36, the natural frequency of the pressure sensor is corrected to detect an appropriate frequency change. You can also

  A computer (not shown) connected to the control unit 40 can set various measurement conditions by an operator. Examples of the setting items include a moving speed (1 to 4 mm / s) and a moving amount (1 to 10 mm) of the probe 22. The computer performs measurement by controlling the movement of the probe 22 by the motor 30 via the control unit 40 based on these setting conditions. Detection information from each sensor unit is transmitted to the control unit 42, and the detection information is recorded in synchronization with a storage unit (not shown).

  In the hardness measuring apparatus 10, the hardness of the cultured tissue cultured in vitro is measured. The cultured tissue that can be measured here may be any tissue that has been cultured in vitro and can be measured by the hardness measurement apparatus 10, but the hardness or elasticity of the cultured tissue changes as the culture period elapses. It is necessary to be an organization. Examples of cells contained in such cultured tissues include chondrocytes, osteoblasts, fibroblasts, endothelial cells, epithelial cells, myoblasts, adipocytes, hepatocytes, nerve cells, or precursor cells thereof. Can do.

  In addition, if a sufficient structure is maintained as a cultured tissue, a tissue regeneration substrate is not necessarily required. However, a cultured tissue to be measured includes a tissue regeneration substrate having a three-dimensional structure, and the tissue regeneration. It is preferable that it is composed of at least one of cells and production substrate held on the substrate for use. Such a cultured tissue is suitable for transplantation into a living body as it is, and the measurement by the hardness measuring apparatus 10 can be more appropriately performed. From the viewpoint of such transplantation form and ease of measurement, the cultured cells for which transplantation determination is performed according to the present invention are more preferably chondrocytes, osteoblasts or their precursor cells.

  Applicable tissue regeneration base materials include collagen, gelatin, hyaluronic acid, fibronectin, fibrin, chitin, chitosan, laminin, dermatan sulfate, heparan sulfate, chondroitin sulfate, calcium alginate, calcium phosphate, calcium carbonate, polyglycolic acid And polylactic acid and polyrotaxane. Among these, collagen, gelatin, hyaluronic acid, and fibrin are more preferable from the viewpoint of biocompatibility and bioabsorbability.

  When the cultured tissue is measured by the hardness measuring device 10, the dish D containing the cultured tissue to be measured is placed on the stage 18 on the gantry 12. At this time, it can be changed depending on the size of the contact 32, but from the viewpoint of measuring a more appropriate hardness by the hardness measuring device 10, the surface area of the cultured tissue is preferably larger than the surface area of the contact 32. The thickness of the cultured tissue is preferably larger than the measurement depth at which the tissue characteristics can be obtained. In addition, when the size of the cultured tissue is larger than the size of the contactor 32 and can be measured at a plurality of locations on the cultured tissue, it is preferable to measure at a plurality of locations from the viewpoint of measuring a more appropriate hardness. In addition, when the shape of the cultured tissue is constant, the measurement result is stable, and the measurement of substrate production and the determination of transplantability can be performed appropriately.

  From the viewpoint of measuring more appropriate hardness, it is also preferable to change the size of the contact 32 in addition to changing the size of the cultured tissue or increasing the number of measurement points. Depending on the size of the cultured tissue, if the contact surface of the contact 32 is made larger, hardness information reflecting the hardness of the entire cultured tissue can be obtained, and the diameter of the contact 32 can be reduced. In addition, when the measurement is performed by increasing the number of measurement points, the local hardness of the cultured tissue can be measured, and the hardness can be accurately measured with good reproducibility by obtaining an average value. From such a viewpoint, when it is desired to obtain hardness information reflecting the hardness of the entire cultured tissue, the contact surface of the contact 32 is about 1 to 1/5 with respect to the projected area of the cultured tissue. Preferably it is. On the other hand, when it is desired to obtain local hardness information of the cultured tissue, it is preferable that the contact surface of the contactor 32 is 1/5 or less of the projected area of the cultured tissue, and reproducibility. In order to measure the hardness well and accurately, it is preferable to measure at 5 to 50 measurement points for one cultured tissue.

Next, the operation of the hardness measuring apparatus 10 will be described.
The cultured tissue to be measured is placed on the stage 18, the arm 14 and the support shaft 16 are adjusted, and the probe 20 is placed above the cultured tissue. The moving speed and moving amount of the probe 22 are set, and measurement is started.

When the measurement is started, the motor 30 starts to be driven and the vibrator 34 starts to vibrate, the measuring element 22 approaches the culture tissue at the set moving speed, and the contact 32 of the measuring element 22 is cultured. Contact with tissue. When the contact 32 comes into contact with the cultured tissue, the vibration frequency of the vibrator 34 changes. After contact with the cultured tissue, the probe 22 moves at the set moving speed, and when the movement of the moving amount from the set reference position is completed, the controller 42 instructs the motor 30 to reverse. The measuring element 22 is moved upward by the reversal of the motor 30, the contact element 32 is separated from the cultured tissue, returns to the reference position, and the measurement is finished.
The measurement of hardness by this series of operations is performed by using one or a plurality of output signals from the tactile sensor unit 24, the pressure sensor unit 26, and the displacement sensor unit 28 obtained in synchronization with the vertical movement of the probe 22. It is performed based on numerical values, changes, relationships, etc. by sensors.

  The principle of the hardness measuring apparatus 10 is based on the fact that when the contact 32 that is naturally oscillating is brought into contact with the measurement object, the frequency of the contact 32 changes according to the measurement object. By applying a predetermined calculation process based on the amount of change in the frequency, it is possible to measure the hardness information of the measurement object. Details of such a hardness measuring device are described in Japanese Patent Application Laid-Open No. 09-145691 (Title of Invention: Frequency Deviation Detection Circuit and Measuring Instrument Using the Same). The operator operates the computer to set measurement conditions and perform hardness measurement.

  In the present invention, the transplantability of the cultured tissue is determined by measuring the hardness using the hardness measurement apparatus 10 described above. In addition, by measuring aseptically and non-destructively with the measuring device 10, the cultured tissue for transplantation itself can be measured, and the transplantability can be determined.

<Transplantability determination>
The determination of the transplantability of the cultured tissue according to the present invention is based on the fact that the hardness of the cultured tissue changes with the culture period. Such a hardness of the cultured tissue is one of the requirements for the transplanted cultured tissue to appropriately act on the application site of the patient when the cultured tissue is transplanted into the living body, and the ease of handling the cultured tissue. It is also one of the requirements. The change in the hardness of the cultured tissue depends on the number of cells in the cultured tissue, the amount of substrate produced from the cells, and the like, and varies depending on the nature of the cell type constituting the cultured tissue. Therefore, the mass of the production base of the cultured tissue can also be measured by measuring the hardness of the cultured tissue. For this reason, the specific determination of the transplantability based on the hardness of the cultured tissue is performed for each type of the cultured tissue. Here, the method for determining the transplantability of the cultured tissue according to the present invention using the cultured cartilage tissue as an example. Will be explained.

  “Determining transplantability” in the present invention refers to determining whether or not the cultured tissue is suitable for transplantation at the time of transplantation. In the present invention, the time until transplantation at a specific time point is taken into consideration. It also includes determining whether the state is appropriate. For example, since the shipping time is a predetermined period earlier than the time of transplantation, it is preferable to determine whether or not the cultured tissue is in an appropriate state in consideration of storage / preservation conditions and the like for that period. . For this reason, it is preferable to determine whether or not the cultured tissue is in an appropriate state at the time of shipment at the time of shipment, and the present invention can also be used in such a case.

  The transplantability of the cultured tissue is determined based on the frequency information obtained from the tactile sensor of the hardness measuring device 10, the load information obtained from the pressure sensor, and the displacement information obtained from the displacement sensor. The determination is made based on various hardness information from a change or a relationship between a plurality of sensor information.

(1) Determination based on frequency information of tactile sensor The hardness of the cultured cartilage is measured by the hardness measuring device 10, and the transplantability of the cultured cartilage is determined depending on whether or not the measurement result is within an appropriate range. . That is, the frequency change amount Δf obtained from the tactile sensor when a predetermined displacement or load is applied to the probe 22 of the hardness measuring device 10 and brought into contact with the cultured tissue is within an appropriate range for the tissue for transplantation. Whether transplantation is appropriate or not is determined. This appropriate range varies depending on various factors such as the cell type, the type and shape of the carrier, the measurement conditions such as contact diameter, and the target disease. It is necessary to decide.

  For example, in the case of a cultured cartilage tissue in which chondrocytes are embedded and cultured in a collagen gel and cultured in a dome shape having a diameter of 10 mm and a thickness of 3 mm, the probe 20 having a hemispherical contactor 32 having a diameter of 5 mm is used. Consider a case where the hardness measurement is performed by setting a position about 0.5 mm above the zenith and giving a moving amount of 2 mm (indentation amount of about 1.5 mm from the cultured tissue zenith) (hereinafter, cultured cartilage) Called tissue measurement model). In such a cultured cartilage tissue measurement model, the appropriate range of hardness when the contact 32 is pushed down about 1.0 mm below the zenith is −200 to 0 Hz in terms of the frequency change Δf, preferably -150 to 0 Hz. When the frequency is lower than −200 Hz, the cultured tissue is not sufficiently hard, and transplantation in this state is not preferable. When the frequency is higher than 0 Hz, the cultured tissue is too hard, so the affinity at the transplantation destination is low, and the possibility of dropping off is considered. The frequency change amount Δf in the present embodiment is obtained by subtracting the natural frequency of the vibrator from the measured frequency.

Further, the determination criterion may be set based on the value of Δf at a predetermined load. For example, in the case of the above-described cultured cartilage tissue measurement model, −150 to 0 Hz is preferable at a load of 0.067 N, and −100 to 0 Hz is more preferable. Outside this range, it is too soft or too hard as described above, which is not preferable for transplantation.
On the other hand, the collagen gel-embedded cultured cartilage tissue produced in a dome shape with a diameter of 30 mm and a thickness of 3 mm is measured from the zenith direction with the probe 20 having a hemispherical contactor 32 with a diameter of 1 mm. In this case, the appropriate range when a load of 0.0049 N (0.5 gf) is applied is −15 to 0 Hz, preferably −10 to 0 Hz in terms of the frequency change Δf. Thus, the appropriate range varies depending on the measurement conditions and the state of the cultured tissue.

(2) Comparison and judgment based on frequency information of tactile sensor Since the change in hardness is almost proportional to the growth of cells and the amount of production substrate, the measurement of the mass of the production group and the transplantability of cultured tissue The determination can be performed by defining a reference model in advance.
In this case, the transplantation suitability is determined by comparing the frequency information of only the carrier that has been cultured under the same conditions as the cultured tissue without seeding the cells as a determination reference model. In addition, since the comparison reference | standard for judging transplant suitability changes variously by the object carrier, cell, culture conditions, measurement conditions, etc., it is necessary to define the suitability range corresponding to each culture tissue.

A predetermined load or displacement is applied to the probe 22 of the hardness measuring apparatus 10 and brought into contact with the determination reference model, and the amount of change in frequency (Δf ₀ ) is measured in advance. At the time of examination of transplantability, the frequency change amount Δf of the cultured tissue is measured under the same conditions as those of the determination reference model. The suitability of the transplanted cultured tissue is determined by comparing the measured Δf with the Δf _{0 of the} criterion model.

Here, in the case of the cultured cartilage tissue measurement model, the comparison standard for determining that the transplantability is in an appropriate range is that when the contactor 32 is pushed downward about 1.5 mm from the zenith in the cultured cartilage tissue measurement model. The indication is that the hardness information (Δf) of the cultured tissue is harder (Δf / Δf ₀ is less than 1.0) with respect to the hardness information (Δf ₀ ) of the criterion model. Preferably, the frequency change ratio Δf / Δf ₀ is ₀ to 0.9, more preferably ₀ to 0.7. Outside this range, it is too soft or too hard as described above, which is not preferable for transplantation.

(3) Judgment based on the relationship between tactile sensation and load information (displacement information) After the contact 32 is brought into contact with the cultured tissue or the carrier, when the displacement is gradually applied, the load L increases accordingly, and the vibration frequency The change amount Δf changes. This change can be grasped as an inclination from the relationship between tactile sensation and load information. Since this inclination includes hardness information, it can be used as a judgment guideline for a transplant aptitude test. Since the relationship between the tactile sensation and the displacement information shows the same tendency as that of the tactile sensation and load information, the relationship based on the tactile sensation and displacement information can be used instead of the tactile sense and load information. The tilt may be the tangential slope at a predetermined load or predetermined displacement in the tactile-load relationship diagram or the tactile-displacement relationship diagram, or a pseudo load or a predetermined displacement value is connected to a reference position value. It may be a straight line slope.

  Here, in the case of the cultured cartilage tissue measurement model, when a tactile-load relationship diagram is created when the vertical axis is Δf and the horizontal axis is the load L, the slope up to 0.067 N load (least square method) is −1200. ˜0 Hz / N is preferable, and −800 to −300 Hz / N can be more preferable. Outside this range, it is too soft or too hard as described above, which is not preferable for transplantation.

  The value of the load for determining the inclination may be anywhere as long as the characteristics of the cultured tissue can be obtained, but measurement with a load affected by a dish (bottom surface) other than the cultured tissue is not preferable.

  Furthermore, the suitability for transplantation can be determined based on the change in elasticity of the cultured tissue. In this case, the elasticity of the cultured tissue is observed from the relationship between the sense of touch and the load information, and the transplantability is determined based on the elastic state of the cultured tissue.

  In the tactile sense-load relationship, elasticity is observed as the difference (residual deformation) in the amount of change in frequency between the forward path and the return path (up and down movement of the measuring element 22) under the same load. When the criterion model is measured, a large difference is observed in the amount of change in the frequency of the forward path and the return path at the same load, but when elasticity is added due to cell growth and substrate production, the frequency change of the forward path and the return path is changed. The difference in quantity is reduced. As described above, when the difference between the forward path and the return path (residual deformation) at a predetermined load is within a predetermined aptitude range, it is possible to derive a test result that the transplantation aptitude is appropriate. In addition, the determination of the suitability for transplantation based on such elasticity information can be performed in the same manner in the relationship of tactile sensation and displacement.

  In this way, using the hardness measurement apparatus 10 capable of measuring hardness nondestructively, the transplantability of the cultured tissue itself for transplantation can be improved without using many other cultured tissues in the same lot. It is also possible to inspect. Moreover, transplantability can be easily determined by examining whether or not the measurement result is within an appropriate range.

  In addition, by defining a criterion model in advance, the state of cell growth and substrate production in the cultured tissue can be easily grasped, and the transplantability test of the cultured tissue can be easily performed.

<Quality control of cultured tissue>
In the quality control method for cultured tissue of the present invention, the culture state is grasped by performing the hardness measurement as described above over time, the cultured state after measurement is predicted from these information, and based on this, the cultured tissue is The quality of the cultured tissue is controlled by appropriately controlling the culture conditions.
That is, the hardness of the cultured tissue changes as the seeded cells grow and produce a substrate with the growth. Moreover, since the time-dependent state of cell growth and substrate production changes by changing the culture conditions such as the seeding density and the culture medium, the time-dependent change in hardness depending on these changes. Based on this, the quality control method obtains information on the hardness of the cultured tissue during the culturing process, predicts subsequent changes in the state of the cultured tissue, and is suitable as a cultured tissue for transplantation in the desired number of culture days. The culture condition of the cultured tissue is controlled based on the prediction so as to be firm.

  Here, “quality of cultured tissue” means that it has a requirement for efficient engraftment after transplantation or efficient tissue regeneration in the transplanted individual, and “having transplantability”. Says that it has such ability.

  In addition, here, “quality control of cultured tissue” means that the number of cells and / or the basic mass in the cultured tissue is within a predetermined range in relation to the provision time in order to be able to provide a cultured tissue having transplantability. Say to adjust. This quality control of the cultured tissue means that only a cultured tissue in an appropriate state can be selected at a specific time. That is, in the culture process, the culture state at the time of measurement is ascertained, and the culture period and culture conditions are adjusted thereafter so that the culture tissue is in an appropriate state at a specific time, for example, at the time of transplantation or shipment. It means controlling the state of the organization. At the time of shipment of the cultured tissue, it means that only the cultured tissue in an appropriate state at the time of shipment is selected in consideration of the period and state until transplantation. Thereby, the culture tissue provided can always be maintained at a constant quality.

  As described in detail below, for such quality control, the state of the cultured tissue being cultured is grasped, and it is determined whether or not it is suitable for the market depending on the grasped state of the cultured tissue. Or adjusting various factors related to the culture to control the state of the cultured tissue. In addition, the control of the culture conditions for quality control of the present invention does not necessarily require changing the culture conditions. Continuation of the same culture condition when it is determined that the change of the culture condition is unnecessary based on the temporal change in the hardness information is also included in the control of the culture condition. The adjustment of various factors relating to the culture includes not only the type of medium and the medium composition such as addition of nutrient factors, but also the extension, shortening or maintenance of the culture period.

  The measurement of hardness for predicting the culture state varies depending on the growth rate of the cell types constituting the culture tissue, but the culture state can be accurately predicted as the number of times increases including the start of the culture. When the culture state can be grasped to some extent from the seeding density and the culture speed, it may be measured at least once after the start of the culture. For example, in the case of chondrocytes, it is predicted to some extent by measuring 14 days after the start of the culture. be able to. Thereby, quality can be managed more simply.

  The measurement of the hardness of the cultured tissue is preferably performed using the hardness measurement apparatus 10 used for the determination of the aptitude for transplantation. Thereby, quality control can be performed using an existing apparatus while determining transplantability based on hardness information.

  The culture control of the cultured tissue is performed by changing the culture conditions to promote or delay cell growth. Such adjustment of the cell growth rate includes changes in the medium composition such as addition and removal of nutrient factors, timing of medium replacement, culture temperature, and the like. Thus, cell growth or substrate production of the cultured tissue can be promoted or delayed, and a cultured tissue in an appropriate state can be obtained at a desired time. In addition, based on hardness information, if the cell growth is slow, the culture period is lengthened. If the cell growth is fast, the culture period is shortened. If appropriate, the culture period is maintained as planned. The quality of the cultured tissue can also be stabilized by controlling the period.

  In addition, the hardness measurement at the time of quality control needs to be measured by applying a load or a displacement amount that does not cause destruction in the cultured tissue. As a result, it is possible to continuously measure the hardness of the same cultured tissue in a non-destructive manner, and thus to perform quality control for supplying a cultured tissue with stable quality.

  As described above, since the culture conditions of the cultured tissue are controlled based on the hardness information of the cultured tissue, the quality of the cultured tissue can be controlled with high accuracy and stability without destroying the cultured tissue. The cultured tissue in a state can be supplied, and the quality control of the cultured tissue with stable quality can be performed.

  Further, by culturing the cultured tissue while measuring the hardness as described above, a cultured tissue having transplantability can be easily produced. When manufacturing a cultured tissue by such a manufacturing method, measure the hardness of the cultured tissue every predetermined period after the start of culture, and grasp the change over time of the cultured tissue, using the hardness as an index. The cultured tissue is cultured while determining transplantability.

  As a result, the culture is performed while judging the suitability of transplantation over time using hardness as an index, so the culture may be stopped before it becomes suitable for transplantation, or it may be cultured beyond the appropriate time. Can be avoided. In addition, since the cultured tissue having transplantability can be produced without destroying the cultured tissue by using the hardness measuring apparatus 10, a large number of transplantable aptitudes can be efficiently obtained from the cultured tissue for transplantation which has been cultured. Can be produced. Moreover, by combining with the adjustment of the culture conditions described in the quality control method, a cultured tissue having transplantability can be efficiently produced in an appropriate amount at an appropriate time.

In the above description, the frequency change amount Δf based on the tactile sensor unit 24 is used as the hardness information. However, the stiffness (hardness) S obtained from the frequency change amount Δf is calculated to measure or transplant the production substrate. It may be used in the same manner as the frequency change amount Δf for the determination of aptitude. The stiffness S can be obtained by the following equation (1).
Δf = a · logS + b formula (1)
However, a and b are constants determined by the apparatus. As described above, since the relationship between Δf and stiffness S varies depending on constants that vary depending on the measurement apparatus, the probe configuration, probe diameter, and probe even in the same cultured tissue. The measured Δf is easily changed by the difference in the material and the like. Therefore, the preferable range of Δf shown in the embodiment of the present invention is based on the following examples, and the technical idea of the present invention is limited to these preferable ranges by these descriptions. is not.

  Hereinafter, a cultured cartilage tissue cultured based on chondrocytes is taken as an example, and an inspection method based on the hardness information of the cultured tissue and a quality control method by measuring the hardness information over time will be described. In the present invention, a biosensor (manufactured by AXIOM) was used to measure the hardness of the cultured tissue.

[Example 1]
<Production of cultured cartilage tissue>
Articular cartilage was collected from the knee, hip and shoulder joints of Japanese white rabbits and subjected to enzyme treatment with trypsin EDTA solution and collagenase solution to separate and collect chondrocytes. After washing the obtained chondrocytes, 10 v / v% FBS (fetal bovine serum) -DMEM (Dulbecco's modified Eagle's minimum essential medium) was added, and the cell suspension was made so that the cell density was 1 × 10 ⁷ cells / ml. A liquid was prepared. The cell suspension and 3% atelocollagen implant (manufactured by Koken Co., Ltd.) were mixed at a ratio of 1: 4, and 100 μl of the mixed solution was mounted in a dome shape on the culture dish. The seeding density at this time is 2 × 10 ⁶ cells / ml.

The mounted mixed solution was allowed to stand for 0.5 to 1 hour under conditions of 5% CO ₂ and 37 ° C. for gelation, a medium was added, and culture was started. As the medium, 10 v / v% FBS-DMEM adjusted to contain 50 μg / ml ascorbic acid and 100 μg / ml hyaluronic acid was used, and cultured for 3 weeks under conditions of 5% CO ₂ and 37 ° C.

  The hardness of the cultured cartilage tissue produced by the above culture method was measured using the hardness measuring apparatus 10 described above. The moving speed and moving amount of the probe 22 at the time of measurement were set at a moving speed of 2 mm / s and a moving amount of 2 mm.

<Measurement of hardness of cultured cartilage>
The probe 20 is fixed to the gantry 12 and the computer is operated to set the above moving speed and moving amount. The cultured cartilage tissue washed with PBS (phosphate buffer) is placed at the measurement position together with the dish, and the probe 20 is set so as to be positioned a predetermined amount above the apex of the cultured cartilage tissue. Measurement is started by the operator operating the computer, and the probe 22 is lowered by the motor 30 at the set moving speed. When the set movement amount is lowered, the motor 30 is reversed, and the motor 30 is raised at the set movement speed, and the hardness measurement is finished. The amount of movement of the probe 22 by the motor 30 is always detected by the displacement sensor as the amount of displacement from the reference position.

  Transplantability is examined based on the hardness information of the cultured cartilage tissue obtained from a single or a plurality of relationships of displacement, pressure, and tactile data obtained during the series of descending / rising measurement steps.

<Determination of transplantability of cultured cartilage>
FIG. 2 is a graph showing temporal changes in tactile information (frequency change Δf) at each seeding density when a predetermined displacement (a point 1.5 mm downward from the top of the cultured tissue) is applied. FIG. 2 shows data of a carrier (collagen gel) on which cells are not seeded as a determination reference model. FIG. 3 is a graph showing a change with time of Δf / Δf ₀ obtained by making the tactile information of various seeding densities in FIG. 2 dimensionless with the tactile information of only the collagen gel carrier.

  FIG. 2 shows that the hardness increases as the frequency change amount Δf increases, and it is observed that the hardness increases earlier as the sowing density is higher. On the other hand, when the seeding density is low, the effect on cell hardness is small. This is considered to be because the growth rate increases as the seeding density decreases, and the production amount of the substrate decreases due to the influence of dedifferentiation and the like as compared with the seeding density high.

Usually, the cultured cartilage tissue has sufficient suitability as a cultured tissue for transplantation after 3 weeks of culture (about 21 days), so that the frequency change Δf is preferably −200 Hz or more, −150 It can be observed that ˜0 Hz is more preferable. From FIG. 3, it can be observed that Δf / Δf ₀ is preferably ₀ to 0.9, more preferably ₀ to 0.7, as a determination criterion for transplantability.

FIG. 4 is a graph showing the change over time in the frequency change amount Δf when a predetermined load (0.067 N) is applied. In the figure, a seeding density of 2 × 10 ⁶ cells / ml and a collagen carrier alone are shown.

  As for the tendency of the frequency change amount Δf when a predetermined load is applied, it is observed that the hardness increases with the number of culture days, as in the case of measuring with the predetermined displacement shown in FIGS. . The transplantability range when a load of 0.067 N is applied is preferably −150 or more, more preferably −100 to 0 Hz from the figure.

  FIG. 5 is a tactile-load relationship diagram, and FIG. 6 is a tactile-displacement diagram. In the tactile-load relationship diagram of FIG. 5, the hardness information can be obtained as an inclination. In the figure, 1W, 2W, 3W, and 5W indicate the first week, the second week, the third week, and the fifth week, respectively. It can be observed that the inclination becomes gentle as the number of culture days elapses and the hardness of the cultured tissue increases. By analyzing the inclination at the time of a predetermined load, it can be used as a judgment criterion for transplantation aptitude. For example, the inclination from the start of load detection to the 0.067N load is preferably −1200 to 0 Hz / N, more preferably −800 to −300 Hz / N.

  The elasticity information in the tactile-load relationship diagram can be obtained as the difference between the forward path and the return path at a predetermined load. In FIG. 5, there is a large difference in the data of the forward path and the return path (the vertical movement of the measuring element 22). This is because the moving speed of the measuring element 22 is fast, so that the restoration of the cultured tissue can cope with the return of the measuring element 22. It is thought that it is not. By setting the moving speed of the probe 20 at the time of measurement to an appropriate state or reducing the applied load, it is possible to determine the suitability for transplantation based on the elasticity of the cultured tissue.

  In the tactile-displacement relationship diagram of FIG. 6, the difference between the forward path and the backward path appears remarkably. Compared to the case of collagen gel carrier alone, the difference between the outward path and the return path of the cultured cartilage tissue is clear, and it is clear that the cultured cartilage tissue is given elasticity.

  Thus, from the tactile-load or tactile-displacement relationship diagram, not only the frequency change amount Δf at the time of a predetermined displacement or load, but also the elasticity information by the inclination and the difference between the forward path and the return path can be obtained. These can be used alone or in association to determine the criteria for transplantation characteristics. Therefore, it is possible to easily and appropriately determine the transplantability of the cultured cartilage similarly cultured using the determination criteria obtained here.

<Change in hardness of cultured cartilage tissue over time>
As shown in the time-dependent change graphs of FIGS. 2 to 4 and the relationship diagram of FIG. 5, the culture in which the cells were incorporated and cultured as compared to the case where the hardness of the collagen gel carrier alone was almost flat and unchanged. In the cartilage tissue, it can be observed that the hardness gradually increases with the passage of culture days.

The hardness of the cultured tissue is almost the same as the reference model of collagen carrier alone until around the 7th day (1 week) of culture, but depending on the seeding density, after the 14th day (2nd week) of culture Then, the difference in hardness can be observed remarkably. Even for cultured cartilage with low seeding density and little difference compared to collagen gel carrier alone, it is confirmed that the cells are proliferating by preparing a section from the cultured tissue at this point and checking the stained image it can. On the 21st day (3 weeks) of culture, the difference is remarkably different from that of the collagen gel carrier alone, and the difference in hardness due to the difference in seeding density can be observed. When the seeding density was 2 × 10 ⁷ cells / ml, no significant change was observed in the change in hardness after the 21st day of culture.

<Quality control based on time-dependent data>
As shown in FIGS. 2 to 4, in the cultured cartilage tissue in this example, no change in hardness is observed immediately after the start of culture, and a change in hardness can be observed remarkably on the 14th to 21st days of culture. Based on the time-dependent change of the cultured cartilage tissue obtained here, the transplantability is predicted, and the culture conditions such as the seeding density and the culture medium are changed. By changing the culture conditions, the rate of change in hardness, the rate of cell growth, etc. can be changed, so the culture can be controlled to achieve the appropriate hardness for the transplanted tissue in the desired number of culture days. Therefore, it is possible to stably provide a cultured tissue having an appropriate hardness.

[Example 2]
Next, Embodiment 2 of the present invention will be described with reference to FIGS. In Example 2, the measurement was performed by partially changing the above-described cultured cartilage tissue measurement model and the measurement conditions of Example 1. In the measurement of the cultured cartilage tissue of this example, the probe 20 having a hemispherical contactor 32 having a diameter of 1 mm is measured using a cultured cartilage tissue having a diameter of 2.8 to 3.0 cm and a thickness of 3 mm. And measured. The hardness measurement apparatus 10 was the same as that used in the cultured cartilage tissue measurement model and Example 1 except that the size of the contact 32 was different.

For preparation of cultured cartilage tissue, the concentration of the cell suspension was mixed with 3% atelocollagen solution at 1 × 10 ⁷ cells / ml, and 1500 μl of this mixed solution (seeding density was 2 × 10 ⁶ cells / ml) was hemispherical. The same procedure as in Example 1 was performed except that mounting was performed. The measurement of hardness was performed at a total of 30 points including the zenith portion of the cultured cartilage tissue and the other 29 points. The moving speed of the probe 22 during measurement was 2 mm / s, and the moving amount was 2 mm. The density of the measurement points was about 5 per 1 cm ² of the surface of the cultured cartilage tissue. The other measurement conditions were the same as the measurement conditions of the cultured cartilage tissue measurement model and Example 1.

FIG. 7 is a graph showing the change over time in the amount of change in frequency Δf when a predetermined load (4.9 × 10 ⁻³ N (0.5 gf)) is applied. In addition, the graph has shown the average of 30 measurement points. The frequency change amount Δf when a predetermined load was applied showed the same tendency as in Example 1, and it was observed that the hardness of the cultured cartilage tissue increased with the passage of the culture period. In addition, by evaluating with the average value based on the multipoint measurement in this way, it is possible to surely grasp the change in the hardness of the cultured cartilage, and it is shown that it is suitable for accurate measurement and determination of transplantability. It was. Under the measurement conditions of this example, preferably, the change in frequency Δf of −15 Hz or more, more preferably −10 to 0 Hz is set as the transplant suitability range, the transplant suitability determination of cultured cartilage tissue, quality control, and the production of cultured cartilage tissue are performed. Can be done.

  FIG. 8 is a graph showing a tactile-displacement relationship. Similar to Example 1, as the culture period elapses, the amount of change in frequency Δf accompanying the increase in displacement decreases, which indicates that the hardness has increased. In addition, on the seventh day of culture, there is a difference in the tactile information of going (contacting element descending) and returning (contacting element ascending), and it can be seen that the cultured tissue does not yet have sufficient elasticity. After the 14th day of culture, there is almost no difference between going and returning, and it can be seen that the cultured tissue has elasticity and is restored to its original shape so as to follow the rise of the contact. In FIG. 8, the amount of displacement indicates the amount of movement of the measuring element 22 from the reference position. For this reason, when the movement amount is 0.5 mm, the contact 32 comes into contact with the cultured cartilage tissue.

  As described above, in the present invention, since the proliferation state of the cells in the cultured cartilage tissue, that is, the culture state is measured as hardness information, it is possible to determine whether or not the culture tissue is suitable for transplantation without destroying the cultured tissue. Can be determined. In addition, by measuring and predicting changes over time, quality control for providing a cultured tissue with a stable quality for transplantation becomes possible. Furthermore, by performing such quality control, a cultured tissue having transplantability can be efficiently and systematically manufactured.

  In the present specification, the present invention has been described by taking cultured cartilage as an example. However, as long as it is a cultured tissue capable of determining the suitability for transplantation based on hardness information, as described above depending on the cell type constituting each cultured tissue. It will be apparent to those skilled in the art that the same information can be applied by obtaining hardness information.

  In addition, in the hardness measurement apparatus 10 according to the present embodiment, the pressure sensor unit 26 and the displacement sensor unit 28 are provided in addition to the tactile sensor unit 24. However, in order to determine the transplantability by measuring the hardness of the cultured tissue. It is sufficient that only the tactile sensor unit 24 is disposed. Thereby, the transplantability of the cultured tissue can be determined with a simple apparatus configuration.

  In the hardness measuring apparatus 10 according to the present embodiment, the probe 20 is fixed to the gantry 12 by the arm 14 and the support shaft 16, but if the probe 20 can contact the cultured tissue with a constant load, the probe 20 is It does not have to be fixed. For example, the present invention can be implemented by measuring the hardness of a cultured tissue even with a pen-type hardness measuring device having the same function.

It is the figure which showed an example of the hardness measuring apparatus applicable to this invention. It is a graph which shows the time-dependent change of the tactile information (frequency variation | change_quantity (DELTA) f) in each seeding density in the Example of this invention. Tactile information in each seeding density in the embodiment of the present invention is a graph showing the time course of Delta] f / Delta] f ₀ which is dimensionless tactile information only collagen gel carrier. It is a graph which shows the time-dependent change of the tactile information in the predetermined load in the Example of this invention. It is a tactile-load relationship diagram according to an embodiment of the present invention. It is a tactile-displacement relationship diagram according to an embodiment of the present invention. It is another graph which shows a time-dependent change in the tactile information (frequency change amount Δf) in another example of the present invention. It is a tactile-displacement relationship diagram in another embodiment of the present invention.

Explanation of symbols

10 Hardness measuring device 20 Probe (detector)
22 Measuring Element 24 Tactile Sensor Unit 26 Pressure Sensor Unit (Load Detection Unit)
28 Displacement sensor (displacement detection unit)
32 Contact (contact part)
34 vibrator 36 pickup (vibration detector)
40 Control unit (calculation means)
42 Controller 44 Self-excited oscillation circuit

Claims (8)

Measures the hardness information of the cultured tissue that has been cultured in vitro and has a three-dimensional structure whose hardness or elasticity changes with the passage of the culture period , and based on the measurement results, the state of cell growth and the amount of production substrate The quality control method of the culture tissue which grasps | ascertains and determines the transplantability of a culture tissue based on the said state . A contact unit, a detection unit including a vibration connected to the contact unit and a vibration detection unit that detects vibration of the vibration unit, and a calculation unit that calculates hardness information based on a detection result from the vibration detection unit When the hardness measuring apparatus using said quality control method for culturing tissue according in contact with the contact portion in the cultured tissue to 請 Motomeko 1 you measure the hardness of the cultured tissue with. The detection unit further includes a load detection unit that detects a load applied to the contact unit, and is based on a relationship between the vibration of the vibrator detected by the vibration detection unit and the load detected by the load detection unit. The quality control method of the cultured tissue according to claim 2 , wherein the hardness of the cultured tissue is measured. The detection unit further includes a displacement amount detection unit that detects a displacement amount from a reference position of the contact unit, and vibration of the vibrator detected by the vibration detection unit and a displacement amount detected by the displacement amount detection unit The quality control method of the cultured tissue according to claim 2 , wherein the hardness of the cultured tissue is measured based on the relationship between the cultured tissue and the tissue.   The method for quality control of a cultured tissue according to any one of claims 1 to 4, wherein the cultured tissue is produced by seeding and culturing cells on a tissue regeneration substrate having a three-dimensional structure.   The tissue regeneration base material is collagen, gelatin, hyaluronic acid, fibronectin, fibrin, chitin, chitosan, laminin, dermatan sulfate, heparan sulfate, chondroitin sulfate, calcium alginate, calcium phosphate, calcium carbonate, polyglycolic acid, polylactic acid and polyrotaxane. The quality control method for cultured tissue according to claim 5, comprising any one of them.   The cell that is the source of the cultured tissue is at least one of chondrocytes, osteoblasts, fibroblasts, endothelial cells, epithelial cells, myoblasts, adipocytes, hepatocytes, nerve cells, or precursor cells thereof. The quality control method for cultured tissue according to any one of claims 1 to 6. Method for producing cultured tissue containing either quality control method of claims 1 to 7.

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