JP2011115080A - Sheet-like cell culture peeling system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sheet-like cell culture peeling system that easily reduces dangerousness such as wrinkle, break, damage, and contamination, and easily peels even a sheet-like cell culture having low strength from a culture vessel. <P>SOLUTION: The system includes (i) a storage part for storing the culture vessel in which a sheet-like cell is cultured, (ii) a vibration part for vibrating the culture vessel in the storage part and peeling the sheet-like cell culture, (iii) a sensor part for measuring the initial state of the sheet-like cell culture in the culture vessel and a state after vibration and (iv) an analysis part for analyzing a case as a peeling state in which the difference between a value in an initial state measured by the sensor part and a value in a state after vibration exceeds a set value. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a system for peeling a sheet-shaped cell culture from a culture vessel.

  Despite recent advances in the treatment of heart disease, a treatment system for severe heart failure has not yet been established. For the treatment of heart failure, medical treatment with β-blockers or ACE inhibitors is performed. For heart failure that has become so severe that these treatments are not successful, replacement treatment such as auxiliary artificial heart and heart transplantation, that is, surgical treatment Is done.

Severe heart failure that is the subject of such surgical treatment includes those caused by advanced valvular disease and severe myocardial ischemia, acute myocardial infarction and its complications, acute myocarditis, ischemic cardiomyopathy (ICM), dilation There are a wide variety of causes such as chronic heart failure due to dilated cardiomyopathy (DCM) and acute aversion.
Depending on these causes and severity, valvuloplasty and replacement, coronary artery bypass surgery, left ventricular plastic surgery, mechanical assisted circulation, etc. are applied.

  Of these, only heart transplantation or replacement treatment with an artificial heart has been regarded as an effective treatment for those who have suffered heart failure due to a severe decrease in left ventricular function caused by ICM or DCM. However, replacement therapy for these patients with severe heart failure has many problems to be solved, such as chronic donor shortages, the need for continuous immunosuppression, and the development of complications. Is hard to say.

  Due to this severe situation of heart transplantation, other surgical treatments such as Batista surgery have been tried for a while, and it has attracted much attention as an alternative treatment for heart transplantation. However, efforts have been made to improve surgical procedures and establish indications.

On the other hand, recently, development of a new regenerative treatment method is considered indispensable as a solution for the treatment of severe heart failure.
In severe myocardial infarction and the like, cardiomyocytes become dysfunctional, and fibroblast proliferation and interstitial fibrosis progress, resulting in heart failure. As the heart failure progresses, the cardiomyocytes are damaged and fall into apoptosis, but the cardiomyocytes hardly undergo cell division, so the number of cardiomyocytes decreases and the cardiac function further decreases.
Cell transplantation is considered useful for the recovery of cardiac function in such patients with severe heart failure, and clinical applications using autologous skeletal myoblasts have already begun in the United States and Europe.

  However, in order to actually improve the cardiac function sufficiently by the cell transplantation method, 70-80% of the transplanted cells are lost by the direct intramyocardial injection method, and the effect cannot be fully exhibited. For example, dilated cardiomyopathy because it is indispensable to solve problems such as side effects such as securing a large and safe cell source, as well as causing inflammation to the local site of cell injection and only local cell transplantation. There is a limit when the heart function of the whole heart is reduced.

  In recent years, in response to these problems, a three-dimensional cell culture that can be applied to the heart including cells derived from parts other than the adult myocardium by using a temperature-responsive culture dish applying tissue engineering, and The manufacturing method was provided (patent document 1).

  In order to transplant such a cell culture to the target affected area (transplant site), for example, the end of the cell culture is picked with tweezers, the cell culture is taken out of the packaging container, and transferred to the affected area. However, a series of operations such as transplantation (attachment) to the affected area is required. However, cell cultures have low absolute physical strength and are prone to wrinkling, tearing, and breakage. Requires advanced technology and requires careful attention.

  Although the temperature-responsive culture dish described in Patent Document 1 facilitates the exfoliation of the cell culture from the culture dish, it cannot be completely exfoliated, and for complete exfoliation, an artificial process such as pipetting is required. Operation is required, causing contamination, wrinkles, tearing, and damage.

  On the other hand, there has also been provided an apparatus for measuring luminance information in a culture container when detaching cells from the culture container and determining the detachment state based on the luminance information (Patent Document 2). Such an apparatus is used in a system in which trypsin is added for detachment in a cell culture container to detach the cells.

JP 2007-528755 A WO2007 / 136073

  It is an object of the present invention to provide a system that can easily peel a sheet-shaped cell culture used for treatment of human and animal diseases and injuries without causing wrinkles, tears, breakage, or the like. .

  The present inventors have found that the sheet-shaped cell culture can be easily detached without causing contamination by applying vibration to the container in which the sheet-shaped cell culture is cultured. However, it was also found that excessive vibration can cause damage to the sheet-like cell culture, and as a result of extensive research, the present invention has been completed.

That is, the present invention is a system for peeling a sheet-shaped cell culture from a culture vessel,
(I) A storage section for storing a culture container in which sheet-like cells are cultured (ii) A vibration section for applying vibration to the culture container in the storage section to separate the sheet-shaped cell culture (iii) A sheet in the culture container A sensor unit for measuring the state of the cell culture (iv), including an analysis unit for analyzing that the difference between the measured value measured by the sensor unit and the initial value exceeds a set value, and a detached state; It relates to the system.

In the present invention, the analysis unit outputs a signal to stop vibration of the vibration unit to the vibration unit when the difference between the measured value and the initial value exceeds the set value, and outputs the signal when the difference does not exceed the set value. The present invention relates to the system having a signal output unit that does not output.
The present invention further relates to the system as described above, wherein the analysis unit has an input unit for inputting an initial value and / or a set value.
The present invention further relates to the system described above, wherein the sensor unit measures the distance.
The present invention still further relates to the above system, wherein the storage unit has a control unit capable of controlling the surrounding environment of the culture vessel.
The present invention also relates to the above system, wherein the control unit includes a temperature control unit, and the temperature around the culture vessel can be kept constant.
Furthermore, this invention relates to the said system which can maintain the environment suitable for cell culture by a control part.

Furthermore, the present invention is a method for producing a sheet-shaped cell culture,
(I) a step of culturing cells in a sheet form on the surface of the culture vessel;
(Ii) applying vibration to the culture vessel of (i) and peeling the sheet-shaped cell culture, and (iii) measurement measured by a sensor that measures the peeled state of the sheet-shaped cell culture in the culture vessel A step of stopping the vibration when the difference between the value and the initial value before applying the vibration exceeds a set value;
The method.

  According to the system of the present invention, the sheet-like cell culture in the culture vessel can be easily peeled without causing contamination, wrinkles, tearing, breakage, or the like. In addition, by integrating with a culture incubator, it is possible to automate the process from creation of a sheet by culture to peeling.

FIG. 1 shows a flow diagram of a stripping process in one embodiment of the present invention. FIG. 2 shows a flow diagram of temperature management in one aspect of the present invention. FIG. 3 shows a flow diagram of an analysis subroutine in one aspect of the present invention. FIG. 4 shows a block diagram in one aspect of the invention.

The present invention is a system for peeling a sheet-shaped cell culture from a culture vessel,
(I) A storage section for storing a culture container in which sheet-like cells are cultured (ii) A vibration section for applying vibration to the culture container in the storage section to separate the sheet-shaped cell culture (iii) A sheet in the culture container A sensor unit for measuring the state of the cell culture (iv), including an analysis unit for analyzing that the difference between the measured value measured by the sensor unit and the initial value exceeds a set value, and a detached state; It relates to the system.

  The sheet-shaped cell culture is typically used for the treatment of diseases and injuries of mammals such as humans and domestic animals, but is not limited thereto. The cell culture is typically composed of cultured cells and / or products thereof, but various materials (such as supplementary materials and the like) for supplementing and / or supporting a predetermined part (for example, an affected part) of a living body. Support material).

  The sheet-like cell culture is derived from any cell type, such as, but not limited to, myoblasts (eg, skeletal myoblasts), cardiomyocytes, fibroblasts, synoviocytes, epithelial cells, endothelial cells, etc. May be. In the present invention, for example, a sheet-like cell culture composed of skeletal myoblasts is preferable from the viewpoint of therapeutic utility and difficulty in handling by conventional techniques. The animal from which the cells constituting the sheet-shaped cell culture are derived is not particularly limited, and includes, for example, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep and the like.

  The sheet-shaped cell culture may be cultured on a so-called temperature-responsive culture dish (for example, see JP-A-2-21865) and peeled off from the culture dish. Also good. Therefore, the culture vessel is preferably a temperature-responsive culture dish, but is not limited thereto.

The shape of the storage part of the culture vessel is not limited, and may be, for example, a warehouse-like shape, or may be only one that fixes the culture vessel to the shaking table. The warehouse shape means that one or more placement shelf members can be provided in a sealed space where the temperature and humidity can be controlled as a storage unit, and a plurality of culture vessels can be placed on the shelf members. The vibration unit may be inside or outside the storage unit, and may vibrate only the culture vessel or may vibrate the entire storage unit. The type of vibration is not limited, but is preferably horizontal vibration, more preferably circular vibration. Examples of vibrators that can be used in the present invention include TAITEC rotary shaker NR-2.
From the viewpoint of reducing damage to the sheet, it is preferable not to apply vibration more than necessary. Therefore, the vibration unit may be provided with a mechanism that stops the vibration after a certain time, or the vibration and / or vibration time may be controlled by an analysis unit or a control unit described later.

  Examples of the state measured by the sensor unit include, but are not limited to, the distance between the sensor and the cell culture on the sheet, the refractive index, the transmittance, and the reflectance. Examples of the sensor that can be used in the present invention include a laser displacement sensor LT-9000 manufactured by Keyence Corporation. The number of sensors present in the sensor unit is not limited, but preferably has two or more sensors. In the case of having a plurality of sensors, the measurement value may be analyzed for each sensor, or may be analyzed using an average value of the measurement values of all the sensors. The measurement by the sensor unit may be always performed, or may be measured after the vibration is temporarily stopped after a certain period of vibration.

  The measurement value may be recorded on a recording medium in the analysis unit before being analyzed by the analysis unit. Therefore, the analysis unit may have a recording unit. The recording unit may be an electrical recording unit such as a magnetic tape, a magnetic disk, an optical disc, a magneto-optical disc, or a flash memory, or may be a physical recording unit such as paper or a photo. There may be.

  The measurement value measured by the sensor unit is analyzed by comparison with the initial value in the analysis unit. The initial value is a numerical value representing the state before peeling, and may be, for example, a measured value before the start of vibration measured at the sensor unit, or may be an arbitrary value input according to the form of use. Alternatively, the value recorded in advance in the recording unit may be used. Therefore, the analysis unit may include an input unit. As the form of the input unit, for example, any input unit known to those skilled in the art such as a keyboard, a sensor (the sensor may be the same as the sensor of the sensor unit), a touch panel, or the like may be used.

  After the start of vibration, the sensor unit measures the state after the start of vibration, and analyzes the peeled state by comparing the measured value with the initial value. The measured value is compared with the initial value, and when the difference exceeds the set value, it is determined that the peeling is completed. Such a determination may be made individually for all the sensors, or may be made using the average value of all the sensors. For example, the difference between the average values of all the sensors exceeds the set value and the measurement of the central sensor is performed. The difference between the value and the initial value may be determined in combination, for example, when the set value is exceeded, it is determined that the peeling is completed.

  Since the set value changes depending on a value measured by the sensor and a desired peeling state, the set value can be arbitrarily input at the time of use. The setting value input unit may be the same as or different from the initial value input unit. Therefore, the analysis unit may have an input unit for inputting an initial value and / or a set value. The recording unit may also serve as an input unit. For example, when there is a difference between the initial value and the measured value, it is determined that peeling occurs (that is, the set value = 0), and a fixed initial value and / or set value corresponding to the sensor is recorded in the recording unit. The value may be used for analysis. Further, the vibration unit may be controlled in the analysis unit, and the input unit may also serve as a setting value input unit for the control.

  In the analysis unit, preferably, the difference between the initial value and the measured value exceeds or does not exceed the set value, or the degree of progress of peeling is output to the outside as information. Therefore, the analysis unit may include an output unit for the information. As information output means, for example, an external monitor, lamp, buzzer, transmission to a pre-registered mobile phone or Internet e-mail address, etc. may be used to notify the person operating the information on the completion of peeling, a printer, etc. It may be output to the recording unit. Preferably, when it is determined that peeling is completed, a signal for stopping vibration is output from the analysis unit to the vibration unit. Further, the information may be simultaneously output to a plurality of locations such as an external monitor and a vibration unit.

  As described above, the sensor unit can measure distance, refractive index, transmittance, reflectance, and the like. From the viewpoint of convenience and accuracy, the distance is preferably measured. The distance measured here differs depending on the position of the sensor and the like, and for example, the distance between the shaking table and the cell culture can be mentioned.

The storage unit may have a control unit that can control the surrounding environment of the culture vessel. The surrounding environment is not limited to this, and examples thereof include temperature, pressure, humidity, and CO ₂ concentration. Therefore, the control unit outputs an input unit that inputs settings to be controlled, a recording unit that records setting values, a measurement unit that measures the current state of the surrounding environment, and information that is measured by the measurement unit. May have a part. Such a control unit may share the analysis unit and the CPU, the input unit, the output unit, and / or the recording unit. For example, in one embodiment of the present invention, the above-described temperature-responsive culture dish can be used as a culture container, and it is possible to increase the peeling efficiency by controlling the temperature around the culture container to a range suitable for peeling. It becomes.

  Moreover, in one aspect of the present invention, the storage unit can keep the periphery of the culture vessel in an environment suitable for cell culture. Therefore, the storage part in this aspect can function as a cell culture incubator. In this embodiment, cells can be cultured using the storage portion as an incubator, and after completion of the culture, it is possible to proceed directly to the step of peeling the cell culture.

Therefore, the present invention also includes a method for producing a sheet-like cultured cell product that automatically performs the process from culturing to detachment of the sheet-like cell culture. Specifically, the method includes:
(I) a step of culturing cells in a sheet form on the surface of the culture vessel;
(Ii) applying vibration to the culture vessel of (i) and peeling the sheet-shaped cell culture, and (iii) measurement measured by a sensor that measures the peeled state of the sheet-shaped cell culture in the culture vessel A step of stopping the vibration when the difference between the value and the initial value before applying the vibration exceeds a set value;
including.

In this method, in order to peel the sheet-shaped cell culture on the surface of the culture container, the culture container is vibrated and peeled. If this vibration is applied too much, it will damage the sheet-shaped cell culture. Therefore, when the detachment of the sheet-shaped cell culture is monitored by one or more sensors and the detachment is completed, the vibration To stop. When the difference between the measured value by the sensor and the initial value before applying the vibration exceeds a certain threshold value, it can be determined that the peeling is completed, but the initial value and the threshold value may be set arbitrarily.
Specific embodiments of the present invention are listed below, but the present invention is not limited to these specific examples.

FIG. 1 illustrates a flowchart of processing in one aspect of the present invention. In this example, storage unit is a storage-shaped housing part provided with a temperature control unit, is set the temperature in the housing portion so as to control the T _1. The sensor unit includes a sensor for measuring the distance. The analysis unit has an input unit common to that of the temperature control unit, and it is possible to input an initial set value and a set value for determination of peeling, vibration time, and the like by the input unit. .
When the system of the present invention receives a peeling start instruction sheet cell cultures, the control unit the temperature in the storage unit is measured first, temperature adjustment to a set temperature T ₁ (temperature adjustment subroutine). When the temperature adjustment is completed, it is determined whether or not the peeling completion indicator is lit (<H001>).

If the completion indicator is on, it is determined whether a stop instruction of the release process, the end if there is an instruction, the process returns to the no If temperature adjustment subroutine, the temperature T ₁ until it receives a stop instruction keep.
When the completion indicator is not lit, the peel state of the cell sheet is measured by the distance sensor (<H002>). In an embodiment that does not have an input unit of the analysis unit, the first measured value can be stored and used as an initial value for determining the peeled state.

In this example, when the peeling state (measured value) of the sheet satisfies the set value (that is, the difference between the measured value and the initial value exceeds the set value), the sheet peeling completion indicator is turned on and a stop instruction is given. The process proceeds to presence / absence determination (<H003>). When the peeling state of the sheet does not satisfy the set value, a vibration start signal is output to the vibration unit (<H005>). After a predetermined time t ₁ has elapsed, and outputs a signal of stopping the vibrating and returns to tempering subroutine.

In this embodiment, the temperature control subroutine is operated only while the vibration is stopped. However, the temperature control subroutine may operate independently of vibration. For example independently of the vibration of the route, so as to always keep the temperature around the container to a set temperature T _1, may also operate in vibration.
In this embodiment, the analysis subroutine also operates while the vibration is stopped. However, the analysis subroutine may also operate independently of vibration. For example, it may operate to always monitor the peeled state during vibration. In this case, a process of outputting a vibration stop signal to the vibration unit is performed in parallel with the sheet peeling completion indicator lighting.

In the figure, T ₁ is the temperature to be kept constant near the culture vessel, and can be set arbitrarily. Fixed time t ₁ is the vibration duration, which also can be arbitrarily set. Moreover, in a certain aspect, the threshold value for judging the peeling state of a cell sheet can also be set arbitrarily. The step of inputting these arbitrarily settable values may be optionally included. In the case of including an input step, all the set values may be input by the time when the set values are referred to, but are preferably input after the start instruction until the inside temperature is measured.

  Since the storage unit of the present invention may include a control unit, in one embodiment, the cell sheet before and / or after peeling can be stored at a constant temperature. FIG. 2 shows a flow chart of a process of keeping the periphery of the culture vessel at a constant temperature for storage. This is a flowchart corresponding to the temperature control subroutine in FIG. Therefore, in the mode in which the temperature control subroutine operates independently of the peeling work as described above, the temperature control subroutine operates based on the flowchart of FIG. The stop instruction in this embodiment may be the same signal as the stop instruction in the embodiment of FIG. 1 or a different signal.

FIG. 3 shows a flow diagram of the analysis subroutine in an independently operating manner. In the subroutine, when the peeling state satisfies the set value, a vibration stop signal is output to the vibration unit after the sheet peeling completion indicator is turned on or simultaneously with the lighting, and the subroutine is finished. If the peeling state does not satisfy the set value, after a predetermined time t ₂ has elapsed, measure cell sheet peeling again. Time t ₂ can be set arbitrarily, it may be input from the input unit included in the analysis unit. For example, t ₂ by setting to zero, it is also possible to monitor continuously peeled state without waiting.

  FIG. 4 depicts a block diagram of one aspect of the present invention. In this aspect, the storage unit is a heat storage. A tray is placed inside the heat insulation box, and a vibration machine that is a vibration unit, a temperature sensor that is a measurement unit of the control unit, and a Peltier element of the control unit are attached to the tray. The vibration is transmitted to the culture vessel by vibrating the tray. The sensor unit is a distance sensor, and the distance between the tray and the cell sheet can be measured at a plurality of locations. Information measured by the distance sensor is input to a CPU that is an analysis unit. The temperature sensor and the Peltier element are elements constituting a control unit provided in the storage unit. The temperature sensor and the Peltier element are simultaneously controlled by the CPU, manage the internal temperature by the temperature sensor, and adjust the temperature by the Peltier element. Therefore, in this aspect, the CPU of the analysis unit and the CPU of the control unit are shared.

  The CPU also outputs information obtained by analyzing input information from the sensor. In addition to being connected to the display unit functioning as the output unit, the information is output to the display unit and the vibrator in conjunction with the vibrator. An operation unit is provided as an input unit in the analysis unit and the control unit.

  As mentioned above, although the system in this invention was demonstrated, various forms can be considered about each part. Therefore, within the scope of achieving the object of the present invention, modified embodiments of the present invention are also included in the scope of the present invention, and such modifications can be easily understood by those skilled in the art.

  According to the system of the present invention, even a low-strength sheet-like cell culture, which has been difficult to collect until now, can be easily collected in a short time without being broken, torn or damaged. In addition, since there is no need for operations such as pipetting, the risk of contamination can be reduced.

Claims (8)

A system for peeling a sheet-shaped cell culture from a culture vessel,
(I) A storage section for storing a culture container in which sheet-like cells are cultured (ii) A vibration section for applying vibration to the culture container in the storage section to separate the sheet-shaped cell culture (iii) A sheet in the culture container A sensor unit for measuring the state of the cell culture (iv), including an analysis unit for analyzing that the difference between the measured value measured by the sensor unit and the initial value exceeds a set value, and a detached state; Said system.   When the difference between the measured value and the initial value exceeds the set value, the analysis unit outputs a signal for stopping the vibration of the vibrating unit to the vibrating unit, and when the difference does not exceed the set value, the signal output unit that does not output the signal The system of claim 1, comprising:   The system according to claim 1, wherein the analysis unit includes an input unit for inputting an initial value and / or a set value.   The system according to claim 1, wherein the sensor unit measures a distance.   The system in any one of Claims 1-4 in which a storage part has a control part which can control the surrounding environment of a culture container.   The system according to claim 5, wherein the control unit includes a temperature control unit, and the temperature around the culture vessel can be kept constant.   The system according to claim 5 or 6, wherein an environment suitable for cell culture can be maintained by the control unit. A method for producing a sheet-shaped cell culture comprising:
(I) a step of culturing cells in a sheet form on the surface of the culture vessel;
(Ii) A step of detaching the sheet-shaped cell culture by applying vibration to the culture container of (i), and a sensor for measuring the detachment state of the sheet-shaped cell culture in the culture container of (iii) (i) Stopping the vibration when the difference between the measured value and the initial value before applying vibration exceeds a set value;
Said method.