JP2007229249A - Method of supplying cell sheet - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To easily provide a cell sheet in a manner that the cell sheet reaches an affected site transorally transanally through the inside of a thin and long body cavity without bringing the adhesive surface into contact with the other site. <P>SOLUTION: A method of supplying a cell sheet includes the steps of immersing a temperature-responsive member 2 having temperature-responsive properties to switch between the hydrophilicity and hydrophobicity depending on a temperature in a cell suspending liquid A to culture the temperature-responsive member 2, of forming the cell sheet on the surface of the temperature-responsive member, of conveying the temperature-responsive member, on which the cell sheet is formed, through the body cavity by taking the temperature-responsive member out of the cell suspending liquid, and of separating the cell sheet in the neighborhood of an affected site in the body cavity by cooling the temperature-responsive member. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell sheet supply method.

  Conventionally, cultured epithelial cell sheets have been used for the treatment of moderate or severe corneal diseases such as scar keratoconjunctivitis (see, for example, Patent Document 1). Further, in order to treat skin damage such as burns, cell sheets are used to remove epidermis killed by burns and mask the removed portions (see, for example, Patent Document 2). Since these cell sheets are manufactured by collecting and culturing normal cells of the patient himself, there is an advantage that there is no problem such as immune rejection.

  Patent Document 2 discloses a method of using a culture container whose surface is coated with a temperature-responsive polymer as a method for producing a cell sheet. A temperature-responsive polymer has a property of rapidly changing from hydrophobic to hydrophilic depending on temperature. For example, when cultured at 37 ° C., it exhibits hydrophobicity and grows while maintaining adherent cells in an adherent state. When promoting and exfoliating, for example, by cooling to about 20 ° C., it becomes hydrophilic and cells can be easily exfoliated from the culture vessel.

Advantages of using a temperature-responsive polymer to produce a healthy cell sheet with less damage to cells compared to the case of exfoliation using a conventional proteolytic enzyme such as trypsin. There is.
And since the cell sheet peeled in this way is peeled by the action of the temperature-responsive polymer provided on the bottom surface of the culture vessel, there is no change in the adhesion of the cell itself, Can be used for masking or the like by pasting the surface adhered to the culture vessel on the affected area as an adhesive surface.
JP 2002-331005 A International Publication No. 02/10349 Pamphlet

  However, in the treatment of a portion exposed to the outside of the human body, such as the treatment of corneal disease described above or the treatment of skin at the time of burns, the adhesive surface of the cell sheet is spread so as not to adhere to other portions. Can be easily placed on the affected area, but for example, to place a cell sheet on the ulcer formed in the inner wall of the esophagus, stomach or intestine, or on the site where the mucosa has been removed endoscopically, It is necessary to insert the cell sheet through the elongated body cavity in a targeted or transanal manner, and it is difficult to reach the affected area without adhering the adhesive surface of the cell sheet to other sites.

  The present invention has been made in view of the above-described circumstances, and allows a cell sheet to reach an affected part through an oral or transanal body cavity without adhering its adhesive surface to another site. A cell sheet supply method capable of easily supplying a cell sheet is provided.

In order to achieve the above-mentioned object, the present invention provides the following means: The present invention is a culture in which a temperature-responsive member having temperature responsiveness that switches between hydrophilicity and hydrophobicity depending on temperature is immersed in a cell suspension. A step of forming a cell sheet on the surface of the temperature responsive member, a step of removing the temperature responsive member formed with the cell sheet from the cell suspension and transporting the body cavities, and in the vicinity of the affected part in the body cavity, And a step of cooling the temperature-responsive member to peel the cell sheet.

According to the present invention, the cell sheet is formed on the surface of the temperature responsive member by immersing and culturing the temperature responsive member in the cell suspension. By inserting and transporting the temperature-responsive sheet on which the cell sheet is formed into the body cavity and cooling in the vicinity of the affected part, the cell sheet can be peeled off and applied to the affected part.
In this case, according to the present invention, since the cell sheet formed by culturing on the surface of the temperature-responsive member is inserted into the body cavity as it is without being peeled off, the cell sheet is not handled alone until it is released in the vicinity of the affected part. The work is simple and easy. During transportation, the cell sheet adhesive surface can be kept adhered to the temperature-responsive member, and inconveniences such as adhesion to each part in the body cavity and adhesion between the cell sheet itself can occur. Can be prevented. At the time of release, the cell sheet can be easily peeled off and released to the affected area simply by cooling the temperature-responsive member.

In the above invention, the temperature-responsive member may be in the form of a sheet.
By doing in this way, it becomes possible to convey the cell sheet in an unfolded state without bringing it together, and the adhesion work to the affected part can be facilitated.

Moreover, in the said invention, the said temperature-responsive member is good also as being comprised by the member which can deform | transform.
By doing in this way, in the narrow body cavity from the mouth or anus to the vicinity of the affected part, the temperature-responsive member to which the cell sheet is adhered can be thinly deformed to be easily transported in the body cavity. Further, by deforming the temperature-responsive member in the vicinity of the affected area, the cell sheet can be expanded, and the adhesion work to the affected area can be facilitated.

Further, in the above invention, the temperature responsive member may include a shape memory actuator.
By doing in this way, a temperature change can be given to a shape memory actuator, and it can change to a desired form easily. Shape memory actuators include shape memory alloys or shape memory polymers.

In the invention described above, the temperature-responsive member may be a metal stent.
By doing in this way, in a narrow body cavity from the mouth or anus to the vicinity of the affected part, the metal stent to which the cell sheet is adhered can be thinly deformed to be easily transported in the body cavity. In addition, by deforming the metal stent so as to expand in the vicinity of the affected area, the cell sheet can be expanded, and the adhesion work to the affected area can be facilitated.

In the above invention, the temperature-responsive member may be a balloon.
In this way, in a narrow body cavity from the mouth or anus to the vicinity of the affected part, the balloon to which the cell sheet is adhered is contracted, so that the body cavity can be easily transported. Further, by inflating the balloon in the vicinity of the affected area, the cell sheet can be spread and flowed into a shape suitable for the affected area, and the adhesion work to the affected area can be facilitated.

  According to the present invention, it is possible to easily supply a cell sheet that allows the cell sheet to reach the affected area without being adhered to other sites orally or transanally through an elongated body cavity. There is an effect.

Hereinafter, the supply method of the cell sheet 1 which concerns on the 1st Embodiment of this invention is demonstrated below with reference to FIGS.
The supply method of the cell sheet 1 which concerns on this embodiment is equipped with the culture | cultivation step, the conveyance step, and the release step.

  As shown in FIG. 1, the culture step uses a sheet-like temperature responsive member (hereinafter referred to as a temperature responsive sheet) 2. The temperature-responsive sheet 2 includes an adhesive portion 2a having a surface subjected to temperature-responsive processing. The temperature-responsive treatment is, for example, applied with a polymer made of N-isopropylacrylamide, and exhibits hydrophobicity at 32 ° C. or higher and hydrophilicity at a temperature lower than 32 ° C.

  Thereby, the cell sheet 1 (refer FIG. 2) can be hold | maintained to the adhesion | attachment part 2a by maintaining at 32 degreeC or more, and the cell of the adhesion | attachment part 2a can be maintained by switching to temperature lower than it. The sheet 1 can be peeled off. Further, the surface other than the bonding portion 2a is treated so as to have hydrophilicity. This prevents cells from adhering to the surface other than the adhesive portion 2a at any temperature.

In the culturing step, the temperature-responsive sheet 2 is immersed in the culture vessel 3 in which the cell suspension A obtained by collecting the oral mucosa and putting it in a predetermined medium is stored, at a temperature of 37 ° C. and at a CO ₂ concentration. Maintain culture conditions of 0.5%. As a result, cells floating in the cell suspension A adhere to and grow on the surface of the adhesive portion 2a of the temperature-responsive sheet 2 exhibiting hydrophobicity at 37 ° C., and the cell sheet 1 is formed after a predetermined time. The Thereby, as shown in FIG. 2, a composite sheet 4 in which the temperature-responsive sheet 2 and the cell sheet 1 are overlapped is formed.

  As shown in FIG. 3, the conveyance step is performed by rolling the composite sheet 4 so as to wind up, and as shown in FIG. 4, the channel 6 (provided along the longitudinal direction in the insertion portion 5 of the endoscope). The composite sheet 4 is accommodated in the distal end portion of the flexible tube 7 having an outer diameter that can be inserted into FIG. Thereby, by inserting the tube 7 containing the composite sheet 4 along the channel 6 of the insertion portion 5 of the endoscope, the distal end portion of which is disposed in the vicinity of the affected part in the body cavity, orally or transanally. The composite sheet 4 can be conveyed to the vicinity of the affected part.

  Then, in the release step, as shown in FIG. 5, the composite sheet 4 is pushed out of the channel 6 by pressing means 8 such as a wire introduced from the rear of the channel 6. Then, the cooling water 9 introduced through the channel 6 is applied to the composite sheet 4 to cool to a temperature lower than 32 ° C. Thereby, the adhesive part 2a of the temperature-responsive sheet 2 constituting the composite sheet 4 is changed to hydrophilic, and the cell sheet 1 adhered to the adhesive part 2a is easily peeled and released to the vicinity of the affected part. be able to.

  As described above, according to the method for supplying the cell sheet 1 according to the present embodiment, the cell sheet 1 is formed in an adhesive state directly by culturing on the temperature-responsive sheet 2 conveyed to the vicinity of the affected part in the body cavity. There is no need to peel the cell sheet 1 until the cell sheet 1 is released in the vicinity of the affected part, and there is an advantage that the handleability can be improved. In addition, since the cell sheet 1 is maintained in the adhesive state to the temperature-responsive sheet 2 during transportation, the adhesive surface 1a of the cell sheet 1 is not exposed, so that the cell sheet 1 adheres to other parts, Generation | occurrence | production of the problem that the adhesion surfaces 1a of the cell sheet 1 adhere | attach can be prevented beforehand.

  In addition, according to the present embodiment, since the cell sheet 1 is adhered to the temperature-responsive sheet 2, the cell sheet 1 does not adhere to other parts even if it is rolled up multiple times so as to be wound up, and can be easily Can be spread. And by rounding in multiple, the cell sheet 1 of a large area can be accommodated in the tube 7, and the wide cell sheet 1 for repairing a comparatively big affected part can be supplied.

  In the present embodiment, the composite sheet 4 in which the cell sheet 1 is superimposed on the temperature-responsive sheet 2 having the adhesive portion 2a subjected to the temperature-responsive process is illustrated, but instead, a flexible sheet is used. You may employ | adopt the temperature responsive sheet 2 comprised by the shape memory alloy (illustration omitted) embedded, or the shape memory polymer. By doing so, it is possible to easily switch between the flatly spread state in FIG. 1 and the wound state as in FIG. 2 by giving a temperature change.

  Thereby, the work of inserting the composite sheet 4 into the channel 6 can be facilitated, and the deployment at the time of release can be performed smoothly. Further, when the cell sheet 1 is released, it can be spread as shown in FIG. 2, wound after the release as shown in FIG. 3, and collected again in the channel 6.

  In this embodiment, the composite sheet 4 is accommodated in the channel 6 and transported and released in the vicinity of the affected part. However, the endoscope can be inserted without accommodating the composite sheet 4 in the channel 6. You may decide to convey in the body cavity in the state hold | gripped by the forceps etc. which are not shown in figure at the front-end | tip of the part 5. FIG. By doing in this way, the process of extruding from the inside of the channel 6 becomes unnecessary, and the cell sheet 1 can be released to the vicinity of the affected part simply by cooling.

  Further, in place of the flat temperature-responsive sheet 2, an expandable metal stent (not shown) or the like is provided inside the composite sheet 4 in which the cell sheet 1 is attached to the outer surface of the temperature-responsive sheet 2 formed in a cylindrical shape. It is good also as arrange | positioning the expansion means. By doing in this way, the composite sheet 4 comprised in the state which expanded the metal stent can be drawn together and can reduce the outer diameter size by shrinking | reducing a metal stent to radial inside. In this state, it can be accommodated in the channel 6 of the insertion portion 5.

  Therefore, it can be transported to the vicinity of the affected area without adhering into the body cavity through the channel 6 of the insertion section 5 of the endoscope and without adhering the adhesive surfaces 2a of the endoscope itself. Moreover, after releasing from the channel 6 of the insertion part 5, by expanding the metal stent, the composite sheet 4 that has been gathered can be expanded so as to stretch, and the cell sheet 1 can be expanded. By cooling the temperature-responsive sheet 2 in this state, the spread cell sheet 1 can be easily peeled and released to the vicinity of the affected part.

  Similarly, a balloon (not shown) in which the cell sheet 1 is attached to the surface of a balloon whose surface has been subjected to a temperature responsive treatment may be employed. The cell sheet 1 is adhered in a state where the balloon is expanded, and the balloon is contracted, so that it can be easily accommodated in the channel 6 of the insertion portion 5.

  Therefore, it can be transported to the vicinity of the affected area without adhering into the body cavity through the channel 6 of the insertion section 5 of the endoscope and without adhering the adhesive surfaces 2a of the endoscope itself. Then, after being released from the inside of the channel 6, the cell sheet 1 that has been gathered can be expanded so as to be stretched by inflating the balloon. By cooling the balloon in this state, the spread cell sheet 1 can be easily peeled and released to the vicinity of the affected area.

  Similarly, the temperature-responsive sheet 2 may be stretched in the form of a film inside an annular snare (not shown) made of wire, and the cell sheet 1 may be attached to the temperature-responsive sheet 2. By making the snare into the storage form, the composite sheet 4 can be easily configured to have a thickness that can be stored in the channel 6, and by projecting the snare out of the channel 6, the composite sheet can be made by the elasticity of the snare. 4 can be expanded.

Next, the supply method of the cell sheet 1 which concerns on the 2nd Embodiment of this invention is demonstrated below with reference to FIGS.
The method for supplying the cell sheet 1 according to the present embodiment uses a cap 10 shown in FIG.

  As shown in FIG. 6, the cap 10 is formed in a substantially cylindrical shape, and includes a fitting portion 11 that can be fitted to the distal end of the insertion portion 5 of the endoscope on one end side in the longitudinal axis direction. . Further, the other end side of the cap 10 is provided with an adhesive portion 10a whose inner surface is subjected to a temperature responsive process.

  Accordingly, the cell sheet 1 can be held in the bonded state by being maintained at 32 ° C. or higher, and the cell sheet 1 of the bonded portion 10a can be peeled by switching to a lower temperature. Can be done. In addition, the surface other than the bonding portion 10a is treated to have hydrophilicity. This prevents cells from adhering to the surface other than the adhesive portion 10a at any temperature.

In order to adhere the cell sheet 1 to such a cap 10, for example, as shown in FIG. 7, a cell suspension A in which cells to be adhered are suspended in a cell suspension A having a longitudinal axis substantially horizontal. Rotating culture is performed by immersing the part and maintaining the predetermined culture conditions such as a temperature of 37 ° C., a humidity of 100%, and a CO ₂ concentration of 0.5% while rotating the cap 10 around the longitudinal axis in this state. As a result, cells adhere to and grow on the bonded portion 10a that has been subjected to the temperature responsive treatment, and cells do not adhere to the other surface, so that the cell sheet 1 is formed only on the bonded portion 10a. become.

  Thus, the cap 10 to which the cell sheet 1 is adhered is taken out from the cell suspension A, and the fitting portion 11 is fitted to the distal end of the insertion portion 5 of the endoscope as shown in FIGS. For example, the cutting line 1b is formed along the longitudinal axis. And the cell sheet 1 will be conveyed in a body cavity by inserting the insertion part 5 of an endoscope into a body cavity orally or transanally.

  In this case, according to the method for supplying the cell sheet 1 according to the present embodiment, the cell sheet 1 is conveyed while its adhesive surface 1a is held in an adhesive state on the adhesive portion 10a formed on the inner surface of the cap 10. Therefore, it is possible to prevent the occurrence of inconvenience that the cell sheet 1 adheres to other parts in the body cavity or the adhesive surfaces 1a adhere to each other during conveyance. Further, since the cell sheet 1 is disposed on the inner surface of the cap 10, it is possible to avoid contact with a site in the body cavity during transportation, and to transport the cell sheet 1 while protecting it. And by conveying in this way, the cell sheet 1 can be arrange | positioned in the vicinity of an affected part in a healthy state.

  When the cap 10 to which the cell sheet 1 is adhered is disposed in the vicinity of the affected part, by supplying cooling water having a temperature lower than 32 ° C. through the channel 6 provided in the insertion part 5, the outlet of the channel 6 The cap 10 arranged around is cooled to change the property of the bonding portion 10a to hydrophilic. Thereby, the cell sheet 1 adhered to the adhesion part 10a is easily separated and supplied toward the affected part.

  In this case, according to the present embodiment, since the cell sheet 1 held in the adhesive portion 10a provided on the inner surface of the cap 10 is released, the cell sheet 1 has a cylindrical shape with the adhesive surface 1a facing outward. Released in form. Therefore, it can be easily adhered to the affected part arranged near the release position.

  That is, when the cell sheet 1 adhered to the inner surface of the cylindrical cap 10 is peeled off from the adhesion part 10a, the cell sheet 1 expands to expand its diameter, so that it fits to the affected part arranged in a flatter part. Will come to do.

  In this case, the cutting line 1b formed on the cell sheet 1 is conveyed so as to be arranged on the opposite side with respect to the affected part to which the cell sheet 1 is attached. Thereby, when the cell sheet 1 is peeled from the adhesive part 10a of the cap 10, it can be applied so as to cover the affected part simply by moving toward the affected part.

  In the present embodiment, the cap 10 is cooled by supplying cooling water via the channel 6 provided in the insertion portion 5 of the endoscope. As shown, a flow path 12 passing through the inside of the wall surface of the cap 10 is provided, and an external water supply tube 13 led through the outside of the insertion portion 5 is connected to the flow path 12 so that the water supply tube 13 is interposed. Then, the cooling water may be circulated in the flow path 12 of the cap 10 to cool the cap 10.

  Further, in the above embodiment, the adhesive portion 10a is provided on the inner surface of the cap 10, but instead of this, when contact with other parts in the body cavity is not a problem, it is shown in FIG. As described above, the cell sheet 1 may be adhered to the adhesive portion 10 a provided on the outer surface of the cap 10. By doing in this way, the operation | work which forms the cutting line 1b in the cell sheet 1 can be made easy.

  In this case, the cutting line 1b formed on the cell sheet 1 is conveyed so as to be arranged on the affected part side to which the cell sheet 1 is attached. Thereby, when the cell sheet 1 is peeled from the adhesive part 10a of the cap 10, it can be applied so as to cover the affected part simply by moving toward the affected part.

  Further, instead of adhering the cell sheet 1 to the outer surface of the cap 10, a temperature-responsive process is performed on the outer peripheral surface of the insertion portion 5 itself of the endoscope to form an adhesion portion, and the insertion portion 5 is used as the cell suspension A. The cell sheet 1 may be adhered to the adhesion part by immersing the cell sheet and culturing.

It is a perspective view explaining the culture | cultivation step in the supply method of the cell sheet which concerns on the 1st Embodiment of this invention. It is a perspective view which shows the composite sheet used in the supply method of the cell sheet | seat of FIG. It is a perspective view which shows the state which rounded the composite sheet | seat of FIG. It is a perspective view explaining the state which accommodates the composite sheet rounded by FIG. 3 in the tube for conveyance. It is a perspective view explaining the release step in the supply method of the cell sheet of FIG. 1, (a) The composite sheet accommodated in a tube and conveyed in the channel, (b) The composite sheet partially extruded from the tube (C) The composite sheet | seat released from the tube is each shown. It is a perspective view which shows the cap used for the supply method of the cell sheet which concerns on the 2nd Embodiment of this invention. It is explanatory drawing which shows the adhesion method of the cell sheet to the adhesion part of the cap in the culture | cultivation step of the supply method of FIG. It is a perspective view which shows the state which attached the cap to which the cell sheet was adhere | attached with the adhesion | attachment method of FIG. 7 to the front-end | tip part of an endoscope. It is a longitudinal cross-sectional view which shows the state of FIG. It is a longitudinal cross-sectional view which shows the modification of FIG. It is a longitudinal cross-sectional view which shows the other modification of FIG.

Explanation of symbols

A cell suspension 1 cell sheet 2 temperature responsive sheet (temperature responsive member)

Claims (6)

A step of immersing and culturing a temperature-responsive member having temperature responsiveness that switches between hydrophilicity and hydrophobicity depending on the temperature in a cell suspension, and forming a cell sheet on the surface of the temperature-responsive member;
Removing the temperature-responsive member on which the cell sheet is formed from the cell suspension and transporting it in the body cavity;
A method of supplying a cell sheet, comprising: cooling a temperature-responsive member and peeling the cell sheet in the vicinity of an affected part in a body cavity.   The method for supplying a cell sheet according to claim 1, wherein the temperature-responsive member has a sheet shape.   The cell sheet supply method according to claim 1, wherein the temperature-responsive member is formed of a deformable member.   The cell sheet supply method according to claim 3, wherein the temperature-responsive member includes a shape memory actuator.   The cell sheet supply method according to claim 3, wherein the temperature-responsive member is a metal stent.   The cell sheet supply method according to claim 3, wherein the temperature-responsive member is a balloon.

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