JP5825571B2 - Cryopreservation tool for cryopreserving biological samples - Google Patents

Description

  The present invention relates to a cryopreservation tool for cryopreserving biological samples such as tissue pieces and cells.

  In recent years, with the progress of multidisciplinary treatment for cancer, the survival rate after treatment of cancer patients has been improved. On the other hand, abolition of ovarian function caused by treatment is a problem in young female cancer patients after treatment remission. In other words, ovarian function is lost due to chemotherapy, surgery, radiotherapy, etc., and QOL as a woman is reduced due to loss of fertility, early menopause, menopause or osteoporosis, etc. It has become. When treating young female cancer patients, maintaining ovarian function is important not only for fertility preservation but also for maintaining QOL as a woman.

  As a method for maintaining ovarian function even after treatment, it has been proposed to remove ovarian tissue from the body prior to treatment, cryopreserve it, and autograft thawed ovarian tissue after remission. In 2004, it was reported that a live child was obtained from a woman who maintained ovarian function by this method (see Non-Patent Document 1). Since then, attempts have been made to cryopreserve ovarian tissue, mainly in Europe, and there are a number of reports that children were obtained after autotransplantation. However, it cannot be said that the rate of obtaining a baby by this technology is high, and there are many problems to be solved to improve the rate of obtaining a baby.

  The ovarian tissue cryopreservation method is roughly classified into a slow freezing method and a vitrification freezing method. The slow freezing method is a method in which ovarian tissue is slowly frozen over time using a program freezer. At present, the slow freezing method is recognized as the standard cryopreservation method. However, in the slow freezing method, cells in the ovarian tissue are damaged due to physical damage caused by ice crystals formed inside and outside the cells and salt damage caused by freezing of water in the solution to increase the salt concentration. Has been pointed out. As a result, in ovarian tissue cryopreserved by the slow freezing method, the balance between oocyte development and granulosa cell maturation in the follicular development process after thawing may be impaired.

  On the other hand, the vitrification freezing method is a method of rapidly freezing ovarian tissue immersed in a freezing solution containing a cryoprotectant at a high concentration in order to suppress the formation of ice crystals (see, for example, Patent Document 1). . The vitrification freezing method is simpler than the slow freezing method without requiring a program freezer or ice planting operation. There is also a basic experimental report using human ovarian tissue, and it is reported that the ratio of morphologically normal atomic follicles was almost the same between the slow freezing method and the vitrification freezing method. The vitrification freezing method is expected to be a cryopreservation method that replaces the slow freezing method because it is a method that can be easily performed with little damage to the ovarian tissue.

  In the vitrification freezing method, ovary tissue immersed in a freezing solution is directly immersed in liquid nitrogen and rapidly frozen. Currently, a metal plate provided with a large number of through-holes is used as a cryopreservation tool used when ovarian tissue is immersed in liquid nitrogen (see, for example, Patent Document 2).

  FIG. 1 is a schematic diagram showing a cryopreservation tool currently used (substantially the same as the tissue piece cryopreservation plate described in Patent Document 2). As shown in FIG. 1A, the conventional cryopreservation tool 10 includes a sample mounting part 20 made of a metal plate provided with a large number of through holes 22 and a holding fixed to the base end of the sample mounting part 20 The unit 30 and the tube main body 40 are included. The holding portion 30 is provided with a screw cap 32 that can seal the tube body 40. As shown in FIG. 1B, the ovarian tissue 50 to be cryopreserved is immersed in liquid nitrogen while being placed on the sample placement unit 20. The frozen ovarian tissue 50 is accommodated and stored in the tube body 40 while being placed on the sample placement unit 20.

  As shown in FIG. 1A, in the conventional cryopreservation tool 10, a large number of through-holes 22 are provided in a metal plate (sample placement unit 20) on which ovarian tissue is placed. In Patent Document 2, by providing a large number of through holes 22 in this way, liquid nitrogen can directly contact the back side of the ovary tissue (the contact surface side of the ovarian tissue and the metal plate), and the metal plate itself. It has been described that ovarian tissue can be frozen more rapidly because it is more susceptible to cooling.

JP 2003-284546 A JP 2008-222640 A

Donnez, J., et al., "Livebirth after orthotopic transplantation of cryopreserved ovarian tissue", Lancet, Vol.364, pp.1405-1410.

  However, in the conventional cryopreservation device described in Patent Document 2, when the biological sample is cryopreserved by vitrification freezing, the cooling rate of the biological sample may not be sufficiently increased.

  As described above, the conventional cryopreservation tool is provided with a number of through holes in the metal plate (sample mounting portion) in order to increase the contact area between the biological sample and liquid nitrogen. When a metal plate provided with a through hole in this way is immersed in liquid nitrogen, nitrogen gas (bubbles) that are constantly generated in liquid nitrogen may be trapped in the through hole. When nitrogen gas is trapped in the through hole in this way, nitrogen gas is interposed between the biological sample to be cryopreserved and liquid nitrogen. As a result, it becomes impossible to take heat energy directly from the biological sample with liquid nitrogen at −196 ° C. Further, since the gas such as nitrogen gas has a very low thermal conductivity, the cooling rate is significantly reduced. When the cooling rate of the biological sample is insufficient, the biological sample may be damaged when frozen.

  Further, the conventional cryopreservation device described in Patent Document 2 has a problem that a biological sample (for example, a cell) smaller than the through hole cannot be frozen.

  The present invention has been made in view of this point, and can be frozen even if the biological sample is small, and the biological sample can be frozen more quickly by preventing the heat insulation effect due to the gas such as nitrogen gas. An object of the present invention is to provide a cryopreservation tool that can be used.

  The inventor has found that a biological sample can be frozen more quickly while preventing a heat insulating effect due to a gas such as nitrogen gas by using a graphite sheet not provided with a through-hole as a sample placement portion, Further studies were made to complete the present invention.

That is, the present invention relates to the following cryopreservation tools.
[1] A cryopreservation tool for cryopreserving a biological sample: a sample placement unit including a graphite sheet on which a biological sample is placed, and a holding unit fixed to a base end portion of the sample placement unit A cryopreservation tool in which the region on which the biological sample of the graphite sheet is placed does not have a through-hole.
[2] The cryopreservation tool according to [1], wherein the graphite sheet does not have a through hole.
[3] The graphite sheet is a liquid solution containing 2 to 6 mol / L of cell membrane permeation-type cryoprotectant, 0 to 100 g / L of cell protectant, and 0 to 1 mol / L of cell membrane-impermeable cryoprotectant. The cryopreservation device according to [1] or [2], which is coated with a membrane.
[4] The cryopreservation device according to [3], wherein the cell membrane permeation cryoprotectant is ethylene glycol, dimethyl sulfoxide, glycerol, propanediol, acetamide, propylene glycol, butanediol, or a combination thereof.
[5] The cryopreservation device according to [3] or [4], wherein the cytoprotective agent is polyvinylpyrrolidone, hyaluronan, polyvinyl alcohol, fibronectin, antifreeze protein, antifreeze amino acid, or a combination thereof.
[6] The cryopreservation device according to any one of [3] to [5], wherein the cell membrane impermeable cryoprotectant is sucrose, trehalose, lactose, raffinose, or a combination thereof.
[7] The cryopreservation tool according to any one of [1] to [6], wherein the sample mounting portion further includes a reinforcing member that supports the graphite sheet.

  ADVANTAGE OF THE INVENTION According to this invention, the cryopreservation tool which can freeze biological samples, such as a tissue piece and a cell, more rapidly can be provided. By using the cryopreservation tool of the present invention, biological samples such as tissue pieces and cells can be cryopreserved more simply and reliably.

Schematic diagram showing a conventional cryopreservation tool The schematic diagram which shows the cryopreservation tool which concerns on one embodiment of this invention The schematic diagram which shows the usage condition of the cryopreservation tool which concerns on one embodiment of this invention

  The cryopreservation device of the present invention is a cryopreservation device for freezing biological samples such as tissue pieces and cells by vitrification freezing (including rapid vitrification freezing and ultrarapid vitrification freezing). The cryopreservation tool of the present invention has a sample mounting part including a graphite sheet, and a holding part fixed to the base end part of the sample mounting part.

  In the cryopreservation tool of the present invention, 1) the through hole is not provided in the region where the biological sample of the sample mounting portion (graphite sheet) is mounted, and 2) the graphite sheet is used as the sample mounting portion. Is the main feature. Hereinafter, each component of the cryopreservation tool of the present invention will be described.

  The sample placement unit has a graphite sheet on which a biological sample (for example, a tissue piece or a cell) is placed. The graphite sheet is obtained by processing graphite (graphite) into a sheet shape and has a very high thermal conductivity. In the cryopreservation tool of the present invention, a graphite sheet is employed as the sample mounting portion in order to freeze the biological sample more quickly.

  Graphite sheets are roughly classified into natural graphite sheets and artificial graphite sheets depending on the production method. The natural graphite sheet is obtained by chemically treating natural graphite and then pressurizing it into a sheet shape. On the other hand, the artificial graphite sheet is obtained by pyrolyzing a polymer film to be graphitized. The graphite sheet used for the sample mounting part may be either natural graphite or artificial graphite, but from the viewpoint of freezing the biological sample more quickly, the structure is closer to a single crystal than the natural graphite sheet with low orientation. An artificial graphite sheet having the above is more preferable. An artificial graphite sheet having a structure close to a single crystal has a very high thermal conductivity of, for example, 700 to 1600 W / (m · K). The value of this thermal conductivity is about 2 to 4 times the thermal conductivity of copper, which is known as a material having high thermal conductivity. In addition, an artificial graphite sheet having a structure close to a single crystal is lightweight while having high strength (about 1/10 to 1/4 times that of copper) and excellent in low temperature resistance.

  In order to prevent trapping of a gas such as nitrogen gas, a through hole is not provided in a region on which the biological sample of the graphite sheet is placed. By using a flat plate (sheet) without a through-hole in this way as the sample mounting portion, gas such as nitrogen gas is prevented from being trapped in the sample mounting portion, and heat insulation of the gas such as nitrogen gas is prevented. A decrease in the cooling rate due to the effect can be prevented. On the other hand, when a flat plate (sheet) without a through hole is used as the sample mounting portion, the biological sample is cooled with liquid nitrogen or the like on the back side of the biological sample (the contact surface side between the biological sample and the sample mounting portion). There is a concern that the cooling rate may decrease because the medium cannot be contacted. However, in the cryopreservation tool of the present invention, this problem is solved by using a graphite sheet having a very high thermal conductivity as the sample mounting portion.

  As described above, the graphite sheet does not have to be provided with a through hole in the region where the biological sample is placed. Therefore, a through hole may or may not be provided in a region other than the region on which the biological sample is placed. In the latter case, no through hole is provided in the entire graphite sheet.

  The size of the graphite sheet may be appropriately set according to the size of the biological sample to be frozen. The graphite sheet needs to have a certain thickness in order to secure the strength capable of holding the biological sample, but is preferably thinner from the viewpoint of thermal conductivity. For example, the thickness of the graphite sheet should just be in the range of 10-100 micrometers.

  The surface of the graphite sheet is preferably smooth. As will be described later, when freezing a biological sample using the cryopreservation device of the present invention, the sample placement part is immersed in a cooling medium such as liquid nitrogen. There is a possibility that gas (for example, nitrogen gas) generated in the medium tends to adhere to the surface of the graphite sheet. If gas adheres to the surface of the graphite sheet around the biological sample, the cooling rate of the biological sample may be reduced due to the heat insulation effect of the gas. The method for smoothing the surface of the graphite sheet is not particularly limited. For example, the surface of the graphite sheet may be covered with a liquid film of a frozen solution to eliminate the irregularities on the surface of the graphite sheet.

  The type of the frozen solution that coats the surface of the graphite sheet is not particularly limited as long as it is a solution that improves the sliding of a gas (for example, nitrogen gas) on the surface of the graphite sheet, and is a known frozen solution used in the vitrification freezing method. May be used. For example, the freezing solution covering the graphite sheet contains 2 to 6 mol / L of cell membrane permeation-type cryoprotectant, 0 to 100 g / L of cell protection agent, and 0 to 1 mol / L of cell membrane non-permeability cryoprotectant. An aqueous solution can be used. Examples of cell membrane permeation cryoprotectants include ethylene glycol, dimethyl sulfoxide, glycerol, propanediol, acetamide, propylene glycol, butanediol, and the like. These cell membrane permeation cryoprotectants may be used alone or in combination of two or more. Examples of cytoprotective agents include polyvinylpyrrolidone, hyaluronan, polyvinyl alcohol, fibronectin, antifreeze protein, antifreeze amino acid and the like. These cytoprotective agents may be used alone or in combination of two or more. Examples of cell membrane non-permeating cryoprotectants include sucrose, trehalose, lactose, raffinose and the like. These cell membrane non-permeating cryoprotectants may be used alone or in combination of two or more.

  Since the graphite sheet is flexible, the biological sample may not be held at the same position as it is. Therefore, the sample placement unit may have a reinforcing member that supports the graphite sheet so that the graphite sheet can hold the biological sample at the same position. The shape of the reinforcing member and the material constituting the reinforcing member are not particularly limited as long as the cooling rate of the biological sample is not lowered. For example, the reinforcing member is a fine metal wire fixed around the graphite sheet (see the embodiment).

  The holding part is a grip fixed to the base end part of the sample placement part. A user (for example, a medical worker) holds the holding unit directly by hand or using an instrument such as tweezers, and immerses the sample mounting unit on which the biological sample is mounted in a cooling medium such as liquid nitrogen. . The size and shape of the holding portion are not particularly limited, and may be any size and shape that can be easily held by the user.

  The method for freezing a biological sample using the cryopreservation device of the present invention is not particularly limited. For example, 1) a biological sample to be frozen on a graphite sheet (a region where no through hole is provided) of the sample mounting portion. 2) The sample placement part on which the biological sample is placed may be gently immersed in a cooling medium (for example, liquid nitrogen, slush nitrogen, liquid helium, liquid propane, ethane slash, etc.). When a biological sample is cryopreserved by vitrification freezing, the biological sample to be cryopreserved is immersed in a frozen solution. Therefore, the biological sample placed on the graphite sheet in step 1) is a biological sample immersed in a frozen solution. Further, as described above, before placing the biological sample on the graphite sheet in step 1), the graphite sheet may be covered with a liquid film of a frozen solution.

  As described above, since the cryopreservation tool of the present invention does not have a through hole in the portion on which the biological sample is placed, gas (for example, nitrogen gas) is not trapped on the back surface of the biological sample. In addition, a small sample can be placed. Further, the cryopreservation tool of the present invention freezes the biological sample more quickly because the graphite sheet on which the biological sample is placed has a very high thermal conductivity in addition to the fact that the gas is not trapped on the back surface of the biological sample. be able to.

[Embodiment]
Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Here, an example of the cryopreservation tool of the present invention integrated with an inner cap type cryotube is shown.

  FIG. 2 is a schematic diagram showing a cryopreservation tool according to an embodiment of the present invention. As shown in FIG. 2, the cryopreservation tool 100 of the present invention includes a sample placement unit 110, a holding unit 120, and a tube body 130.

  The sample mounting unit 110 includes a graphite sheet 112 and a reinforcing member 114. On the graphite sheet 112, a biological sample to be frozen (for example, a tissue piece or a cell) is placed. The graphite sheet 112 is not provided with a through hole. For example, the graphite sheet 112 is a graphite sheet having a width of 8 mm, a length of 20 mm, and a thickness of 25 to 100 μm. On the other hand, the reinforcing member 114 is a metal plate (metal thin wire) provided on both ends (opposing long sides) of the graphite sheet 112. For example, the reinforcing member 114 is a metal plate (copper plate, gold plate, or silver plate) having a width of 1 mm, a length of 20 mm, and a thickness of 1 mm or less.

  The holding unit 120 is a resin molded product (grip) fixed to the base end of the sample mounting unit 110. The holding unit 120 also functions as an inner cap that seals the tube main body 130. The type of resin constituting the holding unit 120 is not particularly limited, and may be selected from resins (for example, polypropylene) used in general cryotubes.

  The tube main body 130 is a container made of resin having the same shape as a general cryotube. The shape of the tube body 130 is not particularly limited, and may be, for example, a round bottom type, a conical bottom type, or a self-standing type. The type of resin constituting the tube main body is not particularly limited, and may be selected from resins (for example, polypropylene) used in general cryotubes.

  FIG. 3 is a schematic diagram showing how the cryopreservation tool 100 of this embodiment is used.

  As shown in FIG. 3A, a biological sample 200 to be frozen (for example, ovarian tissue immersed in a frozen solution) is placed on the graphite sheet 112 of the sample placement unit 110. At this time, before the sample 200 is placed, the surface of the sample placing unit 110 may be covered with a liquid film of a frozen solution.

  Next, the user holds the holding unit 120 and immerses the sample mounting unit 110 in a cooling medium such as liquid nitrogen. At this time, the holding unit 120 may be held with tweezers, and not only the sample mounting unit 110 but also the holding unit 120 may be immersed in the cooling medium. By immersing the sample mounting part 110 in the cooling medium, the biological sample 200 is also immersed in the cooling medium and frozen. Of the surface of biological sample 200, the surface that is not in contact with graphite sheet 112 is in direct contact with the cooling medium. On the other hand, the surface in contact with the graphite sheet 112 is in contact with the graphite sheet 112. In the cryopreservation tool 100 of the present embodiment, since the graphite sheet 112 is not provided with a through hole, gas (for example, nitrogen gas) is not trapped between the biological sample 200 and the graphite sheet 112. The biological sample 200 and the graphite sheet 112 are in direct contact. Moreover, since the thermal conductivity of the graphite sheet 112 is very high, the biological sample 200 is quickly cooled.

  As shown in FIG. 3B, the frozen biological sample 200 is housed and stored in the tube main body 130 in a state of being placed on the sample placing portion 110 (graphite sheet 112).

[Reference experiment]
Hereinafter, as a reference experiment, measurement results of a cooling rate and a heating rate of a graphite sheet (used in a cryopreservation tool of the present invention) and a metal plate (used in a conventional cryopreservation tool) are shown.

  As the graphite sheet, a crystalline graphite sheet (PGS graphite sheet; Panasonic) having a thickness of 100 μm was used. As the metal plate, a copper plate (Taifeng Trading Co., Ltd.) having a thickness of 100 μm was used. The graphite sheet and the copper plate were both cut to 0.8 cm × 2 cm. For the measurement of the cooling rate and the heating rate, a recorder (EB2205; Chino Co., Ltd.) connected with a surface temperature sensor was used.

  First, the surface temperature sensor was directly immersed in liquid nitrogen, and the time from 0 ° C. to −100 ° C. was measured. The cooling rate was 26500 ° C./min. When this surface temperature sensor was taken out from liquid nitrogen into the air atmosphere and the time from -100 ° C. to 0 ° C. was measured, the heating rate was 64500 ° C.

  Next, the surface temperature sensor was fixed between two graphite sheets or two copper plates, and then immersed in liquid nitrogen, and the time from 0 ° C. to −100 ° C. was measured. . As a result, the surface temperature sensor fixed to the copper plate had a cooling rate of 2524 ° C./min, whereas the surface temperature sensor fixed to the graphite sheet had a cooling rate of 10021 ° C./min. Moreover, the graphite sheet or copper plate to which the surface temperature sensor was fixed was taken out from liquid nitrogen into the air atmosphere, and the time from -100 ° C. to 0 ° C. was measured. As a result, the heating rate of the surface temperature sensor fixed to the copper plate was 4957 ° C./min, whereas the heating rate of the surface temperature sensor fixed to the graphite sheet was 34667 ° C./min. The measurement results are shown in Table 1.

  From the above results, the cryopreservation device of the present invention using a graphite sheet as a sample mounting part is superior in thermal conductivity to a conventional cryopreservation device using a metal plate as a sample mounting part, It turns out that it is more suitable for cryopreservation of biological samples, such as a tissue piece and a cell.

  The cryopreservation tool of the present invention can more easily and reliably freeze biological samples such as tissue pieces and cells. For example, the cryopreservation device of the present invention is useful when cryopreserving ovarian tissue, induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, and the like.

DESCRIPTION OF SYMBOLS 10 Cryopreservation tool 20 Sample mounting part 22 Through-hole 30 Holding part 32 Screw cap 40 Tube main body 50 Ovarian tissue 100 Cryopreservation tool 110 Sample mounting part 112 Graphite sheet 114 Reinforcement member 120 Holding part 130 Tube main body 200 Biological sample

Claims (4)

A cryopreservation tool for cryopreserving a biological sample,
A sample placement unit including a graphite sheet on which a biological sample is placed;
A holding part fixed to the base end part of the sample mounting part,
The area where the biological sample of the graphite sheet is placed does not have a through hole,
Cryopreservation tool.   The cryopreservation tool according to claim 1, wherein the graphite sheet does not have a through hole. The graphite sheet is coated with a liquid membrane of a solution containing 2 to 6 mol / L of a cell membrane permeation-type cryoprotectant, 0 to 100 g / L of a cell protective agent, and 0 to 1 mol / L of a cell membrane non-permeability cryoprotectant. Has been
The cell membrane permeation cryoprotectant is ethylene glycol, dimethyl sulfoxide, glycerol, propanediol, acetamide, propylene glycol, butanediol or a combination thereof,
The cytoprotective agent is polyvinylpyrrolidone, hyaluronan, polyvinyl alcohol, fibronectin, antifreeze protein, antifreeze amino acid or a combination thereof,
The cell membrane impermeable cryoprotectant is sucrose, trehalose, lactose, raffinose or a combination thereof.
The cryopreservation tool according to claim 1.   The cryopreservation tool according to claim 1, wherein the sample mounting portion further includes a reinforcing member that supports the graphite sheet.

Images