Cryopreservation method for tublar cell structure, cryopreservation tool and frozen substance

Abstract

To make cryopreservation of a tublar cell structure possible.SOLUTION: A cryopreservation method has a structure holding step, a structure enclosure step, a preservation liquid immersion step, and a freezing step. In the structure holding step, a tublar cell structure 11 is held in a middle part in a longitudinal direction of the rod-like holding core 32 supporting an inner peripheral surface of the tublar cell structure 11. In the structure enclosure step, the holding core 32 is held in a shaft center part of a protective sleeve 33 having cylindrical communication holes 33a, 33b that have larger diameters than the tublar cell structure 11 and communicate inside and outside. In the preservation liquid immersion step, the tublar cell structure 11 covered with the protective sleeve 33 is encapsulated in an inner bag 34 together with cryopreservation liquid 13 for each holding core 32.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to the cryopreservation of tubular cell structures. [Background technology]

[0002] A tubular cell structure is a tubular structure that contains cells or other substances in whole or in part, and is of an appropriate length with a penetrating lumen, such as a blood vessel, digestive tract, trachea, or lymphatic vessel. Tubular cell structures include cell products formed solely from biological cells (see Patent Document 1 below). In addition, there are hybrid products obtained by mixing biological materials such as biological cells, biological tissue-derived substances, and plasma component-derived substances with artificial materials, as well as artificial products derived from biological materials obtained by processing human or animal biological tissue.

[0003] The tubular cell structure is placed in a container together with a cell preservation solution, packed in a temperature-controlled transport container, and transported while maintained at a constant temperature. However, simply immersing the structure in a cell preservation solution causes the lumen to collapse over time, making it impossible to maintain the normal tubular shape for a long period of time. Furthermore, since tubular cell structures take time to manufacture, it is difficult to ship them in time for transplant surgery.

[0004] Cryopreservation is used for long-term storage of cells. However, when tubular cell structures are placed in a freezing container, such as a bottomed cylindrical resin tube or bag container of a certain size, together with a cryoprotectant, and then frozen, the tubular cell structures cannot maintain their shape due to their tubular shape, and the cells are physically and biochemically damaged. While containers such as those described in Patent Document 2 below have been proposed for cryopreserving preservation solutions containing biological samples, such as 3D cell aggregates or cell sheets, no containers suitable for freezing three-dimensional tubular cell structures have been developed to date. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2020-202785 [Patent Document 2] Patent No. 7082377 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, the main object of this invention is to enable cryopreservation of tubular cell structures. [Means for solving the problem]

[0007] To this end, the present invention provides the following cryopreservation method.

[0008] This cryopreservation method comprises, in order, a structure holding step, a structure surrounding step, a preservation liquid immersion step, and a freezing step. In the structure holding step, the tubular cell structure is held in the longitudinal middle portion of a rod-shaped holding core that supports the inner surface of the tubular cell structure. In the structure surrounding step, the holding core is held in the axial center portion of a protective sleeve that is cylindrical and has a larger diameter than the tubular cell structure and has a communicating hole on its side that connects the inside and outside. In the preservation liquid immersion step, the tubular cell structure covered with the protective sleeve is enclosed together with the holding core in an inner container together with a cryopreservation liquid.

[0009] In this configuration, the retaining core supports the inner circumferential surface of the tubular cell structure, preventing deformation of the lumen of the tubular cell structure during and after the freezing process. On the other hand, the protective sleeve maintains a space around the tubular cell structure, preventing pressure on the outer circumferential surface of the tubular cell structure due to contact with the protective sleeve or inner container. In this way, the shape of the tubular cell structure is maintained.

[0010] At the same time, the protective sleeve and the inner container covering it reduce the amount of cryopreservation solution immersing the tubular cell structure, and the communicating holes in the protective sleeve allow the transfer of liquid and heat, resulting in rapid and uniform freezing and thawing. [Effects of the Invention]

[0011] As described above, the present invention employs a configuration that allows for more uniform freezing and thawing while maintaining the internal and external shapes of the tubular cell structure, thereby enabling the tubular cell structure to be appropriately cryopreserved. [Brief explanation of the drawings]

[0012] [Figure 1] A partially cutaway front view of a main part of a frozen tubular cell structure. [Figure 2] A perspective view of a tubular cell structure. [Figure 3] FIG. 1 is a front view of the cryopreservation device in a separated state. [Figure 4] FIG. [Figure 5] 1A and 1B are a front view and a cut-away end view of a protective sleeve; [Figure 6] FIG. [Figure 7] An explanatory diagram of a preservative solution immersion process. [Figure 8] FIG. 1 is a perspective view of a canister and a cryopreservation device. [Figure 9] Cross-sectional view of the cryopreservation device sandwiched between canisters. [Figure 10] 10A and 10B are front views of a holding core according to another example in a separated state and in a used state. DETAILED DESCRIPTION OF THE INVENTION

[0013] An embodiment of the present invention will be described below with reference to the drawings.

[0014] Figure 1 shows a partially broken front view of a main part of a frozen body 12 of a tubular cell structure 11. The frozen body 12 is formed by placing the tubular cell structure 11 in a cryopreservation device 31 and freezing it. As shown in Figure 2, the tubular cell structure 11 is tubular in shape and has a lumen 11a running through it in the longitudinal direction. The tubular cell structure 11 varies in diameter, length, and structure. Examples of tubular cell structures 11 include blood vessels, digestive tracts, tracheas, lymphatic vessels, as well as the intestines, esophagus, ureter, and fallopian tube.

[0015] The cryopreservation device 31 is composed of, in order from the inside that contacts the tubular cell structure 11, a holding core 32, a protective sleeve 33, and an inner bag 34 as an inner container. In addition, it has a stopper 35 attached to the holding core 32, a connector cap 36 that connects the holding core 32 and the protective sleeve 33, and a protective bag 37 shown in Figure 3. In Figure 1, 13 is a cryopreservation solution, shown in a frozen state. These components are made of synthetic resin that has properties suitable for cryopreservation of cells, such as impact resistance, cold resistance, chemical resistance, and non-toxicity.

[0016] The retaining core 32 is rod-shaped and supports the inner circumferential surface of the tubular cell structure 11, and its cross-sectional shape is circular to match the shape of the lumen 11a of the tubular cell structure 11. The thickness of the retaining core 32 is sized to fit into the inner circumferential surface of the tubular cell structure 11, and its length is longer than that of the tubular cell structure 11. An introduction surface 32a that tapers or has rounded ends is formed at one or both longitudinal ends. In the illustrated example, the introduction surface 32a is formed at one end, and this introduction surface 32a consists of an inclined surface that tapers toward the end. The retaining core 32 may be either hollow or solid. Furthermore, since the retaining core 32 is the part that comes into direct contact with the tubular cell structure 11, a material that is not only non-cytotoxic but also non-cell-adhesive is used to form the retaining core 32.

[0017] The stopper 35 is also an accessory member of the holding core 32, and is configured to be detachable from the holding core 32. Specifically, it is a short cylinder with a thickness that fits onto the outer surface of the holding core 32, with a portion of the side open, and a cross-sectional shape that is roughly C-ring shaped. It is formed from a material that is flexible and has contact resistance with the holding core 32, and is configured to be fixed to any portion of the holding core 32 other than the introduction surface 32a. Two stoppers 35 are provided for one holding core 32. As shown in Figure 4, by attaching them to both sides of the tubular cell structure 11 held in the middle of the longitudinal direction of the holding core 32, they have the function of preventing the tubular cell structure 11 from shifting position on the holding core 32.

[0018] The protective sleeve 33 is formed in a cylindrical shape with a larger diameter than the tubular cell structure 11, and stores and holds the holding core 32 holding the tubular cell structure 11 in its axial center. It is cylindrical overall, and has two types of communication holes 33a and 33b on its side that connect the inside and outside. The communication holes 33a and 33b improve the transfer of liquid and heat, and the multiple communication holes 33a and 33b are distributed as evenly as possible in the longitudinal and circumferential directions of the protective sleeve 33.

[0019] 5, a plurality of communication holes 33a are formed in the longitudinal middle portion of the protective sleeve 33, and a plurality of communication holes 33b are formed on both longitudinal sides of the protective sleeve 33. In the illustrated example, the communication holes 33a and 33b are oval in shape that are long in the longitudinal direction of the protective sleeve 33, but they may have other shapes, and may be mesh-like as long as they at least allow liquid to pass through.

[0020] Regarding the size of the communicating holes 33a, 33b, they can be set to a size that allows the tubular cell structure 11 inside to be visible. Here, "a size that allows visibility" means a size that allows the surface of the tubular cell structure 11 to be seen through one communicating hole 33a, 33b, and is not too small like a mesh. It is also acceptable for a wide area of ​​the tubular cell structure 11 to be visible at once, and it is preferable that the communicating hole 33a in the middle is formed to have a length comparable to or longer than the length of the tubular cell structure 11 to be held.

[0021] The protective sleeve 33 must be strong enough to avoid contact with the tubular cell structure 11 even when compressed during freezing. Therefore, the communicating holes 33a, 33b in this example are oval as shown in Figure 5, but are distributed in the middle part in the longitudinal direction and on both sides in a manner that the longitudinal ends of the communicating holes 33a, 33b partially overlap. The communicating holes 33a in the middle part and the communicating holes 33b on both sides are formed in pairs at opposing positions on the circumferential surface, and are arranged 90 degrees offset in the circumferential direction, as can be seen in the three cross-sectional end views of Figure 5.

[0022] The length of the protective sleeve 33 is longer than the tubular cell structure 11 and shorter than the holding core 32. At both ends of the protective sleeve 33, there are cylindrical portions 33c without communication holes 33a, 33b.

[0023] Regarding the thickness of the protective sleeve 33, it is preferable to make it as thin as possible. This is to minimize the space created outside the tubular cell structure 11, thereby achieving high-quality, uniform freezing and thawing, and to reduce the amount of cryopreservation solution 13 used. In other words, the thickness of the protective sleeve 33 is set to the smallest or near-smallest thickness within the range that can avoid contact with the tubular cell structure 11 even when the protective sleeve 33 is compressed during freezing. In other words, it is the smallest or near-smallest thickness within the range that can prevent anything other than the cryopreservation solution 13 from touching the outer surface of the tubular cell structure 11, even when the maximum deformation that can occur due to compression during freezing occurs.

[0024] The protective sleeve 33, like the aforementioned retaining core 32, is made of a synthetic resin that has properties such as impact resistance, cold resistance, chemical resistance, and non-toxicity, which make it suitable for cryopreservation of cells.However, it is particularly desirable that the protective sleeve 33 be made of a transparent resin to ensure visibility of the tubular cell structure 11.

[0025] The connector cap 36 is a component that can be handled as an accessory to the retaining core 32 or the protective sleeve 33. It is detachably fitted onto the cylindrical portions 33c at both ends of the protective sleeve 33, positioning the retaining core 32 at the axial center of the protective sleeve 33 during installation. To this end, the connector cap 36 has a fitting portion 36a on one side that fits into the cylindrical portion 33c and a contact step portion 36b on the other side that protrudes outward from the cylindrical portion 33c and contacts the end surface of the protective sleeve 33. The fitting portion 36a and the contact step portion 36b are plate-shaped with flush front and back surfaces and have the same overall thickness. The thickness is thinner than the inner diameter of the protective sleeve 33 and thicker than the diameter of the retaining core 32. A retaining hole 36c is located at the center of a cross section cut in the thickness direction, through which the retaining core 32 is inserted and held, passing through the fitting portion 36a and the contact step portion 36b.

[0026] A gap 36d is formed in the middle of the plate-shaped fitting portion 36a and the abutting step portion 36b in the thickness direction, extending from the retaining hole 36c to the end so as to divide the plate-shaped fitting portion 36a into two in the thickness direction, and the gap 36d is open in the fitting portion 36a. The width of this gap 36d is narrower than the diameter of the retaining hole 36c, and it has the function of allowing the cryopreservation solution 13 to pass through.

[0027] Two connector caps 36 are provided, both of which have the same shape.

[0028] The inner bag 34 is made of a flexible, transparent sheet material that can accommodate the protective sleeve 33 to which the holding core 32 is joined, and is filled with the cryopreservation solution 13. It has a flat structure with no gusset, consisting of two sides, a front and a back, and has an opening 34a on one side, and one or more tubes 34b formed at one end for filling with the cryopreservation solution 13 and for removing air. Because this is the part that will be filled with the cryopreservation solution 13, the inner bag 34 does not need to be unnecessarily large, and it is preferable to make it as small as possible within the necessary limits.

[0029] The protective bag 37 is made of a flexible, transparent sheet material having a space for accommodating the inner bag 34, and has an opening 37a formed on one side, which serves as an opening for accommodating the inner bag 34. Like the inner bag 34, the protective bag 37 has a flat structure with no gusset, consisting of two surfaces, a front and a back.

[0030] The cryopreservation device 31 configured as described above is used for cryopreserving the cultured tubular cell structure 11 through the following steps: a structure holding step, a structure surrounding step, a preservation solution immersion step, and a freezing step.

[0031] In the structure holding process, as shown in Figure 4, the tubular cell structure 11 is held in the longitudinal middle of the holding core 32. Specifically, the rod-shaped holding core 32 that supports the inner surface of the tubular cell structure 11 is inserted into the lumen 11a of the tubular cell structure 11, or the tubular cell structure 11 is fitted into the holding core 32. An introduction surface 32a is formed at the end of the holding core 32, which prevents damage to the tubular cell structure 11 due to insertion or fitting. After the tubular cell structure 11 is moved to the longitudinal middle of the holding core 32, a stopper 35 is attached to the holding core 32. The stoppers 35 are attached to both sides of the held tubular cell structure 11 with an appropriate gap between them and the tubular cell structure 11.

[0032] In the structure-enclosing step, the holding core 32 holding the tubular cell structure 11 is held in the axial center of the protective sleeve 33. That is, as shown in Figure 6, connector caps 36 are fitted onto the cylindrical portions 33c at both ends of the protective sleeve 33, and at the same time, both ends of the holding core 32 are inserted and held in the holding holes 36c of the connector caps 36. The connector caps 36 can also be fitted onto the ends of the holding core 32 and then fitted into the protective sleeve 33.

[0033] When the holding core 32 is set, a space is created around the periphery of the tubular cell structure 11 held on the outer surface of the holding core 32, large enough that the protective sleeve 33 will not come into contact with it even when pressure is applied during freezing. The tubular cell structure 11 is then located at a position corresponding to the intermediate communicating hole 33a of the protective sleeve 33, and even if the tubular cell structure 11 attempts to move on the holding core 32 during operation, the stopper 35 prevents it from shifting beyond a predetermined position, maintaining the tubular cell structure 11 at a predetermined intermediate position. At this time, the connector cap 36 is oriented along the surface direction of the inner bag 34.

[0034] In the preservation solution immersion step, the tubular cell structure 11 covered with the protective sleeve 33 is sealed together with the holding core 32 in an inner bag 34 together with the cryopreservation solution 13, and air is removed. That is, as shown in Figure 7, an assembly consisting of the protective sleeve 33 etc. is placed in the inner bag 34, and the opening 34a is sealed as shown by the imaginary line. The holding core 32 is placed in the inner bag 34 with its longitudinal direction parallel or nearly parallel to the longitudinal direction of the inner bag 34.

[0035] Thereafter, the cryopreservation liquid 13 is poured into the tube 34b and air is removed, leaving only the assembly and a predetermined amount of the cryopreservation liquid 13 inside the inner bag 34, and the tube 34b is then sealed.

[0036] The inner bag 34 is stored in a protective bag 37 (see FIG. 3) and then transferred to a subsequent freezing process. When the inner bag 34 is stored in the protective bag 37, the longitudinal direction of the inner bag 34 becomes parallel or nearly parallel to the longitudinal direction of the protective bag 37, and therefore the longitudinal direction of the holding core 32 also becomes parallel or nearly parallel to the longitudinal direction of the protective bag 37. After storing the inner bag 34, a vacuum is drawn when the opening 37a of the protective bag 37 is sealed. In other words, air is removed from between the protective bag 37 and the inner bag 34.

[0037] In the freezing process, the holding core 32 is frozen while supported with its longitudinal direction oriented horizontally. Here, "horizontal" does not mean horizontal in the strict sense, but rather includes a position that is quasi-horizontal. In the cryopreservation device 31 having the inner bag 34 that matches the size of the assembly as described above, if the longitudinal directions of the inner bag 34 and the protective bag 37 are horizontal or nearly horizontal, as described above, then the holding core 32 can also be said to be horizontal or horizontal.

[0038] The freezing is performed using a canister 51 to protect the protective bag 37 and the inner bag 34, to ensure uniform and rapid freezing in the programmable freezer, and to ensure that the thickness of the inner container is consistent. That is, the freezing device 31 is sandwiched between canisters 51 made of metal plates 52 and frozen.

[0039] Canister 51 is composed of two metal plates 52 made of aluminum alloy or stainless alloy, and fasteners 53 that connect these metal plates 52 so that the distance between them can be adjusted. Metal plate 52 is rectangular and larger than protective bag 37, and has insertion holes 52a formed in at least four corners and other necessary positions through which fasteners 53 can be inserted. Fasteners 53 are composed of bolts 53a and nuts 53b.

[0040] When the cryopreservation tool 31 is held in the canister 51, it is confirmed that there is no moisture on either of them, and then the cryopreservation tool 31 and the metal plate 52 are clamped together with their longitudinal directions aligned as shown in Figures 8 and 9. Even if some pressure is applied when clamping, the tubular cell structure 11 is protected because the cryopreservation tool 31 has a protective sleeve 33. The clamping is performed so that the metal plates 52 are parallel to each other, and in this state, the specified freezing is performed using a programmable freezer.

[0041] Cooling is carried out quickly and evenly via the metal plate 52 of the canister 51. During cooling, the tubular cell structure 11 is prevented from deformation or compression by the holding core 32 and protective sleeve 33. Although the protective sleeve 33 surrounds the tubular cell structure 11, liquid and heat move through the communicating holes 33a and 33b, so freezing proceeds quickly. As the cryopreservation solution 13 freezes, it expands, but the metal plate 52 holds it down, so the thickness of the inner bag 34 does not exceed a certain level. This prevents uneven temperature and concentration distributions in the cryopreservation solution 13 inside, allowing for uniform freezing. Freezing at a predetermined cooling rate suppresses the formation of intracellular ice crystals and promotes moderate dehydration within the cells, resulting in a good frozen state.

[0042] The frozen body 12 of the tubular cell structure 11 obtained by such a cryopreservation method can be stored for a long period of time and can be thawed and used when necessary.

[0043] Since the communication holes 33a, 33b of the protective sleeve 33 are arranged in a dispersed manner, the liquid and heat are transferred evenly and quickly during thawing.

[0044] As described above, the shape of the tubular cell structure 11 is maintained by the cryopreservation device 31, and the long tubular cell structure 11 can be quickly frozen and thawed under the same conditions.

[0045] Therefore, damage to the tubular cell structure 11 can be suppressed and appropriate cryopreservation can be achieved.

[0046] In particular, since the cryopreservation device 31 is sandwiched between the canisters 51 and frozen during the freezing process, the thickness of the inner bag 34 is kept below a certain level, thereby allowing freezing and thawing to occur quickly and uniformly.

[0047] Uniformity in freezing and thawing is also achieved by minimizing the thickness of the protective sleeve 33 while avoiding contact with the tubular cell structure 11, thereby thinning the layer of cryopreservation solution 13 surrounding the tubular cell structure 11. Furthermore, the communication holes 33a and 33b are configured as elongated holes and are distributed along the longitudinal direction of the protective sleeve 33, which not only allows the protective sleeve 33 to function as an enclosure for the tubular cell structure 11, but also allows for more uniform transfer of liquid and heat throughout the entire longitudinal direction of the protective sleeve 33. This also enables uniform freezing and thawing. In particular, the connector cap 36 is formed in a plate shape with a gap 36d provided in the middle in the thickness direction, which further facilitates the transfer of liquid and heat at both ends of the protective sleeve 33.

[0048] If the communicating holes 33a, 33b of the protective sleeve 33 are sized to allow the tubular cell structure 11 inside to be visible, the tubular cell structure 11 can be easily visually observed when storing it in the cryopreservation device 31, ensuring that appropriate work can be carried out.

[0049] The connector cap 36, which connects the holding core 32 holding the tubular cell structure 11 to the protective sleeve 33, is formed in a roughly square plate shape, so it does not roll when handled and is easy to operate with tweezers, making it easy to perform appropriate work.

[0050] The above configuration is one embodiment for carrying out the present invention, and the present invention is not limited to the above configuration, and other configurations can be adopted.

[0051] The shape, size, and number of the communication holes 33a, 33b of the protective sleeve 33 may be other than those described above, such as being formed into a circle and arranged in a staggered pattern. If strength can be ensured, communication holes may also be formed in the cylindrical portions 33c at both ends of the protective sleeve 33 into which the connector caps 36 are fitted.

[0052] A member equivalent to the connector cap 36 may be formed on the retaining core 32 or the protective sleeve 33. Furthermore, the two connector caps 36 may have different configurations in consideration of operability.

[0053] The stopper 35 can also be formed on the retaining core 32. FIG. 10 shows an example. That is, as shown in FIG. 10(a), the retaining core 32 is composed of a rod-shaped base-end member 38 and a free-end member 39 joined together in the longitudinal direction. The base-end member 38 has a stopper 38a integrally formed in the middle of its length and an introduction surface 38b at one end. The introduction surface 38b has rounded corners. An insertion hole 38c is formed inside the end having the introduction surface 38b. The free-end member 39 has a stopper 39a integrally formed near one end, and an insertion shaft 39b is formed at the tip of this stopper 39a to be inserted into the insertion hole 38c of the base-end member 38. Both stoppers 38a, 39a are hexagonal, like nuts, to improve handling with tweezers.

[0054] In this holding core 32, the tubular cell structure 11 is held on the side having the introduction surface 38b of the base-end side member 38. Then, as shown in Figure 10(b), when the insertion shaft 39b of the free-end side member 39 is fitted into the insertion hole 38c of the base-end side member 38 holding the tubular cell structure 11, the base-end side member 38 and the free-end side member 39 become integrated. At the same time, stoppers 38a, 39a on both sides of the held tubular cell structure 11 prevent it from shifting position.

[0055] A holding core 32 having such a configuration is easy to operate because it is possible to shorten the movement distance of the tubular cell structure 11 on the holding core 32 when holding and separating the tubular cell structure 11. Operation is also simple in that there is no need to hold a stopper separately.

[0056] In the above example, the inner container was described using the inner bag 34, which has a bag structure made entirely of a flexible sheet material and in which the tubular cell structure 11 and the like are placed and the opening is closed, but the inner container is not limited to a bag structure. For example, the inner container may have other structures or shapes, such as a combination of a molded product and a sheet material, and may be configured as a container having at least a partially flexible portion.

[0057] The canister 51 used in the freezing step may be made up of metal plates 52 joined together by a hinge or the like.

[0058] A plurality of protective bags, such as two, may be used instead of one protective bag as described above. The protective bag 37 is not limited to a bag structure and may be covered with a sheet material, for example. [Explanation of symbols]

[0059] 11...Tubular cell structure 12...Frozen body 13... Cryopreservation solution 31...Freezing storage device 32...Holding core 33...Protective sleeve 33a, 33b…Communication hole 34...Inner bag 51...Canister 52...Metal plate

Claims

1. a structure holding step of holding the tubular cell structure in a longitudinal middle portion of a rod-shaped holding core that supports the inner peripheral surface of the tubular cell structure; a structure-enclosing step of holding the holding core at the axial center of a protective sleeve having a cylindrical shape with a larger diameter than the tubular cell structure and a communicating hole on the side that communicates with the inside and outside; a preservation solution immersion step of sealing the tubular cell structure covered with the protective sleeve together with the holding core in an inner container together with a cryopreservation solution; A freezing process is performed. A method for cryopreserving tubular cell structures.

2. In the freezing step, the cryopreservation device having the inner container is sandwiched between canisters made of metal plates and frozen. A method for cryopreserving the tubular cell structure according to claim 1.

3. The freezing step is carried out in a state where the longitudinal direction of the holding core is supported horizontally. A method for cryopreserving the tubular cell structure according to claim 1 or 2.

4. a rod-shaped holding core that supports the inner circumferential surface of the tubular cell structure and is longer than the tubular cell structure; a protective sleeve having a cylindrical shape with a larger diameter than the tubular cell structure, having a communicating hole on the side that communicates with the inside and outside, and storing and holding the holding core in its axial center; The inner container is capable of accommodating a protective sleeve to which a holding core is bonded, and is at least partially made of a flexible material and is filled with a cryopreservation solution. Cryopreservation device for tubular cell structures.

5. The thickness of the protective sleeve is set to the minimum within a range that allows the protective sleeve to avoid contact with the tubular cell structure even when compressed during freezing. A device for cryopreserving tubular cell structures according to claim 4.

6. A plurality of communication holes are formed in the protective sleeve and are distributed. A device for cryopreserving tubular cell structures according to claim 5.

7. The tubular cell structure according to claim 4 or 5, which has been frozen using the cryopreservation device for cryopreservation. Cryostat of tubular cell structures.

Images