CELL STRUCTURE CONNECTION METHOD AND CONNECTION SUPPORT DEVICE

The connection support device includes a rod-shaped member insertable into respective hollow portions of two or more cell structures, the rod-shaped member being inserted into the hollow portions, the rod-shaped member including a circular cross section having an outer diameter capable of adhering to inner surfaces of the cell structures when the rod-shaped member contracts after maturing, and a total length longer than a sum of respective lengths of the two or more cell structures, and two presser devices including clamp portions capable of being fixed to the rod-shaped member by clamping and fitting to the rod-shaped member, the rod-shaped member being made of a material with oxygen permeability.

TECHNICAL FIELD

The present invention relates to a connection method and a connection support device for connecting two or more tubular cell structures including hollow portions inside.

BACKGROUND ART

Conventionally, as disclosed in PTL 1, a technique has been known that produces a three-dimensional structure by utilizing the characteristic that cell aggregates contacting each other in an adjacent manner are fused to each other, and three-dimensionally stacking the cell aggregates (spheroids) so that cell aggregates are adjacent to each other, by utilizing a support body formed by a plurality of needle-like bodies fixed in advance to extend in the normal direction of a substrate.

In this technique, the technique is disclosed that extracts cell aggregates51, and sticks each of the cell aggregates51to each of the needle-like bodies of the support body to manufacture the state where the cell aggregates are skewered. For example, as a cell structure5for blood vessel, needle-like bodies are arranged in a tubular shape, and the cell aggregates51are stuck to each of the needle-like bodies. Then, when cultivated for a certain time period, as shown inFIG. 7B, the adjacent cell aggregates51are fused to each other, and the tubular cell structure5is formed on the needle-like bodies52. Then, as shown inFIG. 7C, when the cell structure5is pulled out from the needle-like bodies, the tubular cell structure5including a hollow portion inside the tube having a length L can be extracted.

CITATION LIST

Patent Literature

PTL 1: Specification of Japanese Patent No. 4517125

SUMMARY OF INVENTION

Technical Problem

In this technique, in order to increase the length L of the tubular cell structure5, the length of the needle-like body52will be increased, and the number of the cell aggregates51stuck to it will be increased. However, there are limitations to the technique of increasing the length of the needle-like body52, since when the length of the needle-like body52is increased, there are problems of the straightness of the needle-like body52, and the parallelism to the adjacent needle-like body52, or due to manufacturing problems.

Therefore, a method and an apparatus that can increase the length L of the cell structure5, without increasing the length of the needle-like body52, are required.

The present invention has been made in view of these problems, and provides a method and a connection support device that can easily increase the length L of the cell structure5.

Solution to Problem

It is solved by a connection support device for connecting two or more tubular cell structures by maturing the cell structures through cultivation for a predetermined time period, each of the cell structures having a hollow portion inside the each of the cell structures, the connection support device including a rod-shaped member insertable into the hollow portion of the each of the two or more cell structures, the rod-shaped member being inserted into the hollow portions, the rod-shaped member including a circular cross section having an outer diameter closely contactable to inner surfaces of the tubular cell structures by shrinkage of the rod-shaped member after the maturing, and a total length longer than a sum of respective lengths of the two or more cell structures; and two presser devices each including a clamp portion capable of being fixed to the rod-shaped member by clamping and fitting to the rod-shaped member, wherein the rod-shaped member is made of a material with oxygen permeability, and wherein in each of the two presser devices, in a case where the rod-shaped member is inserted into the two or more cell structures in a state where one ends of the two or more cell structures contacts with each other, each of the clamp portions makes a contact with each of end surfaces of another ends of the two or more cell structures that are not in a contact state.

It is solved by a connection support device for connecting two or more tubular cell structures by maturing the cell structures through cultivation for a predetermined time period, each of the cell structures having a hollow portion inside the each of the cell structures, the connection support device including a rod-shaped member insertable into the hollow portion of the each of the two or more cell structures, the rod-shaped member being inserted into the hollow portions, the rod-shaped member including a circular cross section having an outer diameter closely contactable to inner surfaces of the tubular cell structures by shrinkage of the rod-shaped member after the maturing, and a total length longer than a sum of respective lengths of the two or more cell structures, and a presser device including a clamp portion capable of being fixed to the rod-shaped member by clamping and fitting to the rod-shaped member, the rod-shaped member including a conduit through which a culture solution flows along an axial direction of the rod-shaped member from one end to the other end of the rod-shaped member, the presser device including a projection, in a case where the rod-shaped member is inserted into the two or more cell structures in a state where one ends of the two or more cell structures contacts with each other, the projection arranged along a circumference contacting an end surface of one of another ends of the two or more cell structures that are not in a contact state, the rod-shaped member being made of a material with oxygen permeability, and at the other of the both ends of the cell structures that are not in the contact state, the presser device contacting an end surface of the other end.

It is solved by a cell structure connection method for connecting two or more tubular cell structures with a rod-shaped member made of a material with oxygen permeability, each of the two or more tubular cell structures including a hollow portion inside, with a rod-shaped member having a conduit inside the each of the cell structures, the rod-shaped member having a conduit penetrating along an axial direction of the rod-shaped member from one end to another end of the rod-shaped member, the rod-shaped member being made of a material with oxygen permeability, the rod-shaped member having a total length longer than a sum of respective lengths of the two or more cell structures, wherein the cell structure connection method includes an insertion step of inserting the rod-shaped member into the hollow portion of the each of the two or more cell structures, a fixing step of fixing the two or more cell structures to the rod-shaped member with two presser devices each of which includes a clamp portion capable of being fixed to the rod-shaped member and has an inner diameter substantially a same as an outer diameter of a cross section of the rod-shaped member, wherein in each of the two presser devices, in a case where the rod-shaped member is inserted into the two or more cell structures in a state where one ends of the two or more cell structures contacts with each other, each of the clamp portions makes a contact with each of end surfaces of another ends of the two or more cell structures that are not in a contact state, so that the two presser devices contacts the one ends of the two or more cell structures that contact with each other, a maturing step of cultivating and maturing the two or more cell structures by flowing a culture solution into the conduit of the rod-shaped member.

It is solved by a cell structure connection method for connecting two or more tubular cell structures with a rod-shaped member made of a material with oxygen permeability, each of the two or more tubular cell structures including a hollow portion inside, with a rod-shaped member having a conduit inside the each of the cell structures, the rod-shaped member having a conduit penetrating along an axial direction of the rod-shaped member from one end to another end of the rod-shaped member, the rod-shaped member being made of a material with oxygen permeability, the rod-shaped member having a total length longer than a sum of respective lengths of the two or more cell structures, wherein the cell structure connection method includes an insertion step of inserting the rod-shaped member into the hollow portion of the each of the two or more cell structures, a fixing step of fixing the two or more cell structures to the rod-shaped member with a presser device which includes a clamp portion capable of being fixed to the rod-shaped member and has an inner diameter substantially a same as an outer diameter of a cross section of the rod-shaped member, the presser device including a projection, in a case where the rod-shaped member is inserted into the two or more cell structures in a state where one ends of the two or more cell structures contacts with each other, the projection arranged along a circumference contacting an end surface of one of another ends of the two or more cell structures that are not in a contact state, so that the presser devices contacts the one ends of the two or more cell structures that contact with each other, a fixing step of fixing the two or more cell structures to the rod-shaped member by causing, at one of both ends of the cell structures that are not in a contact state when one ends of the cell structures are brought into the contact state, and the cell structures are inserted into the rod-shaped member, a projection arranged along a circumference of the rod-shaped member to contact an end surface of the one end, and causing, at an end surface of the other end, a presser device to contact the end surface of the other end, a maturing step of cultivating and maturing the two or more cell structures by flowing a culture solution into the conduit of the rod-shaped member.

Advantageous Effects of Invention

According to the connection method and the connection support device of the present invention, in a case where a long cell structure is manufactured, it can be manufactured by creating and connecting a plurality of short cell structures.

DESCRIPTION OF EMBODIMENTS

First, usingFIG. 1toFIG. 4, a connection method for a cell structure5of the present invention and Embodiment 1 of a connection support device1for realizing it will be described.FIG. 1illustrates the connection support device1to which two cell structures5are attached.FIG. 2illustrates the state where bonding surfaces of the two cell structures5are separated, and a gap6is created.FIG. 3is a diagram illustrating one embodiment of a presser device3constituting the connection support device1. The left-hand diagram inFIG. 3is a diagram seen from the axial direction of the cell structure5, and the right-hand diagram inFIG. 3is a diagram seen from the direction perpendicular to the left-hand side ofFIG. 3.FIG. 4is a diagram illustrating another embodiment of the presser device3constituting the connection support device1.

The connection support device1includes a rod-shaped member2and the presser device3. The cell structure5was formed in, for example, the steps ofFIG. 7AtoFIG. 7Cand the step as described above, and has a tubular structure including a hollow portion having a circular cross section inside the cell structure5. Here, a cell structure5aand a cell structure5bare prepared, and they are connected to each other. In this specification, it is assumed that the inner diameters of the cell structures5aand5bare R.

The rod-shaped member2is an elongated cylindrical member having a circular cross section. The outer diameter of the rod-shaped member2is R. In the step for connecting the cell structures5aand5bto each other, when the rod-shaped member2is inserted into the cell structures5aand5b, the inner diameters of the cell structures5aand5bare slightly larger than the outer diameter R of the rod-shaped member2. However, since the inner diameters of the cell structures5aand5bhave the characteristic of gradually contracting in the course of maturation of cells that constitute the cell structures5aand5bwith the passage of time in the state where cultivation is being performed, when time elapses in the state where cultivation is being performed, the rod-shaped member2will be in the state where the rod-shaped member2is tightly inserted and fit into the cell structures5aand5bwithout a gap between the cell structures5aand5band the rod-shaped member2. In the inside, a conduit2ais provided so as to penetrate along the axial direction in the longitudinal direction of the rod-shaped member2from one end to the other end of the rod-shaped member2. As for the length of the rod-shaped member2, the rod-shaped member2is longer than at least a combined total length of the cell structure5aand the cell structure5b. That is, when the rod-shaped member2is fit into in the hollow portions of the cell structure5aand the cell structure5b, even if one end of the cell structure5aand one end of the cell structure5bare in a contact state, the connected cell structures5aand5bare long enough to expose a certain amount of the rod-shaped member2at both ends of the connected cell structures5aand5b. A top end2bof the rod-shaped member2has a tapered shape so as to be easily inserted into the hollow portion of the cell structure5a.

The rod-shaped member2has a structure with oxygen permeability between the conduit2aand the outer surface of the rod-shaped member2. For example, small through-holes for oxygen penetration may be arranged between the conduit2aand the outer surface of the rod-shaped member2. Additionally, a material having oxygen permeability such as dimethylpolysiloxane (PDMS) may be used, without arranging the small through-holes for oxygen permeation. By introducing an oxygen-dissolved culture solution into the conduit2a, as the culture solution passes, oxygen reaches the outer surface of the rod-shaped member2, and the cell structure5acan accept oxygen from the inner surface over the entire length of the cell structure5a.

The presser device3includes a clamp portion31and a grip portion32. The clamp portion31can be fixed to the rod-shaped member2by clamping and fitting to the rod-shaped member2. The clamp portion31is divided into two portions, a fitting portion31aand a fitting portion31b, and when the fitting portion31aand the fitting portion31bare combined, a circular shape having the inner circumference radius of R is formed. The fitting portion31aand the fitting portion31bof the clamp portion31are coupled to an arm32aand an arm32bof the grip portion32, respectively. The grip portion32includes a spring portion33, and biases the grip portion32, so that the arm32aand the arm32bare spread to combine the fitting portion31aand the fitting portion31b, and the inner circumference constitutes the circular shape having the inner circumference radius of R. In this state, the fitting portion31aand the fitting portion31bclamp the outer circumference of the rod-shaped member2, and also fit to the rod-shaped member2(the state of solid lines inFIG. 3). By narrowing the arm32aand the arm32b, and applying force in the opposite direction to the bias direction of the grip portion32, the fitting portion31aand the fitting portion31bare divided into two portions, and the fitting portion31aand the fitting portion31bare released from the state of clamping the rod-shaped member2(one-dot-chain lines inFIG. 3).

As illustrated inFIG. 3, in the state where the fitting portion31aand the fitting portion31bclamp the rod-shaped member2, the fitting portion31aand the fitting portion31bclose at an end surface7of an end of the cell structure5aand the cell structure5b, and a side surface8of the clamp portion31contacts the end surface7. In this state, the presser devices3regulate at the both ends of the cell structure5aand the cell structure5b, so that the cell structure5aand the cell structure5bdo not move on the rod-shaped member2. At this time, ideally, as illustrated inFIG. 3, the presser device3has the structure in which the side surface8of the clamp portion31and the end surface7of each of the both ends of the cell structures5aand5bcontact each other over the entire circumferences of the end surface7. Especially, it is preferable that the side surface8of the clamp portion31of the presser device3is a flat surface. Accordingly, in the state where cultivation has advanced, the flatness of the end surface7of each of the both ends of the cell structures5aand5bbecomes high. However, when the side surface8of the clamp portion31and the end surface7of each of the both ends of the cell structures5aand5bhave the structures to at least partially contact each other, even if not contacting each other over the entire circumference of the end surface7, as long as the presser device3can regulate at the both ends of the cell structure5aand the cell structure5b, so that the ends of the cell structure5aand the cell structure5bdo not move on the rod-shaped member2, an effect required for connection of the cell structures5aand5bis produced. Additionally, insertion holes34aand34binto which top ends2bof tweezers can be inserted can be arranged in the arms32aand32b, respectively, so that the arms32aand32bof the presser device3can be expanded and narrowed with the tweezers (FIG. 4).

Subsequently, how to connect the cell structure5aand the cell structure5bto each other by using the connection support device1, a cell structure connection method in Embodiment 1, will be described. First, the rod-shaped member2is inserted into the hollow portions of the cell structure5aand the cell structure5b(insertion step). Subsequently, a contact state is made so that there is no gap6between the opposing ends of the cell structure5aand the cell structure5b. In this state, the rod-shaped member2is clamped by the two presser devices3aand3bat the both ends of the cell structure5aand the cell structure5bthat are not in a contact state, and the presser device3aand the presser device3bare made to fit to the rod-shaped member2. At this time, the cell structures5aand5bare fixed by clamping the rod-shaped member2with the two presser devices3aand3b, at the positions where the respective side surfaces8of the two presser devices3aand3bcontact the end surfaces7of the both ends of the cell structures5aand5b, and pressing forces are applied in directions along which the cell structures5aand5brelatively approach to the end surfaces of the cell structures5aand5bin the contact state, respectively (fixing step).

In this state, a solution allowing permeation of oxygen is passed through the inside of the conduit2aof the rod-shaped member2, and the inside and outside of the cell structures5aand5bare immersed in the solution, and cultured and matured for a predetermined time period (maturing step). Referring toFIG. 5, an example of using a cultivation maintaining apparatus41will be described as an example of supplying a culture solution to the rod-shaped member2inserted into the cell structures5aand5bin the maturing step.FIG. 5is a schematic diagram of the cultivation maintaining apparatus41. The cultivation maintaining apparatus41includes a sealed culture chamber43, and the inside is filled with a culture solution43a. The culture chamber43includes a first inlet46dand a second inlet47dfor introduction into the culture chamber43, and a first exit46cand a second exit47cfor discharging the culture solution43afrom the culture chamber43. Additionally, the cultivation maintaining apparatus41includes a first conduit46that fluidly connects the first exit46cto the first inlet46dvia a pump44, and a second conduit47that fluidly connects the second exit47cto the second inlet47dvia the pump44. The second inlet47d, the first exit46c, and the second exit47care open ends, and the rod-shaped member2is attached to the first inlet46dvia an adapter.

The culture solution43aflows back inside the first conduit46and the second conduit47with the driving force of the pump44. When the culture chamber43filled with the culture solution43ais seen as a starting point, the culture solution43aflows out of the first exit46cinto a first conduit46b, and travels toward the pump44. Additionally, the culture solution43aflows out of the second exit47cinto a second conduit47b, and travels toward the pump44. Thereafter, the culture solution43ato which required nutrients and oxygen have been supplied at the pump44returns to the culture chamber43from the first conduit46avia the first inlet46dwith the driving force of the pump44. Additionally, it returns to the culture chamber43from the second conduit47avia the second inlet47d. The first conduit46is mainly for supplying the culture solution43ato the inside of the cell structures5aand5bthrough a penetration conduit inside the rod-shaped member2. Additionally, the second conduit47is mainly for spreading the culture solution43aover the outside of the cell structures5aand5b. By circulating the culture solution43awithin the cultivation maintaining apparatus41for a predetermined time period, the cell structures5aand5bare cultivated and matured.

Since the cell structures5aand5bcontract in the axial direction of the rod-shaped member2with the progress of the maturing step, the pressing forces at the end surfaces of the cell structures5aand5bin the contact state are decreased, and in some cases, the end surfaces in the contact state may be separated to create the gap6. In that case, the distance of each of the two presser devices3aand3bis reduced to brought the separated gap6into the contact state again, and the rod-shaped member2is pressed and clamped again by the two presser devices3aand3bat the positions where the pressing forces are maintained at the end surfaces of the cell structures5aand5bin the contact state.

With the completion of the maturing step in which the cells of the cell structures5aand5bmature through cultivation for a predetermined time period, the cells at the portions of the cell structures5aand5bin the contact state are fused to each other, and the cell structures5aand5bare connected to each other.

Subsequently, usingFIG. 6, a connection method for the cell structure5of the present invention and Embodiment 2 of the connection support device1for realizing it will be described. In Embodiment 1, the two presser devices3aand3bare used. A connection support device4of Embodiment 2 is different in that the rod-shaped member2and one presser device3are used to connect the cell structures5aand5bto each other. Hereinafter, regarding Embodiment 2, the differences from Embodiment 1 will be described.FIG. 6is a diagram illustrating the connection support device4and the connection method for the cell structure in Embodiment 2 of the present invention.

In the rod-shaped member2of Embodiment 2, a projection2cis arranged in the vicinity of an end of the rod-shaped member2opposite to the tapered top end2b. The projection2chas a flange-shape arranged to project in the radial direction of the rod-shaped member2along the circumference of the rod-shaped member2in the vicinity of its end. The radial distance is greater than the thicknesses of the cell structure5aand the cell structure5b, and an end of the cell structure5acan be made to abut against the projection2c. In the other respects, the rod-shaped member2is the same as in Embodiment 1. The presser device3of Embodiment 2 is completely the same as in Embodiment 1 as illustrated inFIG. 3andFIG. 4. However, although the two presser devices3aand3bare required in Embodiment 1, only one presser device3is used in Embodiment 2.

Subsequently, how to connect the cell structure5aand the cell structure5bto each other by using the connection support device4, a cell structure connection method in Embodiment 2, will be described. First, the rod-shaped member2is inserted into the hollow portions of the cell structure5aand the cell structure5b(insertion step). Then, a contact state is made so that there is no gap6between the opposing ends of the cell structure5aand the cell structure5b. At this time, an end surface of the cell structure5ais made to abut against the projection2c. The order of the step of making the contact state so that there is no gap between the opposing ends of the cell structure5aand the cell structure5b, and the step of making the end surface of the cell structure5ato abut against the projection2cdoes not matter.

In the state where the end surface of the cell structure5aabuts against the projection2c, the rod-shaped member2is clamped by the presser device3at the end of the cell structure5bthat is not in the contact state to make the presser device3fit to the rod-shaped member2. At this time, the cell structures5aand5bare fixed by clamping the rod-shaped member2with the presser device3at the position where the side surface of the presser device3contacts the end surface of the end of the cell structure5b, and a pressing force is applied in a direction along which each of the cell structures5aand5brelatively approaches to the end surface of the cell structure5bin the contact state (fixing step). Then, the end surface of the cell structure5aon the side opposite to the presser device3abuts and is pressed against the projection2c. As a result, the end surfaces of the cell structures5aand5bin the contact state are pressed against each other.

In this state, a solution allowing permeation of oxygen is passed through the inside of the conduit2aof the rod-shaped member2, and the inside and outside of the cell structures5aand5bare immersed in the solution, and cultured and matured for a predetermined time period (maturing step). The maturing step is performed in the same apparatus as illustrated inFIG. 5of Embodiment 1. Since the cell structures5aand5bcontract in the axial direction of the rod-shaped member2with the progress of the maturing step, the pressing forces at the end surfaces of the cell structures5aand5bin the contact state are decreased, and in some cases, the end surfaces in the contact state may be separated to create the gap6. In that case, as in Embodiment 1, the presser device3is moved closer to the projection2cto bring the separated gap6into the contact state again, and the rod-shaped member2is pressed and clamped again by the presser device3at the position where the pressing forces are maintained at the end surfaces of the cell structures5aand5bin the contact state.

With the completion of the maturing step in which the cells of the cell structures5aand5bmature through cultivation for a predetermined time period, the cells at the portions of the cell structures5aand5bin the contact state are fused to each other, and the cell structures5aand5bare connected to each other.

REFERENCE SIGNS LIST