JP6782002B2 - Method for manufacturing a microchannel structure having a capillary network-like microchannel - Google Patents

Description

The present invention relates to a method for manufacturing a microchannel structure having a three-dimensional network-like microchannel, particularly a microchannel structure having a capillary network-like microchannel.

Organ transplantation is the only curative treatment for intractable organ diseases. However, due to the shortage of donors of organs for transplantation, there are not many patients with intractable organ diseases who can be treated by organ transplantation. If an artificial organ can be produced in vitro, the number of patients who can be treated by organ transplantation can be increased, and as a result, the lifesaving rate of the patient can be improved.

Blood vessels play an important role in the living body. In particular, capillaries play an extremely important role in supplying oxygen and nutrients to cells in living tissues. Capillaries usually have a diameter through which blood cells can pass, eg, about 10 μm in diameter. Further, in the living body, the capillaries constitute a capillary network having a three-dimensional network-like or cotton candy-like shape. The microscopic capillary network allows cells in living tissue to receive sufficient amounts of oxygen and nutrients. By the action of such a capillary network, the living body can maintain a large organ of 0.1 to 1.5 L. Therefore, when manufacturing an artificial organ in vitro, a means for forming a microchannel having a capillary network-like shape is required.

For example, Non-Patent Document 1 describes a method in which vascular endothelial cells covering the lumen of blood vessels are cultured in a gel made of a biological protein such as collagen, and the cells spontaneously form a capillary-like structure. Describe.

Non-Patent Document 2 describes a method of creating a flow path in a collagen gel by arranging a gelatin gel fiber in a collagen gel, raising the temperature of the gelatin gel fiber, and dissolving only the fiber by applying 3D printing technology. To do.

In Non-Patent Document 3, a gel made of polymethyl methacrylate (PMMA) having a diameter of about 10 μm is prepared, a bundle of the fibers is embedded in an agarose gel, and only the fibers are specifically dissolved. It is described that a capillary network-like flow path network was prepared therein.

Patent Document 1 describes at least one inlet A1 to Am (m ≧ 1) for introducing a first aqueous solution in which sodium alginate and a protein are dissolved, and at least one inlet G1 for introducing a gelling agent aqueous solution. ~ Gn (n ≧ 1), inlet channels CA1 to CAm and CG1 to CGn connected to inlets A1 to Am and inlets G1 to Gn, respectively, and inlet channels CA1 to CAm and inlet channels CG1 to CGn at the same time. Alternatively, for the flow path structure X having a merging flow path M that merges stepwise and an outlet O existing downstream of the merging flow path M, the first aqueous solution and the gelling agent aqueous solution are added to the flow path structure X, respectively. It is continuously introduced to form a hydrogel by continuously gelling the first aqueous solution inside the flow path structure X, and then contained in the hydrogel outside or inside the flow path structure X. A method for producing a fibrous protein material, which chemically crosslinks the protein and further removes alginic acid contained in the hydrogel, will be described.

International Publication No. 2016/021498

Folkman J, Haudenschild C., Angiogenesis in vitro, Nature Vol. 288, p. 551-556, 1980 Lee W, Lee V, Polio S, Keegan P, Lee JH, Fischer K, Park JK, Yoo SS., On-demand three-dimensional freeform fabrication of multi-layered hydrogel scaffold with fluidic channels, Biotechnol Bioeng, Vol. 105, p. 1178-1186, 2010 Grant-in-Aid for Scientific Research (Grants-in-Aid for Scientific Research) Research Results Report, Takayuki Takei, "Development of Capillary Network Similar to Living Body and Heavy Three-Dimensional Organ Creation Technology for Regenerative Medicine", Issue No. 23760752, 2013

As described above, several means for forming microchannels having a capillary network-like shape are known. However, there was room for improvement in known means.

In the case of the method described in Non-Patent Document 1, it takes several days to form a flow path having a dense shape. During this period, it becomes difficult to supply sufficient amounts of oxygen and nutrients to the organ cells placed in the gel, which may result in the death of the organ cells.

In the case of the method described in Non-Patent Document 2, the diameter of the flow path produced can vary depending on the diameter of the gelatin gel fiber. The gelatin gel fiber described in the document has a diameter of 100 μm or more. Therefore, the resulting flow path also has a diameter of 100 μm or more. It is difficult for a channel having such a large diameter to function as a capillary network in an artificial organ. In addition, it takes a very long time to produce a dense shape such as a capillary network by 3D printing technology.

In the case of the method described in Non-Patent Document 3, a fat-soluble organic solvent such as dichloromethane is used to dissolve the PMMA fiber. The use of fat-soluble organic solvents is not preferred in the production of biocompatible artificial organs.

In the case of the method described in Patent Document 1, a mixed solution of sodium alginate and a protein is gelled to form a fibrous gel, the protein is chemically crosslinked, and then alginic acid is removed to prepare a fibrous protein. However, chemically crosslinked fibrous proteins are difficult to dissolve again. It is also conceivable to increase the protein content in order to improve the strength of the fibrous protein without chemically cross-linking the protein. In this case, the removal of alginic acid becomes insufficient, and alginic acid may remain in the fibrous protein. If alginic acid remains in the fibrous protein, the fibrous alginic acid may remain even if the protein is dissolved. Therefore, even if the fibrous protein obtained by the method described in Patent Document 1 is applied as a fiber in the method described in Non-Patent Document 2 or 3, for example, it is difficult to use it as a template for a flow path. In addition, in the method described in Patent Document 1, it is necessary to use a microchannel structure having a special and complicated shape produced by using microfabrication technology in order to obtain a fibrous protein having a minute diameter. is there. Therefore, the method described in the document may be costly to implement.

Therefore, an object of the present invention is to provide a means for forming a microchannel structure having a capillary network-like microchannel in a short time.

The present inventors have studied various means for solving the above problems. The present inventors contain gelatin by coextruding a core component containing gelatin and a sheath component containing an alkali metal salt of alginic acid into a fiber-forming liquid containing divalent cations under specific conditions. It has been found that a gelatin core-sheath fiber having a core and a sheath containing a divalent cation salt of alginic acid can be obtained. Further, the present inventors embed a gelatin fiber having a diameter of about 10 μm obtained by dissolving the sheath portion of the gelatin core sheath fiber with an alginic acid removing agent such as a chelating agent in a gel. It was found that by dissolving gelatin fibers, a three-dimensional network-like microchannel having a diameter of about 10 μm can be formed in a gel in a short time. The present inventors have completed the present invention based on the above findings.

That is, the gist of the present invention is as follows.

［式中、
Dは、ゼラチン芯鞘繊維の芯部の直径（cm）であり、
Qは、芯成分及び鞘成分の総押出流量（ml/分）であり、
Rは、芯成分及び鞘成分の総押出流量に対する芯成分の押出流量の比であり、
vは、ゼラチン芯鞘繊維の引取速度（cm/分）であり、
πは、円周率である。］
で表される関係を満たす条件で実施される、前記ゲルとゲル中に2〜25 μmの範囲の直径の三次元網状の微小流路とを有する、微小流路構造体の製造方法。
（2） ゼラチン芯鞘繊維紡糸工程において、
ゼラチン芯鞘繊維の芯部の直径Dが、2〜25 μmの範囲であり、
芯成分及び鞘成分の総押出流量Qが、0.5〜6 ml/分の範囲であり、
芯成分及び鞘成分の総押出流量に対する芯成分の押出流量の比Rが、0.0000293〜0.00283の範囲であり、且つ
ゼラチン芯鞘繊維の引取速度vが、300〜900 cm/分の範囲である、前記実施形態（1）に記載の方法。
（3） ゼラチン繊維形成工程で得られたゼラチン繊維を、エタノール、2-プロパノール若しくはメタノール、又はそれらの混合物である低融点有機溶媒中で脱水し、次いで脱水したゼラチン繊維を、低融点有機溶媒、又はtert-ブチルアルコール若しくはジメチルスホキシド、又はそれらの混合物である中融点有機溶媒中で凍結乾燥して、三次元網状のゼラチン繊維を得る、凍結乾燥工程をさらに含む、前記実施形態（1）又は（2）に記載の方法。
（4） 共押出が、同心円状に配設された2個の吐出口を有するダイの中心部吐出口から芯成分を、外周部吐出口から鞘成分を、それぞれ吐出することによって実施され、中心部吐出口の直径が、200〜600 μmの範囲であり、外周部吐出口の直径が、200〜1000 μmの範囲である、前記実施形態（1）〜（3）のいずれかに記載の方法。
（5） ゼラチン芯鞘繊維紡糸工程で使用される芯成分が、1〜50 w/v％ ゼラチン水溶液である、前記実施形態（1）〜（4）のいずれかに記載の方法。
（6） ゼラチン芯鞘繊維紡糸工程で使用される鞘成分が、0.1〜10 w/v％ アルギン酸ナトリウム水溶液である、前記実施形態（1）〜（5）のいずれかに記載の方法。
（7） ゼラチン芯鞘繊維紡糸工程で使用される二価陽イオンを含む繊維形成液が、10〜6000 mM 塩化カルシウム水溶液である、前記実施形態（1）〜（6）のいずれかに記載の方法。
（8） アルギン酸除去剤が、10〜1000 mM クエン酸水溶液である、前記実施形態（1）〜（7）のいずれかに記載の方法。
（9） コラーゲン及びフィブリンからなる群より選択される1種以上のゲル化剤を含むゲルと、該ゲル中に2〜25 μmの範囲の直径の三次元網状の微小流路とを有する、微小流路構造体。 (1) The core component containing gelatin and the sheath component containing an alkali metal salt of alginic acid are coextruded into a fiber forming liquid containing divalent cations at a temperature of 25 ° C. or lower, and the core containing gelatin and alginic acid are coextruded. A gelatin core-sheath fiber spinning step in which a gelatin core-sheath fiber having a sheath portion containing a divalent cation salt of is taken from a fiber-forming liquid and spun.
The gelatin core sheath fiber is brought into contact with a chelating agent or an alginic acid removing agent which is an alginic acid hydrolyzing enzyme to dissolve the sheath containing the divalent cation salt of alginic acid to obtain a gelatin fiber having a diameter of 2 to 25 μm. , Gelatin fiber forming step;
Immersion step of immersing gelatin fibers in one or more gelling agents selected from the group consisting of collagen and fibrin;
A gelling step in which a gelling agent in which gelatin fibers are immersed is gelled and the gelatin fibers are embedded in a gel;
A microchannel formation step in which gelatin fibers embedded in a gel are melted at a temperature exceeding 25 ° C. to form microchannels in the gel;
Including
The gelatin core-sheath fiber spinning process is based on the following formula:

[During the ceremony,
D is the diameter (cm) of the core of the gelatin core sheath fiber.
Q is the total extrusion flow rate (ml / min) of the core component and sheath component.
R is the ratio of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component.
v is the take-up rate (cm / min) of gelatin core sheath fiber,
π is the pi. ]
A method for producing a microchannel structure, which comprises the gel and a three-dimensional network microchannel having a diameter in the range of 2 to 25 μm in the gel, which is carried out under the condition of satisfying the relationship represented by.
(2) In the gelatin core-sheath fiber spinning process
The diameter D of the core of the gelatin core sheath fiber is in the range of 2 to 25 μm.
The total extrusion flow rate Q of the core component and the sheath component is in the range of 0.5 to 6 ml / min.
The ratio R of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component is in the range of 0.0000293 to 0.00283, and the take-up speed v of the gelatin core and sheath fiber is in the range of 300 to 900 cm / min. The method according to the embodiment (1).
(3) The gelatin fibers obtained in the gelatin fiber forming step are dehydrated in a low melting point organic solvent which is ethanol, 2-propanol or methanol, or a mixture thereof, and then the dehydrated gelatin fibers are subjected to a low melting point organic solvent. Alternatively, the embodiment (1) or the above embodiment (1), further comprising a lyophilization step of lyophilizing in a medium melting point organic solvent which is tert-butyl alcohol or dimethyl succide, or a mixture thereof, to obtain a three-dimensional network gelatin fiber. The method described in (2).
(4) Coextrusion is carried out by discharging the core component from the central discharge port and the sheath component from the outer peripheral discharge port of the die having two discharge ports arranged concentrically, and the center. The method according to any one of the above-described embodiments (1) to (3), wherein the diameter of the partial discharge port is in the range of 200 to 600 μm, and the diameter of the outer peripheral discharge port is in the range of 200 to 1000 μm. ..
(5) The method according to any one of the above-described embodiments (1) to (4), wherein the core component used in the gelatin core-sheath fiber spinning step is a 1 to 50 w / v% gelatin aqueous solution.
(6) The method according to any one of the above-described embodiments (1) to (5), wherein the sheath component used in the gelatin core-sheath fiber spinning step is an aqueous solution of 0.1 to 10 w / v% sodium alginate.
(7) The above-described embodiment (1) to (6), wherein the fiber-forming liquid containing a divalent cation used in the gelatin core-sheath fiber spinning step is an aqueous solution of 10 to 6000 mM calcium chloride. Method.
(8) The method according to any one of the above-described embodiments (1) to (7), wherein the alginic acid removing agent is an aqueous solution of 10 to 1000 mM citric acid.
(9) A micro with a gel containing one or more gelling agents selected from the group consisting of collagen and fibrin, and a three-dimensional network microchannel having a diameter in the range of 2 to 25 μm in the gel. Channel structure.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a means for forming a microchannel structure having a capillary network-like microchannel in a short time.
Issues, configurations and effects other than the above will be clarified by the following description of the embodiments.

FIG. 1 is a schematic view showing an embodiment of a microchannel structure according to an aspect of the present invention. FIG. 2 is a schematic view of an embodiment of the gelatin core-sheath fiber spinning step included in the method for producing a microchannel structure according to one aspect of the present invention. A: Outline of one embodiment of the gelatin core-sheath fiber spinning process, B: Cross-sectional view of one embodiment of the apparatus used in the gelatin core-sheath fiber spinning process. FIG. 3 shows the relationship between the ratio of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component in coextrusion (extrusion flow rate ratio) and the diameter of the resulting gelatin core-sheath fiber and the diameter of the core portion. It is a graph which shows. Black-painted square: the diameter of the gelatin core-sheath fiber, black-painted circle: the diameter of the core of the gelatin core-sheath fiber. FIG. 4 is a phase-contrast micrograph of the obtained gelatin core-sheath fiber. A: Gelatin core-sheath fiber obtained when the extrusion flow rate ratio is 0.167, B: Gelatin core-sheath fiber obtained when the extrusion flow rate ratio is 0.000833. FIG. 5 is a stereomicroscope and a scanning electron micrograph of the obtained gelatin fiber. A: Photograph showing the overall appearance of gelatin fiber, B: Enlarged photo of gelatin fiber. FIG. 6 is a schematic view showing an outline of manufacturing a microchannel structure having linear microchannels. FIG. 7 is a fluorescence micrograph of the obtained microchannel structure having linear microchannels. FIG. 8 is a schematic view showing an outline of the production of a microchannel structure having a capillary network-like microchannel. FIG. 9 is a photograph showing the appearance of a microchannel structure having a capillary network-like microchannel in which rat blood is injected. A: Photograph 5 seconds after the start of injection, B: Photograph 10 seconds after the start of injection.

Hereinafter, preferred embodiments of the present invention will be described in detail.
<1: Manufacturing method of capillary network-like microchannel structure>
Capillaries play a vital role in supplying oxygen and nutrients to cells in living tissues. Therefore, when manufacturing an artificial organ in vitro, a means for forming a capillary network-like microchannel is required. However, a means for forming a capillary network-like microchannel in a short time has not been known.

The present inventors have formed a gelatin core sheath fiber having a core containing gelatin and a sheath containing a divalent cation salt of alginic acid, and then using an alginic acid removing agent such as a chelating agent to form the gelatin core sheath. By dissolving the gelatin fiber while embedding the gelatin fiber having a diameter of about 10 μm obtained by dissolving the sheath of the fiber in the gel, a three-dimensional network-like minute having a diameter of about 10 μm is dissolved in the gel. It was found that the flow path can be formed in a short time. Therefore, one aspect of the present invention relates to a method for producing a capillary network-like microchannel structure. The method for producing a micropath structure according to this aspect includes a gelatin core-sheath fiber spinning step, a gelatin fiber forming step, a dipping step, a gelation step, and a microchannel forming step. In addition, the method according to this aspect can further include a freeze-drying step, if desired. The process of the method according to this aspect will be described in detail below.

In each aspect of the invention, the "capillary network-like" shape means a shape similar to the capillary network present in animal tissues or organs, eg, a three-dimensional network or cotton candy-like shape. .. The capillaries that make up the capillary network have a diameter, for example, in the range of 2 to 25 μm, usually in the range of 5 to 20 μm, typically in the range of 5 to 15 μm, particularly about 10 μm. The diameter means the average value of the inner diameters of capillaries. Also, in the capillary network, the distance between adjacent capillaries is usually in the range of 50-200 μm, especially about 100 μm. The distance means the average value of the distances between adjacent capillaries in the capillary network. In the capillary network, the diameter of the capillaries and the distance between adjacent capillaries are determined by, for example, observing the capillary network with an optical microscope or a scanning electron microscope, and the diameter and distance of several places (for example, 10 to 50 places). Can be determined by measuring and calculating the average value thereof.

[I-1: Gelatin core sheath fiber spinning process]
In the method according to this embodiment, the core component containing gelatin and the sheath component containing an alkali metal salt of alginic acid are coextruded into a fiber-forming liquid containing divalent cations, and the core containing gelatin and the divalent alginic acid are coextruded. It is necessary to include a gelatin core-sheath fiber spinning step in which a gelatin core-sheath fiber having a sheath portion containing a cation salt is taken from a fiber-forming liquid and spun.

In each aspect of the invention, "gelatin" means a protein obtained by heat denaturing collagen. Collagen is an adhesive protein secreted from cells of multicellular organisms such as mammals, and may be a major component of the extracellular matrix together with glycoproteins such as fibronectin, hydronectin, osteonectin and proteoglycan. Are known. Collagen consists of many types of protein superfamily, and is classified as type I, type II, type III, ..., Etc., respectively. Each type of collagen molecule consists of three homogeneous or heterologous polypeptide chains. Collagen solutions usually have the property of gelling at temperatures in the range of 25-37 ° C. On the other hand, the gelatin solution usually has the property of gelling at a temperature of 25 ° C. or lower. The gelatin used in each aspect of the present invention is not particularly limited, and examples thereof include gelatin derived from pig or bovine.

The gelatin contained in the core component used in this step is usually preferably in the form of water or a water-miscible organic solvent such as ethanol, 2-propanol or dimethyl sulfoxide, or a solution in a mixture thereof. , It is more preferable that it is in the form of an aqueous solution. In this case, the concentration of gelatin contained in the core component is preferably in the range of 1 to 50 w / v%, more preferably in the range of 5 to 20 w / v%, and is about 10 w / v%. Is more preferable. When the concentration of gelatin contained in the core component is less than the lower limit, the diameter of the core of the resulting gelatin core-sheath fiber may be smaller than the range specified in the present specification, or the core may not be formed. There is sex. Further, when the concentration of gelatin contained in the core component exceeds the upper limit value, the diameter of the core portion of the resulting gelatin core-sheath fiber may be larger than the range specified in the present specification. Therefore, by using the core component containing gelatin under the above conditions, gelatin core-sheath fibers having desired characteristics can be obtained.

The alkali metal salt of alginic acid contained in the sheath component used in this step is preferably one or more compounds selected from the group consisting of sodium alginate, potassium alginate and lithium alginate, and is preferably sodium alginate. More preferred. By using the compound as an alkali metal salt of alginic acid contained in the sheath component, a sheath portion containing a divalent cation salt of alginic acid can be formed.

The alkali metal salt of alginic acid contained in the sheath component used in this step is usually in the form of water or a water-miscible organic solvent such as ethanol, 2-propanol or dimethyl sulfoxide, or a solution in a mixture thereof. It is preferably present, and more preferably in the form of an aqueous solution. In this case, the concentration of the alkali metal salt of alginic acid contained in the sheath component is preferably in the range of 0.1 to 10 w / v%, more preferably in the range of 0.5 to 3 w / v%, and is about 1 It is more preferably w / v%. By co-extruding the sheath component containing an alkali metal salt of alginic acid into a fiber-forming liquid containing divalent cations, two carboxyl groups of alginic acid are ion-crosslinked via one divalent cation. By forming a divalent cation salt of alginic acid having this ionic crosslink, the sheath component gels to form a sheath. When the concentration of the alkali metal salt of alginic acid contained in the sheath component is less than the above lower limit, ionic cross-linking is not sufficiently formed, and the diameter of the resulting gelatin core sheath fiber is smaller than the range specified in the present specification. Or the sheath may not be formed. Further, when the concentration of the alkali metal salt of alginic acid contained in the sheath component exceeds the upper limit value, the diameter of the resulting gelatin core sheath fiber may be larger than the range specified in the present specification. Therefore, by using a sheath component containing an alkali metal salt of alginic acid under the above conditions, a gelatin core sheath fiber having desired characteristics can be obtained.

The core component and sheath component used in this step preferably do not contain the divalent cations disclosed as the divalent cations contained in the fiber forming liquid below. Thereby, gelation of the sheath component can be substantially prevented before the core component and the sheath component are co-extruded into the fiber forming liquid.

In this step, the divalent cation contained in the fiber forming liquid that coextrudes the core component and the sheath component is one or more cations selected from the group consisting of calcium ion, magnesium ion, barium ion and strontium ion. Is preferable, and calcium ion is more preferable. The divalent cation contained in the fiber-forming liquid is preferably in the form of a salt with a halide ion, and is in the form of one or more compounds selected from the group consisting of calcium chloride, strontium chloride and barium chloride. More preferably, and even more preferably in the form of calcium chloride. By using the fiber-forming liquid containing the divalent cation, a sheath portion containing a divalent cation salt of alginic acid can be formed.

The divalent cation or the compound containing the divalent cation contained in the fiber forming solution used in this step is usually water, or a water-miscible organic solvent such as ethanol, 2-propanol or dimethyl sulfoxide, or It is preferably in the form of a solution in a mixture thereof, more preferably in the form of an aqueous solution. In this case, the concentration of the divalent cation or the compound containing the divalent cation contained in the fiber forming liquid is preferably in the range of 10 to 6000 mM, more preferably in the range of 50 to 1000 mM. , More preferably about 100 mM. When the concentration of the divalent cation contained in the fiber-forming liquid or the compound containing the divalent cation is less than the lower limit, the ion cross-linking of alginic acid contained in the sheath component is not sufficiently formed, and the resulting gelatin is obtained. The diameter of the core-sheath fiber may be smaller than the range specified herein, or the sheath may not be formed. Therefore, a gelatin core-sheath fiber having desired characteristics can be obtained by using a fiber-forming solution containing a divalent cation or a compound containing the divalent cation under the above conditions.

The temperature of the fiber-forming liquid that coextrudes the core component and the sheath component needs to be 25 ° C. or lower, preferably in the range of 1 to 25 ° C., and preferably in the range of 1 to 10 ° C. More preferably, it is further preferably about 4 ° C. When the temperature of the fiber forming liquid is not more than the above upper limit value, gelatin contained in the core component may gel. Therefore, a gelatin core-sheath fiber having a core portion containing gelatin and a sheath portion containing a divalent cation salt of alginic acid by co-extruding the core component and the sheath component into a fiber forming liquid having a temperature in the above range. Can be formed.

［式中、
Dは、ゼラチン芯鞘繊維の芯部の直径（cm）であり、
Qは、芯成分及び鞘成分の総押出流量（ml/分）であり、
Rは、芯成分及び鞘成分の総押出流量に対する芯成分の押出流量の比であり、
vは、ゼラチン芯鞘繊維の引取速度（cm/分）であり、
πは、円周率である。］
で表される。それ故、本工程を、前記数式で表される関係を満たす条件で実施することにより、結果として得られるゼラチン芯鞘繊維の直径を本明細書で規定される範囲とすることができる。 In this step, the total extrusion flow rate of the core component and the sheath component, the ratio of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component, and the take-up speed at which the gelatin core-sheath fiber is taken from the fiber forming liquid and spun It was found that there is a certain correlation with the diameter of the core of the resulting gelatin core-sheath fiber. This correlation is based on the following formula:

[During the ceremony,
D is the diameter (cm) of the core of the gelatin core sheath fiber.
Q is the total extrusion flow rate (ml / min) of the core component and sheath component.
R is the ratio of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component.
v is the take-up rate (cm / min) of gelatin core sheath fiber,
π is the pi. ]
It is represented by. Therefore, by carrying out this step under the conditions that satisfy the relationship represented by the above formula, the diameter of the resulting gelatin core-sheath fiber can be within the range specified in the present specification.

In this step, the total extrusion flow rate Q of the core component and the sheath component is preferably in the range of 0.5 to 6 ml / min. When the total extrusion flow rate Q of the core component and the sheath component exceeds the above upper limit value, the diameter of the core portion of the resulting gelatin core sheath fiber may exceed the upper limit value specified in the present specification. Further, when the total extrusion flow rate Q of the core component and the sheath component is less than the above lower limit value, the diameter of the core portion of the resulting gelatin core sheath fiber may be less than the lower limit value specified in the present specification. .. Therefore, by carrying out this step under the conditions that the total extrusion flow rate Q of the core component and the sheath component is within the above range and the relationship expressed by the above formula is satisfied, the core portion of the gelatin core sheath fiber obtained as a result The diameter can be in the range specified herein.

In this step, the ratio R of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component is preferably in the range of 0.0000293 to 0.00283. When the flow rate ratio R of the core component and the sheath component exceeds the upper limit value, the diameter of the core portion of the resulting gelatin core sheath fiber may exceed the upper limit value specified in the present specification. Further, when the flow rate ratio R of the core component and the sheath component is less than the lower limit value, the diameter of the core portion of the resulting gelatin core sheath fiber may be less than the lower limit value specified in the present specification. Therefore, the diameter of the core of the gelatin core-sheath fiber obtained as a result by carrying out this step under the conditions that the flow rate ratio R of the core component and the sheath component is within the above range and satisfies the relationship expressed by the above mathematical formula. Can be in the range specified in this specification.

In this step, the total extrusion flow rate Q of the core component and the sheath component and the ratio R of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component are determined by an extrusion means such as a syringe for extruding the core component and the sheath component. , It can be adjusted by operating at a predetermined flow rate using a means such as a syringe pump.

In this step, the take-up speed v when the gelatin core-sheath fiber is taken up from the fiber-forming liquid is preferably in the range of 300 to 900 cm / min. If the take-up rate v of the gelatin core-sheath fiber is less than the lower limit, the diameter of the core of the resulting gelatin core-sheath fiber may exceed the upper limit specified in the present specification. Further, when the take-up speed v of the gelatin core-sheath fiber exceeds the upper limit value, the diameter of the core portion of the resulting gelatin core-sheath fiber may be less than the lower limit value specified in the present specification. Therefore, by carrying out this step under the condition that the take-up speed v of the gelatin core-sheath fiber is within the above range and the relationship expressed by the above mathematical formula is satisfied, the diameter of the core portion of the gelatin core-sheath fiber obtained as a result can be obtained. It can be in the range specified in this specification.

In this step, the take-up speed v can be adjusted by operating a take-up means such as a take-up shaft for taking up the gelatin core-sheath fiber from the fiber-forming liquid at a predetermined speed.

The diameter of the gelatin core-sheath fiber obtained by this step, that is, the outer diameter of the gelatin core-sheath fiber, is usually in the range of 150 to 650 μm, typically in the range of 200 to 500 μm, and particularly about 450 μm. Is. The diameter D of the core of the gelatin core-sheath fiber obtained by this step, that is, the outer diameter of the core of the gelatin core-sheath fiber is, for example, in the range of 2 to 25 μm, and usually in the range of 5 to 20 μm. Yes, typically in the range of 5 to 15 μm, especially about 10 μm. Since the gelatin core-sheath fiber obtained by this step, particularly the core portion, has a minute diameter in the above range, the core portion of the gelatin core-sheath fiber can be used as a template for a capillary network-like microchannel. it can.

Preferably,
The diameter D of the core of the gelatin core sheath fiber is in the range of 2 to 25 μm.
The total extrusion flow rate Q of the core component and the sheath component is in the range of 0.5 to 6 ml / min.
The ratio R of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component is in the range of 0.0000293 to 0.00283, and the take-up speed v of the gelatin core and sheath fiber is in the range of 300 to 900 cm / min. By carrying out this step under the above conditions, the diameter of the resulting gelatin core-sheath fiber can be within the range specified in the present specification.

The diameter of the gelatin core-sheath fiber and the diameter of the core portion obtained by this step can be determined by observing the gelatin core-sheath fiber with an optical microscope or a scanning electron microscope, for example, in several places (for example, 10 to 50 places). It can be determined by measuring the diameter and the diameter of the core and calculating the average value thereof.

A schematic diagram of an embodiment of this step is shown in FIG. As shown in FIG. 2A, the core component containing gelatin and the sheath component containing an alkali metal salt of alginic acid are coextruded into a fiber-forming liquid containing divalent cations at a temperature of 25 ° C. or lower. Alginic acid contained in the sheath component extruded into the fiber-forming liquid immediately forms an ionic crosslink via divalent cations contained in the fiber-forming liquid. As a result, the sheath component extruded into the fiber-forming liquid can immediately gel from its surface to form a sheath portion of the gelatin core-sheath fiber. Gelation of the sheath surface usually proceeds within 0.1 seconds of extrusion into the fiber-forming solution. On the other hand, the gelation of the core portion usually progresses gradually several seconds after being extruded into the fiber forming liquid. That is, at the time when the gelation of the surface of the sheath portion progresses, the ion cross-linking of alginic acid is not sufficiently formed inside the sheath portion, and the gelation of the core component does not sufficiently proceed. Here, the diameter of the gelatin core-sheath fiber, that is, the diameter of the sheath can be reduced by taking over the gelatin core-sheath fiber whose surface is gelling. This makes it possible to reduce the diameter of the flow path of the non-gelled sheath component and core component. After that, ionic cross-linking of alginic acid is formed inside the sheath to form the sheath, and gelatin contained in the core component is gradually also formed in the fiber forming liquid having a temperature below the gelation temperature (25 ° C or lower). Can gel to form a core. Since the formation of gelatin core-sheath fibers proceeds by such a mechanism, even when the core component and the sheath component are co-extruded into the fiber-forming liquid from a relatively large-diameter discharge port, it is expressed by the above formula. Adjust the total extrusion flow rate Q of the core component and the sheath component, the ratio R of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component, and the take-up rate v of the gelatin core-sheath fiber so as to satisfy the conditions. By doing so, the diameter of the sheath portion and the diameter of the flow path of the non-gelled sheath component and the core component are reduced, and the diameter of the gelatin core sheath fiber obtained as a result and the diameter of the core portion are defined in the present specification. It can be in the range to be.

FIG. 2B shows a cross-sectional view of an embodiment of the apparatus used in this step. As shown in FIG. 2B, the apparatus used in this step usually includes a means 21 for coextruding a core component and a sheath component, and a fiber forming liquid tank 22 for containing a fiber forming liquid containing divalent cations. , The taking-up means 23 for taking up the gelatin core-sheath fiber from the fiber-forming liquid tank 22 is provided. The means 21 for co-extruding the core component and the sheath component is not particularly limited as long as it is a means capable of forming core-sheath fibers having a core-sheath structure. For example, coextrusion is performed by ejecting a core component from a central discharge port 24 of a die having two discharge ports 24 and 25 arranged concentrically, and a sheath component from an outer peripheral discharge port 25, respectively. It is preferable to carry out. In the case of the above embodiment, the means 21 for co-extruding the core component and the sheath component includes the core component extrusion means 26 for extruding the core component to the central discharge port 24 and the sheath for extruding the sheath component to the outer peripheral discharge port 25. It is preferable to further include the component extrusion means 27. By using a die having two discharge ports arranged concentrically, the core component can be discharged from the central discharge port 24 and the sheath component can be discharged from the outer peripheral discharge port 25, respectively. In this case, the diameter (that is, the inner diameter) of the central discharge port 24 is preferably in the range of 200 to 600 μm, more preferably in the range of 300 to 500 μm, and preferably in the range of 450 to 500 μm. Is even more preferable. The diameter (that is, the inner diameter) of the outer peripheral discharge port 25 is preferably in the range of 200 to 1000 μm, and more preferably in the range of 250 to 950 μm. The means 21 for co-extruding the core component and the sheath component is not particularly limited in shape and size of other parts as long as the discharge port has the above-mentioned characteristics. For example, the shape of the central discharge port 24 and the outer peripheral discharge port 25 can be cylindrical or truncated cone, or a combination thereof. As described above, since the central discharge port 24 and the outer peripheral discharge port 25 have relatively large diameters, the means 21 for co-extruding the core component and the sheath component is the central discharge port 24 and the outer peripheral discharge port. 25, and as the core component extrusion means 26 and the sheath component extrusion means 27, for example, syringe needles and syringes commonly used in the art can be used. As described above, in the method according to this embodiment, the core component and the sheath component are put into the fiber forming liquid from the discharge port having a large diameter as compared with the diameter of the resulting gelatin core-sheath fiber and the diameter of the core portion. Even in the case of co-extrusion, the diameter of the gelatin core-sheath fiber and the diameter of the core portion can be within the ranges specified in the present specification. Therefore, the method according to this aspect does not use as a means for coextruding a microchannel structure having a special and complicated shape produced by using a microfabrication technique, and a simple apparatus having the above-mentioned characteristics is used. By carrying out this step using this, gelatin core-sheath fibers having desired characteristics can be easily obtained.

[I-2: Gelatin fiber forming process]
The method according to this aspect is a gelatin fiber forming step in which the gelatin core sheath fiber obtained in the gelatin core sheath fiber spinning step is brought into contact with an alginic acid removing agent to dissolve the sheath portion containing calcium alginate to obtain gelatin fiber. It is necessary to include.

The alginate remover used in this step is usually a chelating agent or an alginate hydrolase. The chelating agent preferably contains one or more ligands selected from the group consisting of citric acid (eg, trisodium citrate), ethylenediaminetetraacetic acid (EDTA) and glycol etherdiaminetetraacetic acid (EGTA). More preferably, it contains an acid. The alginate hydrolase is preferably alginate lyase or the like. The alginic acid remover is preferably in the form of a solution containing the ligand or enzyme in water, a water-miscible organic solvent such as ethanol, 2-propanol or dimethyl sulfoxide, or a mixture thereof. More preferably, it is in the form of an aqueous solution containing a ligand or an enzyme in water. In this case, the concentration of the ligand contained in the chelating agent is preferably in the range of 10 to 1000 mM, more preferably in the range of 50 to 800 mM. The pH of the alginic acid remover is preferably in the range of 3-10, more preferably in the range of 5-8. When the gelatin core sheath fiber and the chelating agent containing the ligand exemplified above are brought into contact with each other, the ligand and the divalent cation contained in the sheath portion of the gelatin core sheath fiber form a chelate complex. As a result, the ionic cross-linking of the divalent cation salt of alginic acid contained in the sheath is cleaved, and the sheath is dissolved. If the concentration of the ligand contained in the chelating agent is less than the above lower limit value, it may not be possible to form a chelate complex with a sufficient amount of divalent cations contained in the sheath portion of the gelatin core sheath fiber. When the pH of the alginic acid remover is less than the lower limit value or exceeds the upper limit value, the divalent cation contained in the sheath portion of the gelatin core sheath fiber and the ligand contained in the chelating agent cannot form a chelate complex. Alternatively, the rate of enzymatic reaction by alginate hydrolase may decrease. In these cases, the sheath portion of the gelatin core sheath fiber may not be sufficiently dissolved. Therefore, by using the alginic acid removing agent having the above-mentioned characteristics, the sheath portion of the gelatin core sheath fiber can be sufficiently dissolved to obtain the gelatin fiber.

In this step, under the condition that the gelatin core sheath fiber and the alginic acid removing agent are brought into contact with each other, the ligand contained in the chelating agent and the divalent cation can form a chelate complex, or the enzymatic reaction by the alginate hydrolase proceeds. There is no particular limitation as long as it is within the possible range. For example, it may be carried out by immersing the gelatin core sheath fiber in the alginic acid removing agent in the form of a solution, or by dropping the alginic acid removing agent in the form of a solution onto the gelatin core sheath fiber. The temperature at which the gelatin core-sheath fiber and the alginic acid removing agent are brought into contact is preferably 25 ° C. or lower, more preferably 1 to 20 ° C., and even more preferably 2 to 8 ° C. The time for contacting the gelatin core sheath fiber with the alginic acid removing agent is preferably in the range of 0.1 to 72 hours, more preferably in the range of 12 to 48 hours. When the temperature at which the gelatin core-sheath fiber and the alginic acid remover are brought into contact with each other exceeds the upper limit value, the gelatin contained in the core portion of the gelatin core-sheath fiber may be dissolved. Therefore, by contacting the gelatin core sheath fiber with the alginic acid removing agent under the above conditions, the sheath portion of the gelatin core sheath fiber can be dissolved to obtain the gelatin fiber.

The gelatin core sheath fibers may be washed with a cleaning solvent before, after, or before and after contact with the alginic acid remover. In this case, the cleaning solvent used is preferably water, a water-miscible organic solvent such as ethanol, 2-propanol or dimethyl sulfoxide, or a mixture thereof, and more preferably water. The temperature at which the gelatin core-sheath fibers and / or the resulting gelatin fibers are washed with a cleaning solvent is preferably the same as the temperature at which the gelatin core-sheath fibers and the alginic acid remover are brought into contact with each other. By washing the gelatin core-sheath fibers in this step, the purity of the resulting gelatin fibers can be improved.

The gelatin fiber obtained by this step corresponds to the core portion of the gelatin core sheath fiber. Therefore, the diameter of the gelatin fiber obtained in this step is usually in the same range as the diameter of the core portion of the gelatin core-sheath fiber obtained in the gelatin core-sheath fiber spinning step. The diameter of gelatin fibers is, for example, in the range of 2 to 25 μm, usually in the range of 5 to 20 μm, typically in the range of 5 to 15 μm, especially about 10 μm. Since the gelatin fiber obtained by this step has a minute diameter in the above range, the gelatin fiber can be used as a template for a capillary network-like microchannel.

For the diameter of the gelatin fiber obtained by this step, for example, the gelatin fiber is observed with an optical microscope or a scanning electron microscope, the diameters of several places (for example, 10 to 50 places) are measured, and the average value is calculated. Can be determined by

[I-3: Freeze-drying process]
In the method according to this embodiment, if desired, the gelatin fibers obtained in the gelatin fiber forming step are dehydrated in a low melting point organic solvent, and then the dehydrated gelatin fibers are freeze-dried in a low melting point organic solvent or a medium melting point organic solvent. The lyophilization step of obtaining a three-dimensional network gelatin fiber can be further included.

In each aspect of the present invention, "medium melting point organic solvent" means an organic solvent having a melting point near room temperature, for example, in the range of 20 to 26 ° C. Further, in each aspect of the present invention, the "low melting point organic solvent" means, for example, an organic solvent having a melting point in the range of 4 ° C. or lower, preferably −114 to -89.5 ° C. The medium melting point organic solvent used in this step is preferably tert-butyl alcohol, dimethyl sulfoxide, or a mixture thereof, and more preferably tert-butyl alcohol. The medium melting point organic solvent exemplified above may contain a small amount of water, for example, 5 v / v% or less, particularly 2 v / v% or less. The low melting point organic solvent used in this step is preferably ethanol, 2-propanol or methanol, or a mixture thereof, and more preferably ethanol. The low melting point organic solvent exemplified above may contain a small amount of water, for example, 5 v / v% or less, particularly 2 v / v% or less.

In this step, the gelatin fiber can be dehydrated by immersing the gelatin fiber obtained in the gelatin fiber forming step in the low melting point organic solvent exemplified above. The temperature at which the gelatin fiber is immersed in the low melting point organic solvent may be a temperature at which the gelatin fiber does not melt, preferably 25 ° C. or lower, more preferably 1 to 20 ° C., 2 It is more preferably in the range of ~ 8 ° C, particularly preferably about 4 ° C. Water contained in the gelatin fibers can be removed by immersing the gelatin fibers in the low melting point organic solvent exemplified above. This makes it possible to obtain gelatin fibers having a desired shape by substantially avoiding the undesired effects of water in the subsequent freeze-drying, for example, the effects of the surface tension of the remaining water.

In each aspect of the present invention, "lyophilization" involves freezing a mixture containing a target substance and a solvent, reducing the pressure of the frozen product to below the vapor pressure of the solvent to sublimate and remove the solvent in the frozen product. It means a process for obtaining a dried target substance. For example, when dehydrated gelatin fibers are freeze-dried in a low melting point organic solvent, the temperature of the frozen mixture containing the gelatin fibers and the low melting point organic solvent may be a temperature exceeding the melting point of the low melting point organic solvent, and is -114 ° C. The above is preferable, and the range of −114 to -89.5 ° C. is more preferable. When the dehydrated gelatin fibers are freeze-dried in a medium melting point organic solvent, the temperature of the frozen mixture containing the gelatin fibers and the medium melting point organic solvent may be a temperature exceeding the melting point of the medium melting point organic solvent, and is 20 to 30 ° C. It is preferably in the range. For example, when gelatin fibers are immersed in a low melting point organic solvent such as water or ethanol and the resulting mixture is frozen and then vacuum dried, when carried out at a temperature exceeding the upper limit, for example, room temperature, water having a low melting point or Low melting point organic solvents such as ethanol usually melt into a liquid. In such a case, the surface tension of the liquid contained inside the gelatin fiber may crush the gelatin fiber, and the gelatin fiber having a desired shape may not be obtained. On the other hand, the medium melting point organic solvent exemplified above has a relatively high melting point. For example, the melting point of tert-butyl alcohol is 25.7 ° C. When a mixture containing gelatin fibers in such a medium melting point organic solvent is frozen and then vacuum dried, the medium melting point organic solvent can be melted into a liquid even if it is carried out at a temperature exceeding the lower limit, for example, room temperature. Low sex. Therefore, by using the low melting point organic solvent or the medium melting point organic solvent exemplified above at a temperature in the above range, the influence of the surface tension of the liquid generated by melting is substantially avoided, and the desired shape is obtained. Gelatin fiber having the above can be obtained.

In this step, the conditions for immersing the gelatin fibers in the low melting point organic solvent or the medium melting point organic solvent, the conditions for freezing the mixture containing the gelatin fibers in the low melting point organic solvent or the medium melting point organic solvent, and the conditions for freezing the mixture are , Can be appropriately set based on the low melting point organic solvent or the medium melting point organic solvent used. For example, the temperature at which the gelatin fiber is immersed in the low melting point organic solvent or the medium melting point organic solvent may be a temperature exceeding the melting point of the low melting point organic solvent or the medium melting point organic solvent, and is preferably 26 ° C. or higher. The range of 50 ° C. is more preferable, and the range of 26 to 40 ° C. is further preferable. The time for immersing the gelatin fiber in the low melting point organic solvent or the medium melting point organic solvent is preferably in the range of 0.1 to 48 hours, more preferably in the range of 0.1 to 24 hours. The temperature at which the gelatin fiber is frozen in the low melting point organic solvent or the medium melting point organic solvent may be a temperature equal to or lower than the melting point of the low melting point organic solvent or the medium melting point organic solvent. For example, in the case of the medium melting point organic solvent. , 26 ° C. or lower, more preferably -80 to 26 ° C., and even more preferably -30 to 26 ° C. The time for freezing the mixture containing the gelatin fiber in the low melting point organic solvent or the medium melting point organic solvent is preferably in the range of 0.1 to 48 hours, more preferably in the range of 0.1 to 24 hours. Further, the pressure for freeze-drying the mixture is preferably in the range of 0.1 to 500 Pa, more preferably in the range of 0.1 to 50 Pa. The time for freeze-drying the mixture is preferably in the range of 1 to 48 hours, more preferably in the range of 1 to 24 hours.

In this step, it is preferable that the gelatin fiber immersed in the low melting point organic solvent or the medium melting point organic solvent is formed into a desired shape, and then the mixture containing the gelatin fiber is frozen. By freeze-drying the mixture containing the molded gelatin fibers, the low melting point organic solvent or the medium melting point organic solvent is sublimated and removed, and gelatin fibers having a desired shape can be obtained. The gelatin fiber preferably has a three-dimensional net-like or cotton candy-like shape. In each aspect of the present invention, "three-dimensional network" means a shape in which a network formed by fibers or microchannels is arranged three-dimensionally. Further, in each aspect of the present invention, "cotton candy-like" means a shape in which fibers or microchannels are arranged with a constant spatial spread while being separated by a predetermined distance. FIG. 5 shows a stereomicroscope and a scanning electron micrograph of gelatin fibers having a three-dimensional net-like or cotton candy-like shape obtained by this step. In the figure, A is a photograph showing the overall appearance of the gelatin fiber, and B is an enlarged photograph of the gelatin fiber. In this case, the distance between adjacent gelatin fibers is usually in the range of 50-200 μm, typically in the range of 50-150 μm, especially about 100 μm. When the gelatin fiber obtained by this step has a three-dimensional network-like or cotton candy-like shape, the gelatin fiber can be used as a template for a capillary network-like microchannel.

For the shape of the gelatin fibers obtained by this step and the distances between the fibers, for example, the gelatin fibers are observed with a stereomicroscope or a scanning electron microscope, and the distances between the fibers are measured at several places (for example, 10 to 50 places). Then, it can be determined by calculating the average value.

[I-4: Immersion process]
The method according to this aspect needs to include a dipping step of immersing the gelatin fiber obtained in the gelatin fiber forming step in a gelling agent.

The gelling agent used in this step needs to contain one or more compounds selected from the group consisting of collagen and fibrin, and preferably contains collagen, and type I collagen derived from pig or bovine. It is more preferable to include it. As mentioned above, collagen usually has the property of gelling at a temperature in the range of 25 to 37 ° C. Therefore, by using a gelling agent containing collagen, gelatin fibers can be embedded in the gel in the steps disclosed below.

In this step, the temperature at which the gelatin fiber is immersed in the gelling agent is preferably 25 ° C. or lower, more preferably 1 to 10 ° C., and 1 to 8 ° C. Is even more preferable. If the temperature at which the gelatin fiber is immersed in the gelling agent exceeds the above upper limit value, the gelatin contained in the gelatin fiber may dissolve. Therefore, by immersing the gelatin fiber in the gelling agent at a temperature in the above range, the gelatin fiber can be immersed in the gelling agent while substantially avoiding the dissolution of gelatin.

[I-5: Gelation process]
The method according to this aspect needs to include a gelling step of gelling a gelling agent in which gelatin fibers are immersed and embedding the gelatin fibers in a gel.

In this step, the conditions for gelling the gelling agent can be appropriately set by those skilled in the art according to the gelling conditions of the gelling agent used in the dipping step. For example, when collagen is used as the gelling agent, the gelling agent in which gelatin fibers are immersed is allowed to stand at a temperature in the range of 25 to 37 ° C, preferably in the range of 30 to 37 ° C. In this case, the time for standing at the temperature is preferably in the range of 10 to 60 minutes, more preferably in the range of 10 to 40 minutes. When the temperature at which the gelling agent in which the gelatin fiber is immersed exceeds the above upper limit value, the gelatin contained in the gelatin fiber may be rapidly dissolved faster than the gelling agent gels. In addition, collagen used as a gelling agent may be thermally denatured. Therefore, when collagen is used as the gelling agent, by carrying out this step under the above conditions, the gelling agent is gelled while substantially avoiding the rapid dissolution of gelatin to obtain gelatin fibers. It can be embedded in gel. Alternatively, when fibrin is used as the gelling agent, the gelling agent and the thrombin aqueous solution in the form of an aqueous solution in which gelatin fibers are immersed are placed in the range of 1 to 37 ° C, preferably in the range of 1 to 10 ° C, more preferably 1 to 1. Mix at a temperature in the range of 4 ° C and let stand. In this case, the time for allowing the mixture to stand at the temperature is preferably in the range of 10 to 60 minutes, more preferably in the range of 20 to 40 minutes. When the temperature at which the gelling agent in which the gelatin fiber is immersed exceeds the above upper limit value, the gelatin contained in the gelatin fiber may be rapidly dissolved faster than the gelling agent gels. In addition, fibrin used as a gelling agent may be thermally denatured. Therefore, when fibrin is used as the gelling agent, by carrying out this step under the above conditions, the gelling agent is gelled while substantially avoiding the rapid dissolution of gelatin to obtain gelatin fibers. It can be embedded in gel.

This step may be carried out separately from the microchannel forming step disclosed below, and may be carried out simultaneously with or continuously with the microchannel forming step, if desired. For example, when collagen is used as the gelling agent, by simultaneously performing this step and the microchannel forming step, the gelling agent gels and embeds the gelatin fiber in the gel, and at the same time, it is contained in the gelatin fiber. The dissolution of gelatin can proceed. Thereby, the gelling step and the microchannel forming step can be carried out substantially simultaneously or continuously.

[I-6: Microchannel formation process]
The method according to this aspect needs to include a microchannel forming step in which the gelatin fibers embedded in the gel are dissolved to form microchannels in the gel.

In this step, the temperature at which the gelatin fibers embedded in the gel are dissolved needs to be a temperature exceeding 25 ° C, preferably in the range of 25 to 37 ° C, and preferably in the range of 30 to 37 ° C. More preferably. The gelatin fibers embedded in the gel are preferably allowed to stand at a temperature in the above range for a certain period of time, for example, in the range of 10 to 60 minutes, preferably in the range of 10 to 40 minutes. The gelatin used in the method according to this aspect usually dissolves at a temperature above 25 ° C. Therefore, the gelatin fiber can be dissolved by setting the temperature of the gelatin fiber to exceed the lower limit value. When collagen or fibrin is used as the gelling agent, if the upper limit is exceeded, collagen or fibrin used as the gelling agent may be thermally denatured. Therefore, when collagen or fibrin is used as the gelling agent, the gelatin fibers can be dissolved while maintaining the gel substantially stable by carrying out this step under the above conditions.

In the method according to this aspect, by using a gelatin fiber having a three-dimensional net-like or cotton candy-like shape as a template, a microchannel structure having a capillary network-like microchannel in the gel can be obtained. .. As disclosed above, the method according to this aspect can form a microchannel in a very short time, for example, a dozen minutes to a few hours. In addition, the microchannels formed by the method according to this aspect have a three-dimensional net-like or cotton candy-like shape, and have a diameter similar to that of capillaries. Therefore, according to the method according to this aspect, a microchannel structure that can be used as a capillary network when producing an artificial organ can be produced in a very short time as compared with the prior art.

<2: Microchannel structure with capillary network-like microchannels>
Another aspect of the present invention relates to a microchannel structure having a three-dimensional network microchannel. An embodiment of the microchannel structure according to this aspect is shown in FIG. As shown in FIG. 1, the microchannel structure 1 according to this embodiment has a gel 11 and a microchannel 12 in the gel 11. The microchannel structure according to this aspect can be manufactured by the method according to one aspect of the present invention described above.

The microchannel structure according to this embodiment has a gel containing one or more gelling agents. One or more gelling agents can be appropriately selected from the gelling agents used in the method according to one aspect of the present invention described above. The shape and size of the gel are not particularly limited as long as the microchannels can be arranged inside the gel. The gel can have any shape and size in consideration of the use of the microchannel structure according to this aspect.

The microchannel structure according to this embodiment has microchannels in the gel. The specific shape of the microchannel is not particularly limited. The shape of the microchannel is, for example, a three-dimensional net-like shape or a cotton candy-like shape. The diameter of the microchannels is, for example, in the range of 2 to 25 μm, usually in the range of 5 to 20 μm, typically in the range of 5 to 15 μm, especially about 10 μm. Also, the distance between adjacent microchannels is usually in the range of 50-200 μm, typically in the range of 50-150 μm, especially about 100 μm. The shape and dimensions are similar to those of the capillary network. Therefore, the microchannel structure obtained by the method according to this embodiment can be used as a capillary network when producing an artificial organ.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the technical scope of the present invention is not limited to these examples.

<I: Manufacture of microchannel structure>
[I-1: Spinning of gelatin core sheath fiber]
Using gelatin (Gelatin from porcine skin (Type A), Sigma-Aldrich), a 10 w / v% gelatin aqueous solution was prepared as a core component. In addition, a 1 w / v% sodium alginate aqueous solution was prepared as a sheath component. Prepare a double cylindrical tube-shaped die (center discharge port diameter: 0.48 mm, outer circumference discharge port diameter: 0.94 mm) having two discharge ports arranged concentrically, and connect to the center discharge port. Syringes were connected to the inner cylinder and the outer cylinder connected to the outer peripheral discharge port, respectively. The central discharge port and the outer peripheral discharge port of the die were manufactured using a commercially available syringe needle (Terumo Syringe, Terumo Corporation) (Fig. 2B). The core component (80 ° C.) was injected into the syringe connected to the inner cylinder, and the sheath component (20 ° C.) was injected into the syringe connected to the outer cylinder. Using a syringe pump, the core component maintained at 80 ° C from the central discharge port and the sheath component maintained at 20 ° C from the outer peripheral discharge port are discharged at a predetermined extrusion flow rate (core component and sheath component). Total extrusion flow rate: 3 ml / min), coextruded into 100 mM calcium chloride aqueous solution at about 4 ° C. The two carboxyl groups of alginic acid can be ionic crosslinked via a single calcium ion. Therefore, alginic acid contained in the sheath component is co-extruded into an aqueous calcium chloride solution to gel. In addition, gelatin contained in the core component is co-extruded into a low-temperature calcium chloride aqueous solution to gel. Therefore, the core component and the sheath component co-extruded into the calcium chloride aqueous solution formed gelatin core-sheath fibers. The formed gelatin core-sheath fiber was taken up and spun from the calcium chloride aqueous solution while being taken up on a take-up shaft (diameter: 7.5 cm) arranged at a position of 30 cm from the discharge port at a take-up speed of 900 cm / min ( Figure 2A). FIG. 3 shows the relationship between the ratio of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component in coextrusion (extrusion flow rate ratio) and the diameter of the core portion of the resulting gelatin core-sheath fiber. In the figure, the black-painted square indicates the diameter of the gelatin core-sheath fiber, and the black-painted circle indicates the diameter of the core of the gelatin core-sheath fiber. In addition, the obtained gelatin core-sheath fiber was observed using a phase-contrast microscope (DM IL LED, Leica). A photograph of the obtained gelatin core-sheath fiber is shown in FIG. In the figure, A is a gelatin core-sheath fiber obtained when the extrusion flow rate ratio is 0.167, and the dimension line represents the diameter of the core portion of the gelatin core-sheath fiber. B is the gelatin core-sheath fiber obtained when the extrusion flow rate ratio is 0.000833, and the arrow represents the gelatin core-sheath fiber.

As shown in FIG. 3, the larger the extrusion flow rate ratio, the larger the diameter of the core portion of the gelatin core-sheath fiber. When the extrusion flow rate ratio was 0.000833 or less, especially in the range of 0.0000587 to 0.000833, the diameter of the core of the gelatin core sheath fiber was 25 μm or less (Fig. 4B). On the other hand, when the extrusion flow rate ratio exceeded 0.05, the diameter of the core of the gelatin core-sheath fiber became 100 μm or more (Fig. 4A).

In the above procedure, the gelatin core-sheath fiber spinning step was carried out in the same manner as described above, except that the total extrusion flow rate, the extrusion flow rate ratio and the take-up speed of the core component and the sheath component were changed. Table 1 shows the conditions of the gelatin core-sheath fiber spinning process and the diameter of the core of the obtained gelatin core-sheath fiber.

［式中、
Dは、ゼラチン芯鞘繊維の芯部の直径（cm）であり、
Qは、芯成分及び鞘成分の総押出流量（ml/分）であり、
Rは、押出流量比であり、
vは、ゼラチン芯鞘繊維の引取速度（cm/分）であり、
πは、円周率である。］
で表される。 As shown in Table 1, between the total extrusion flow rate, extrusion flow rate ratio and take-up rate of the core component and the sheath component in the gelatin core-sheath fiber spinning process and the diameter of the core portion of the resulting gelatin core-sheath fiber. It became clear that there was a certain correlation. This correlation is based on the following formula:

[During the ceremony,
D is the diameter (cm) of the core of the gelatin core sheath fiber.
Q is the total extrusion flow rate (ml / min) of the core component and sheath component.
R is the extrusion flow rate ratio
v is the take-up rate (cm / min) of gelatin core sheath fiber,
π is the pi. ]
It is represented by.

As a comparative example, gelatin fibers were spun based on the method described in Patent Document 1. An aqueous solution containing 1 w / v% sodium alginate and 10 w / v% gelatin (gelled protein) was prepared as a fiber component. Using a commercially available syringe needle (Terumo Syringe, Terumo Corporation), prepare a die with one discharge port (discharge port diameter: 0.48 mm), and apply the fiber component (80 ° C) to the syringe connected to the discharge port. Infused. Using a syringe pump, the fiber component maintained at 80 ° C. was extruded from the discharge port into a 100 mM calcium chloride aqueous solution at about 4 ° C. while being discharged at a predetermined extrusion flow rate. The fiber components extruded into the calcium chloride aqueous solution formed gelatin fibers. The formed gelatin fibers were taken up and spun from an aqueous calcium chloride solution while being taken up on a take-up shaft (diameter: 7.5 cm) at a predetermined take-up speed. The diameter of the obtained gelatin fiber was 200 μm or more. The extrusion flow rate and the take-up speed were adjusted in order to reduce the diameter of the gelatin fiber, but the fiber broke during the take-up and the fiber could not be spun.

[I-2: Formation of gelatin fiber]
The gelatin core-sheath fibers of Example 1 (2) obtained in I-1 (core diameter: 11 μm, length: 2 × 10 ³ m) were washed with distilled water at 4 ° C. The gelatin core-sheath fiber was then immersed in 5000 mL of 100 mM citric acid aqueous solution (pH 7.4) at 4 ° C. for 48 hours. Citric acid and calcium ions contained in the sheath portion of the gelatin core sheath fiber form a chelate complex. Therefore, by immersing the gelatin core sheath fiber in the citric acid aqueous solution, the ion cross-linking of calcium alginate contained in the sheath portion is cut, and the sheath portion is dissolved. Therefore, the sheath portion of the gelatin core sheath fiber was dissolved to form the gelatin fiber. Gelatin fibers were removed from the aqueous citric acid solution and washed with distilled water at 4 ° C. Gelatin fibers were dehydrated by immersing them in 99 v / v% ethanol at 4 ° C. for 24 hours. The gelatin fibers were then immersed in tert-butyl alcohol at 30 ° C. The tert-butyl alcohol mixture containing gelatin fibers was cooled to 4 ° C and frozen. The mixture was lyophilized to give gelatin fibers (0.06 g). The obtained gelatin fibers were observed using a stereomicroscope (SMZ1500, Nikon Corporation) and a scanning electron microscope (SEM) (S-3000N, Hitachi High-Technologies Corporation). A photograph of the obtained gelatin fiber is shown in FIG. In the figure, A is a photograph showing the overall appearance of the gelatin fiber, and B is an enlarged photograph of the gelatin fiber.

As shown in FIG. 5A, the obtained gelatin fibers had a three-dimensional net-like or cotton candy-like shape. As a result of measurement based on the SEM photograph shown in FIG. 5B, the average value of the diameters of gelatin fibers was 10 ± 2 μm (n = 50). In gelatin fibers having a three-dimensional net-like or cotton candy-like shape, the average value of the distances between adjacent gelatin fibers was 88 ± 68 μm (n = 50).

[I-3: Fabrication of microchannel structure with linear microchannels]
FIG. 6 shows an outline of the production of a microchannel structure having linear microchannels. In I-1 and I-2, fluorescently labeled polystyrene fine particles (Fluoresbrite Carboxylate Microspheres, Catalog No .: 15702, Polysciences) having a diameter of 1 μm were added to the gelatin aqueous solution used as the core component at a concentration of 0.1 w / v%. Gelatin fibers (diameter: 10 μm, length: 5 × 10 ² m) containing fluorescently labeled polystyrene fine particles were obtained under the same procedure and conditions as in Example 1 except for the addition. This gelatin fiber was cut to obtain a 3 cm piece of fiber. 0.3% Cellmatrix® Type IA Aqueous Collagen (Nitta Gelatin), 10x Eagle Minimum Essential Medium and Reconstitution Buffer (47.7 g / L HEPES, 1 M Sodium Hydroxide and 22 g / L Carbonate An aqueous solution containing sodium hydrogen hydrogen) was mixed in an ice water at about 4 ° C. at a ratio of 8: 1: 1 (volume ratio) to obtain a collagen mixed solution (pH 7.4). Gelatin fiber pieces containing fluorescently labeled polystyrene fine particles were allowed to stand in a circular plastic petri dish (10 cm). A 2 mL collagen mixture at 4 ° C was placed in this petri dish, and gelatin fiber pieces were immersed in collagen at 4 ° C.

A circular petri dish containing gelatin fiber pieces soaked in collagen was allowed to stand at 20 ° C. for 20 minutes. Collagen gelled, and gelatin fiber pieces containing fluorescently labeled polystyrene fine particles were embedded in the collagen gel. Next, a circular petri dish containing gelatin fiber pieces containing fluorescently labeled polystyrene fine particles embedded in collagen gel was placed in an incubator at 37 ° C. and allowed to stand at 37 ° C. for 20 minutes. The dissolution of gelatin contained in the gelatin fiber pieces proceeded. As a result, microchannels using gelatin fiber pieces as a template were formed in the collagen gel, and a collagen gel and a microchannel structure having linear microchannels in the gel were obtained. The obtained microchannel structure was observed using a fluorescence microscope (ECLIPSE Ti, Nikon Corporation). A fluorescence micrograph of the microchannel structure is shown in FIG. In addition, using a fluorescence microscope, the diameters of the gelatin fiber pieces containing the fluorescent particles and the obtained microchannels were measured at several points, and the average value was calculated. The results are shown in Table 2.

As shown in FIG. 7, microchannels through which fluorescently labeled polystyrene fine particles flow were formed in the collagen gel. The diameter of the formed microchannel was substantially the same as the diameter of the gelatin fiber piece used as the template.

[I-4: Preparation of microchannel structure having capillary network-like microchannels]
FIG. 8 shows an outline of the production of a microchannel structure having a capillary network-like microchannel. As shown in Figure 8, on the side of a clear plastic container (opening: 3 cm x 0.5 cm, depth: 2 cm), two stainless needles (terumo syringe,) with an outer diameter of 0.7 mm and a length of 70 mm. Terumo) was penetrated. The two stainless needles were placed parallel to each other and to the bottom of the plastic container and at regular intervals (1.3 cm) in the depth direction. The three-dimensional net-like or cotton candy-like gelatin fibers (diameter: 10 μm, length: 2 × 10 ³ m, mass: 0.06 g) of Example 1 obtained according to the procedures of I-1 and I-2 were used. It was placed inside the plastic container so that it touched the two needles. A 4 ° C collagen mixture (pH 7.4) prepared in the same procedure as I-3 was placed in a plastic container, and gelatin fibers were immersed in collagen at 4 ° C. A plastic container containing gelatin fibers soaked in collagen was allowed to stand at 20 ° C. for 20 minutes. Collagen gelled and gelatin fibers were embedded in the collagen gel. Then, a plastic container containing gelatin fibers embedded in collagen gel was placed in an incubator at 37 ° C. and allowed to stand at 37 ° C. for 20 minutes. The dissolution of gelatin contained in the gelatin fiber proceeded. As a result, microchannels using gelatin fibers as a template were formed in the collagen gel, and a collagen gel and a microchannel structure having a three-dimensional network-like microchannel in the gel were obtained.

The two needles were gently pulled out of the plastic container. Heparinized rat blood was injected using a syringe from one of the through holes in the plastic container from which the needle was pulled out. FIG. 9 shows a photograph showing the appearance of a microchannel structure having a capillary network-like microchannel in which rat blood was injected. In the figure, A is a photograph 5 seconds after the start of injection, and B is a photograph 10 seconds after the start of injection. As shown in FIG. 9, 10 seconds after the start of infusion, rat blood circulated over approximately the entire microchannel inside the microchannel structure.

1 ... Microchannel structure
11 ... Gel
12 ... Microchannel
21 ... Means for coextruding core component and sheath component
22… Fiber forming liquid tank
23… Means of collection
24 ... Central discharge port
25 ... Outer peripheral discharge port
26 ... Core component extrusion means
27… Sheath component extrusion means

Claims (8)

The core component containing gelatin and the sheath component containing an alkali metal salt of alginic acid are coextruded into a fiber-forming liquid containing divalent cations at a temperature of 25 ° C. or lower, and the core containing gelatin and the divalent alginic acid are coextruded. A gelatin core-sheath fiber spinning step in which a gelatin core-sheath fiber having a sheath containing a cation salt is taken from a fiber-forming liquid and spun.
The gelatin core sheath fiber is brought into contact with a chelating agent or an alginic acid removing agent which is an alginic acid hydrolyzing enzyme to dissolve the sheath containing the divalent cation salt of alginic acid to obtain a gelatin fiber having a diameter of 2 to 25 μm. , Gelatin fiber forming step;
Immersion step of immersing gelatin fibers in one or more gelling agents selected from the group consisting of collagen and fibrin;
A gelling step in which a gelling agent in which gelatin fibers are immersed is gelled and the gelatin fibers are embedded in a gel; and the gelatin fibers embedded in the gel are dissolved at a temperature exceeding 25 ° C. to form a microchannel. To form a microchannel in the gel;
Including
The gelatin core-sheath fiber spinning process is based on the following formula:

[During the ceremony,
D is the diameter (cm) of the core of the gelatin core-sheath fiber obtained by the gelatin core- sheath fiber spinning process .
Q is the total extrusion flow rate (ml / min) of the core component and sheath component.
R is the ratio of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component.
v is the take-up rate (cm / min) of gelatin core sheath fiber,
π is the pi. ]
A method for producing a microchannel structure, which comprises the gel and a three-dimensional network microchannel having a diameter in the range of 2 to 25 μm in the gel, which is carried out under the condition of satisfying the relationship represented by. In the gelatin core sheath fiber spinning process
The diameter D of the core of the gelatin core sheath fiber is in the range of 2 to 25 μm.
The total extrusion flow rate Q of the core component and the sheath component is in the range of 0.5 to 6 ml / min.
The ratio R of the extrusion flow rate of the core component to the total extrusion flow rate of the core component and the sheath component is in the range of 0.0000293 to 0.00283, and the take-up speed v of the gelatin core and sheath fiber is in the range of 300 to 900 cm / min. The method according to claim 1. The gelatin fibers obtained in the gelatin fiber forming step are dehydrated in a low melting point organic solvent which is ethanol, 2-propanol or methanol, or a mixture thereof, and then the dehydrated gelatin fibers are dried in a low melting point organic solvent or tert-. butyl alcohol or Jimechirusu Le sulfoxide, or lyophilized with the melting point organic solvent in a mixture thereof, to obtain a gelatin fibers three dimensional network, further comprising the freeze-drying process a method according to claim 1 or 2. Coextrusion is carried out by ejecting the core component from the central discharge port of the die having two concentrically arranged discharge ports and the sheath component from the outer peripheral discharge port, respectively, and the central discharge port. The method according to any one of claims 1 to 3, wherein the diameter of the outer peripheral discharge port is in the range of 200 to 600 μm, and the diameter of the outer peripheral discharge port is in the range of 200 to 1000 μm. The method according to any one of claims 1 to 4, wherein the core component used in the gelatin core-sheath fiber spinning step is a 1 to 50 w / v% gelatin aqueous solution. The method according to any one of claims 1 to 5, wherein the sheath component used in the gelatin core-sheath fiber spinning step is an aqueous solution of 0.1 to 10 w / v% sodium alginate. The method according to any one of claims 1 to 6, wherein the fiber-forming liquid containing a divalent cation used in the gelatin core-sheath fiber spinning step is an aqueous solution of 10 to 6000 mM calcium chloride. The method according to any one of claims 1 to 7, wherein the alginic acid removing agent is an aqueous solution of 10 to 1000 mM citric acid.

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