JP2019198255A - Production method of collagen tube - Google Patents

Abstract

To provide a method for producing a collagen tube whose diameter is 100 micrometer or less, whose thickness is 20 micrometer or less, and which can introduce cells into a lumen in an alive state, the method is simple and has good reproductivity.SOLUTION: There is provided a method for continuously introducing four kinds of solutions A-D, to a channel structure X comprising at least four introduction holes and at least one discharge hole. When the solutions A-D are continuously introduced to the channel structure X, in a cross section S vertical to the flow direction, the channel structure X has at least one point P where, the solution B surrounds a periphery of the solution A, the solution C contacts at least partially to a periphery of the solution B, and the solution D contacts at least partially a periphery of the solution C without contacting to a periphery of the solution B. The solution B includes 0.1% or greater of collagen dissolved therein, the solution D includes a component for gelatinizing the solution B.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a method for producing a collagen tube suitable for cell culture.

  Technology for culturing cells in a three-dimensional environment similar to that in vivo is extremely important in a wide range of fields such as regenerative medicine, cell-based drug assays, bioartificial organ development, and cell biochemistry research. It has been reported that culturing cells in a three-dimensional environment makes it possible to maintain long-term cell function, maintain survival rate, control cell differentiation, and the like.

  Hepatocytes, which are the main cells that make up the liver, play important roles such as drug metabolism, detoxification, and protein production. Therefore, the in vitro hepatocyte culture system is particularly important in the evaluation of drug efficacy and toxicity in the development of new drugs. It is also indispensable for the development of bioartificial liver that replaces the liver function of the living body or for the production of hepatocyte organoids aimed at organ regeneration.

  However, when hepatocytes are removed from the living body and cultured in a flat culture system, the function is rapidly lost. Therefore, cell culture systems that can maintain the function and survival rate of hepatocytes are being actively developed.

  In vivo, hepatocytes are regularly and linearly (string-shaped) arranged, and surrounded by sinusoidal endothelial cells via a gap containing an extracellular matrix component such as collagen called a disserum space. It is expected that the function and survival rate of hepatocytes can be maintained by imitating the microenvironment of hepatocytes in vivo and culturing in three dimensions.

  As a culture method that satisfies the characteristics of hepatocytes in vivo, that is, a culture method that arranges cells three-dimensionally and linearly, a culture method using hydrogel fibers as shown in Patent Document 1, Patent Document 2 and a culture method using a hollow fiber as shown in Patent Document 3 have been proposed.

  Furthermore, as shown in Patent Document 4, by producing a core-shell type fiber formed by a shell made of alginate hydrogel and a core made of collagen and culturing cells inside, various linear shapes can be obtained. Techniques for forming cell clumps have been reported.

  In addition, as shown in Patent Document 5 and Non-Patent Document 1 made of collagen, a method for producing a collagen tube having a hollow fiber-like structure has also been proposed.

Japanese Patent No. 5945802 Japanese Patent No. 3725147 Japanese Patent Application Laid-Open No. 2018-50498 Japanese Patent No. 5633077 Special table 2009-540896

“ACS Applied Materials & Interfaces”, 2015, 7 (35), 19789-97.

  As described above, hepatocytes are arranged in a linear / string shape in a living body and are three-dimensionally surrounded by a thin film-like gap containing an extracellular matrix component such as collagen called a disserum cavity. If a cell culture system that mimics such a microenvironment can be constructed, it will be possible to maintain the survival rate and function of hepatocytes over a long period of time. However, the technology for realizing an in vivo hepatocyte environment has been established at this stage. Not.

  Patent Document 1 discloses a technique in which hepatocytes are introduced into microfibers made of sandwich-shaped alginate hydrogel having a diameter of about 100 micrometers using a microfluidic device. Further, Patent Document 2 and Patent Document 3 show a method of filling cells in the lumen of a hollow fiber. In these methods, it is possible to arrange hepatocytes linearly and perform culture. However, these methods use alginate hydrogel and synthetic polymer materials that are not present in the liver tissue of living organisms, and it is impossible to reproduce the hepatocyte environment surrounded by a thin film of collagen. Met. Moreover, in the case of a hollow fiber having a diameter of about 100 micrometers or less, there is a problem that introduction of cells into the inside becomes difficult.

  Further, Patent Document 4 reports a method of producing a linear cell tissue using a core-shell type alginate hydrogel tube whose core is made of collagen, but it is a hollow shape made of a thin film made of collagen. It was impossible to make a structure.

  In Patent Document 5, after repeating the two-stage operation of adhering solubilized collagen to a cylindrical column and drying, the collagen is chemically crosslinked, and the column is removed by removing the column. The technique of making is shown. However, since the collagen tube obtained by this method is not intended for cell culture, it was impossible to introduce cells into the interior of the collagen tube when it was produced. In addition, there are problems such as that it takes time to produce a collagen tube and that the inner diameter cannot be reduced to about 50 micrometers or less.

  Further, in Non-Patent Document 1, a sodium alginate aqueous solution in which cells are suspended and a collagen aqueous solution are caused to flow around the center portion of the microchannel, and a sodium alginate aqueous solution portion and a collagen aqueous solution are provided at the outlet of the channel. The method of producing the collagen tube which introduce | transduced the cell into the lumen | bore by simultaneously bridge | crosslinking this part using a chemical crosslinking agent is shown. In this method, cells can be introduced into the inside, but chemical crosslinking for stabilizing the collagen is necessary, so that the cells are damaged. Furthermore, there are problems that a multi-step process is required to produce a hollow structure and that the outer diameter of the tube is difficult to control. In addition, there is a problem that it is impossible to produce a tube having a diameter of about 100 micrometers or less and a tube having a thickness of 20 micrometers or less.

  In summary, the above-described existing methods can introduce cells into the lumen without damaging the cells, have a diameter of at least partially 100 micrometers or less, and a thickness of at least partially. It was impossible to simultaneously produce a collagen tube having a size of 20 micrometers or less at the same time.

  The present invention has been made in view of the above-mentioned problems of the prior art, and the object is to have a diameter of about 100 micrometers or less and a thickness of 20 micrometers or less. Furthermore, the present invention intends to provide a novel method for easily and reproducibly producing a collagen tube capable of introducing cells alive into the lumen.

  In addition, the present invention aims to provide a cell culture technique that has not been reported so far and can arrange hepatocytes in a line and coat the hepatocytes with a thin layer of collagen.

  In order to achieve the above object, the invention according to one aspect of the present invention is such that four types of aqueous solutions A to D are continuously applied to the channel structure X having at least four inlets and at least one outlet. In the flow path structure X, when the aqueous solutions A to D are continuously introduced into the flow path structure X, the aqueous solution B crosses the outer periphery of the aqueous solution A in the cross section S perpendicular to the flow direction. The aqueous solution C is arranged so as to surround and at least partially contacts the outer periphery of the aqueous solution B, and the aqueous solution D does not contact the outer periphery of the aqueous solution B and Possesses at least one point P that is arranged to be at least partially in contact with the aqueous solution B. Further, the aqueous solution B contains 0.1% or more of collagen, and the aqueous solution D contains the aqueous solution B. Gelled That component is contained, a method for manufacturing a collagen tube. By using such a method, the collagen contained in the aqueous solution B gels while containing the aqueous solution A inside the flow path, so that a collagen tube having a liquid center part can be produced. It is possible to produce a collagen tube with a small diameter, and furthermore, a stable and continuous collagen tube can be formed by preventing direct contact between the aqueous solution B containing collagen and the aqueous solution D containing a component that gels collagen. And the diameter of the collagen tube can be controlled.

  Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is desirable for the aqueous solution A to suspend 1000 or more cells per mL. By doing so, it is possible to produce a collagen tube in which cells are introduced into the lumen.

  Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is desirable that the said cell is an adherent cell derived from a mammal. In this way, a collagen tube into which mammalian cells are introduced into the lumen, as represented by hepatocytes, can be produced.

  Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is desirable that pH of the aqueous solution D exists in the range of 7-10. By doing so, the aqueous solution B can be efficiently gelled, and damage to cells contained in the aqueous solution A can be minimized.

  Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is desirable that the aqueous solution D contains a phosphate ion in the range of 1 mM to 200 mM. By doing in this way, a collagen tube can be produced more efficiently and a collagen tube with high strength can be produced.

  Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is desirable that at least any one among the aqueous solution A, the aqueous solution C, and the aqueous solution D contains a thickener. By doing so, it becomes possible to improve the stability of the flow in the flow channel structure X, so it is possible to produce a collagen tube more stably and with good reproducibility, and also improve the operability of production. It becomes.

  Further, in the invention according to this aspect, the thickener is at least one of polyethylene glycol, dextran, carboxymethyl cellulose, polyvinyl alcohol, sodium alginate, and propylene glycol alginate. It is desirable. By doing in this way, it becomes possible to produce a collagen tube more stably.

  In the invention according to this aspect, although not limited, at least one of the values such as the width, depth, and diameter of the channel structure X is at least partially 500 micrometers or less. It is desirable. In this way, a collagen tube having a diameter of about 100 micrometers or less can be efficiently and easily produced.

  Further, in the invention according to this aspect, although not limited, the flow channel structure X may be configured at least partially by a capillary tube. By doing in this way, aqueous solution AD can be arrange | positioned inside a capillary tube, and it becomes possible to produce a collagen tube efficiently.

  Further, in the invention according to this aspect, although not limited, the flow channel structure X may be configured at least partially by a micro flow channel structure manufactured using a microfabrication technique. By doing so, it becomes possible to arrange the aqueous solutions A to D more accurately inside the flow channel structure, and it becomes possible to efficiently produce a collagen tube, and a collagen tube with a small diameter can be produced. It can be easily obtained.

  Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, in the cross section S in the point P, it is preferable that the width | variety of the aqueous solution C is at least partially 50 micrometers or less. By doing so, it becomes possible to efficiently form a collagen tube in which the thickness of the collagen portion is at least partially about 20 micrometers or less.

  Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is desirable that the diameter of the collagen tube obtained is at least partially 100 micrometers or less. By doing in this way, it becomes possible to supply oxygen and nutrients uniformly and efficiently to the cells introduced into the inside.

  Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is desirable that the film thickness of the collagen tube obtained is at least partially 10 micrometers or less. In this way, in particular, it becomes possible to provide a cell culture substrate that highly mimics the cell environment in a living body, and also from the viewpoint of efficiently supplying oxygen and nutrients to internal cells. Is also advantageous.

  Since the present invention is configured as described above, a small collagen tube having a diameter of about 100 micrometers or less and a film thickness of about 20 micrometers or less, in which cells are introduced into the lumen, can be easily and easily formed. A method for manufacturing with good reproducibility can be provided. In addition, the diameter of the collagen tube can be freely adjusted, and since the collagen tube can be produced without using a chemical cross-linking agent, damage to cells introduced into the lumen is minimized. be able to.

  In addition, since the present invention is configured as described above, in particular, by introducing hepatocytes into the interior, hepatocytes are arranged linearly or in a string shape, and the periphery thereof is three-dimensionally formed by collagen components. A cell culture environment coated with can be provided. By imitating the environment in the living body in this way, it becomes possible to maintain the function of hepatocytes, and it can be applied as a model for drug metabolism tests and a liver tissue model in vitro.

It is the schematic which showed the state of the flow of aqueous solution AD in the cross-section S of the example of the flow-path structure X for collagen tube preparation based on embodiment, and FIG. The structure X is shown in FIGS. 1B and 1C, respectively, and the channel structure X having a rectangular cross section is shown. It is the schematic which showed the state of the flow of the multilayer flow path structure X for collagen tube preparation which concerns on embodiment, and the aqueous solution AD inside it. The schematic which showed the state of the flow of the flow path structure X formed by connecting the capillary with a circular cross section for producing the collagen tube based on embodiment, and the aqueous solution AD in the cross section S in the figure is there. FIG. 4 is a schematic diagram showing a microfluidic device having a flow channel structure X, which is produced by superimposing four acrylic plates used to produce a collagen tube in Examples. FIG. 4D shows a flow path structure formed on the lower surface of the first acrylic board from the top, a flow path structure formed on the lower surface of the second acrylic board, and a third acrylic board. FIG. 4B is a schematic diagram showing the channel structure formed on the lower surface of the third-layer channel structure and the arrow B in the microfluidic device shown in FIG. FIG. FIG. 4E is an enlarged view of the region e in FIG. 4D, and is a B arrow view of the microfluidic device in FIG. 4F. FIG. 4F is a cross-sectional view of the microfluidic device taken along the line A-A ′ in FIGS. 4A to 4E. FIG. 5 is a micrograph of a collagen tube having HepG2 cells introduced into the lumen, produced using the flow channel structure X shown in FIG. 4 in Examples, and FIG. ) Is a photomicrograph after 2 days of cell culture and FIG. 5 (c) after 6 days of cell culture. FIG. 6A is a micrograph of a frozen section of a collagen tube having HepG2 cells introduced into its lumen, prepared using the channel structure X shown in FIG. FIG. 6B shows a cross section of a collagen tube prepared by adjusting the flow ratio of aqueous solution A and aqueous solution B to 1:20. It is a microscope picture shown. FIG. 7A is a micrograph of a collagen tube prepared by using the flow channel structure X shown in FIG. 4 and having HepG2 cells introduced into the lumen, and FIG. 7A shows an aqueous solution A and an aqueous solution B, respectively. FIG. 7B is a micrograph of a collagen tube prepared by adjusting the total flow rate to 5 microliters per minute, and FIG. 7B is a collagen tube prepared by adjusting the total flow rate of aqueous solution A and aqueous solution B to 25 microliters per minute. FIG.

  Hereinafter, the best mode relating to the method for producing a collagen tube according to the present invention will be described in detail. However, the present invention can be implemented in many different forms, and is not limited only to the embodiments and examples described below.

  FIG. 1 shows an example of a flow path structure X for producing a collagen tube and a schematic diagram showing a flow state of the aqueous solutions A to D in the cross section S. FIG. 1A shows a circular cross section. FIGS. 1B and 1C show the channel structure X having a rectangular cross section.

  Each of the flow channel structures X shown in FIGS. 1A to 1C has at least four inlets, and four types of aqueous solutions A to D continuously introduced from the inlets. Flow at the point P so as to merge.

  In the cross section S at the point P, the aqueous solution B completely flows around the aqueous solution A, so that the aqueous solution B part finally gels and the aqueous solution A part has a hollow structure. A tube can be obtained.

  Further, in any case, the flow channel structure X shown in FIGS. 1A to 1C flows so that the aqueous solutions B and C are in contact with each other in the cross section S, and the aqueous solutions C and D are in contact with each other. On the other hand, the aqueous solution D flows so as not to come into direct contact with the aqueous solution B. As long as these conditions can be satisfied, these aqueous solutions may be arranged in the cross section S. As shown in FIG. 1C, there are a plurality of portions of the aqueous solution C and a plurality of portions of the aqueous solution D. You may do it. Furthermore, the shape of the cross section S can be any other shape that is not circular or rectangular.

  FIG. 2 is a schematic view showing another example of the multilayered channel structure X for producing a collagen tube and the flow state of the aqueous solutions A to D therein.

  The flow path structure X shown in FIG. 2 is formed of a patterned nozzle portion present in the lower part and a flow path network present in the upper part. The aqueous solution A is passed through the central nozzle, and the aqueous solution B is passed through the surrounding nozzles. Are introduced into the upper channel structure, the aqueous solution C flows so as to sandwich the flow of the aqueous solution B in the cross section S, and the aqueous solution D flows further outside. ing.

  The flow path width in the flow path structure X as shown in FIG. 2 is about 100 to 500 micrometers. By using such a flow path structure X, the shape of the cross section, the diameter of the lumen, and the outer diameter were controlled. A collagen tube can be produced.

  FIG. 3 is a schematic diagram schematically showing a flow channel structure X formed by connecting capillaries having a circular cross section for producing a collagen tube, and the flow states of the aqueous solutions A to D in the cross section S. It is shown.

  The flow channel structure X shown in FIG. 3 has four inlets and one outlet, and is configured by connecting capillary structures. Further, the diameter of the cross section S near the outlet is 500 micrometers or less. By using such a structure, it is possible to easily achieve the state where the aqueous solutions A to D are arranged concentrically in the cross section S, and the diameter of the collagen tube can be controlled by controlling the flow rate. Can be controlled accurately.

  In the channel structure X as shown in FIGS. 1 to 3, the width of the aqueous solution C is desirably at least partially 50 micrometers or less. By doing in this way, the collagen tube in which the film thickness of the collagen layer is at least partially 20 micrometers or less can be easily formed.

  Furthermore, in the channel structure X, it is preferable that any one of the width, diameter, and depth is at least partially 500 micrometers or less. By using a channel structure of such a size, a collagen tube having a diameter of 100 micrometers or less can be easily produced.

  Further, the flow channel structure X may be configured by a micro flow channel structure manufactured using a fine processing technique. By doing in this way, since the flow path structure X in which the size and shape are arbitrarily and accurately controlled can be used, the reproducibility of the production of the collagen tube can be easily ensured.

  In addition, when the flow path structure X is manufactured using a microfabrication technique, the material of the device includes various polymer materials such as PDMS (polydimethylsiloxane) and acrylic, various metals such as glass, silicon, ceramics, and stainless steel, etc. In addition, it is also possible to use any of a plurality of types of these materials in combination. As the processing technology of the channel structure, machining is preferable in that the channel structure can be easily manufactured, but in addition, a manufacturing technology using a mold such as molding or embossing, wet etching, dry etching, Manufacturing techniques such as laser processing, direct electron beam drawing, three-dimensional modeling, and three-dimensional optical modeling can also be used.

  In order to adjust the osmotic pressure, glucose or the like can be added to the aqueous solutions A to D used. Such an operation makes it possible to minimize damage to cells introduced into the lumen.

  By introducing the cells into the aqueous solution A in advance, a collagen tube into which the cells have been introduced into the lumen can be produced in one step. In this case, in order to perform cell culture with high efficiency, the concentration of the cells suspended in the aqueous solution A is preferably 1000 or more per mL. Further, for example, when primary hepatocytes are used, When it is necessary to efficiently form an interaction, it is more preferable that the number of cells is 1 million or more per 1 mL of the aqueous solution A.

  Furthermore, the cells to be used are not limited, but it is desirable to use adherent cells derived from mammals. In particular, the use of hepatocytes may bring about innovation in drug discovery and regenerative medicine. When hepatocytes are used, primary hepatocytes derived from experimental animals, human-derived primary hepatocytes, cultured hepatocytes including hepatoma cells, stem cell-derived differentiated hepatocytes including iPS cells, and the like can be used. it can. However, as cells other than hepatocytes, it is also possible to produce collagen tubes into which various cells such as nerve cells, vascular cells, cells forming luminal structures, skeletal muscle cells, cardiomyocytes, etc. are introduced. .

  The concentration of collagen contained in the aqueous solution B is desirably 0.1% or more. By doing in this way, a high intensity | strength collagen tube can be formed easily. As collagen, collagen derived from animals such as humans, cows, horses, pigs and mice can be used, and collagen-like peptides and atelocollagen can also be used.

  As the aqueous solution C, various buffers can be used as long as they do not contain a component that gels the aqueous solution B. It is possible to use pure water containing only a thickener, or pure water not containing a thickener or the like may be used. It is also possible to use an aqueous solution that is made isotonic with components such as glucose.

  As the gelling agent contained in the aqueous solution D which is a gelling agent, any solution can be used as long as it is a solution capable of gelling collagen. However, the pH is preferably in the range of 7 to 10 in the sense of reducing damage to cells and efficiently forming a high strength collagen tube. Furthermore, a buffer containing phosphate ions in the range of 1 mM to 200 mM is more preferable.

  In addition, since the aqueous solution B containing collagen has a relatively high viscosity, the addition of a thickener to at least one of the aqueous solution A, the aqueous solution C, and the aqueous solution D enables stable liquid feeding, and the collagen tube It can be formed efficiently. As the thickener, for example, polyethylene glycol, dextran, carboxymethyl cellulose, polyvinyl alcohol, sodium alginate, propylene glycol alginate, or any combination thereof can be used.

  When the aqueous solutions A to B are continuously introduced individually from the respective inlets to the channel structure X as shown in FIGS. 1 to 3, the aqueous solution B is contained in the aqueous solution D from the outside via the aqueous solution C. By supplying the gelling agent component by diffusion, the aqueous solution B is gelled in the flow path X and continuously discharged from the outlet. If the aqueous solution A is a solution that is not gelled, the portion of the aqueous solution A remains in a liquid state, so that a hollow structure is formed.

  In the channel structure X, it is preferable that all of the aqueous solution A, the aqueous solution B, the aqueous solution C, and the aqueous solution D flow while maintaining a laminar flow, and more specifically, the Reynolds number is preferably 1000 or less. Since an aqueous solution containing collagen is generally high in viscosity, it is easy to maintain a laminar flow when the diameter of the channel structure is 1 mm or less.

  It is also possible to perform a culture operation on the cells introduced into the collagen tube lumen. As a culture solution to be used, a general culture solution for cell culture according to the target cell type can be used. Moreover, at the time of culture | cultivation, it is preferable to immerse a cell introduction | transduction collagen tube in the culture solution with which the petri dish, the flask, etc. were filled, and culture | cultivate using culture apparatuses, such as a CO2 incubator. Moreover, it is also possible to perform shaking operation as needed.

  Hereafter, the effect of this invention was confirmed by actually performing the collagen tube preparation method concerning the said embodiment. This will be described below.

  FIG. 4 is a schematic view showing a microfluidic device having a flow channel structure X, which is produced by superposing four acrylic plates, used to produce a collagen tube. 4 (d) passes through the channel structure formed on the lower surface of the first acrylic plate from the top, the channel structure formed on the lower surface of the second acrylic plate, and the third acrylic plate. FIG. 4B is a schematic view showing the formed channel structure and the channel structure formed on the lower surface of the third-layer channel structure, and is a view as viewed from the arrow B of the microfluidic device shown in FIG. It is. FIG. 4E is an enlarged view of the region e in FIG. 4D, and is a B arrow view of the microfluidic device in FIG. 4F. FIG. 4F is a cross-sectional view of the microfluidic device taken along the line A-A ′ in FIGS. 4A to 4E.

  The flow channel structure shown in FIG. 4 includes three 1.5 mm-thick acrylic flat plates cut using a microfabrication technique and 1.5 mm thick acrylic plates that have not been cut. It is formed by laminating flat plates by thermocompression bonding.

  The flow path structure shown in FIG. 4 has six inlets A, inlet B, inlet B ′, inlet B ″, inlet C, inlet D, one outlet O, and inlet flow paths AF1 connected to the respective inlets. It has an inlet channel BF1, an inlet channel B′F1, an inlet channel B ″ F1, an inlet channel CF1, and an inlet channel DF1. Further, the inlet channel AF1, the inlet channel BF1, the inlet channel B′F1, the junction P1 where the inlet channel B ″ F1 merges, the junction P2 where the inlet channel CF1 merges downstream, and further downstream. , The merging point P3 where the inlet channel DF1 merges, and the merging channel G existing between the merging point P3 and the outlet O.

  The width of the channel existing on the lower surface of the first flat plate was, for example, 400 micrometers for the merged channel. The depth was uniform and 300 micrometers. The length of the confluence channel was 35 millimeters. In addition, it becomes possible to control the diameter of the collagen tube obtained by controlling the diameter of the merging channel.

  4, the aqueous solution A is introduced from the inlet A, the aqueous solution B containing 0.1% or more of collagen is introduced from the inlet B, the inlet B ′ and the inlet B ″, and the aqueous solution C is introduced from the inlet C to the inlet. When each of the aqueous solutions D is continuously introduced from D, the aqueous solution A and the aqueous solution B come into contact at the junction P1, and the laminar flow is patterned so that the aqueous solution B surrounds the aqueous solution A. The patterned solution flows inside the merging channel G so that the aqueous solution C is in contact with the outside thereof, and further, the aqueous solution D is arranged on the outside thereof. Then, the gelling agent component contained in the aqueous solution D is supplied from the outside through the aqueous solution C by diffusion, whereby the flow of the aqueous solution B is continuously gelled, and the collagen tube is continuously formed. It will be collected.

  As the cells, HepG2 cells, which is a human liver cancer-derived cell line, were used. The cells were proliferated by culturing in advance on a normal cell culture dish, detached from the plate by enzyme treatment, and used after removing the culture solution components by centrifugation.

  As the aqueous solution A, an aqueous solution in which 5 g of glucose and 10 g of dextran having a molecular weight of 500,000 were dissolved in 100 mL of water was used. About 1 trillion hepatocytes were suspended per mL including the cell volume. Of these components, dextran was used to adjust the viscosity of the solution, but other components can be added or replaced with other components as necessary.

  As the aqueous solution B, an aqueous solution in which 0.4 g of collagen, 0.1 mM of hydrochloric acid and 5 g of glucose were dissolved in 100 mL of water was used. In addition, it is possible to control the intensity | strength of the collagen tube obtained by changing a collagen density | concentration.

  As the aqueous solution C, an aqueous solution in which 5 g of glucose and 10 g of dextran having a molecular weight of 500,000 were dissolved in 100 mL of water was used. In addition to confirming that dextran can be used as a thickener, it is possible to send liquids stably, and it is confirmed that similar effects can be obtained when other water-soluble polymers such as polyethylene glycol are used. It was done.

  As the aqueous solution D, an aqueous solution in which 0.3 g of disodium hydrogen phosphate, 5 g of glucose, and 10 g of dextran having a molecular weight of 500,000 as a thickener was dissolved in 100 mL of water. Similar to the aqueous solution C, it was confirmed that dextran can be used as a thickener to enable stable liquid feeding, and the same effect can be obtained when other water-soluble polymers such as polyethylene glycol are used. It was confirmed that it was obtained.

  Note that all these aqueous solutions were sterilized by heating or filtering in advance.

  These solutions were continuously introduced into the channel structure X shown in FIG. 4 using a syringe pump. In addition, in order to connect each inlet in the syringe and the flow path structure X, a PTFE tube was used.

  The flow rate of the aqueous solution introduced from each inlet was changed according to the size of the collagen tube to be produced. As for the introduction flow rate from each inlet, when the width of the confluence channel G is 400 micrometers and the depth is 300 micrometers, for example, the aqueous solution A introduced from the inlet A is 1 to 10 microliters per minute, the inlet B, the inlet B ′ and the aqueous solution B introduced from the inlet B ″ are 5 to 50 microliters per minute, the aqueous solution C introduced from the inlet C is 1 to 50 microliters per minute, and the aqueous solution D introduced from the inlet D is 30 to 200 Microliter per minute.

  The aqueous solution A introduced from the inlet A passes through the inlet channel AF1, is introduced into the upward channel structure formed on the lower surface of the third-layer flat plate, and exists in the second-layer flat plate. At the merge point P1, the solution B merges with the aqueous solution B introduced from the inlet B, the inlet B ′, and the inlet B ″. Furthermore, the aqueous solution C joins at the junction P2 existing on the lower surface of the first flat plate, and the aqueous solution D joins at the further downstream junction P3. And inside the confluence | merging flow path G, the flow of the aqueous solution B gelatinized, the collagen tube was formed continuously, and it collect | recovered from the exit O. FIG.

  FIG. 5 shows micrographs of collagen tubes prepared by using the flow channel structure X shown in FIG. 4 and introducing HepG2 cells into the lumen in the examples. FIG. 5B is a photomicrograph after 2 days of cell culture, and FIG. 5C is a photomicrograph after 6 days of cell culture.

  The diameter of the collagen tube shown in FIG. 5 was about 50 micrometers, and it was confirmed that the cells adhered to the lumen of the collagen tube and proliferated with the passage of the culture days. It was confirmed that cells can be introduced into the lumen in a living state.

  In the collagen tube shown in FIG. 5, the flow rate of the aqueous solution A introduced from the inlet A is 5 microliters per minute, and the total flow rate of the aqueous solution B introduced from the inlets B, B ′ and B ″ is every 20 microliters. This is obtained when the flow rate of the aqueous solution C introduced from the inlet C is 5 microliters per minute and the flow rate of the aqueous solution D introduced from the inlet D is 70 microliters per minute.

  FIG. 6 shows micrographs of frozen sections of collagen tubes prepared using the flow channel structure X shown in FIG. 4 and having HepG2 cells introduced into the lumen. 6 (a) is a collagen tube prepared by adjusting the flow rate ratio of the aqueous solution A and the aqueous solution B to 1: 4, and FIG. 6 (b) shows the flow rate ratio of the aqueous solution A and the aqueous solution B adjusted to 1:20. It is a microscope picture which shows the cross section of the produced collagen tube.

  From the cross-sectional observation result shown in FIG. 6, it was observed that the collagen tube was deformed by the adhesive force of the cells introduced into the lumen. In addition, it was confirmed that the collagen tube thickness could be 20 micrometers or less, and that a suitable environment for cell culture could be provided from the viewpoint of nutrition and oxygen supply. It was also demonstrated that the film thickness can be controlled by changing the flow rate condition.

  FIG. 7 shows micrographs of collagen tubes prepared by using the flow channel structure X shown in FIG. 4 and having HepG2 cells introduced into the lumen in the examples. FIG. 7 is a micrograph of a collagen tube prepared by adjusting the total flow rate of aqueous solution A and aqueous solution B to 5 microliters per minute, and FIG. 7B shows the total flow rate of aqueous solution A and aqueous solution B at 25 microliters per minute. It is the microscope picture of the collagen tube produced by adjusting to.

  As shown in FIG. 7, it was possible to adjust the diameter of the collagen tube by changing the flow rate of each solution introduced into the channel structure. In particular, it is possible to produce a collagen tube having a diameter of 100 micrometers or less, and it is also possible to obtain a tube having a diameter of about 20 micrometers, which is the thinnest and can align cells in a row. there were.

  Since the present invention is configured as described above, a micro collagen structure in which cells are introduced into a lumen without using a complicated device or operation is used in one step by using a microchannel structure. And the diameter can be freely adjusted. Moreover, a collagen tube can be produced without using a cross-linking agent, and damage to cells can be further reduced. Therefore, it is possible to provide a method for producing a new material useful in hepatocyte culture, which is impossible with conventional tissue preparation methods and collagen tube preparation methods for cell culture. Such materials are useful in the evaluation of new drugs in drug discovery, as well as the construction of transplanted tissues for regenerative medicine, the development of bioartificial organs such as artificial livers, and tissue models for biochemistry. Applicable for industrial use.

  In addition, since the present invention is configured as described above, it is possible to construct a cell culture system in which cells are linearly aligned and three-dimensionally covered with collagen. Therefore, when constructing a tissue body in vitro using cells differentiated from stem cells, for example, a state closer to a living tissue can be reproduced. Therefore, it is widely used in the field as a meaningful technique in regenerative medicine. Conceivable.

Claims (13)

A method for producing a collagen tube, wherein four types of aqueous solutions A to D are successively introduced into a flow path structure X having at least four inlets and at least one outlet,
The channel structure X is
When the aqueous solutions A to D are continuously introduced into the channel structure X, the aqueous solution B is disposed so as to surround the outer periphery of the aqueous solution A in the cross section S perpendicular to the flow direction, and the aqueous solution C is the aqueous solution B. At least one point P is disposed so as to be at least partially in contact with the outer periphery, and is disposed so that the aqueous solution D is at least partially in contact with the outer periphery of the aqueous solution C without contacting the outer periphery of the aqueous solution B. Or own
Furthermore, 0.1% or more of collagen is dissolved in the aqueous solution B,
Furthermore, the method for producing a collagen tube, wherein the aqueous solution D contains a component that gels the aqueous solution B. The method for producing a collagen tube according to claim 1, wherein 1000 or more cells are suspended in 1 mL of the aqueous solution A. The method for producing a collagen tube according to claim 2, wherein the cell is an adherent cell derived from a mammal. The method for producing a collagen tube according to any one of claims 1 to 3, wherein the pH of the aqueous solution D is in the range of 7 to 10. The method for producing a collagen tube according to any one of claims 1 to 4, wherein the aqueous solution D contains phosphate ions in a range of 1 mM to 200 mM. The method for producing a collagen tube according to any one of claims 1 to 5, wherein at least one of the aqueous solution A, the aqueous solution C, and the aqueous solution D includes a thickener. The method for producing a collagen tube according to claim 6, wherein the thickener is at least one of polyethylene glycol, dextran, carboxymethyl cellulose, polyvinyl alcohol, sodium alginate, and propylene glycol alginate. The production of the collagen tube according to any one of claims 1 to 7, wherein at least one of the width, depth, diameter, etc. of the channel structure X is at least partially 500 micrometers or less. Method. The method for producing a collagen tube according to any one of claims 1 to 8, wherein the channel structure X is at least partially constituted by a capillary tube. The method for producing a collagen tube according to any one of claims 1 to 9, wherein the channel structure X is at least partially configured by a microchannel structure manufactured using a microfabrication technique. The method for producing a collagen tube according to any one of claims 1 to 10, wherein in the cross section S at the point P, the width of the aqueous solution C is at least partially 50 micrometers or less. The method for producing a collagen tube according to any one of claims 1 to 11, wherein a diameter of the obtained collagen tube is at least partially 100 micrometers or less. The method for producing a collagen tube according to any one of claims 1 to 12, wherein a film thickness of the obtained collagen tube is at least partially 20 micrometers or less.