JP2016131551A - Method for perfusing fluid in vascular bed, and apparatus for conducting the method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a perfusion method for a fluid to be perfused through a vascular bed, and to provide an apparatus for conducting the same.SOLUTION: Perfusion culture of a vascular bed is conducted under fluctuating pressure, e.g. under pulse pressurization. A conducting device comprises a pressurization part which applies, for pressurization, cycle-variation pressure to the inside of a culture vessel into which the vascular bed is put. Preferably the pressurization part is configured to use the pressure of gas for cell culture to be supplied to the culture vessel 3. Specifically, a unitized gas supplying part 41 and a gas discharge part 63 are connected to the culture vessel 3, and the pressure of gas is fluctuated by control which uses a timer 73.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a method for perfusing fluid in a vascular bed and an apparatus for performing the method.

In recent years, regenerative research has started in which cells are organized in vitro and transplanted into a defective part as a regenerative tissue. As part of this, cell-cell adhesion and ECM structure and function are investigated using temperature-responsive culture dishes. A collection technique for collecting cells as a sheet without destroying cells has been developed.
According to this collection method, the obtained cell sheet has a characteristic of being rapidly engrafted in a living tissue, and if the cell sheet can be laminated, it is expected that a thick tissue or organ can be produced. ing.

International Publication WO2012 / 036225 International Publication WO2012 / 036224

By the way, with regard to the laminated cell sheets, conventionally, when four or more sheets are laminated, oxygen / nutrient cannot sufficiently permeate inside the cell sheet laminate, resulting in necrosis. Was the limit.
However, as described in Patent Document 1, recently, a perfusionable blood vessel network using a collagen gel with a microchannel, placing a cell sheet thereon and culturing, and using the collagen gel as a blood vessel bed In addition, the cell sheet is laminated on the medium, and the medium is perfused to form a capillary network in the cell sheet, and stacking is performed while supplying oxygen and nutrients to the upper cell sheet. A layering method in vitro has been proposed, and it has been reported that this method has been used to break the conventional stacking limit.
However, even when this layering method is used, the number of layers is limited to 12 at present, and it has been reported that it takes 5 days from angiogenesis to the next cell sheet.

  In Patent Document 2, an animal-derived tissue fragment having an arterial / venous loop is used as a vascular bed to construct a perfusable vascular network, and a cell sheet is laminated on the vascular network. A method has been proposed in which culture medium is also fed into cell sheets laminated by perfusion culture to create a layered structure while regenerating a capillary network, and it has been reported that this method has been used to break the conventional stacking limit. Has been. However, even when the layering method of Patent Document 1 is used, at present, the number of layers is limited to 12 and there is an interval of 3 days before the next cell sheet is stacked. That was a problem. Further, in this method, since the culture fluid perfused from the artery side leaks from the vascular bed, the ratio of the culture fluid discharged from the venous side becomes 40% or less, and the vascular bed and the cells stacked thereon There was also a problem that the culture solution was not efficiently supplied into the sheet.

  Therefore, as a result of diligent efforts regarding the perfusion method of the medium to be perfused into the vascular bed, the present inventors have found that the fluid in the vascular bed is efficient when a test is performed with variable pressure applied to the vascular bed. It was found to be perfused. The present invention has been completed based on such findings.

Therefore, an object of the present invention is to provide a novel and useful method for perfusing fluid in a vascular bed and an apparatus for performing the method, characterized by utilizing fluctuating pressure.
Another object of the present invention is to provide a unit in which the pressurizing unit is made detachable from the vascular bed perfusion culture apparatus.

The present invention solves the above problems. That is, the present invention is as follows.
[1] A method for perfusing a fluid in a vascular bed, wherein a perfusion culture is performed while applying a variable pressure from the outside of the vascular bed and applying the pressure.
[2] The perfusion method according to [1], wherein the fluctuating pressure is a cyclic fluctuating pressure that fluctuates periodically.
[3] The perfusion method according to [1] or [2], wherein the vascular bed is derived from a biological tissue piece.
[4] The perfusion method according to [3], wherein the biological tissue piece is a flap.
[5] The perfusion method according to [1] to [4], wherein the vascular bed is a vascular bed in which cell sheets are laminated.
[6] In the perfusion culture apparatus that performs the perfusion method according to [1] to [5], a culture container that houses a blood vessel bed, and a fluid inflow portion that is connected to the culture container and flows fluid into the blood vessel bed And a fluid discharge unit that is connected to the culture vessel and discharges fluid from the vascular bed, and a pressurization unit that is connected to the culture vessel and applies a cyclically varying pressure to the inside of the culture vessel. A perfusion culture apparatus characterized by the above.
[7] The perfusion culture apparatus according to [6], wherein the pressurizing unit pressurizes the cell culture gas by changing the pressure of the cell culture gas passing through the culture vessel.
[8] The perfusion culture apparatus according to [6] or [7], wherein the cyclically varying pressure is a pulse pressurization.
[9] The perfusion culture apparatus according to any one of [6] to [8], wherein the pressurizing unit is unitized and an apparatus connected to the culture container is constructed.
[10] A pressurizing unit that is detachably attached to the perfusion culture apparatus described in [9].

According to the method for fluid perfusion in a vascular bed of the present invention, it is possible to efficiently supply a medium or the like into a vascular bed having capillaries and a cell sheet laminated on the vascular bed. This makes it possible to efficiently supply oxygen and nutrients to the vascular bed and to the laminated cell sheets, and to obtain a vascular bed and laminated cell sheets that are more active than the prior art.
This method can also be performed using the perfusion culture apparatus of the present invention. In this apparatus, if the pressurizing part related to pressurization is unitized, it can be separated from the culture container side, which is convenient for handling.

1 is an overall configuration diagram of a vascular bed perfusion culture apparatus according to an embodiment of the present invention. It is a block diagram of the pulse pressurization part of FIG. It is explanatory drawing of the pulse pressurization in FIG. It is a figure which shows the preparation methods of the vascular bed derived from the biological body in an Example. It is a graph which shows the test result (comparison under constant pressure and pulse pressurization) of the perfusion rate of an Example. It is a graph which shows the test result (time dependence of the perfusion rate under pulse pressurization) of an Example. It is a graph which shows the standard deviation of the perfusion rate of an Example. It is a graph which shows the variation coefficient of the perfusion rate of an Example.

A perfusion culture apparatus 1 used for carrying out a method for perfusing a fluid in a blood vessel bed according to an embodiment of the present invention will be described with reference to the drawings.
First, the configuration will be described.
As shown in FIG. 1, the perfusion culture apparatus 1 includes a culture vessel 3.
The container body 5 of the culture container 3 is a rectangular box that opens upward, and a fluid inflow connection port 7 and a fluid outflow connection port 9 are provided side by side on the side that can be seen from the front. Further, a gas inflow connection port 11 is provided near one corner of the left and right sides, and a gas outflow connection port 13 is provided near the other diagonal. In addition, female screw holes 15 are formed in four corners of the upper surface of the square frame shape formed on the upper surface of the side portion.
Reference numeral 17 denotes a lid, which has a rectangular plate shape corresponding to the container body 3 described above. The lid 17 is provided with a large transparent rectangular observation window 19 on the inner side. Further, through holes 21 are formed at the four corners corresponding to the female screw holes 15 on the container body 3 side.

When the upper opening of the container body 5 is closed with the lid 17, the culture container 3 is substantially a sealed container except for the above-described port portion. Since a temperature controller (not shown) is also provided, the inside of the culture vessel 3 becomes a culture space in which the temperature is adjusted.
The lid body 17 is fixed by connecting the through hole 21 on the lid body 17 side to the female screw hole 15 on the container body 5 side and tightening with the screw 23.
In this culture vessel 3, the blood vessel bed B and the cell sheet C are cultured.

In the case of this embodiment, a culture solution is used as a perfusate for supplying oxygen and nutrients to the vascular bed, and a perfusion system for perfusing a medium in the culture space of the culture vessel 3 is provided. Yes. The perfusate is not particularly limited, but may be any kind of medium such as medium used for cell culture, blood, phosphate buffered saline (PBS), physiological saline, serum-free medium, etc. What is necessary is just to select suitably according to. The perfusate may contain a protein such as a cell growth factor or a drug as necessary.
Reference numeral 25 denotes a tube-like fluid supply line, one end of which is connected to the fluid inflow connection port 7 described above. A supply needle 27 is connected to the other end, and the supply needle 27 descends through the lid 31 of the culture medium tank 29, and the tip of the medium B stored in the culture medium tank 29. It extends below the liquid level. In the middle of the fluid supply line 25, a pump 33 as a fluid feeding means is interposed.

Reference numeral 35 denotes a tube-like fluid discharge line, one end of which is connected to the fluid outflow connection port 9 described above. A weight measuring scale 37 is connected to the other end. The scale 37 is provided with a discharge bag, and is configured to measure the weight of the medium accumulated therein.
The perfusion system is constructed using the above-described medium supply / discharge line, and the scale 37 is provided to enable evaluation of the perfusion rate described later. One end portion of the fluid supply line 25 and one end portion of the fluid discharge line 35 may be connected to the same culture solution tank 29. In this case, the perfusate used for culturing the vascular bed B is discharged from the fluid discharge line 35 to the culture medium tank 29 and mixed with the medium B. The culture tank 29 is provided with a dialysis membrane or the like. Thus, the discharged perfusate can be reused, and the amount of perfusate used can be saved.

  The vascular bed is provided with an infinite number of capillary networks, and the capillaries branched from the artery converge again and return to the veins. In the living body, blood flowing into the blood vessel bed returns from the artery through the capillaries to the vein at a rate of almost 100%. Therefore, oxygen and nutrients can be efficiently supplied into the tissue, and waste products such as carbon dioxide and lactic acid produced in the tissue can be efficiently discharged. However, the vascular bed used in the present invention is supplied from the artery because it is taken out of the living body and used as a vascular bed by cutting a part of the capillary network connected to the tissue in the living body. The perfusate leaks from the cut capillary network or tissue, and the perfusate returning to the vein is significantly reduced. When cell sheets are laminated on such a vascular bed, it is possible to supply nutrients and oxygen up to a certain number, but if more than 12 sheets, the amount of medium supplied to the entire cell sheet is insufficient. And partly necrotic.

In order to solve such problems, the perfusion culture apparatus 1 of the present invention is provided with a pulse pressurizing unit. By pressurizing the vascular bed from the outside, it is possible to promote the flow of fluid in the vascular bed, not only can promote the construction of the capillary network in the vascular bed, but also laminated on the vascular bed It is possible to efficiently supply oxygen and nutrients to the cell sheet. The perfusion culture apparatus 1 of the present invention is provided with a cell culture gas supply mechanism necessary for culturing cells and the like, and a pulse pressurizing unit is configured using this gas supply mechanism. In this embodiment, the cell culture gas is a mixed gas (CO ₂ : 5%, air: balance), but can be appropriately selected depending on the application, such as oxygen, nitrogen, air, or the like.
The pulse pressurizing unit is divided into a gas supply side and a discharge side. On the gas supply side, reference numeral 39 denotes a tube-like mixed gas supply line, one end of which is connected to the gas inflow connection port 11 described above. A gas supply unit 41 is connected to the other end.

The inside of the housing 42 of the gas supply unit 41 is configured as shown in FIG.
The supply gas is a mixture of air and CO ₂ gas. One end of the tube-like air inflow line 43 is opened to the outside, and a gas pump 45 and a throttle valve 47 for adjusting the flow rate are sequentially provided from the open side. The outside air is taken in and is sent at a regulated flow rate.

On the CO ₂ gas side, a gas cylinder 49 (not shown in FIG. 1) in which CO ₂ gas is stored is disposed outside the housing. One end side of the tube-like CO ₂ inflow line 51 extends to the outside of the housing and is connected. Inside, a mass flow controller 53 is interposed so as to be sent at an adjusted flow rate.
The air inflow line 43 and the CO ₂ inflow line 51 are merged and connected to the mixed gas inflow line 55. The mixed gas inflow line 55 is connected to the above-described mixed gas supply line 39 outside the housing through the connector. .
Accordingly, the mixed gas of air and CO ₂ gas taken into the housing 42 is supplied into the culture vessel 3 through the mixed gas supply line 39. Although not shown, the gas supply line 39 is provided with a sterilization filter to prevent contaminants (bacteria, viruses, dust, etc.) from flowing into the culture vessel. The means for preventing the contaminants from flowing into the culture vessel is not particularly limited as long as it is provided in the flow path connecting the outside air and the culture vessel.

A CO ₂ sensor 57 is interposed in the mixed gas inflow line 55 inside the housing. The CO ₂ sensor 57 is CO ₂ concentration regulator 59 and the electrical connection, the CO ₂ concentration regulator 59 is a mass flow controller 53 and the electrical connection described above.
With this configuration, when the CO ₂ sensor 57 constantly detects the CO ₂ concentration of the mixed gas and the CO ₂ concentration controller 59 receives this concentration information, the target concentration (5% in this embodiment) is obtained. A control signal is appropriately generated and sent to the mass flow controller 53 so as to be within the range, and the mass flow controller 53 is controlled to maintain a desired CO ₂ gas concentration.

On the gas discharge side, reference numeral 61 denotes a tube-like gas discharge line, and one end thereof is connected to the gas outflow connection port 13 described above. The other end is connected to a gas suction line 65 in the housing 64 of the gas discharge part 63. Although not shown, the gas discharge line 61 is provided with a sterilization filter to prevent contaminants (bacteria, viruses, dust, etc.) from flowing into the culture vessel. The means for preventing the contaminants from flowing into the culture vessel is not particularly limited as long as it is provided in the flow path connecting the outside air and the culture vessel.
The inside of the housing 64 of the gas discharge unit 63 is configured as shown in FIG. 2, and the gas discharge line 61 described above is connected to the gas suction line 65.
The distal end of the gas suction line 65 is open to the outside, and a pressure adjusting valve 67 is interposed closer to the open side. In the perfusion culture device 1, when the pressure adjusting valve 67 is closed, the culture space inside the culture vessel 3 is placed under pressure, and the pressure value to be applied is controlled by adjusting the degree of opening of the valve. Yes.

  An electromagnetic valve (switching valve) 69 is interposed on the side where the mixed gas from the culture vessel 3 is sent to the pressure adjusting valve 67. The solenoid valve 69 is a three-port type, and one port is for pressure release, and is connected to a tube-shaped pressure release line 71. This pressure release line 71 is open to the outside.

A timer 73 is electrically connected to the electromagnetic valve 69.
In the setting unit 75 of the timer 73, the switching mode of the electromagnetic valve 69 can be set by manual operation.
The switching mode of the timer 73 is intermittently repeated ON / OFF, and in this ON state, the gas discharge line 61 communicates with the pressure adjusting valve 67 side via the electromagnetic valve 69, so that the gas supply unit A gas line from 41 to the gas discharge part 63 via the culture vessel 3 is established, and the inside of the culture vessel 3 is pressurized with the pressure adjusted by the pressure adjusting valve 67.

  On the other hand, in the OFF state, the gas suction line 65 extending from the culture vessel 3 side communicates with the pressure release line 71 side via the electromagnetic valve 69, so that the gas in the culture vessel 3 goes out to the outside as it is. The pressurization is released in the culture vessel 3.

In this perfusion culture apparatus 1, using this ON / OFF, a cyclic fluctuation pressure is applied in the culture vessel 3, and the fluctuation form is a pulse (rectangular) mode.
In this embodiment, the pressure (P), the pulse width (W), and the interval (D) are set by the pressurization by the pressure adjusting valve 67 and the switching timing of the electromagnetic valve 69. For example, as shown in FIG. The inside of the culture vessel 3 is pressurized in the pressure mode.

  In the housing, a branch line 77 is provided on the culture vessel 3 side from the electromagnetic valve 69, and a pressure sensor 79 is attached thereto. The pressure sensor 79 is provided with a digital display 81. The display 81 is disposed on the front surface of the housing 64 as shown in FIG. Therefore, the pressurization condition to the culture vessel 3 can be visually confirmed.

As described above, the perfusion culture apparatus 1 includes a plurality of elements with the culture vessel 3 as the center, and the gas supply unit 41 and the gas discharge unit 63 are unitized.
Since the lines extending from the respective casings 42 and 64 are detachably connected to the ports of the culture vessel 3, the devices can be constructed by connecting them in use.

  When laminating cell sheets using the perfusion culture apparatus 1, a new cell sheet is placed on the cell sheet with a vascular bed in the culture vessel 3, under the adjusted temperature and pulse pressure. Then, after culturing with perfusion of blood and vascularization in the cell sheet, a new cell sheet is further stacked thereon.

  The vascular bed used in the present invention refers to a structure in which capillaries found in living tissues and organs and surrounding tissues are combined. The vascular bed functions to exchange oxygen, glucose, and other nutrients in living tissue through the thin walls of capillaries that are infinitely stretched in the vascular bed, and waste products such as carbon dioxide are vascularized. It functions to exude into the capillaries of the floor. The reason why the tissue fluid is kept relatively constant in the living tissue is that there is substance exchange in the capillaries.

  The vascular bed used in the present invention is not particularly limited as long as it is provided with arteries and veins, and can be any biological tissue piece with blood flow, skin flap, or artificial vascular bed with arteries and veins. . For example, muscle, mesentery, omentum, etc., as biological tissue pieces, are already convenient because the vascular network is highly constructed. Among them, the muscle is a site where the nutrient and oxygen are sufficiently supplied on the living tissue, that is, the vascular network is more highly constructed, which is more convenient (FIG. 4). In the present invention, a vascular network may be further constructed for these tissue pieces, skin flaps, and an artificial vascular bed provided with arteries and veins. The method is not particularly limited. For example, for a tissue piece or a flap, a method in which some blood vessels are closed, a blood vessel network is further constructed, and processing is performed in vivo until the blood flow circulates. A method in which the tissue pieces and flaps are confined in a sterile container, the state where the living body and the arteries and veins are connected to each other is created, and the vascular network is further constructed and placed in the living body until the blood flow circulates, VEGF And a method of administering a cytokine that promotes angiogenesis such as FGF. Although the origin of the animal species of the vascular bed used in the present invention is not particularly limited, for example, human or rat, mouse, guinea pig, marmoset, rabbit, dog, cat, sheep, pig, goat, monkey, chimpanzee Or those immunodeficient animals etc. are mentioned.

  An arterial-venous loop is an arterial-venous loop, and when a liquid component such as blood or culture medium flows from one or more blood vessels, the liquid component from one or more blood vessels other than the blood flowed in. It means a blood vessel flow channel in which a flow channel is constructed so that the spillage flows out. The length of the arterial-venous loop is not particularly limited, but can be determined appropriately depending on the size of the vascular bed. The method for connecting the artery and the vein is not particularly limited, but a capillary network may be constructed between the artery and the vein, and one end of the cut artery and one end of the cut vein are anastomosed. May be produced. The method for constructing the capillary network between the artery and the vein is not particularly limited. For example, the biological tissue piece in which the artery and the vein are connected to each other is separated with an electric knife or the like, and the biological tissue piece and the artery are separated. One example is a method in which one vein is connected to each other, and the indwelling state is maintained in the living body for about one week while maintaining the state. As a result, a capillary short circuit (arteriovenous loop) occurs between the artery and the vein, and a flow path from the artery to the vein via the capillary network is constructed.

  The vascular bed used in the present invention may be one that has been decellularized. Decellularization treatment refers to a method in which biological tissue is physically or chemically treated to remove cells constituting the tissue and antigenic components and leave only the proteins constituting the extracellular matrix. . The extracellular matrix thus obtained maintains the skeleton of tissues and organs before decellularization and the skeleton of capillaries, and this skeleton (structure) is seeded with vascular endothelial cells and other cells of interest. Thus, a three-dimensional tissue having a capillary network can be obtained. A method for performing the decellularization treatment is not particularly limited, and there are perfusion-type decellularization treatment, immersion-type decellularization treatment, and the like, which may be appropriately selected.

  The artificial vascular bed in the present invention refers to a vascular bed-like structure in which a capillary network is constructed and the space between the capillary networks is filled with gel and / or cells. A vascular bed derived from a living tissue includes various cells derived from a tissue in addition to cells constituting a capillary vessel. For this reason, when the cell sheet laminate is perfused on a vascular bed derived from a living tissue, a cell metabolite derived from the vascular bed may also be mixed into the cell sheet laminate. The contaminating cell metabolite may be preferable for the maintenance culture of the cell sheet on the vascular bed, or may promote the formation of a vascular network in the cell sheet and may be preferable. On the other hand, depending on the type of cells that constitute the cell sheet, the contaminated cell metabolites may adversely affect the maintenance culture, or may be used for stacked cell sheets (for example, used for transplantation or cell sheet activity). In some cases, it is not preferable. Whether to select a vascular bed derived from a living body or an artificial vascular bed may be selected according to the purpose, and is not particularly limited.

  A method for producing an artificial vascular bed having arteries and veins is not particularly limited. For example, a living tissue having an arterial-venous loop in a region partitioned in a sterile container (such as omentum) And a method for constructing a capillary network at a high level by placing it in a living body. The container for producing the artificial blood vessel bed is not particularly limited as long as it is a biocompatible base material, and a general material in the field of medical instruments and the like can be used. Specific examples include polyethylene, polypropylene, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyvinyl acetate, poly (meth) acrylic acid, poly (meth) acrylic acid derivatives, Examples thereof include polyacrylonitrile, poly (meth) acrylamide, poly (meth) acrylamide derivatives, polysulfone, polycarbonate, cellulose, cellulose derivatives, polysilicone, glass, ceramic, and metal. These materials may be used alone or in combination. The size of the artificial blood vessel bed can be optimized as appropriate depending on the cell sheet laminate to be produced, the cell sheet laminate to be transplanted, the site of the biological tissue from which the artificial blood vessel bed is to be produced, and the size is not particularly limited.

  The gel to be filled in the artificial blood vessel bed is not particularly limited, but a gel made of a biodegradable polymer is preferably used for the purpose of being used for transplantation into a living body. The type of biodegradable polymer is not particularly limited, and examples thereof include collagen, fibrin, gelatin, polysaccharides, elastin, fibronectin, laminin, chitin, chitosan and the like alone or a mixture of two or more.

You may mix a cell with the gel with which an artificial blood vessel bed is filled. Examples of the cells mixed in the gel include cells directly collected from biological tissues, cells directly collected and differentiated, and cell lines, but the type is not limited at all. For example, in the case of a method for evaluating myocardial tissue regeneration or myocardial function, the cells used include cardiomyocytes, myocardial blasts, myoblasts, mesenchymal stem cells, vascular endothelial cells, vascular endothelial progenitor cells, Any one of fibroblasts, bone marrow-derived cells, fat-derived cells, or a mixture of two or more cells can be used. For the purpose of regeneration of liver tissue, production of artificial liver simulating liver tissue, or method for evaluating liver tissue function, liver parenchymal cells, sinusoidal endothelial cells, Kupffer cells, stellate cells, pit cells, Examples include biliary epithelial cells, vascular endothelial cells, vascular endothelial progenitor cells, fibroblasts, bone marrow-derived cells, fat-derived cells, mesenchymal stem cells, or a mixture of two or more cells. When aiming at regeneration of kidney tissue, preparation of artificial kidney simulating kidney tissue, or evaluation of kidney function, for example, kidney cells, granule cells, collecting duct epithelial cells, wall epithelial cells, podocytes, mesangial Cells, smooth muscle cells, tubular cells, interstitial cells, glomerular cells, vascular endothelial cells, vascular endothelial progenitor cells, fibroblasts, bone marrow derived cells, adipose derived cells, mesenchymal stem cells, or Examples include a mixture of two or more types of cells. For the purpose of regeneration of adrenal tissue, production of an artificial adrenal that simulates the adrenal gland, or a method for evaluating adrenal function, for example, adrenal medullary cells, adrenal cortical cells, spherical layer cells, bundled layer cells, reticulolayer cells, Examples include vascular endothelial cells, vascular endothelial precursor cells, fibroblasts, bone marrow-derived cells, adipose-derived cells, and mesenchymal stem cells, or a mixture of two or more cells. For the purpose of evaluating skin regeneration or skin function, for example, epidermal keratinocytes, melanocytes, napped muscle cells, hair follicle cells, vascular endothelial cells, vascular endothelial progenitor cells, fibroblasts, bone marrow-derived cells , Any one of fat-derived cells and mesenchymal stem cells, or a mixture of two or more cells. For the purpose of regenerating mucosal tissue or evaluating the function of mucosal tissue, cells collected from the tissue constituting the mucous membrane may be used. For example, buccal mucosa, gastric mucosa, intestinal mucosa, olfactory epithelium , Oral mucosa, uterine mucosa and the like.
The content ratio of the cells is not particularly limited. At that time, if vascular endothelial cells, vascular endothelial progenitor cells, and the like are mixed in the artificial blood vessel bed, the construction of the blood vessel network in the artificial blood vessel bed is efficiently performed, which is convenient.

  A vascular network may be constructed by previously mixing a vascular growth factor that promotes angiogenesis with a gel filled in the artificial blood vessel bed. The method of performing blood vessel induction on the artificial blood vessel bed is not particularly limited. For example, a method of directly mixing a blood vessel growth factor in a gel, FGF which is a blood vessel growth factor is embedded in a microsphere, and the microsphere is Examples of the method include mixing with a gel and releasing FGF from the microsphere for a long time.

  The flow rate of the medium to be perfused into the vascular bed is not particularly limited, but the maximum flow rate is such that the flow path in the vascular bed is not destroyed, and the perfused medium oozes into the vascular bed, The flow rate at which the medium can reach the vascular bed surface may be set to the minimum flow rate.

  The present invention efficiently perfuses a perfusate even in an in vitro environment on a vascular bed such as a biological tissue piece having a vascular network, a flap, or an artificial vascular bed having an artery and a vein. And a perfusion culture device that implements the method. As a result, it becomes possible to effectively perfuse the perfusate into the vascular bed and maintain the state of the vascular bed close to that in the living body, as well as cells engrafted on the vascular bed. A perfusate can be efficiently supplied to a sheet or the like, and a vascular bed having a thicker cell sheet than before can be produced.

  The cell sheet in the present invention refers to a sheet-like cell group composed of one layer or a plurality of layers obtained by culturing on a cell culture device. The method for obtaining the cell sheet is not particularly limited. For example, the cells are cultured on a cell culture device coated with a polymer whose molecular structure is changed by stimulation of temperature, pH, light, etc. By changing the conditions of light, etc., and changing the surface of the cell culture equipment, the method of peeling the cell layer from the surface of the cell culture equipment while maintaining the adhesion state between the cells, the cells can be removed with any cell culture equipment Examples include a method of culturing and physically peeling from the end of the cell culture equipment with tweezers. It is particularly preferable that cells are cultured in a temperature range where the hydration power of the polymer is weak on a cell culture support whose surface is coated with a polymer whose hydration power changes in the temperature range of 0 to 80 ° C. This is a method of culturing the liquid by changing the temperature to a temperature at which the hydration power of the polymer is strong, and peeling the cells into a sheet. At that time, the cells are cultured in a temperature range where the hydration power of the polymer is weak on a cell culture support on which the polymer whose surface changes in hydration power in the temperature range of 0 to 80 ° C. is coated. Usually, the temperature is preferably 37 ° C., which is a temperature for culturing cells. The temperature-responsive polymer used in the present invention may be either a homopolymer or a copolymer. Examples of such a polymer include polymers described in JP-A-2-21865. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, or a vinyl ether derivative. Two or more of these can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer. In this case, since the cells to be cultured and peeled are cells, the separation is performed in the range of 5 to 50 ° C. Therefore, as the temperature-responsive polymer, poly-Nn-propylacrylamide (lower limit of homopolymer) Critical dissolution temperature 21 ° C), poly-Nn-propylmethacrylamide (27 ° C), poly-N-isopropylacrylamide (32 ° C), poly-N-isopropylmethacrylamide (43 ° C), poly-N -Cyclopropylacrylamide (at about 45 ° C), poly-N-ethoxyethylacrylamide (at about 35 ° C), poly-N-ethoxyethylmethacrylamide (at about 45 ° C), poly-N-tetrahydrofurfurylacrylamide (at about the same) 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (about 35 ° C.), poly-N, N-ethylmethylacrylate Amide (the same 56 ° C.), poly -N, N-diethyl acrylamide (the 32 ° C.), and the like. As monomers for copolymerization used in the present invention, polyacrylamide, poly-N, N-diethylacrylamide, poly-N, N-dimethylacrylamide, polyethylene oxide, polyacrylic acid and salts thereof, polyhydroxyethyl methacrylate, Examples thereof include water-containing polymers such as polyhydroxyethyl acrylate, polyvinyl alcohol, polyvinyl pyrrolidone, cellulose, carboxymethyl cellulose, and the like, but are not particularly limited.

  The method for producing a laminated cell sheet in the present invention is not particularly limited. For example, cells are seeded on a cell culture device, and a protein constituting an extracellular matrix protein (laminin, collagen, After applying a gel containing gelatin, cadherin, hyaluronic acid, fibronectin, fibrin, elastin, chitin, chitosan, vitronectin, etc.) If necessary, it is obtained by a method of laminating cultured cell sheets using a cultured cell moving jig. At that time, the temperature of the culture medium is not particularly limited if the polymer coated on the surface of the culture equipment has an upper critical solution temperature or lower, and if the polymer has a lower critical solution temperature or higher, the temperature is particularly limited. Not. However, it goes without saying that culturing in a low temperature range where cultured cells do not proliferate or in a high temperature range where cultured cells die is inappropriate. The culture conditions other than the temperature may be in accordance with conventional methods and are not particularly limited. For example, the medium to be used may be a medium to which serum such as known fetal calf serum (FCS) is added, or a serum-free medium to which such serum is not added. Moreover, you may utilize the jig | tool for moving a cell sheet as needed. Such a jig is not limited in terms of material and shape as long as it can capture the peeled cell sheet, but such materials are usually polyvinylidene difluoride (PVDF), silicon Polyvinyl alcohol, urethane, cellulose and derivatives thereof, chitin, chitosan, collagen, gelatin, fibrin glue, etc. are used in contact with the cell sheet in the form of a membrane, porous membrane, nonwoven fabric or woven fabric.

As described above, the embodiments of the present invention have been described in detail. However, the specific configuration is not limited to these embodiments, and the invention can be changed even if there is a design change without departing from the gist of the present invention. include.
For example, the form of pulse pressurization is not particularly limited, and can be appropriately set including not only the pulse width and interval but also the case where the pressure value is negative.

  As a means of promoting angiogenesis in the transplanted laminated myocardial tissue, the epidermis of the rat's thigh is incised, and the quadriceps skeletal muscle tissue including the femoral arteriovenous is 1.5 cm × 2.0 cm square using an electric knife. Then, only the femoral artery and vein were connected to the 1.5 cm × 2.0 cm square quadriceps skeletal muscle tissue, and only the incised epidermis was sutured while maintaining this state. A four-muscle skeletal muscle tissue that has been cut into 1.5 cm x 2.0 cm squares is left in the body for one week, causing a short circuit of the capillaries (arteriovenous loop) and a vascular network that returns from the artery to the vein A vascular bed was created (FIG. 4). The blood vessel bed in which the arteriovenous loop has occurred is completely dissected from the thigh of the rat, and the femoral artery and femoral vein are connected to a tube for perfusing the perfusate, allowing the perfusate to circulate in the blood vessel bed and the blood vessel. A tissue perfusion bioreactor capable of sealing the external space of the floor was produced (FIG. 1).

As a method for evaluating the flow of the perfusate in the vascular bed itself, the perfusion rate was defined as follows.

Perfusion rate (%) = Venous perfusate return weight (g) / arterial perfusate feed weight (g) × 100

A state where the perfusion rate is 100% indicates that all the medium sent from the artery side of the vascular bed has returned from the venous side, and there is no leakage of the medium from any place other than the vein of the vascular bed. Indicates.

An arterial vein of the vascular bed is cannulated in a container (hereinafter referred to as a pressurized chamber) having a gas sealing mechanism such as an O-ring, and a closed system of medium supply consisting of the arteriovenous of the vascular bed (hereinafter referred to as a vascular bed vascular system). 2), a closed system for pressurizing the exterior of the vascular bed. The external closed system of the vascular bed was pressurized by blowing 5% CO ₂ (including a low oxygen state) into the closed system for pressurizing the outside of the constructed vascular bed using a gas mixer. The pressure was 10 mmHg, and then a perfusion experiment was conducted in a pulse pressurization space where the pressure in the sealed space was 0 mmHg by automatic opening and closing of the valve. The pressure was adjusted by controlling the flow rate of the gas sent from the gas mixer into the pressurized chamber by a valve at the outlet, and simultaneously the pressure in the chamber was measured with a pressure gauge. In addition, the pulse pressure generated on the outlet side a control line by a valve and an atmospheric release line by a digital timer of an electronic three-way valve to generate a pressure state of 10 mmHg for 20 seconds and an air release state of 0 mmHg for 20 seconds. .

変動係数は、下記の式で算出した（図８）。

各データ取得時間においてパルス加圧、加圧なしの両群のデータの標準偏差および変動係数を比べると、パルス加圧の方が標準偏差、変動係数ともに減少しており、データのばらつきが抑えられたことが明らかとなった（ｎ＝３）（図７、８）。
これらの結果から、加圧チャンバーのパルス加圧により、血管床灌流の高効率化と安定化が同時に可能であることが明らかとなった。つまり、本発明の方法及びそれを用いた装置は、採取する個体や部位、作製手技が異なることにより血管床中の毛細血管網の構築度合が異なっていたとしても、血管床灌流を高効率的かつ安定的に行うことができ、血管床を用いた組織の品質の安定化に寄与する。 The perfusate to be perfused into the vascular bed vasculature used a medium and was fed at a constant rate of 50 μl / min. The perfusion rate after 48 hours increased to an average value of 68% ± 5.8% (n = 5) compared to 35% of the non-pressurized perfusion, and the flow of the vascular bed vasculature was improved. (Fig. 5). It was also suggested that a high perfusion rate was maintained even during long-term perfusion of 168 hours (FIG. 6).

Furthermore, in order to investigate whether or not the variation in reflux rate data is improved by the presence or absence of pulse pressurization, a plurality of independent experiments were performed, and the standard deviation (SD) was calculated by applying the following equation. (FIG. 7).

The coefficient of variation was calculated by the following formula (FIG. 8).

Comparing the standard deviation and coefficient of variation of the data for both groups with and without pulse pressurization at each data acquisition time, both the standard deviation and the coefficient of variation of pulse pressurization are reduced, and data variation can be suppressed. (N = 3) (FIGS. 7 and 8).
From these results, it was clarified that the vascular bed perfusion can be highly efficient and stabilized at the same time by the pulse pressurization of the pressurization chamber. In other words, the method of the present invention and the apparatus using the same enable highly efficient vascular bed perfusion even if the degree of construction of the capillary network in the vascular bed differs due to different individuals, parts, and production techniques. It can be performed stably and contributes to stabilization of the quality of the tissue using the vascular bed.

  According to the fluid perfusion method in the vascular bed of the present invention, it becomes possible to efficiently supply the perfusate into the vascular bed, and it is also possible to increase the number of stacked cell sheets using the vascular bed. . This is expected to actually open the way for the production of thick organs.

DESCRIPTION OF SYMBOLS 1 ... Perfusion culture apparatus 3 ... Culture container 5 ... Container main body 7 ... Fluid inflow connection port 9 ... Fluid outflow connection port 11 ... Gas inflow connection port 13 ... Gas outflow connection port 15 ... Female screw hole 17 ... Cover DESCRIPTION OF SYMBOLS 19 ... Observation window 21 ... Through-hole 23 ... Screw 25 ... Fluid supply line 27 ... Supply needle 29 ... Culture liquid tank 31 ... (Blood reservoir) lid 33 ... Pump 35 ... Fluid discharge line 37 ... Scale 39 ... Gas supply line 41 ... gas supply 42 ... (the gas supply unit) housing 43 ... air inlet line 45 ... gas pump 47 ... throttle valve 49 ... gas canister 51 ... CO ₂ inflow line 53 ... mass flow controller 55 ... mixed gas supply line 57 ... CO ₂ Sensor 59 ... CO ₂ concentration controller 61 ... Gas exhaust line 63 ... Gas exhaust part 64 ... Housing (of gas exhaust part) 65 ... Gas suction line 67 ... Valve for pressure adjustment 69 ... Solenoid valve (switching valve)
71 ... Pressure release line 73 ... Timer 75 ... (Timer) setting section 77 ... Branch line 79 ... Pressure sensor 81 ... Digital display B ... Blood vessel bed C ... Cell sheet

Claims (10)

A method for perfusion of fluid in a vascular bed,
A perfusion method, wherein fluctuating pressure is applied from the outside of the vascular bed and perfusion culture is performed while applying pressure.   The perfusion method according to claim 1, wherein the fluctuating pressure is a cyclic fluctuating pressure that fluctuates periodically.   The perfusion method according to claim 1, wherein the vascular bed is derived from a biological tissue piece.   The perfusion method according to claim 3, wherein the biological tissue piece is a flap.   The perfusion method according to any one of claims 1 to 4, wherein the vascular bed is a vascular bed in which cell sheets are laminated. In the perfusion culture apparatus which implements the perfusion method according to any one of claims 1 to 5,
A culture vessel for storing the vascular bed;
A fluid inflow portion connected to the culture vessel and flowing fluid into the vascular bed;
A fluid discharger connected to the culture vessel and discharging fluid from the vascular bed;
A pressurizing unit connected to the culture vessel and pressurizing the culture vessel by applying a cyclically varying pressure;
A perfusion culture apparatus comprising: In the perfusion culture apparatus according to claim 6,
The perfusion culture apparatus, wherein the pressurizing unit pressurizes the cell culture gas by changing the pressure of the cell culture gas passing through the culture vessel. In the perfusion culture apparatus according to claim 6 or 7,
The perfusion culture apparatus, wherein the cyclically varying pressure is pulse pressurization. In the perfusion culture apparatus according to any one of claims 6 to 8,
An apparatus for perfusion culture in which the pressurizing unit is unitized and an apparatus connected to a culture vessel is constructed.   A pressurizing part detachably attached to the perfusion culture apparatus according to claim 9.

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