JP3919181B2 - Organoid sheet, its use and production method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a sheet-like cell tissue (organoid) that can be used for artificial organs, transplantation materials, and the like.
More specifically, the present invention relates to a sheet-like organoid that can be used in hybrid artificial organs (bioartificial organs) and regenerative medical techniques.
The present invention also relates to the use and production method of the organoid sheet.
[0002]
[Prior art]
In recent years, development of hybrid artificial organs (also referred to as bioartificial organs) that combine cultured cells and biocompatible materials and regenerative medical techniques has attracted attention as a method of treating organs and tissues that have become dysfunctional or defective.
[0003]
For example, there are currently more than 600,000 liver disease patients in Japan, and about 50,000 patients die from liver disease annually. Of these, about 1,000 people die from acute liver failure and the rest from chronic liver failure, including liver cancer. The fundamental treatment for liver diseases such as liver failure is liver transplantation, but a shortage of organ donors (donors) is a major problem, and the development of an artificial liver is required.
However, it is difficult to replace more than 500 types of complicated and diverse liver functions with only artificial means. Recently, bioartificial livers that use hepatocytes themselves have attracted attention as artificial livers.
[0004]
As a bioartificial liver, an in vitro installed treatment system as shown in FIG. 1 is currently mainstream. As shown in FIG. 1, the artificial liver is composed of a biological side circuit that draws and circulates blood from patients with liver failure, an artificial liver module side circuit that circulates plasma on the artificial liver module side and performs metabolism and detoxification, and a plasma separator. The treatment is carried out by exchanging the substance through.
[0005]
It is not sufficient to use dispersed cells for such an artificial organ module. That is, in the monolayer culture method conventionally used for cell culture, it is inevitable that the function of the cell is lost or lowered, and it is important to construct and use a tissue body similar to a living tissue.
[0006]
From this point of view, a new method for culturing organ-like tissues (organoids) such as spherical aggregates (spheroids) and cylindrical structures (cylindroids) has recently been established, and high-functional expression of cells in culture. And its long-term functional maintenance is becoming possible.
[0007]
For example, the present inventors have developed a method for forming spheroids in a polymer substrate such as polyurethane foam (PUF) as a method for culturing spheroids (Japanese Patent Laid-Open No. 10-29951, H. Ijima et al. “Tissue Engineering "Vol. 4, No. 2, pages 213-226 (1998)). PUF is a porous material having a main skeleton and a thin membrane beam structure, and the PUF pores communicate with each other to some extent, so that high-density culture can be achieved in a good material exchange environment. When hepatocytes are cultured in the PUF pores, about 200 hepatocytes gradually gather and spontaneously form many spheroids with a particle size of about 100 μm. The present inventors have already succeeded in developing a human clinical scale short-term application type (about 10 days) bioartificial liver using spheroids utilizing this culture method.
[0008]
Furthermore, the present inventors have sought a more compact and long-term artificial liver, and found that hepatocytes can be filled into the hollow fiber with high density by centrifugal force, and 2.4 × 10 ⁷ cells / module per module. An artificial module having a cell density of cm ³ was obtained (see “Abstracts of the 31st Annual Meeting of the Fiscal Society of Japan Summer Seminar” pages 115-118).
In addition, considering the improvement of the operability at the bedside of the artificial liver and the elimination of chronic donor shortage, an artificial liver that is more compact and capable of maintaining long-term function is required. A higher density hepatocyte organoid (cell tissue) was developed and a patent application was filed (Japanese Patent Application No. 2001-48201).
[0009]
However, these conventional organoids are limited to spherical (spheroids) and cylindrical structures (cylindroids) in hollow fibers.
As a result of further research and development, the present inventors have expanded not only these spherical and columnar organoids but also sheet-like organoids, so that they can be applied to flat membrane and laminated artificial organs. I thought of it. There have been no reports on organoids formed in such a sheet form.
[0010]
[Problems to be solved by the invention]
An object of the present invention is to provide a sheet-like organoid (cell tissue body).
[0011]
[Means for Solving the Problems]
As a result of intensive studies on the above problems, the present inventors have succeeded in developing a sheet-like organoid that can be applied to an artificial organ module.
That is, the present inventors pay attention to the fact that a cell tissue body is formed by increasing the contact frequency between cells, and by applying pressure to the cells by a mechanical technique such as centrifugal force or hydrostatic pressure. Thus, it was possible to obtain a highly packed organoid having a sheet-like shape and a function inherent to the cell tissue.
As a result, it was unexpectedly found that the organoid sheet of the present invention has a further improved cell function than the previously filed cylindrical structure (cylindroid) in hollow fibers (Japanese Patent Application No. 2001-48201). .
[0012]
In addition, the sheet-like organoid of the present invention can provide a transplant material such as a myocardial patch, artificial skin, and liver transplant.
[0013]
Furthermore, the present inventors have found that at least two layers of hepatocytes in the sheet are necessary for the function of the hepatocyte tissue.
Furthermore, the present inventors have also found that when the thickness of the sheet-like hepatocyte organoid exceeds 150 μm, the cells in the central part of the hepatocyte organoid sheet are necrotized, which is not preferable.
The present invention also relates to a production method characterized in that such an organoid sheet is obtained by applying centrifugal force or hydrostatic pressure.
[0014]
More particularly, the present invention provides:
(1) An organoid sheet characterized in that it is a sheet composed of cell aggregates, wherein two or more layers of cells are laminated in the thickness direction of the sheet, and has a function inherent to cell tissue,
(2) The organoid sheet according to (1), wherein the cell is an animal tissue-derived cell,
(3) The organoid sheet according to (2), wherein the cell is a human organ-derived cell,
(4) The organoid sheet according to (2) or (3), wherein the cell is a hepatocyte,
(5) The organoid sheet according to (2) or (3) above, wherein the cells are cardiomyocytes,
(6) The organoid sheet according to any one of (1) to (5), wherein the thickness is 150 μm or less,
(7) The organoid sheet according to any one of (1) to (6), further comprising one or more support layers on at least one surface of the sheet,
(8) The organoid sheet according to (7) above, wherein the support layer includes a porous layer that does not allow the cells of the organoid sheet to pass through but allows the liquid component to permeate.
(9) The organoid sheet for artificial organs described in any one of (1) to (8) above,
(10) A bioartificial organ in which at least one organoid sheet according to any one of (1) to (8) above is enclosed in a container having an inlet and an outlet, and a passage for the liquid to be treated is provided. ,
(11) The above-mentioned (10), wherein the organoid sheet in the container and the passage of the liquid to be treated are completely separated by a porous partition wall that does not pass cells but allows liquid components to permeate. A bioartificial organ described in
(12) The bioartificial organ according to (10) or (11) above, wherein the liquid to be treated is blood, plasma, or a culture medium,
(13) The bioartificial organ according to any one of (10) to (12), wherein the cells of the organoid sheet are hepatocytes,
(14) A transplant material comprising the organoid sheet according to any one of (1) to (8) above,
(15) The transplant material according to (14) above, wherein a support layer composed of a biodegradable polymer is provided on at least one surface of the organoid sheet.
(16) The transplant material according to (15) above, wherein the biodegradable polymer is a synthetic polymer comprising a polyacid.
(17) The transplant material according to (15) above, wherein the biodegradable polymer is a biopolymer,
(18) Sheet-like support materials that do not allow cells to pass through but permeate liquid components are arranged in parallel at minute intervals, and cells are injected into the minute gaps between the sheet-like support materials, and mechanical forces such as centrifugal force or hydrostatic pressure are applied. The method for producing an organoid sheet according to any one of the above (1) to (8), further comprising a step of pressurizing by a technique, and (19) a step of removing the support material after the pressurizing step The method for producing an organoid sheet according to any one of the above (1) to (7), comprising:
It is about.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below.
In the present invention, an organoid refers to a cell tissue body in which cells are accumulated and approximates an organ (organ) having a function inherent to the cell tissue. For example, a tissue body in which hepatocytes obtained in the present invention are accumulated is a cell organoid because it has liver functions such as an ammonia removal effect and an albumin secretion effect inherent in the liver tissue.
[0016]
The cells used in the present invention include hepatocytes, cardiomyocytes, kidney cells, skin cells, epidermal keratinocytes, fibroblasts, vascular endothelial cells, vascular wall cells, nerve cells, chondrocytes, or combinations thereof. However, in the present invention, hepatocytes and cardiomyocytes are particularly preferably used.
The liver is the largest human organ, which synthesizes and stores substances necessary for the body, including proteins and carbohydrates, or metabolic detoxification of ammonia and drugs, and releases bile acids as an exocrine organ. It has a wide variety of functions such as being involved in digestion of fat and absorption of vitamins. The hepatocyte organoid sheet of the present invention has functions inherent to liver tissues such as ammonia metabolism and albumin synthesis.
[0017]
The cell source of the present invention is usually a normal cell derived from mouse, rat, guinea pig, rabbit, dog, pig, baboon, human and the like, but is not limited thereto. In addition, established cell lines are also targeted.
For example, in the case of normal hepatocytes, isolated hepatocytes can be obtained by using a general enzyme digestion method in which the liver is treated with an enzyme solution such as a collagenase solution.
In the case of cardiomyocytes, isolated cardiomyocytes can be obtained by using a general enzyme digestion method in which the heart is treated with an enzyme solution such as trypsin or collagen.
[0018]
The thickness of the cell organoid of the present invention is at least two cell layers. If it has the thickness for two layers, the function as an organoid can be exhibited.
For example, in the case of hepatocytes, the size of the cells is about 20 μm, so the two layers correspond to a thickness of about 40 μm. If the thickness exceeds 150 μm, oxygen cannot be supplied to the cells in the central portion, and the cells may be necrotic, which is not preferable.
Accordingly, in the present invention, the thickness of the organoid requires two or more layers of cells, and is preferably 150 μm or less.
Here, the thickness refers to the thickness from the surface layer to the surface layer of the organoid sheet, but the length and width of the organoid sheet can be arbitrarily set.
[0019]
In the organoid sheet of the present invention, the cell density is 1 × 10 ⁷ cells / cm ³ or more, more preferably 5 × 10 ⁷ cells / cm ³ or more, and further preferably 9 × 10 ⁷ cells / cm ³ or more.
When the cell density of the organoid is high, voids are reduced, so that the thickness cannot be increased too much for the supply of oxygen. good. Therefore, in the organoid of the present invention, it is more preferable that the cell density and the thickness have an inverse correlation.
[0020]
In the organoid of the present invention, it is important that the cells are in contact with each other three-dimensionally except for the surface. It is known that cells in the living body are expressing functions by exchanging information through various intercellular connections between adjacent cells. In the present invention, cells are filled with high density by applying physical force such as centrifugal force or hydrostatic pressure, and a tissue body with high cell density is formed by dramatically improving the contact frequency between cells. I was able to.
[0021]
In the organoid sheet of the present invention, a support layer is preferably provided on at least one surface of the sheet, but depending on the application, it is preferable that there is no support layer.
The support layer can be used as a carrier for producing and culturing the organoid sheet, and can also be used as a partition wall that separates the liquid to be treated and cells when the organoid sheet is applied to an artificial organ.
Furthermore, when an organoid sheet is used as a transplant material, the support layer can be lost after transplantation by using a biodegradable polymer for the support layer.
[0022]
When applied to an artificial organ, the support layer preferably includes a porous layer that does not allow cells to pass but allows liquid components to permeate.
The material of the porous layer may be any material that can be generally used as a selective separation membrane, such as cellulose, polyolefin, polyester, polysulfone, polyethylene, polypropylene, and polyamide.
The porous layer only needs to have a pore size small enough not to allow cells to pass therethrough, and may have a pore size and thickness large enough not to impair the substance permeability. For example, the hole diameter is about 0.001 to 15 μm and the thickness is preferably about 10 to 200 μm, but is not limited thereto.
The shape of the porous layer may be any as long as the above-described permeability is obtained, and examples thereof include a flat membrane, a foam, a woven fabric, and a nonwoven fabric.
[0023]
When applied to the implant material, the support layer is preferably composed of a biodegradable polymer. Biodegradable polymers are roughly classified into synthetic polymers and biopolymers, and can be appropriately selected depending on the transplant site, purpose, and use.
A synthetic polymer as a biodegradable polymer refers to all biodegradable polymer substances obtained by operations such as monomer polycondensation and polyaddition, and includes polyester polymers, polyamide polymers, polycyanoacrylate polymers, etc. Is mentioned. Of these, polyacids are preferable, and polylactic acid, polyglycolic acid, or glycolic acid-lactic acid copolymer is particularly preferable. These polyacids are susceptible to hydrolysis even under mild conditions in vivo. Lactic acid and glycolic acid that are temporarily generated by decomposition are extremely low in toxicity and are decomposed into carbon dioxide and water in vivo. Because.
The biopolymer as a biodegradable polymer refers to all polymer substances known to exist in the living body, including polysaccharides, proteins, nucleic acids, and composites such as glycoproteins. Including. Among these, substances called extracellular matrix such as collagen, laminin, fibronectin, elastin, tenascin, hyaluronic acid, fibrin, proteoglycan and the like can be preferably used because they mainly contribute to cytoskeleton formation and cell-cell adhesion. Fibrin is most preferable in that the support layer can be easily processed into an arbitrary shape.
The shape of the support layer made of the biodegradable polymer may be any shape as long as it can support the organoid sheet, and examples thereof include films, meshes, woven fabrics, nonwoven fabrics, and particles.
[0024]
In order to produce the organoid sheet of the present invention, it is necessary to apply a physical force such as centrifugal force or hydrostatic pressure to the dispersed cells to fill and culture at high density. Specifically, in the method for producing an organoid sheet of the present invention, sheet-like support materials that do not allow cells to pass through but allow liquid components to permeate are arranged in parallel at minute intervals, and the liver is placed in the minute gaps between the sheet-like support materials. It includes a step of injecting cells and pressurizing them by a mechanical method such as centrifugal force or hydrostatic pressure.
[0025]
For example, a scaffold (culture carrier structure) in which flat membranes and non-woven fabrics with good material permeability are regularly arranged with a minute gap of about 100 μm is constructed, and a mechanical method (centrifugal force, hydrostatic pressure) is built in this scaffold. Etc.) can be used to produce an organoid sheet that allows all cells to survive and function.
The formed organoid sheet can be used for culture or treatment in a state where it is fixed to the scaffold or removed from the scaffold.
In FIG. 2, the manufacturing method of this organoid sheet was shown with the conceptual diagram.
[0026]
The flat membrane and the nonwoven fabric used in the production method of the present invention are as described in the above support layer.
[0027]
It is preferred that the cells be in cell suspension and injected into the scaffold so as not to damage the cells. The concentration of the cell suspension is preferably 2 × 10 ⁷ cells / ml or less, more preferably 0.1 to 1 × 10 ⁷ cells / ml in order to increase the density without damaging the cells. In order to fill with high density, it is preferable to perform injection while removing only the culture solution from the membrane pores.
[0028]
The cell density is increased by applying a centrifugal force of 5 to 1500 G for about 30 to 600 seconds on the scaffold into which the cells are injected. Beyond 1500G, the cells are damaged or die. In order to prevent the cells from being killed as much as possible, a centrifugal force of about 60 G × 90 seconds is appropriate.
When a hydrostatic pressure is applied, a hydrostatic pressure of 5 to 25 kPa is applied to the scaffold into which the cells are injected for 4 to 120 hours. Most preferably, a constant hydrostatic pressure of 10 kPa is loaded for 24 hours.
[0029]
As the culture medium, a serum-free medium in which hormones or inorganic salts are added to a basic medium such as William E medium (WE) or Dulbecco's modified Eagle medium (DMEM), or a serum supplement in which serum is added to a basic medium such as WE or DMEM Medium is used.
[0030]
Hereinafter, the present invention will be described more specifically with reference to examples using hepatocytes, but the present invention is not limited thereto.
In the following examples and control examples, hepatocytes were prepared as follows.
[Method for preparing hepatocytes]
In order to prepare primary rat hepatocytes, 150 ml of a 0.5 mg / ml collagenase (Wako Pure Chemical Industries) solution was prepared. A cannula was introduced into the portal vein (blood vessel entering the liver) of a 7-week-old male Wistar rat (body weight 250 g), blood removal was allowed to flow at 30 ml / min for 5 minutes, and then 15 ml of collagenase solution heated to 37 ° C. For 10 minutes. The liver treated with collagenase was placed in the culture medium, and hepatocytes were dispersed using a scalpel and a pipette. The obtained hepatocyte suspension was washed 3 times to remove cells other than hepatocytes (purity of 95% or more). A final density of the hepatocyte suspension of 1.0 × 10 ⁵ cells / ml was prepared and used for culture experiments.
[0031]
In the following Examples and Control Examples, the ammonia removal rate and albumin secretion rate for measuring functional activity were measured as follows.
[Ammonia removal rate]
Ammonia was added to the culture medium to a concentration of 1 mM, the amount of decrease in ammonia concentration with time was measured, and the ammonia removal rate (μmol / 10 ⁶ cells / hr) was calculated.
[0032]
[Albumin secretion rate]
Albumin secreted into the culture medium was quantified by enzyme-labeled immunoassay and converted to albumin secretion rate (μg / 10 ⁶ cells / day).
[0033]
〔Example〕
In this example, an organoid sheet production container according to the conceptual diagram of FIG. 2 was produced, but the present invention is not limited to this. Two polycarbonate porous membranes (3) (thickness 10 μm, open area 23%, pore diameter 1.2 μm, manufactured by Millipore) 100 μm inside a polycarbonate housing (2) having a small hole (1) for discharging culture medium Containers arranged at intervals of were prepared. At this time, the upper part of the housing was opened to form a cell seeding port. The space in which the organoid sheet was formed between the two polycarbonate porous membranes was 1.5 cm long, 2.5 cm wide, and 100 μm thick.
[0034]
0.5 ml of 1.0 × 10 ⁷ cells / ml primary rat hepatocyte suspension (4) prepared in the above [Hepatocyte preparation method] by collagenase digestion was injected into the cell seeding port of the container. Next, by loading the container with centrifuge (60 × G, 90 seconds), cells between the porous membranes are densified while excreting the culture medium from the culture medium discharge hole of the housing, and the organoid sheet formation is induced. did.
[0035]
After completion of the centrifugation, the housing part was removed, and the hepatocyte organoid sheet formed between the porous membranes was cultured in a culture dish (manufactured by Asahi Techno Glass) having a diameter of 100 mm. The culture medium is Dulbecco's modified eagle medium (Gibco) 13.5 g / L, 60 mg / L proline, 50 ng / mL EGF (Funakoshi), 10 mg / L insulin (Sigma), 7.5 mg / L hydrocortisone (Wako Pure Chemicals) ), 0.1 μM copper sulfate pentahydrate (Wako Pure Chemical Industries), 3 μg / L selenate (Wako Pure Chemical Industries), 50 pM zinc sulfate heptahydrate (Wako Pure Chemical Industries), 50 μg / L linoleic acid (Sigma), 58.8mg / L penicillin (Meiji Seika), 100mg / L streptomycin (Meiji Seika), 1.05g / L sodium bicarbonate (Wako Pure Chemical), 1.19g / L HEPES (Dojindo) 18 mL of serum-free medium supplemented with The culture was swirling (45 rpm) in an atmosphere of 5% carbon dioxide and 95% air.
As an evaluation of hepatocyte function, ammonia was added to the culture medium to a concentration of 1 mM, and its activity was evaluated by measuring the amount of decrease in ammonia concentration over time. Moreover, the activity was evaluated by quantifying albumin secreted into the culture medium. Furthermore, the cut section of the cultured organoid sheet was stained by a hematoxylin / eosin staining method and observed using a microscope.
[0036]
As a control, hollow fiber / hepatocyte organoids were cultured, and the respective functions were compared by performing the same evaluation. The hollow fiber / hepatocyte organoid of this control example is a hollow fiber bundle composed of 6 hollow fibers having a length of 3 cm, and a columnar organoid is formed from about 1.2 × 10 ⁶ hepatocytes. is there.
[0037]
FIG. 3 shows a split cross-sectional form of the formed hepatocyte organoid sheet. FIG. 3 is a cross-sectional photomicrograph of the fourth day of culture. It is shown that hepatocytes form an organoid sheet in close contact in three dimensions. In FIG. 3, A is when the thickness is 110 μm, and B is when the thickness is about 150 μm. In A, 5 shows a split section of the organoid sheet, and 6 shows a porous membrane. In B, 7 indicates a necrotic layer of cells. When the organoid thickness is 5 layers (about 110 μm), all cells are alive in (A), but when the thickness is about 150 μm (B), the hepatocytes in the center become necrotic cells whose nuclei have fallen off. You can see that Furthermore, the initial cell packing density of the formed hepatocyte organoid sheet was about 1.2 × 10 ⁸ cells / cm ³ , indicating that the cells were in a densified organoid state.
As a result of the functional evaluation of the organoid sheet, FIG. 4 shows the change over time in the ammonia removal rate per initial number of immobilized cells, and FIG. 5 shows the change over time in the albumin secretion rate. 4 and 5, it was shown that the hepatocyte organoid sheet of the present invention still maintains the functions of ammonia removal activity and albumin secretion activity even after at least one month of culture. In addition, these activities were shown to have more functions than the control hollow fiber / hepatocyte organoid.
[0038]
【The invention's effect】
As described above in detail, according to the present invention, an organoid sheet having an original function of a cell tissue was obtained. The cell organoid sheet of the present invention can be directly applied as an artificial organ or a transplant material.
[Brief description of the drawings]
FIG. 1 shows a conceptual diagram of an artificial liver to which the hepatocyte organoid of the present invention is applied.
FIG. 2 is a conceptual diagram showing a method for producing a hepatocyte organoid sheet of the present invention.
FIG. 3 is a photograph of a cut section of the hepatocyte organoid sheet of the present invention.
FIG. 4 is a graph showing the change over time in the ammonia removal rate by the hepatocyte organoid sheet of the present invention in comparison with the hollow fiber / hepatocyte organoid.
FIG. 5 is a graph showing the change over time in the rate of albumin secretion removal by the hepatocyte organoid sheet of the present invention in comparison with the hollow fiber / hepatocyte organoid.
[Explanation of symbols]
1: Small hole 2: Housing 3: Membrane 4: Cell suspension 5: Split surface of organoid sheet 6: Porous membrane 7: Necrotic layer

Claims (16)

  It comprises a step of arranging sheet-like supporting materials that do not allow cells to pass through but allow liquid components to permeate in parallel at minute intervals, inject cells into the minute gaps between the sheet-like supporting materials, and pressurize them by a mechanical method. A method for producing an organoid sheet.   The method for producing an organoid sheet according to claim 1, wherein as a mechanical technique, pressurization is performed by centrifugal force or hydrostatic pressure.   The method for producing an organoid sheet according to claim 1, further comprising a step of removing the support material after the pressurizing step. An organoid sheet obtained by the method according to any one of claims 1 to 3, wherein the sheet is composed of cell aggregates, wherein two or more cells are laminated in the thickness direction of the sheet, and the thickness is 150 µm or less. , Olga proteinoid sheet characterized by having a function having tissue originally. The organoid sheet according to claim 4 , wherein the cells are hepatocytes. Furthermore, Olga proteinoid sheet according to claim 4 or 5, characterized in that it comprises a supporting lifting layer on at least one surface of the sheet. The organoid sheet according to claim 6 , wherein the support layer includes a porous layer that does not allow the cells of the organoid sheet to pass through but allows the liquid component to pass therethrough. The organoid sheet for artificial organs according to any one of claims 4 to 7 . A bioartificial organ in which at least one organoid sheet according to any one of claims 4 to 7 is enclosed in a container having an inlet and an outlet, and a passage for a liquid to be treated is provided. The bioid according to claim 9 , wherein the organoid sheet in the container and the passage of the liquid to be treated are completely separated by a porous partition wall that does not allow cells to pass but allows liquid components to permeate. Artificial organ. The bioartificial organ according to claim 9 or 10 , wherein the cells of the organoid sheet are hepatocytes. The bioartificial organ according to claim 11 , wherein the liquid to be treated is blood, plasma, or a culture medium. A transplant material comprising the organoid sheet according to any one of claims 4 to 7 . The transplant material according to claim 13 , further comprising a support layer made of a biodegradable polymer on at least one surface of the organoid sheet. The implant material according to claim 14 , wherein the biodegradable polymer is a synthetic polymer comprising a polyacid. The transplant material according to claim 14 , wherein the biodegradable polymer is a biopolymer.

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