WO2019004382A1 - Immunoisolation membrane, method for producing immunoisolation membrane, transplant chamber, and transplant device - Google Patents

Abstract

Provided are: an immunoisolation membrane which is resistant to a drop in substance permeability, and which can be produced inexpensively; and a method for producing same. This immunoisolation memrane contains a surface-modified porous membrane, and the surface-modified porous membrane contains polysulfone or polyethersulfone and preferably polyvinyl pyrrolidone. The ratio of the number of oxygen atoms to the total number of oxygen atoms, nitrogen atoms, carbon atoms and sulfur atoms on at least one surface of the surface-modified porous membrane falls within the range of 11.3% to 31.2%, inclusive, and the ratio of the number of nitrogen atoms to the aforementioned total number of atoms falls within the range of 0.1% to 4.0%. The production method for immunoisolation membrane includes subjecting the porous membrane surface to physical oxygen atom penetration, such as plasma treatment.

Description

Immuno-isolating membrane, method for producing immuno-isolating membrane, chamber for transplantation, and device for transplantation

The present invention relates to an immunoisolation membrane and a method of producing the same. The invention also relates to a grafting chamber having an immunoisolating membrane and a grafting device.

Immunoisolation is one of the methods to prevent the immune response in recipients during transplantation of biological components such as cells, tissues and organs, and the immunoisolation membrane allows water, oxygen, glucose, etc. to permeate. On the other hand, it is a permselective membrane that performs immune isolation by blocking the permeation of immune cells involved in immune rejection. For example, a transplant device utilizing an immunoisolating membrane which allows the physiologically active substance to permeate the transplantation of cells secreting the physiologically active substance can achieve the purpose of transplantation while preventing immune rejection.

In Non-Patent Document 1, a commercially available transplantation chamber formed using a porous membrane which is a laminated membrane of a cell holding membrane with a pore diameter of 0.45 μm and an outer membrane of polytetrafluoroethylene (PTFE) with a pore diameter of 5 μm. It is described that transplantation was performed using (TheraCyte®).

On the other hand, a membrane containing polysulfone as a porous membrane is well known as a precision filtration membrane used for industrial applications and the like (Patent Document 1).

JP-A-4-349927

Transplantation, 67, 665 (1995)

The immunoisolation membrane described in Non-Patent Document 1 is a laminate of a plurality of layers, and is likely to increase costs. An object of the present invention is to provide an immunoisolation membrane which can be manufactured inexpensively.

The present inventors examined the use of various porous membranes as an immunoisolation membrane under the above-mentioned problems, and a porous membrane containing polysulfone as described in Patent Document 1 can be used as an immunoisolation membrane. Yes, I found that I could solve the above problems. On the other hand, a porous membrane containing polysulfone required vacuuming for degassing treatment during use as a grafting device. The present inventors have further studied to improve this, and have found that the surface treatment of the porous membrane containing polysulfone imparts advantageous properties when used in a device for implantation, and this finding The present invention has been completed on the basis of

That is, the present invention provides the following <1> to <21>.
<1> An immunoisolation membrane comprising a surface-modified porous membrane,
The surface modified porous membrane comprises polysulfone or polyethersulfone,
The ratio of the number of oxygen atoms to the total number of oxygen atoms, nitrogen atoms, carbon atoms, and sulfur atoms on at least one surface of the surface-modified porous membrane is 11.3% or more and 31.2% or less. An immunoisolation membrane having a nitrogen atom number ratio of 0.1% or more and 4.0% or less.
<2> The carbon atom number ratio with respect to the said total number of at least one surface of said 2nd is 65.8%-80.9%, and the sulfur atom ratio with respect to said total number is 2.2%-2.9% or less The immunoisolation membrane as described in <1>.
<3> The immunoisolation membrane according to <1> or <2>, wherein the surface modified porous membrane comprises a nitrogen-containing polymer.
<4> The immunoisolation membrane according to <3>, wherein the nitrogen-containing polymer is polyvinyl pyrrolidone.
<5> The immunoisolation membrane according to any one of <1> to <4>, wherein at least one surface of the above is both surfaces.
<6> The immunoisolation membrane according to any one of <1> to <5>, which comprises the surface-modified porous membrane.
<7> The immunoisolation membrane according to any one of <1> to <6>, wherein the thickness of the surface modified porous membrane is 1.0 μm to 200 μm.
<8> The immunoisolation membrane according to any one of <1> to <7>, wherein the surface-modified porous membrane has a layered compact portion in which the pore diameter is minimized.
<9> The immunoisolation membrane according to <8>, in which the pore diameter is continuously increased in the thickness direction from the dense portion toward at least one surface of the surface-modified porous membrane.
<10> The immune isolation membrane according to <8> or <9>, wherein the dense portion is biased to one surface X of the central portion of the thickness of the surface-modified porous membrane.

<11> A transplantation chamber for containing a biological component, the chamber comprising:
The implantation chamber having the immunoisolation membrane according to any one of <1> to <10> on at least a part of the boundary between the inside and the outside of the implantation chamber.
<12> A transplantation chamber for containing a biological composition,
The immune isolation membrane according to <10> is provided on at least a part of the boundary between the inside and the outside of the transplantation chamber,
The implantation chamber, wherein the surface X of the surface modified porous membrane is on the inner side.
<13> The transplantation chamber according to <11> or <12>, wherein the biological construct is a cell.
The transplantation device according to any one of <14><11> to <13>, wherein the biological composition is contained in the transplantation chamber.
<15> The transplantation device according to <14>, wherein the biological component releases a physiologically active substance.
<16> The transplantation device according to <15>, wherein the physiologically active substance is insulin.

It is a manufacturing method of <17> immune isolation film, Comprising: The manufacturing method including performing physical oxygen atom osmosis processing to the porous membrane surface.
It is a manufacturing method of the immuno isolation film in any one of <18><1>-<10>, Comprising: The surface of the porous membrane containing a polysulfone or a polyether sulfone is physically oxygenated, and the said surface is processed. A method of manufacture comprising obtaining a modified porous membrane.
Casting a membrane-forming solution containing <19> polysulfone or polyethersulfone on a support,
Further, it is possible to obtain the above-mentioned porous membrane by a method including the steps of applying temperature-controlled humid air to the surface of the cast liquid film, and immersing the film obtained after applying temperature-controlled air in the coagulating liquid. The manufacturing method as described in <17> or <18> containing.
<20> The method according to <19>, wherein the membrane-forming solution contains polyvinyl pyrrolidone.
<21> The method according to any one of <17> to <20>, wherein the physical oxygen atom permeation treatment is plasma treatment.

The present invention can provide an immunoisolation membrane which can be manufactured inexpensively. The immunoisolation membrane of the present invention can be used as a grafting chamber and a grafting device in which a biological component is enclosed. The immunoisolation membrane of the present invention has advantages such as no need for vacuuming in degassing treatment when used in a device for transplantation.

The surface-modified porous membrane of 2 cm square is embedded under the back skin of a rat and sutured, and after 1 week rearing of the rat, the same site is excised and the image of the tissue stained section prepared.

Hereinafter, the present invention will be described in detail.
In the present specification, “to” is used in the meaning including the numerical values described before and after that as the lower limit value and the upper limit value.

<Immune isolation membrane>
As used herein, immunoisolation membrane means a membrane used for immunoisolation.
Immunoisolation is one of the methods to prevent the recipient's immune rejection during transplantation. Here, the immune rejection is the rejection of the recipient for the biological composition to be transplanted. Immunoisolation isolates the biological composition from the recipient's immune rejection. Immune rejection includes those based on cellular immune responses and those based on humoral immune responses.

The immunoisolation membrane is a selectively permeable membrane that transmits nutrients such as oxygen, water, glucose and the like and blocks the permeation of immune cells and the like involved in immune rejection. The immune cells include macrophages, dendritic cells, neutrophils, eosinophils, basophils, natural killer cells, various T cells, B cells, and other lymphocytes.
The immunoisolation membrane of the present invention preferably blocks permeation of high molecular weight proteins such as immunoglobulins (such as IgM or IgG) and complement depending on the use, and it is preferable to use relatively low molecular weight physiologically active substances such as insulin. It is preferable to make it permeate.

The selective permeability of the immunoisolation membrane may be adjusted according to the application. The immunoisolation membrane of the present invention may be, for example, a selectively permeable membrane that blocks substances having a molecular weight of 500 kDa or more, 100 kDa or more, 80 kDa or more, or 50 kDa or more. For example, the immunoisolation membrane is preferably capable of blocking the permeation of the smallest IgG (molecular weight: about 160 kDa) among antibodies. Furthermore, the immunoisolation membrane of the present invention may be a selectively permeable membrane that blocks substances having a diameter of 500 nm or more, 100 nm or more, 50 nm or more, or 10 nm or more as spheres.

The immunoisolation membrane of the present invention comprises a surface modified porous membrane. The immunoisolation membrane of the present invention may consist only of the surface modified porous membrane, or may include other layers such as a hydrogel membrane in addition to the surface modified porous membrane. The immunoisolation membrane of the present invention may have a protective film which can be easily peeled off for transport and the like. The immunoisolation membrane of the present invention preferably comprises only a surface-modified porous membrane.
In the immunoisolation membrane of the present invention, the surface modified porous membrane comprises polysulfone or polyethersulfone.

The surface modified porous membrane contained in the immunoisolation membrane of the present invention has an oxygen atom number ratio to the total number of oxygen atoms, nitrogen atoms, carbon atoms, and sulfur atoms on at least one surface (hereinafter referred to as "O element ratio") May be 11.3% or more and 31.2% or less, and the nitrogen atom ratio to the total number (hereinafter sometimes referred to as "O element ratio") is 0.1% or more and 4.0% or less As such, it is a porous membrane modified. In the present specification, a surface satisfying the above-described element ratio or a surface to which a physical oxygen atom infiltration treatment is performed as described later may be referred to as a modified surface.
The surface of the film means the main surface (the front or back surface indicating the area of the film), and does not mean the surface in the thickness direction of the end of the film. The surface of the surface-modified porous membrane may be an interface with another layer.

Also, on the modified surface, the carbon atom number ratio (hereinafter sometimes referred to as “C element ratio”) to the total number of oxygen atoms, nitrogen atoms, carbon atoms, and sulfur atoms is 65.8% to 80.9%. It is preferable that the sulfur atom number ratio (hereinafter sometimes referred to as "S element ratio") to the total number is 2.2% or more and 2.9% or less.

In the present specification, the O element ratio, the N element ratio, the C element ratio, and the S element ratio of the film surface are calculated from data of measurement based on X-ray photoelectron spectroscopy. X-ray photoelectron spectroscopy is a method of analyzing the composition of elements constituting a film surface by irradiating the film surface with X-rays and measuring the kinetic energy of photoelectrons emitted from the film surface. Sometimes referred to as -ray Photoelectron Spectroscopy) or ESCA (Electron Spectroscopy for Chemical Analysis). The measurement may be performed, for example, under the conditions using the monochromatized Al-Kα ray described in the examples. Ratios of measured values of oxygen atom, nitrogen atom, carbon atom and sulfur atom to the total of measured values of oxygen atom, nitrogen atom, carbon atom and sulfur atom on the surface, O element ratio, N element ratio and C element respectively The ratio and the S element ratio.

The O element ratio of the modified surface is preferably 13% or more and 31.2% or less, and more preferably 24% or more and 31.2% or less. The elemental ratio of N is preferably 0.1% to 3.9%, and more preferably 0.1% to 3.1%.
The C element ratio of the modified surface is preferably 65.8% to 80.9%, and more preferably 65.8% to 70.5%. Furthermore, it is preferable that it is 2.2% or more and 2.9% or less, and, as for S element ratio of said at least one surface, it is more preferable that it is 2.4% or more and 2.9% or less.

In the immunoisolation membrane of the present invention, either surface of the surface-modified porous membrane may be a modified surface or both surfaces may be modified surfaces, but both surfaces are modified surfaces. Is preferred. That is, it is preferable that the surface-modified porous film satisfy the above-described element ratio on both surfaces thereof.
The surface modified porous membrane in the immunoisolation membrane of the present invention has a surface N element ratio decreased and an O element ratio increased as compared to the modified porous membrane in the prior art, and preferably, The surface C element ratio decreases and the S element ratio increases. Within this range, the immunoisolation membrane of the present invention has the advantage that when it is used as a grafting device, no vacuuming is required when degassing the cells inside the membrane. . That is, there is an advantage that use can be started only by immersion in an aqueous medium such as physiological saline without vacuuming. In addition, when the immunoisolation membrane of the present invention is used as a grafting device that contains pancreatic β cells and the like and releases insulin, there is an advantage that insulin responsiveness (insulin permeability) in the initial stage of use is high. Here, the initial stage of use means within one day after the start of use.
The thickness of the immunoisolation membrane of the present invention is not particularly limited, but may be 1 μm to 500 μm, preferably 10 μm to 300 μm, and more preferably 15 μm to 250 μm.

[Porous membrane, surface modified porous membrane]
Hereinafter, the structure and composition of the porous membrane will be described. In this description, unless stated otherwise, the description of "porous membrane" also applies to "surface modified porous membrane".
(Structure of Porous Membrane) The porous membrane refers to a membrane having a plurality of pores. The holes can be identified, for example, in a scanning electron microscope (SEM) image or a transmission electron microscope (TEM) image of the cross section of the film.
The thickness of the porous membrane is not particularly limited, but may be 1 μm to 200 μm, preferably 10 μm to 180 μm, and more preferably 15 μm to 150 μm.

The porous membrane preferably has a layered dense portion in which the pore size is minimized. In addition, it is preferable that the pore diameter continuously increases in the thickness direction from the dense portion toward at least one surface of the porous membrane. The hole diameter is determined by the average hole diameter of the dividing line described later.
In the immunoisolation membrane of the present invention, the porous membrane has a uniform structure in the in-membrane direction (direction parallel to the membrane surface) with respect to the pore size or pore size distribution (difference in pore size in the thickness direction). preferable.

By the porous membrane having a pore size distribution in the thickness direction, the immunoisolation membrane of the present invention can improve the life. This is because the effect of multistage filtration using a plurality of membranes having substantially different pore sizes can be obtained, and deterioration of the membranes can be prevented.

The pore size may be measured from a photograph of the cross section of the membrane obtained by an electron microscope. The porous membrane is cut by a microtome or the like, and a photograph of the cross section of the porous membrane can be obtained as a section of the thin film whose cross section can be observed.

In the present specification, the comparison of the pore size in the thickness direction of the membrane is made by comparing the pore sizes at 19 dividing lines when the SEM photograph of the cross section of the membrane is divided into 20 in the thickness direction of the membrane. A total of 50 or more holes intersecting or touching the parting line are continuously selected, the respective pore sizes are measured, and an average value is calculated to be an average pore size. Here, the hole diameter is not the length of the portion where the selected hole intersects with the dividing line, but the area of the hole is calculated by image processing from the SEM photograph of the membrane cross section, and the obtained area is taken as the area of a true circle. Use the calculated diameter. At this time, for a dividing line which has a large hole and can not be selected at 50 or more, the field of view of the SEM photograph for obtaining the cross section of the film is expanded to be measured at 50. The average pore diameter obtained is compared for each parting line to compare the pore diameter in the thickness direction of the membrane.

The layered compact portion where the pore size is minimized refers to the layered portion of the porous membrane including the parting line where the average pore diameter is minimized among the parting lines in the film cross-sectional photograph. The compacted site may contain more than one parting line. For example, when two or more continuous parting lines having an average pore size within 1.1 times the minimum average pore size are continuous, the dense portion is assumed to include the two or more continuous parting lines. In the present specification, the thickness of the dense portion is the product of the number of parting lines included in the dense portion and 1/20 of the thickness of the film.

The thickness of the dense portion may be 0.5 μm to 50 μm, and preferably 0.5 μm to 30 μm. In the present specification, the average pore diameter of the dense portion is taken as the minimum pore diameter of the porous membrane. The minimum pore size of the porous membrane is preferably 0.02 μm to 1.5 μm, and more preferably 0.02 μm to 1.3 μm. This is because the minimum pore size of such a porous membrane can at least prevent normal cell permeation. Here, it is assumed that the average pore diameter of the dense portion is measured by ASTM F316-80.

The porous membrane preferably has a dense portion inside. The term "inside" means not in contact with the surface of the membrane, and "having a compact site inside" means that the compact site is not a site including the above-mentioned dividing line closest to any surface of the membrane. Do. By using a porous membrane having a structure having a dense portion inside, the permeability of a substance intended to be permeated is less likely to decrease than when a porous membrane having a dense portion in contact with the surface is used. Although not bound by any theory, it is thought that protein adsorption is less likely to occur due to the presence of a compact site inside.

The dense portion is preferably biased toward one of the surface sides of the central portion of the thickness of the porous membrane. Specifically, the dense portion is preferably located at a distance smaller than one half of the thickness of the porous membrane from any one surface of the porous membrane, and more preferably within two fifths of the porous membrane. . This distance may be determined in the film cross-sectional photograph described above. In the present specification, the surface of the porous membrane on the side closer to the dense portion is referred to as “surface X”.
When only one of the surfaces of the surface-modified porous membrane is a modified surface, the surface X is preferably a modified surface.

In the porous membrane, it is preferable that the pore diameter continuously increases in the thickness direction from the dense portion toward at least one of the surfaces. In the porous membrane, the pore diameter may be continuously increased in the thickness direction from the dense portion toward the surface X, and the pore diameter is continuously increased in the thickness direction from the dense portion toward the surface opposite to the surface X The pore diameter may be continuously increased when going from the dense portion to any surface of the porous membrane in the thickness direction. Among these, it is preferable that the pore diameter continuously increases in the thickness direction from at least the dense portion toward the surface opposite to the surface X, and when the dense portion is directed in the thickness direction to any surface of the porous membrane More preferably, the pore size is continuously increased. “The pore diameter continuously increases in the thickness direction” means that the difference in average pore diameter between the above-mentioned dividing lines adjacent in the thickness direction is the difference between the maximum average pore diameter (maximum pore diameter) and the minimum average pore diameter (minimum pore diameter). It is said to increase to 50% or less, preferably 40% or less, more preferably 30% or less. "Continuously increasing" essentially means increasing uniformly without decreasing, but decreasing sites may occur accidentally. For example, when dividing lines are combined two by two from the surface, if the average value of the combination is uniformly increased (uniformly decreased when going from the surface toward the dense region), The hole diameter is continuously increased in the thickness direction.
The structure of the porous membrane in which the pore diameter continuously increases in the thickness direction can be realized, for example, by the manufacturing method described later.

The maximum pore size of the porous membrane is preferably 1.5 μm or more and 25 μm or less, more preferably 1.8 μm to 23 μm, and still more preferably 2.0 μm to 21 μm. In the present specification, among the dividing lines of the membrane cross section, the average pore diameter of the dividing line at which the average pore diameter is the largest is taken as the maximum pore diameter of the porous membrane.

The ratio of the average pore diameter of the dense portion to the maximum pore diameter of the porous membrane (the ratio of the minimum pore diameter to the maximum pore diameter of the porous membrane, the maximum pore diameter divided by the minimum pore diameter, referred to herein as "anisotropic ratio 3) is preferable, 3 or more is preferable, 4 or more is more preferable, and 5 or more is further preferable. This is to increase the average pore diameter of regions other than the dense region and to increase the material permeability of the porous membrane. The anisotropy ratio is preferably 25 or less, more preferably 20 or less. This is because an effect such as the above-described multistage filtration can be efficiently obtained when the anisotropy ratio is 25 or less.
It is preferable that the parting line where the average pore diameter is the largest is the parting line closest to any surface of the membrane or a parting line in contact with the parting line.
At a dividing line closest to any surface of the membrane, the average pore diameter is preferably more than 0.05 μm and 25 μm or less, more preferably more than 0.08 μm and 23 μm or less, and more than 0.5 μm and 21 μm or less Is more preferred. Further, the ratio of the average pore diameter of the parting line closest to any surface of the membrane to the average pore diameter of the dense part is preferably 1.2 or more and 20 or less, and more preferably 1.5 or more and 15 or less. The number is preferably 2 or more and 13 or less.

(Element distribution of porous membrane)
The porous membrane (before the surface modification by physical oxygen atom permeation treatment) has the formula (I) and the formula on at least one surface, and the surface modified porous membrane has a surface other than the modified surface. It is preferable to satisfy (II).
B / A ≦ 0.7 (I)
A 0.01 0.015 (II)
In the formula, A indicates the ratio of N element (nitrogen atom) to C element (carbon atom) on the surface of the film, and B indicates the ratio of N element to C element at a depth of 30 nm from the same surface.
Formula (II) indicates that a certain amount or more of N element is present on at least one surface of the porous membrane, and in formula (I), the N element in the porous membrane is localized at less than 30 nm on the surface Indicates that the

When the surface satisfies the formulas (I) and (II), the biocompatibility of the porous membrane, in particular, the biocompatibility on the surface side satisfying the formulas (I) and (II) is enhanced.
In the porous membrane, only one of the surfaces may satisfy formula (I) and formula (II), or both surfaces may satisfy formula (I) and formula (II), It is preferred that both surfaces satisfy the formula (I) and the formula (II). When only one of the surfaces satisfies the formulas (I) and (II), the surface may be inside or outside in the implantation chamber described later, but it is inside Is preferred. Also, in the case where only one of the surfaces satisfies the formula (I) and the formula (II) and the porous membrane has the above-mentioned surface X, the formula (I) and the formula (II) The surface to be filled is preferably surface X.

In the present specification, the ratio (A value) of N element to C element on the film surface and the ratio (B value) of N element to C element at a depth of 30 nm from the surface are those calculated using XPS measurement results. Do. The A value was calculated from the result at the start of sputtering under the conditions using monochromatized Al-Kα radiation described in the examples, and the result of the time calculated to be 30 nm from the surface of the film measured from the sputter rate B We shall calculate the value.

B / A may be 0.02 or more, preferably 0.03 or more, and more preferably 0.05 or more.
A is preferably 0.050 or more, more preferably 0.080 or more. Further, A may be 0.20 or less, preferably 0.15 or less, and more preferably 0.10 or less.
B may be 0.001 to 0.10, preferably 0.002 to 0.08, and more preferably 0.003 to 0.07.

The element distribution of the porous film, particularly the distribution of the N element, is the concentration of water contained in the temperature-controlled air, the time for applying the temperature-controlled air, the temperature of the coagulating solution, the immersion time in the method for producing the porous film described later. It can be controlled by the temperature of the diethylene glycol bath for washing, the immersion time in the diethylene glycol bath for washing, the speed of the porous production line, etc. The distribution of the N element can also be controlled by the water content in the stock solution.

(Composition of porous membrane)
The porous membrane comprises polysulfone or polyethersulfone. The porous membrane preferably contains 30% by mass or more, preferably 40% by mass or more of polysulfone or polyethersulfone based on the total mass.
The polysulfone or polyethersulfone preferably has a number average molecular weight (Mn) of 1,000 to 10,000,000, and more preferably 5,000 to 1,000,000.

The porous membrane preferably comprises a nitrogen-containing polymer with polysulfone or polyethersulfone. As examples of nitrogen-containing polymers, mention may be made of polyvinyl pyrrolidone, polypropyl acrylamide, chitin, chitosan, polyacrylamide, polyamines, polylysine. Among these, polyvinyl pyrrolidone is particularly preferred. Biocompatibility can be increased by combining hydrophobic polysulfone or polyethersulfone with hydrophilic polyvinyl pyrrolidone.

The porous membrane may contain other components other than the above components as additives. Additives include metal salts of inorganic acids such as sodium chloride, lithium chloride, sodium nitrate, potassium nitrate, sodium sulfate and zinc chloride, metal salts of organic acids such as sodium acetate and sodium formate, and other polymers such as polyethylene glycol And polyelectrolytes such as sodium polystyrene sulfonate and polyvinylbenzyltrimethyl ammonium chloride; and ionic surfactants such as sodium dioctyl sulfosuccinate and sodium alkylmethyl taurate. The additive may act as a swelling agent for the porous structure. As additives it is preferred to use metal salts, in particular lithium chloride.

The porous membrane is preferably a membrane formed from one composition as a single layer, and is preferably not a laminated structure of multiple layers.

<Method of producing immunoisolation membrane>
The present inventors have carried out a vacuum treatment when degassing the immunoisolation membrane by performing physical oxygen atom permeation treatment on the porous membrane of the immunoisolation membrane including the porous membrane containing polysulfone or polyethersulfone. It has been found that there is an advantage that the pulling becomes unnecessary. Also, in particular, when the manufactured immunoisolation membrane is used as a grafting device that contains pancreatic β cells and the like and releases insulin, there is an advantage that insulin responsiveness (insulin permeability) in the initial stage of use is high. I found it. The physical composition of the porous membrane surface can be modified by physical oxygen atom permeation treatment. The immunoisolation membrane of the present invention having the above-described surface elemental composition can be produced by subjecting a porous membrane containing polysulfone or polyethersulfone to physical oxygen atom permeation treatment to obtain the above surface-modified porous membrane. .

[Method of producing porous membrane]
The method for producing the porous membrane (prior to surface modification by physical oxygen atom permeation treatment) is not particularly limited, and any usual polymer membrane forming method can be used. The polymer film forming method may, for example, be a stretching method or a casting method, and the casting method is preferable.
For example, in the casting method, the porous membrane having the above-described structure can be produced by adjusting the type and amount of the solvent used for the membrane forming solution and the drying method after casting.

The production of a porous membrane using a casting method can be carried out, for example, by a method comprising the following (1) to (4) in this order.
(1) A film-forming stock solution containing polysulfone or polyethersulfone, optionally, a nitrogen-containing polymer (in particular, polyvinyl pyrrolidone), an additive, and, if necessary, a solvent is cast on a support in a dissolved state.
(2) The temperature and humidity are applied to the surface of the cast liquid film.
(3) Immerse the film obtained after application of temperature and humidity air in the coagulating liquid.
(4) Peel the support if necessary.

The temperature of the temperature and humidity air may be 4 ° C. to 60 ° C., preferably 10 ° C. to 40 ° C. The relative humidity of the temperature and humidity style may be 15% to 100%, preferably 25% to 95%. The temperature and humidity air may be applied at a velocity of 0.1 m / sec to 10 m / sec for 0.1 second to 30 seconds, preferably 1 second to 10 seconds.
The average pore diameter and position of the dense portion can be controlled by the concentration of water contained in the temperature-controlled air and the time for applying the temperature-controlled air. The average pore diameter of the dense portion can also be controlled by the water content in the stock solution for membrane formation.

As described above, the evaporation of the solvent can be controlled by applying the temperature and humidity to the surface of the liquid film, and coacervation can be generated from the surface of the liquid film toward the inside. The coacervation phase is fixed as micropores to form pores other than micropores by immersing in a coagulating liquid containing a solvent having low solubility of the polymer but being compatible with the solvent of the polymer in this state. be able to.

The temperature of the coagulating solution may be -10 ° C to 80 ° C in the process of immersing in the above-mentioned coagulating solution. By changing the temperature during this period, it is possible to control the time from formation of the coacervation phase on the support surface side to the solidification from the dense region, and to control the size of the pore diameter to the support surface side. It is. When the temperature of the coagulating solution is increased, the formation of the coacervation phase is accelerated and the time to solidification is prolonged, so the pore diameter toward the surface of the support tends to be large. On the other hand, when the temperature of the coagulating solution is lowered, the formation of the coacervation phase is delayed and the time to solidification is shortened, so the pore diameter toward the surface of the support is hardly increased.

As a support, a plastic film or a glass plate may be used. Examples of plastic film materials include polyesters such as polyethylene terephthalate (PET), polycarbonates, acrylic resins, epoxy resins, polyurethanes, polyamides, polyolefins, cellulose derivatives, silicones and the like. As a support, a glass plate or PET is preferable, and PET is more preferable.

The membrane-forming stock solution may contain a solvent. As the solvent, a solvent having high solubility of the polymer to be used (hereinafter sometimes referred to as “good solvent”) may be used depending on the polymer to be used. The good solvent is preferably one which is quickly replaced with the coagulating solution when immersed in the coagulating solution. Examples of the solvent include N-methyl-2-pyrrolidone, dioxane, tetrahydrofuran, dimethylformamide, dimethylacetamide or a mixed solvent thereof. Of these, N-methyl-2-pyrrolidone is preferred.

It is preferable to use, in addition to the good solvent, a solvent having a low solubility of polysulfone or polyethersulfone but having compatibility with the above-mentioned good solvent (hereinafter sometimes referred to as “non-solvent”). Examples of the non-solvent include water, cellsorbs, methanol, ethanol, propanol, acetone, tetrahydrofuran, polyethylene glycol, glycerin and the like. Of these, water is preferably used.

The concentration of polysulfone or polyethersulfone as a membrane forming solution may be 5% by mass to 35% by mass, preferably 10% by mass to 30% by mass. When the content is 35% by mass or less, sufficient permeability (for example, permeability to water) can be given to the obtained porous membrane, and by setting the content to 5% by mass or more, the porous membrane selectively transmitting the substance Can secure the formation of The additive amount of the additive is not particularly limited as long as the uniformity of the membrane forming solution is not lost by the addition, but it is usually 0.5% by volume or more and 10% by volume or less with respect to the solvent. When the membrane forming solution contains a non-solvent and a good solvent, the ratio of the non-solvent to the good solvent is not particularly limited as long as the mixture can maintain a uniform state, but 1.0 to 50 mass% Preferably, 2.0% by mass to 30% by mass is more preferable, and 3.0% by mass to 10% by mass is more preferable.

When polyvinyl pyrrolidone is used, polyvinyl pyrrolidone is preferably contained in an amount of 50% by mass to 120% by mass with respect to the total mass of polysulfone and polyethersulfone in the membrane-forming solution, and is preferably 80% by mass to 110%. More preferably, it is contained by mass%.
Furthermore, when the membrane-forming solution contains lithium chloride as an additive, lithium chloride is preferably contained in an amount of 5% by mass to 20% by mass with respect to the total mass of polysulfone and polyethersulfone, and 10% by mass More preferably, it is contained at 15% by mass.

As the coagulating liquid, it is preferable to use a solvent having low solubility of polysulfone or polyethersulfone to be used. Examples of such solvents include alcohols such as water, methanol, ethanol and butanol; glycols such as ethylene glycol and diethylene glycol; aliphatic hydrocarbons such as ether, n-hexane and n-heptane; Glycerol etc. are mentioned. Examples of preferred coagulating solutions include water, alcohols or a mixture of two or more thereof. Of these, water is preferably used.

It is also preferable to wash with a solvent different from the coagulation solution used after immersion in the coagulation solution. The washing can be carried out by immersion in a solvent. Diethylene glycol is preferred as the washing solvent. The distribution of N element in the porous film can be controlled by using diethylene glycol as a washing solvent and adjusting one or both of the temperature and the immersion time of diethylene glycol in which the film is immersed. In particular, the remaining amount of polyvinyl pyrrolidone to the membrane can be controlled. After washing with diethylene glycol, it may be further washed with water.

JP-A-4-349927, JP-B-4-64966, JP-A-4-351645, JP-A-2010-235808 and the like can be referred to for the method of producing the porous membrane.

[Physical oxygen atom penetration treatment]
The surface-modified porous membrane can be obtained by physically impregnating the porous membrane obtained as described above. The surface-modified porous membrane obtained by such treatment is suitable for use in the immunoisolation membrane, since the physical oxygen atom permeation treatment does not cause changes on the surface of the porous membrane to be a stimulus to cells etc. It is thought that Also, although the porous membrane containing polysulfone or polyethersulfone is usually highly hydrophobic, the physical oxygen atom permeation treatment increases the hydrophilicity of the surface of the porous membrane, and when defoaming treatment is performed as described above It is considered that the advantage of eliminating the need for evacuation is obtained. In addition, physical oxygen atom penetration treatment removes impurities and the like on the surface of the porous membrane, and has the advantage of eliminating the need for evacuation when degassing treatment and the advantage of increasing the initial permeability of insulin etc. It is considered to be

The physical oxygen atom permeation treatment may be performed on either surface of the porous membrane or on both surfaces, but it is preferable to perform on both surfaces.

Examples of physical oxygen atom permeation treatment include plasma treatment, corona discharge treatment, glow discharge treatment, ozone / ultraviolet irradiation treatment, flame treatment, hot air treatment and the like. Among these, plasma treatment and corona discharge treatment are preferable, and plasma treatment is more preferable.

(Plasma treatment)
The plasma treatment can be performed by disposing a porous film to be treated between opposing electrodes, introducing a plasma-exciting gas into the apparatus, and applying a high frequency voltage between the electrodes. The surface treatment of the porous membrane is performed by causing the gas to be plasma-excited to cause glow discharge between the electrodes.
The frequency of the high frequency voltage is preferably 1 kHz to 100 kHz, and more preferably 1 kHz to 10 kHz.

As said plasma excitation gas, what added the oxygen atom to inert gas, such as argon and neon, for example is preferable. The inert gas flow rate is preferably 5 to 500 cm ³ (STP) / minute, more preferably 50 to 200 cm ³ (STP) / minute, and particularly preferably 80 to 120 cm ³ (STP) / minute. preferable. Further, a limiting oxygen flow rate 45cm ³ (STP) / min or more, preferably 50cm ³ (STP) / min or more, more preferably 50 ~ 100cm ³ (STP) / min.

The plasma treatment is preferably performed under reduced pressure. The degree of vacuum at the time of plasma treatment is preferably 0.6 to 100 Pa, more preferably 1 to 60 Pa, and particularly preferably 2 to 40 Pa. Plasma treatment under such conditions can be performed using a vacuum plasma treatment apparatus. Alternatively, the treatment may be performed using an atmospheric pressure plasma treatment apparatus capable of stably generating high density plasma under an atmospheric pressure atmosphere.

The input power (discharge output) in the plasma treatment may be 1 to 300 W, preferably 3 to 150 W, and particularly preferably 5 to 100 W.
The time of the plasma treatment depends on the input power and the like, but is preferably 3 to 270 seconds, and more preferably 5 to 180 seconds, for example, when the input power is 50 W.
Moreover, it is preferable to use anodic coupling as plasma processing conditions.

(Corona discharge treatment)
Corona discharge treatment can be carried out by any method known in the art, such as Japanese Patent Publication Nos. 48-5043, 47-51905, 47-28067, 49-83767, and 51-41770. The method can be carried out according to the method disclosed in JP-A-51-131576. As the processor, various commercially available corona processors can be applied. For example, a corona processor having a multi-knife electrode manufactured by SOFTAL (Sophtal) can be used.

[Other layer]
The immunoisolation membrane of the present invention may include other layers other than the surface modified porous membrane. Other layers include hydrogel membranes. The hydrogel film is preferably biocompatible, and examples thereof include an alginate gel film, an agarose gel film, a polyisopropylacrylamide film, a film containing cellulose, a film containing a cellulose derivative (such as methyl cellulose), a polyvinyl alcohol film, etc. Can be mentioned. Alginic acid gel membrane is preferred as the hydrogel membrane. Specific examples of alginic acid gel membranes include alginic acid-poly-L-lysine-alginic acid polyion complex membranes.
The immunoisolation membrane of the present invention preferably comprises only a surface-modified porous membrane. It is because the above-mentioned effect obtained by physical oxygen atom permeation treatment can be more easily obtained. However, in the immunoisolation membrane having a surface other than the modified surface, another layer may be provided on the surface side other than the modified surface. Furthermore, even on the modified surface side, a layer or the like made of a biocompatible plastic having a reticulated structure that does not impair the above-described effect of the immunoisolation film may be provided on the surface.

<Use of immunoisolation membrane>
Immunoisolation membranes can be used to prevent immune rejection. In particular, it can be used to prevent the immune rejection of the recipient against the biological composition to be transplanted. That is, the immunoisolation membrane can be used to protect biological constituents from the recipient's immune system. In the present specification, the recipient means a living body to receive transplantation. Preferably, the recipient is a mammal, more preferably a human.

[Biological composition]
By biological composition is meant a structure of biological origin. Organisms include viruses, bacteria, yeast, fungal cells, insects, plants, mammals and the like. It is preferable that the living body is usually a mammal. Mammals include cows, pigs, sheep, cats, dogs, humans and the like. Preferably, the biological construct is a construct derived from any mammal.

Biological compositions include organs, tissues, cells and the like. Of these, cells are preferred as biological constituents. The number of cells may be one or more, preferably two or more. The plurality of cells may be separated from one another or may be aggregates.

The biological composition may be obtained directly from the living body. In addition, especially when the biological component is a cell, the biological component may be obtained directly from the living body, such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), What induced differentiation of cells such as mesenchymal stem cells may be used. The cells may be progenitor cells.

As the biological composition, in one aspect, one that releases a physiologically active substance is preferable. Examples of physiologically active substances include various hormones, various cytokines, various enzymes, and various other in vivo factors. More specific examples include insulin, dopamine, factor VIII and the like.

Here, insulin is a polypeptide (molecular weight about 6000) in which the A chain of 21 amino acid residues and the B chain of 30 amino acid residues are linked via a disulfide bond. In mammalian organisms, insulin is secreted from β cells in the islets of Langerhans of the pancreas. When insulin-secreting cells are used as a biological component in the present invention, the insulin to be secreted may be human insulin or insulin of other mammalian types (eg, pig type). The insulin may be insulin produced by a method of genetic modification. For a method of obtaining genetically modified insulin, for example, the description of Takafumi Kadowaki: Diabetes Navigator (pp. 270-271, Takeshi Tao, Yoshikazu Oka "Present and Future Insulin Preparations", Medical Review, 2002) can be referred to. . Various insulin analogues (see, for example, HC Lee, J. W. Yoon, et al., Nature, 408, 483-488, 2000) may be used.

Preferably the biological construct is an insulin secreting cell. Insulin-secreting cells are cells that can secrete insulin in response to changes in blood glucose level. The insulin-secreting cells are not particularly limited, and examples thereof include pancreatic β cells present in the islets of Langerhans of the pancreas. The pancreatic β cells may be human pancreatic β cells or pancreatic β cells such as pigs and mice. The method of extracting pancreatic β cells from swine can be referred to the description of JP-A-2007-195573. Furthermore, as insulin-secreting cells, cells derived from human stem cells (for example, see Miyazaki Jun-ichi, Regenerative Medicine, vol. 1, No. 2, page 57-61 (2002)), or small intestinal epithelial stem cells An insulinotropic cell, which may be a cell (see, for example, Atsuko Fujimiya et al., Regenerative Medicine, Vol. 1, No. 2, p. 63-68, 2002), and which incorporates a gene encoding insulin. (See, for example, HC Lee, J. W. Yoon, et al., Nature, 408, 483-488, 2000). Furthermore, it may be the islet of Langerhans of the pancreas (see, for example, Horiyo, Inoue Ichichi, Regenerative Medicine, Volume 1, No. 2, pp. 69-77, 2002).

[Chamber for transplantation]
The immunoisolation membrane of the present invention can be used as a component of a transplantation chamber for containing a biological composition. The implantation chamber can be used as a container for containing a biological composition when implanting the biological composition into a recipient. The immunoisolation membrane is disposed at least part of the boundary between the inside and the outside of the implantation chamber. By arranging in this manner, nutrients such as water, oxygen, glucose and the like can be incorporated from the outside to the inside of the transplantation chamber while protecting the biological components contained in the transplantation chamber from the immune cells etc. existing outside. It can be imported.

The immunoisolation membrane may be disposed on the entire surface of the boundary between the inside and the outside of the transplantation chamber, and for example, 1 to 99%, 5 to 90%, 10 to 80%, 20 to 70% of the entire surface. It may be arranged in a part corresponding to the area of 30 to 60%, 40 to 50%, etc. The surface on which the immunoisolation membrane is disposed may be one continuous portion or may be divided into two or more portions.
When the immunoisolation membrane of the present invention is not disposed on the entire surface of the boundary between the inside and the outside of the transplantation chamber, the remaining surface is, for example, nonpermeabilizing not to permeate nutrients such as oxygen, water and glucose in addition to cells. It may be formed of a permeable membrane.

If only one surface of the surface modified porous membrane is a modified surface, in the implantation chamber, the modified surface may be inside or outside.
In addition, when the surface modified porous membrane has a dense portion and has the above-mentioned surface X, it is preferable that the surface X be on the inside side of the implantation chamber. That is, it is preferable that the immunoisolation membrane be disposed so that the compacted portion of the porous membrane in the immunoisolation membrane is closer to the inside of the implantation chamber. By making the surface X inside the implantation chamber, the permeability of the physiologically active substance can be further increased.

The implantation chamber may have a junction where the immunoisolation membranes are oppositely joined. The portion of the immunoisolation membrane to which it is conjugated is not particularly limited, but is preferably the end of the immunoisolation membrane. In particular, it is preferable that the ends be joined. In the present specification, when the film is referred to as an "end", it means an outer peripheral portion or a part of a fixed width substantially in contact with the side surface (edge) consisting of the thickness of the film. It is preferable that the entire periphery of the immunoisolation membrane is joined except for the injection port described later. For example, the implantation chamber has a configuration in which two immunoisolation membranes are opposed and their outer circumferences are joined, or one immunoisolation membrane of axisymmetric structure is folded in two and the facing outer circumferences are joined. Is also preferred.

Bonding can be performed using adhesion or fusion using an adhesive.
For example, a curable adhesive can be used to bond. Examples of the adhesive include known adhesives such as epoxy type, silicon type, acrylic type and urethane type.
Alternatively, a thermoplastic resin may be sandwiched between the porous membranes, and the two may be joined by heating the portion. At this time, as the thermoplastic resin, it is preferable to use a resin having a melting point lower than that of the polymer forming the porous film. Specific examples of the thermoplastic resin include polyethylene, polypropylene, polyurethane, polyvinyl chloride, polytetrafluoroethylene, polyethylene terephthalate, polycarbonate and the like. Among them, polyethylene, polypropylene, polyurethane, polyvinyl chloride and polytetrafluoroethylene are preferable, and polyethylene, polyurethane and polyvinyl chloride are more preferable.

Furthermore, the porous membranes in the immunoisolation membrane may be fused in direct contact with each other without sandwiching other materials between them. By such fusion, it is possible to obtain a transplantation chamber which has no problem derived from a resin to be inserted or the like. When a porous membrane containing a polymer selected from the group consisting of polysulfone and polyethersulfone is used, the porous membranes are fused and integrated by heating to a temperature lower than the melting point of the polymer and higher than the glass transition temperature. Can be Specifically, heating for fusion may be 190 ° C. or more and less than 340 ° C., and preferably 230 ° C. or more and less than 340 ° C.

The form of the implantation chamber is not limited, and may be bag-like, bag-like, tube-like, microcapsule-like, drum-like or the like. For example, a drum-shaped implantation chamber can be formed by adhering an immuno-isolating membrane on the top and bottom of a silicone ring. The shape of the implantation chamber is preferably a shape that can prevent movement of the implantation chamber in the recipient when used as an implantation device described later. Specific examples of the shape of the implantation chamber include a cylindrical shape, a disk shape, a rectangular shape, an oval shape, a star shape, and a circular shape. The implantation chamber may be sheet-like, strand-like, spiral-like or the like. The implantation chamber may contain the biological component, and may have the above-described shape only when it is used as an implantation device described later.

The implantation chamber may include a biocompatible plastic or the like for maintaining the shape and strength of the container. For example, the boundary between the inside and the outside of the implantation chamber may be made of a biocompatible plastic which does not correspond to the immunoisolation membrane and the immunoisolation membrane. Alternatively, the implantation chamber in which the immunoisolation membrane is disposed substantially on the entire inner / outer border is further arranged with a network-like biocompatible plastic outside the inner / outer border in terms of strength. It may be

(Inlet)
It is also preferable that the implantation chamber is provided with an injection port for injecting a biological component or the like into the implantation chamber. As an inlet, a tube may be provided which leads to the interior of the implantation chamber.
The tube may be, for example, one containing a thermoplastic resin. The thermoplastic resin preferably has a melting point lower than that of the porous membrane polymer material.

Specific examples of the thermoplastic resin used for the tube include polyethylene, polypropylene, polyurethane, polyvinyl chloride, polytetrafluoroethylene, polyethylene terephthalate, polycarbonate and the like. Among them, polyethylene, polypropylene, polyurethane, polyvinyl chloride and polytetrafluoroethylene are preferable, and polyethylene, polyurethane and polyvinyl chloride are particularly preferable.

The tube is, for example, sandwiched with the immunoisolation membrane so as to be in contact with a part of the porous membrane and then joined with the part. Bonding can be performed by fusion bonding or adhesion using an adhesive. Among these, it is preferable to perform fusion. The fusion may be heat fusion.
When fusion is performed, the tube preferably comprises a thermoplastic resin having a melting point lower than that of the porous membrane polymer material. When a tube containing a thermoplastic resin having a lower melting point is fused to a porous membrane, it is thought that the tube material can be melted first and then enter into the pores of the porous membrane during heating.

When bonding is performed, the adhesive can be appropriately selected according to the material of the polymer forming the film and the tube, and an adhesive of epoxy type, silicon type, acrylic type, urethane type or the like can be used. For example, when using a tube containing a resin material having a melting point lower than that of the porous membrane polymer material, bonding can be performed by adhesion.

[Porting device]
The implantation device is a complex comprising at least the implantation chamber and the biological composition. In the implanting device, the implanting chamber encloses the biological composition.
In the device for transplantation, the chamber for transplantation may contain only the biological component or may contain other components or components other than the biological component and the biological component. May be For example, the biological composition may be contained in the implantation chamber together with the hydrogel, preferably in the state of being contained in the hydrogel. Alternatively, the implant device may contain a pH buffer, an inorganic salt, an organic solvent, a protein such as albumin, and a peptide.
In the device for transplantation, only one type of biological composition may be contained, or two or more types may be contained. For example, it may contain only biological constituents that release bioactive substances for the purpose of transplantation, or perform other functions of the purpose of transplantation, which support the function of these biological constituents Further biological constituents may be included.

The implanting device may be implanted in, for example, the abdominal cavity or subcutaneously. Alternatively, the implantable device may be a vascular connection device. For example, in the case of using an insulin-secreting cell as a biological component, transplanting blood and a membrane in direct contact with each other enables insulin secretion corresponding to a change in blood glucose level.

The device for transplantation and the chamber for transplantation are described in Protein Nucleic Acid Enzyme, Volume 45, pp. 2307 to 2312 (Okuhara Hisako, 2000), JP 2009-522269, JP 6-507412, etc. You can refer to

The features of the present invention will be more specifically described below with reference to examples and comparative examples. The materials, amounts used, proportions, treatment contents, treatment procedures, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Accordingly, the scope of the present invention should not be construed as limited by the specific examples shown below.

[Preparation of porous membrane]
15 parts by mass of polysulfone (Sorbay Corporation P3500), 15 parts by mass of polyvinyl pyrrolidone (K-30), 1 part by mass of lithium chloride and 2 parts by mass of water dissolved in 67 parts by mass of N-methyl-2-pyrrolidone I got This film-forming solution was cast on the surface of a PET film. Air adjusted to 30 ° C. and relative humidity 80% RH was applied at 2 m / sec for 5 seconds to the surface of the cast liquid film. Immediately thereafter, it was immersed in a 65 ° C. coagulation bath filled with water. The PET film was peeled off to obtain a porous membrane. Then, it was placed in a diethylene glycol bath at 80 ° C. for 120 seconds, and then thoroughly washed with pure water to obtain a porous membrane with a dry thickness of 50 μm.

An SEM photograph (S-5200, Hitachi High-Technologies, 10.0 kBV) of the cross section of the obtained porous membrane was performed. As a result of image analysis, the obtained porous film had a pore diameter distribution in which the pore diameter was continuously increased toward the surface in the thickness direction, and had a dense region inside. The thickness of the porous membrane was 50 μm, the minimum pore size was 0.8 μm, and the maximum pore size was 5.6 μm. The dense portion was located 15 μm from one surface (surface X).

The obtained porous film is placed in a desktop vacuum plasma apparatus (manufactured by U-Tech Co., Ltd.), and the carrier gas conditions are: oxygen flow 20 cm ³ (STP) / min, argon flow 100 cm ³ (STP) / min, vacuum degree 30 Pa, input power The surface X is first plasma-treated at a processing time between 0 and 270 seconds shown in Table 1, and then reversely processed, and the opposite surface is plasma-treated to plasma-treat both surfaces. A porous membrane was obtained.

(Measurement of elemental ratio)
The central part of the obtained surface modified porous membrane was sampled, and the elemental ratio of the surface was calculated using ESCA (Electron Spectroscopy FOR Chemical Analysis).
Surface modified porous membrane was prepared from Physical Electronics, Inc. X-ray source: Al-Kα ray (1490 eV, 25 W, 100 μm diameter), measurement area: 300 μm × 300 μm, Pass Energy 55 eV, Step 0.05 eV, carbon atoms on surface X, oxygen The ratio of each element to the total of atoms, nitrogen atoms, and sulfur atoms was measured. The results are shown in Table 1.

(Insulin permeability)
The membrane of a Corning insert cell (6.5 mm Transwell with 5.0 μm Pore Polycarbonate Membrane Insert) was hollowed out, and instead, the surface-modified porous membrane was fixed to the insert cell with double-sided tape.
The surface-modified porous membrane sample to be subjected to pretreatment for evacuation is an insert cell in which the surface-modified porous membrane is attached to the well of a 24-well plate containing 1 mL of culture medium (medium for pancreatic islet culture, Cosmobio, PNIM3) Was set so that the membrane was immersed in the medium, and vacuum was applied using a vacuum pump for 10 minutes.

After removing the insert cell and quickly removing the medium so as not to dry the membrane, a medium (islet culture) containing 0.2 μg / mL of insulin (Wako Pure Chemical Industries, Insulin Humane recombinant, 097-06474) in the insert cell 100 μL of medium, Cosmobio, PNIM3) was added and inserted into a 24-well plate containing 800 μL of a similar medium without insulin. The mixture was allowed to stand for 1 hour, the medium on the insert cell side and the 24-well plate side was collected, and the amount of insulin was quantified by insulin ELISA (80-INSRT-E01 manufactured by ALPCO). The amount of permeated insulin on the 24 well side was evaluated according to the following criteria.

After 60 minutes, the amount of insulin on the permeation side is 4.5 ng or more ... AA
After 60 minutes, the amount of insulin on the permeation side is 4.0 ng or more and less than 4.5 ng ... A
After 60 minutes, the amount of insulin on the permeation side is 2.0 ng or more and less than 4.0 ng ... B
After 60 minutes, the amount of insulin on the permeation side is 1.0 ng or more and less than 2.0 ng ... C
After 60 minutes, the amount of insulin on the permeation side is 0 ng or more and less than 1.0 ng ... D

[Preparation of chamber for transplantation]
The surface-modified porous membrane subjected to plasma treatment for 90 seconds on both sides was cut into 3 cm × 5 cm, and the surface on the side to which air was applied at the time of production was turned inside to be folded in two. Thereafter, using a tea bag sealer (T-230K) manufactured by Fuji Impulse Co., four long sides of 3 cm × 2.5 cm rectangular long sides and two short sides are heated at 230 ° C. and joined, and the outer peripheral portion is joined The part was cut leaving 1 mm to make a 1 cm × 2 cm implantation chamber.

(Cell infiltration inhibition test)
The inhibition of cell infiltration into the membrane inside of the living body of the produced transplantation chamber was evaluated as follows. The evaluation of cell infiltration inhibition is an index of the function of blocking various cells.
A 1 cm × 2 cm implantation chamber was implanted and sutured subcutaneously on the back of SD rat (Sprague-Dawley rat). After rearing for 2 weeks, the same site was excised and HE (hematoxylin and eosin) tissue stained sections were prepared. An image of this tissue stained section is shown in FIG. As shown in FIG. 1, the cell infiltration-inhibiting layer was observed, and no cell invasion into the inside was observed, which indicates that the cell infiltration was inhibited.

1 surface modified porous membrane 2 cell infiltration inhibition layer

Claims (21)

An immunoisolation membrane comprising a surface modified porous membrane,
The surface modified porous membrane comprises polysulfone or polyethersulfone,
The ratio of the number of oxygen atoms to the total number of oxygen atoms, nitrogen atoms, carbon atoms, and sulfur atoms on at least one surface of the surface-modified porous membrane is 11.3% or more and 31.2% or less, with respect to the total number An immunoisolation membrane having a nitrogen atom number ratio of 0.1% or more and 4.0% or less. The carbon atom number ratio to the total number of the at least one surface is 65.8% to 80.9%, and the sulfur atom number ratio to the total number is 2.2% to 2.9%. The immune isolation membrane according to Item 1. The immunoisolation membrane according to claim 1 or 2, wherein the surface modified porous membrane comprises a nitrogen-containing polymer. The immunoisolation membrane according to claim 3, wherein the nitrogen-containing polymer is polyvinyl pyrrolidone. The immunoisolation membrane according to any one of claims 1 to 4, wherein the at least one surface is both surfaces. The immunoisolation membrane according to any one of claims 1 to 5, comprising the surface-modified porous membrane. The immunoisolation membrane according to any one of claims 1 to 6, wherein the thickness of the surface modified porous membrane is 1.0 μm to 200 μm. The immunoisolation membrane according to any one of claims 1 to 7, wherein the surface-modified porous membrane has a layered compact portion in which the pore size is minimized. The immunoisolation membrane according to claim 8, wherein the pore diameter is continuously increased in the thickness direction from the dense portion toward at least one surface of the surface-modified porous membrane. The immunoisolation membrane according to claim 8 or 9, wherein the dense portion is biased to one surface X of the central portion of the thickness of the surface-modified porous membrane. A grafting chamber for containing biological constituents, wherein
The implantation chamber having the immunoisolation membrane according to any one of claims 1 to 10 at least a part of the boundary between the inside and the outside of the implantation chamber. A grafting chamber for containing biological constituents, wherein
The immunoisolation membrane according to claim 10 is provided on at least a part of the boundary between the inside and the outside of the transplantation chamber,
The implanting chamber, wherein the surface X of the surface modified porous membrane is on the inner side. The implantation chamber according to claim 11 or 12, wherein the biological construct is a cell. A device for transplantation, wherein the biological composition is contained in the chamber for transplantation according to any one of claims 11 to 13. The implantable device according to claim 14, wherein the biological composition releases a biologically active substance. The implantable device according to claim 15, wherein the physiologically active substance is insulin. A method of producing an immunoisolation membrane, which comprises performing physical oxygen atom permeation treatment on the surface of a porous membrane. The method for producing an immunoisolation membrane according to any one of claims 1 to 10, wherein the surface modified porous membrane is subjected to physical oxygen atom permeation treatment on the surface of the porous membrane containing polysulfone or polyethersulfone. A method of manufacture comprising obtaining a quality membrane Casting a casting solution containing polysulfone or polyethersulfone on a support;
The porous membrane is further obtained by a method including applying temperature-controlling temperature / humidity air to the surface of the casted liquid film, and immersing the film obtained after applying temperature-controlling air-conditioning in a coagulating solution. The production method according to claim 17 or 18, comprising. The method according to claim 19, wherein the membrane-forming solution contains polyvinyl pyrrolidone. The manufacturing method according to any one of claims 17 to 20, wherein the physical oxygen atom permeation treatment is plasma treatment.

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