JPH024302B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a metabolic support device for an artificial liver.
More specifically, the present invention relates to a metabolic support device for an artificial liver that allows material exchange between a patient's blood and a medium in which free hepatocytes collected from a different species of animal are suspended, via an ultrafiltration membrane.

[Conventional technology]

The liver is an important organ that has two functions: detoxification, such as conjugation of various compounds, and synthesis of substances such as sugar, ammonia, and lipids, as well as plasma proteins such as albumin. It is broadly classified into so-called metabolic capacity.

When these functions of the liver deteriorate, an artificial device is required to compensate for the decline, and therefore various liver function support devices have been devised. Some adsorbents such as activated carbon and ion exchange resins have been used clinically to aid in detoxification.

However, it is not possible to support complex metabolic functions using adsorbents alone, and there are limits to the therapeutic results of adsorbents. Therefore, in order to support the decreased liver function of patients, it is considered essential to develop a metabolic support device that utilizes the metabolic ability of hepatocytes collected from xenobiotic animals. Studies conducted to date based on this idea include those that use excised animal livers as they are, and those that use them as sections. However, using suspended free hepatocytes is the most convenient method for handling [for example, Japanese Patent Application Laid-Open No. 53-94496,
Olumide, F. et al: Surg. 82 (5), 599 (1977),
Shinichi Kasai et al.: Artificial Organs 14 (1), 228 (1985)].

On the other hand, in a metabolic support device that uses liver collected from a xenogeneic animal, if proteins produced or released from xenogeneic animal liver cells enter the patient's body,
It is obvious that it causes an adverse immunological reaction in the patient.

As suggested by Olumide et al., placing a membrane between the patient's blood and the hepatocyte suspension medium, such as the Cyprofan membrane, which allows only low-molecular substances to pass through, does not fully utilize the metabolic capacity of animal hepatocytes. Therefore, it is unsatisfactory as a metabolic support device.

In addition, in the metabolic support device disclosed in the above-mentioned Japanese Patent Application Laid-open No. 53-94496, approximately 0.01
A membrane material made of a polymeric material with a pore size of ~0.5μ is used, and the hepatocyte suspension medium is passed through this membrane, and the resulting liquid is injected into the patient's body. Under such conditions, proteins produced or released from hepatocytes may enter the patient's body, which is also unsatisfactory as a metabolic support device.

On the other hand, Kasai et al. have focused on the importance of metabolism of medium-molecular weight substances in hepatocytes. In the metabolic auxiliary device devised by them, patient blood or plasma and hepatocyte suspension medium come into contact through an ultrafiltration membrane with a molecular weight cutoff of 20,000 to 300,000, and the gap between the two is mainly caused by diffusion. Material exchange takes place [Artificial Organs 11 (6), 941 (1982); 13 (2), 626 (1984);
14 (1), 228 (1985)]. They connected this metabolic support device to galactosamine-deficient dogs and perfused them, and found it to be effective in extending the dogs' survival.

Although the effectiveness of Kasai et al.'s metabolic support device has been demonstrated, it has various problems due to its structure. That is, in their device, hollow fibers, which are ultrafiltration membranes, are packed in multiple layers at a low packing density from side wall to side wall of the cell suspension culture tank, and are fixed to both walls with potting material. . The cell suspension medium outside the hollow fibers is stirred by air or oxygen bubbling, and material exchange occurs between the cell suspension medium and the blood or plasma flowing inside the hollow fibers.

[Problem that the invention seeks to solve]

Although it is possible to conduct animal experiments using their equipment, if you try to expand the culture tank, ultrafiltration membrane surface area, etc. and use it clinically, you will encounter the following difficulties. The main ones are: (1) It is difficult to achieve uniform circulation of the cell suspension medium by bubbling alone, but it is difficult to design a system that includes other stirring means.

(2) It is difficult to design a blood port to keep the priming volume small.

(3) In order to make it a practical device, it is necessary to make the hollow fiber ultrafiltration membrane and cell culture tank together into a disposable device, which is not suitable for mass production.

(4) Bubbling is harmful to maintaining hepatocyte viability.

The present invention aims to solve the above-mentioned difficulties by moving the place where the cell suspension medium exchanges substances with the patient's plasma via the hollow fiber ultrafiltration membrane from the inside of the cell bath to the outside. It aims to achieve this purpose.

[Means for solving problems]

The above object is achieved by the present invention as described below.
That is, the metabolic support device according to the present invention is characterized by comprising the following parts.

(a) A floating culture tank for free hepatocytes collected from a different species of animal; (b) A cylindrical tube composed of a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 20,000 to 300,000, preferably 60,000 to 150,000. module, (c) Plasma separator, (d) Liquid feed for continuously removing floating hepatocytes along with their culture medium from the culture tank, perfusing the inside of the hollow fibers of the cylindrical module, and then returning it to the culture tank. mechanism, and (e) the blood from the patient is led to a plasma separator, the plasma obtained from this is continuously perfused on the outside of the hollow fiber of the cylindrical module, and then delivered to the patient together with the blood cell components obtained from the plasma separator. Liquid feeding mechanism for returning liquid.

In the present invention, floating hepatocytes are taken out from a culture tank together with their culture medium, passed through a cylindrical module composed of a hollow fiber ultrafiltration membrane, and then returned to the culture tank. sometimes,
This procedure is based on the newly discovered finding that this procedure has little detrimental effect on hepatocyte viability and biochemical indicators.

When passing the cell suspension medium through a cylindrical module composed of a hollow fiber ultrafiltration membrane,
It is necessary to pass the hepatocytes together with their culture medium through the hollow fiber ultrafiltration membrane. If hepatocytes are perfused with their culture medium on the outside of the hollow fiber separation membrane, the hepatocytes will be trapped in areas where the flow is slow and stagnant due to the structure of the cylindrical module. Because culture conditions such as oxygen concentration are poor in areas of stagnation, hepatocytes in these areas die as a result.
This results in a rapid decrease in the survival rate of hepatocytes throughout the culture tank.

As mentioned earlier, Kasai et al.'s metabolic support device has several design difficulties when expanding its capacity. According to the present invention, each of these difficulties can be solved as follows.

(1) Since flexibility is given to the culture tank design, its material, shape, stirring method, ventilation method, etc. can be optimized for free hepatocyte suspension culture.

(2) Any plasma separator can be used. That is, it is possible to select a blood port with a small priming volume, leading to a reduction in the burden on the patient.

(3) The cylindrical module composed of the hollow fiber ultrafiltration membrane, the plasma separator, and the piping part of the liquid feeding mechanism can be easily made into disposable containers, and sterilized products can be mass-produced.

Next, a detailed description will be given of each part constituting the present invention.

(a) Culture tank: The culture tank can be any culture tank capable of culturing free hepatocytes collected from a heterologous animal by suspending them in a medium. However, one of the effects of the present invention is that it provides flexibility in culture tank design. Therefore, the specifications of the culture tank are, of course, to maintain the survival rate of free hepatocytes for as long as possible and to maximize the function of hepatocytes as expressed by biochemical indicators. It can be possible.

One example is the ventilation method. Bubbling oxygen or air directly into the medium in which hepatocytes are suspended in order to maintain the oxygen concentration within the bath is detrimental to hepatocyte viability. Hepatocyte survival rates can be improved over a longer period of time if oxygen is supplied only from the surface of the medium or by bubbling into the medium without hepatocytes suspended in it and then adding the medium to the medium in which hepatocytes are suspended. maintained. Therefore, it is preferable to use such a ventilation method. Such a ventilation method can be employed for the first time in the surrogacy assisting device according to the present invention.

In the case of a culture tank in which a hollow fiber ultrafiltration membrane is fixed inside the culture tank, it is difficult to employ the aeration method described above. Or even if it were possible, it would not be suitable for mass production.

In addition to the aeration method, the metabolic support device according to the present invention also has various aspects such as the shape and material of the culture tank,
The capacity can also be arbitrarily selected depending on the purpose of use of the metabolic support device.

It goes without saying that it is preferable that a device for appropriate stirring, a device for keeping warm, and a device for monitoring and adjusting culture conditions such as pH, temperature, and oxygen concentration are attached.

Here, we will discuss free hepatocytes collected from a xenobiotic animal suspended in a culture tank. Depending on the purpose of use, liver cells from rats, dogs,
Berry and other animals such as pigs, cows, and apes
Friend's collagenase perfusion method or Seglen
It can be prepared using a modified method thereof, etc. Cryopreserved hepatocytes can also be used. The medium used is preferably one that maintains the survival rate of hepatocytes over a longer period of time and can maximize the functions of hepatocytes as expressed by biochemical indicators. Generally, fetal bovine serum is added,
MEM medium or WE medium can be used. Hormones such as insulin and dexamethasone, or fluorocarbons as oxygen transport agents are preferably added. Serum-free media that have been commercially available in recent years can also be used depending on the purpose. The concentration for suspending cells is 10 to 1×
A range of 10 ⁶ pieces/ml is preferable.

(b) Cylindrical module: In the cylindrical module, the hepatocyte suspension medium that perfuses the inside of the hollow fiber ultrafiltration membrane and the plasma that perfuses the outside of the hollow fiber ultrafiltration membrane pass through the ultrafiltration membrane. contact, and an exchange of substances takes place between the two, primarily by diffusion.

Ultrafiltration membranes are used in so-called artificial kidneys to effectively and safely transfer the active ingredients in the hepatocyte suspension medium into the patient's blood and the harmful components in the patient's plasma into the hepatocyte suspension medium. A highly water-permeable membrane module with a molecular weight cut-off of around 20,000 to 300,000, preferably around 60,000-150,000, which is called a protein leak membrane, is used.

Here, the molecular weight cutoff refers to indicator substances with clear molecular weights (MW) such as IgM (MW960000), apoferritin (MW480000), IgG (MW160000), albumin (67000), egg albumin (MW45000),
Pepsin (MW35000”, Inulin (MW5200)
The transmittance is measured using a solution such as, and a graph with transmittance (rejection rate) and MW as axes is drawn, and this is the molecular weight that indicates a transmittance of 10% (rejection rate of 90%).

Furthermore, in the cylindrical module, the inside of the hollow fiber is smooth and the inner diameter is small in order to allow free hepatocytes to pass through the inside of the hollow fiber smoothly.
It is preferable that the thickness is 100μ or more.

The ultrafiltration membrane area of the cylindrical module, other shapes and specifications, the material of the ultrafiltration membrane, etc. can be arbitrarily selected depending on the purpose of use of the metabolic auxiliary device. The material may be any material that is safe for living organisms, such as polymethyl methacrylate, cellulose acetate, polyvinyl alcohol, polycarbonate, and polysulfone.

(c) Plasma separator: In the plasma separator, part of the plasma from the patient's blood is taken out and continuously perfused outside the hollow fibers of the cylindrical module.

As a plasma separator, it has a high plasma separation speed.
Any membrane type plasma separator that also has good permeability to plasma proteins can be used. Hollow fiber separators, which have been recently developed, are preferably used because they have a small amount of blood filling. The membrane material may be any material that is essentially safe, such as polymethyl methacrylate, cellulose acetate, polyvinyl alcohol, polyethylene, polypropylene, polycarbonate, or polysulfone, and its pore diameter is usually 0.05 to 2 microns, preferably 0.1 to 1.0 microns. It is on the order of microns.

(d), (e) Liquid feeding mechanism: In the metabolic auxiliary device according to the present invention, the liquid feeding mechanism connects each of the above-mentioned parts and includes piping, pumps, and other components for guiding plasma and hepatocyte suspension medium. Consists of parts.

As for the piping, a sterilized vinyl chloride blood circuit used in so-called artificial kidneys is preferably used, but it goes without saying that piping made of other materials such as silicone can also be used. Furthermore, the inner diameter of the pipe can be arbitrarily selected depending on the purpose of use of the metabolic support device.

As for the pump for feeding the culture medium and blood, the pressure measuring device, and the heating coil, devices that have been developed for clinical use in artificial kidneys and plasma exchange (plasmapheresis) are preferably used. below,
The present invention will be explained with reference to FIG. 1st
The figure shows one example of the device of the present invention.

A circuit 2 equipped with a pump 1 feeds a hepatocyte suspension medium from a culture tank 3 into the hollow fibers of a cylindrical module 4. This medium exchanges substances with the patient's plasma via the hollow fiber ultrafiltration membrane, and then returns to the culture tank 3 via the circuit 5.

The patient's blood is led to a plasma separator 8 via a circuit 7 by the action of a pump 6. A portion of plasma is taken out from the plasma separator 8 and perfused outside the hollow fibers of the cylindrical module 4 via a circuit 10 by the action of a double pump 9. Thereafter, the plasma passes through a circuit 11 under the action of the dual pump 9, merges with blood cell components, and returns to the patient's body through a circuit 12.

In FIG. 1, only one pressure measuring device 13 is shown, and the heating coil and drip embers are not shown at all. but,
These accessories can be arranged as appropriate depending on the purpose of using the metabolic assist device.

Furthermore, the arrangement of the pumps is not necessarily limited to that shown in FIG. for example,
The dual pump 9 spanning the circuits 10 and 11 in FIG. 1 can also be a series pump.
Instead, if a new pump is placed in the circuit 12, the same flow of culture medium and blood as before can be ensured. In this way, the arrangement of the pumps can be arbitrarily set as long as the flow of the culture medium and blood can be ensured.

Next, the flow rates of the culture medium and blood to be sent by the liquid sending mechanism will be described. These can be set arbitrarily as long as the following conditions are met. In other words, it is necessary to minimize the decline in survival rate and functional damage of hepatocytes, to minimize the burden on patients or experimental animals, and to achieve the most efficient exchange of substances between the culture medium and blood. It is something to do.

More specifically, the ratio of medium flow rate to plasma flow rate in the cylindrical module is preferably greater than 1. Further, the blood flow rate guided to the plasma separator and the plasma flow rate drawn therefrom are set in consideration of the performance of the plasma separator and the burden on the patient.

Further, in the metabolic support device according to the present invention,
It is not necessarily necessary to start perfusion of patient blood at the same time as suspending hepatocytes in the culture tank. When cells are suspended in a medium, they do not immediately exhibit their full potential; some culturing time is required. Therefore, in such a case, in order to reduce the burden on the patient, it is preferable to start perfusion of the patient's blood after some culture time.

EXAMPLES Hereinafter, the effects of the present invention will be explained in more detail with reference to Examples.

Example 1 In order to investigate the metabolic ability of the metabolic assist device according to the present invention, changes in the rate of ammonia removal from 2 Hank's solution used instead of patient blood and the survival rate of hepatocytes in a culture tank were investigated.

Cells were collected from a beagle dog weighing 8 kg by the collagenase perfusion method according to a modification of Seglen, and after washing, they were suspended in MEM medium at a concentration of 4 x 10 ⁶ cells/ml. This medium was previously supplemented with 10% fetal bovine serum, 10 ⁻⁶ M insulin and 10 ⁻⁵ M dexamethasone. The culture tank was made of glass and had a stirring blade, and was kept in a constant temperature bath at 37°C. Oxygen and carbon dioxide gas were fed into the gas phase portion of the culture tank at a flow rate controlled by a flow meter. The dissolved oxygen partial pressure in the medium is in the range of 100-200 mmHg,
The PH in the medium was kept in the range of 7.1-7.3.

As a cylindrical module, it has a molecular weight cutoff of 100,000.
A hollow fiber ultrafiltration membrane made of PMMA was used. The membrane area is 0.4m ² and the hollow fiber inner diameter is
It was 300μ.

The plasma separator is "PlasmaTux" PS-05H (manufactured by Toray Industries, Inc.), which is made of PMMA hollow fibers and has a membrane area of ^0.5m2 , an average pore diameter of 0.3μ, and a maximum pore diameter of 0.5μ.
was used. The inner diameter of the hollow fiber was 330μ.

A vinyl chloride blood circuit with an inner diameter of 3.5 mm and an outer diameter of 6.0 mm was used to connect the above-mentioned parts, and the blood was delivered using a blood pump as follows. That is,
The hepatocyte suspension medium was drawn out from the culture tank at a rate of 100 ml/min, perfused inside the hollow fibers of the cylindrical module, and then returned to the culture tank. From the Hank's solution storage tank, which was placed in place of the patient's blood, 120 ml/min is introduced into the plasma separator, and some of the fluid is drawn out to the plasma side at 40 ml/min. was returned to the Hank's solution storage tank along with Hank's solution.

Ammonium chloride was added to Hank's solution twice, and changes in ammonia concentration in Hank's solution and hepatocyte suspension medium were measured. Measurements were conducted using a modified Fujii-Okuda method. The results are shown in Figure 2. Furthermore, the survival rate of floating hepatocytes counted by trypan blue staining is also shown in FIG. 2.

Furthermore, Fig. 3 shows changes in ATP content of floating hepatocytes measured using a luminoaggregometer.

There was a slight decrease in the survival rate of hepatocytes even after 12 hours of culture. intracellular
The amount of ATP started to increase at the same time as the start of culture, and 7-8
It reached its maximum after an hour. Even after 12 hours, the level was still higher than at the start of the culture. Ammonia added to Hank's solution was removed even after 6 hours at a rate completely comparable to the culture start time. Almost no increase in ammonia concentration was observed on the hepatocyte suspension medium side.

From the above, it can be said that the metabolic support device according to the present invention has metabolic ability and is stable over a long period of time.

〔Effect of the invention〕

The present invention enables a design for capacity expansion and an oxygen supply method that does not damage hepatocytes by extending a cylindrical module made of a hollow fiber ultrafiltration membrane to the outside of the cell culture tank. In addition, by perfusing hepatocytes through the inside of the hollow fibers of the cylindrical module and perfusing plasma through the outside of the hollow fibers, it is possible to maintain favorable culture conditions for hepatocytes, thereby maintaining the survival rate of hepatocytes and improving biochemical indicators. The function of liver cells can be maximized. In addition, by setting the molecular weight cutoff of the hollow fiber ultrafiltration membrane that separates the hepatocyte suspension medium and the patient's plasma from 20,000 to 300,000, the active ingredients in the hepatocyte suspension medium can be transferred into the patient's blood and the patient's plasma. Harmful components can be effectively and safely transferred into the hepatocyte suspension medium.

[Brief explanation of drawings]

FIG. 1 is a schematic explanatory diagram showing an example of an embodiment of the apparatus of the present invention. FIG. 2 shows changes in ammonia concentration and survival rate of floating hepatocytes in Example 1, and FIG. 3 shows changes in ATP content in floating hepatocytes in Example 1. 1, 6, 9...pump, 3...culture tank, 4...
Cylindrical module, 8...Plasma separator.

Claims (1)

[Scope of Claims] 1 (a) A culture tank for free hepatocytes collected from a different species of animal, (b) A cylindrical module composed of a hollow fiber ultrafiltration membrane having a molecular weight cutoff of 20,000 to 300,000, (c) a plasma separator; (d) a liquid delivery mechanism for continuously removing floating hepatocytes from the culture tank along with their culture medium, perfusing the inside of the hollow fibers of the cylindrical module, and then returning the cells to the culture tank; and (e) to lead the blood from the patient to a plasma separator, to continuously perfuse the outside of the hollow fibers of the cylindrical module with the plasma obtained from this, and then to return it to the patient together with the blood cell components obtained from the plasma separator. liquid delivery mechanism. A metabolic support device for an artificial liver, characterized by having the following.