JP3072368B2 - High-density culture method of hepatocytes and apparatus therefor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a technique for culturing hepatocytes.

[0002]

2. Description of the Related Art The liver has a function of metabolizing various substances as a central organ of substance metabolism. In the liver, for example, production and secretion of bile, metabolism of nutrients (sugars, proteins, lipids) and vitamins, inactivation of biologically active substances (toxins, steroids, hormones, etc.), plasma proteins (albumin, blood coagulation factors) Etc.) and immunity (Kupper cells).

[0003] In fulminant hepatitis, these liver functions are rapidly lost. Attempts to medically treat this fulminant hepatitis have resulted in mortality rates of over 70% and there is currently no effective treatment other than liver transplantation. For example, dialysis, filtration,
It is difficult to rescue patients with severe liver failure by purely artificial techniques such as adsorption.

[0004] This is because the function of the liver is centered on the metabolic function which is difficult to substitute by a mechanical method. As a solution to this, development of a hybrid artificial liver in which a hepatocyte culture system is incorporated into an apparatus and its metabolic function is utilized is expected.

[0005]

SUMMARY OF THE INVENTION A packed bed type culture apparatus in which animal cells such as hepatocytes are immobilized at a high density on a particulate carrier having a microporous three-dimensional reticulated porous structure, and the immobilized material is filled. Are bioreactors that produce useful substances,
As an artificial liver function assisting device such as an artificial liver, useful applications can be expected.

The present inventor described one example of a cell immobilized product in JP-A-6-277050 and JP-A-5-76364. In such a cell immobilized product, animal cells can be immobilized at high density and cultured.

[0007] In order to develop a hybrid artificial liver that can be used for clinical application, a technique for culturing hepatocytes at a higher density and in a larger amount is indispensable. The present inventor has developed a packed-bed reactor using a porous polyvinyl formal (PVF) resin as a carrier for cell immobilization, which is a porous resin.

According to the study of the present inventor, hepatocytes can be cultured at a high density of 1 × 10 ⁷ cells / cm ³ in such a reactor, and the metabolic ability of the hepatocytes can be maintained for one week or more. ((1) K. Yanagi, H. Miyoshi, H.
Fukuda and N. Ohshima (1992), A packed-bed reactor
utilizing porous resin enables high density cultu
re of hepatocytes., Appl. Microbiol. Biotechnol.,
37: 316-320, (2) H. Miyoshi, K. Yanagi, H. Fukuda,
N. Ohshima (1994), Long-term continuousculture of
hepatocytes in a packed-bed reactor utilizing por
ous resin., Biotechnol.Bioeng., 43: 635-644, (3)
H. Miyoshi, K. Yanagi, H. Fukudaand N. Ohshima (1
996), Long-term performance of albumin secretion o
f hepatocytes cultured in a packed-bed reactor uti
lizing porous resin., Artif.Organs, 20: 803-807
].

An object of the present invention is to maintain hepatocytes cultured at a high density in an even more optimal state.

[0010]

According to the present invention, hepatocytes are immobilized on the surface and in pores of a porous resin carrier, and the porous resin carrier is arranged in a reactor. And an outlet, the liquid medium is introduced from the inlet of the reactor, the resin porous carrier is immersed in the liquid medium, the liquid medium is brought into direct contact with the hepatocytes, The liquid medium is taken out from the outlet of the reactor, the liquid medium is returned to the inlet of the reactor, and the liquid medium is perfused to culture the hepatocytes. The present invention relates to a high-density culturing method for hepatocytes in which the oxygen concentration of a liquid medium is maintained at 204 to 969 μM.

[0011] The present invention also provides a method for immobilizing hepatocytes on a porous resin carrier, immersing the porous resin carrier in a liquid medium, and culturing the hepatocytes. The present invention relates to a high-density culture method for hepatocytes maintained at 300 to 400 µM.

[0012] The liver is known to exhibit an active regenerative ability in vivo, but under artificial culture conditions, hepatocytes hardly exhibit an ability to proliferate and rapidly lose their activity. The present inventor considers that it is important to overcome these problems in order to develop a hybrid artificial liver.

In order to develop a practical artificial liver,
At a high density of 10 ⁷ or more per cm, a large amount of hepatocytes of the order of 10 ¹⁰ , equivalent to about 1/3 of the liver of a living body, can be artificially produced while maintaining metabolic capacity comparable to that of a living body. Culture technology is essential. For clinical applications, it is essential to develop a reactor that can be easily scaled up and aseptically operated.

As described above, the present inventor has previously used a porous polyvinyl formal resin as a carrier.
Developed packed bed reactor and developed 10 ⁷ cells / cm ³ -P
It has been reported that hepatocytes can be cultured while maintaining the metabolic ability equivalent to that of a monolayer culture using a dish at a hepatocyte density of VF for one week or more (see the above-mentioned reference).

The present inventor has studied various culture conditions in order to further improve such a culture method and culture hepatocytes at a high density under optimal conditions.

As a result, surprisingly, the inventor
It has been found that a condition of high oxygen concentration, which was conventionally considered unfavorable because of damaging cells, is rather preferable for high-density culture of hepatocytes.

The present inventor has found that by controlling the oxygen concentration in the liquid medium within a predetermined range, the ammonia metabolizing ability of hepatocytes can be improved more than twice as much as the conventional one, and completed the present invention. I let it.

[0018] The present invention provides a method for culturing hepatocytes having a predetermined density or higher density and consuming a large amount of oxygen.
This is due to the fact that the supply of oxygen by the liquid medium cannot keep up with its consumption, and the fact that the flow of the liquid medium is biased has a tendency to deteriorate the activity per unit cell of these cells.

According to the present invention, by controlling the oxygen concentration in the liquid medium within a predetermined range, the function of the cultured hepatocytes can be maintained in a more favorable state.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, it is preferable to use a carrier having a microporous three-dimensional network porous structure as a resin porous carrier for immobilizing hepatocytes. Such carriers include those made of various materials such as a porous polyvinyl formal resin.

Examples of the hepatocytes include hepatocytes expected to be applied to an artificial liver function assisting device.

In the present invention, the oxygen concentration in the liquid medium is adjusted to 2
Set in the range of 04-969 μM. If oxygen in this range is provided to the liquid medium, the cultured hepatocytes can maintain good functions. The oxygen concentration of 204 μM corresponds to the dissolved oxygen concentration when oxygen is dissolved in a liquid medium under the usual conditions of 5% CO ₂ -20% O ₂ . Also, 9
The oxygen concentration of 69 μM corresponds to the dissolved oxygen concentration when oxygen is dissolved in the liquid medium under the condition of 5% CO ₂ -95% O ₂ .

Surface oxygen can be used to introduce oxygen into the liquid medium. The treatment of exposing the liquid medium to a gaseous phase having a high oxygen concentration does not require a special device and is less likely to be contaminated.

[0024] The oxygen concentration in the liquid medium is 204 to 969.
The control to μM can be performed by exposing the liquid medium to a gas having an oxygen partial pressure of 152 to 722 mmHg (corresponding to an oxygen concentration of 20 to 95%) under normal atmospheric pressure.

To efficiently introduce oxygen, it is preferable to use an oxygenator. The oxygenator includes various means for dissolving oxygen in the liquid medium. For example, oxygen can be dissolved in the liquid medium by passing the liquid medium through a Teflon tube and exposing the tube to a high concentration of oxygen. At this time, if the surface area of the tube is increased, oxygen can be more easily dissolved in the liquid medium.

In the present invention, the flow velocity of the liquid medium in contact with the cultured hepatocytes is set in a range of a linear velocity of 5.4 to 10.8 cm / min. These linear velocities correspond to, for example, a flow rate of 17 to 34 mL / min for a reactor having an inner diameter of 20 mm. Within this range, the cultured hepatocytes maintain good metabolic capacity. At a linear velocity of less than 5.4 cm / min, sufficient oxygen cannot be supplied to the hepatocytes. On the other hand, when the linear velocity exceeds 10.8 cm / min, the shear stress that rubs the hepatocytes causes damage to the hepatocytes. At these linear velocities, hepatocyte viability decreases, resulting in an increase in alanine transaminase (ALT) concentration in the liquid medium. ALT, also called glutamate-pyruvate transaminase (GPT), is an enzyme released outside hepatocytes when hepatocytes are destroyed.

Hereinafter, the present invention will be described in detail with reference to the drawings. FIG. 1 is a schematic diagram of an example of a culture device. FIG.
Is suitable for the high-density culture method of hepatocytes of the present invention, and can control the metabolic ability of hepatocytes extremely well. In the apparatus 1 of FIG. 1, a packed bed reactor 2 is used. Various types of reactors such as a flat plate type, a stacked type, a packed bed type, and a fluidized bed type can be used in the apparatus of the present invention.

In the packed bed reactor 2 shown in FIG. 1, a cylindrical reactor 3 is filled with a resin porous carrier 4. The reactor 3 has a lower port 3a into which the liquid medium 5 flows.
And an upper port 3b through which the liquid medium 5 flows out. The lower port 3a can be provided with a flow divider for adjusting the flow of the liquid medium.

For example, as shown in FIG.
The lower port 3a is filled with a Teflon ball 6 inside. In addition, a plurality of partition plates 7 are provided in the reactor 3 to partition the space filled with the carrier 4 at appropriate intervals so that the flow of the liquid medium 5 can be uniform. A baffle plate or the like can be used as such a partition plate. Furthermore, the reactor 3 can be designed to have a cock 9 into which hepatocytes 8 to be cultured are injected, which is wrapped in a water jacket 10 and through which hot water 11 circulates.

For example, hepatocytes are immobilized on the resin porous carrier 4. The hepatocytes 8 are immersed in the liquid medium 5 and cultured. The liquid medium 5 is sent from the lower port 3 a into the packed bed reactor 2 by the pump 12, processed by the hepatocytes 8, and then exits from the upper port 3 b and stored in the reservoir 13. The reservoir 13 is provided with an upper port 13a for supplying the liquid medium 5. As with the reactor 3, the reservoir 13 can be kept warm by a water jacket or the like. Liquid medium 5 in reservoir 13
Is again perfused into the packed bed reactor 2 from the lower port 3a by the pump 12.

In the present invention, as shown in FIG. 1, high-concentration oxygen 15 is introduced into the liquid medium 5 via the oxygenator 14 before entering the packed-bed reactor 2. Oxygen 15
Is supplied from the upper port 14a of the oxygenator 14. For the liquid medium line 16 passing through the oxygenator 14, for example, a tube having high oxygen permeability such as Teflon is used.
5 is dissolved in the liquid medium 5. High concentration oxygen is supplied from the upper port 13a of the reservoir 13 to the liquid medium 5
Can also be introduced directly.

In the present invention, the liquid medium 5 is caused to flow on the surface of the hepatocytes 8 at a predetermined flow rate. The flow of the liquid medium 5 is rectified by the flow divider, the carrier 4 between the partition plates 7 is stirred, and the bias of the flow of the liquid medium 5 is reduced.
Can be perfused into the packed bed reactor 2.

The culture device of the present invention can be expected to be applied as an artificial liver for culturing hepatocytes. FIG. 2 is a schematic diagram of an example of an artificial liver. As shown in FIG. 2, the culture device of the present invention can function as an artificial liver for a patient with impaired liver function via a plasma separator. At this time, as shown in FIG.
6, a pump 27 and the culture apparatus 1 of the present invention. Blood drawn from the patient's artery is sent through line 28 to plasma separator 26 to separate plasma from the blood.
The plasma is sent to the reservoir 14 of the culture device 1 by the pump 27 via the line 29 and is processed. The processed plasma passes through line 30 in combination with line 31 that returns components other than plasma to the patient and is pumped back to the patient's vein by pump 27.

According to the method of the present invention, the function as an artificial liver is remarkably improved by introducing a predetermined concentration of oxygen into a liquid medium and keeping the flow rate of the liquid medium uniform at a predetermined rate. Can be. By using such a reactor, the activity of hepatocytes per unit cell can be significantly improved.

[0035]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to the drawings based on experimental examples. Experimental Example 1 1. Experimental method In this experimental example, the purpose of this study was to improve the metabolic capacity of hepatocytes by improving the operating conditions in a packed-bed reactor using a porous polyvinyl formal resin. The effect of the perfusion rate of the lip was examined.
First, the effect of oxygen concentration on cultured hepatocytes was examined in a monolayer culture system using a collagen-coated dish, and then examined in a perfusion culture system using a packed-bed reactor.

1-1. Experimental Materials As a carrier for hepatocyte immobilization, polyvinyl formal (PVF resin, manufactured by Kanebo Kasei Co., Ltd.) having an average pore diameter of 250 μm was used after being finely cut into 2 × 2 × 2 mm ³ . As the reactor, a tubular reactor and a flat reactor were used. 250 carriers were filled in a cylindrical reactor having an inner diameter of 20 mm, sterilized in an autoclave, and treated with Hanks' solution.
(ed salt solution) to give a packed bed reactor. Further, a plate-shaped reactor was coated with a 35 mm Petri dish (Petri dish, Falcon 1008) using a 0.06% type I collagen solution to form a monolayer culture.

Hepatocytes were released from male Wistar rats weighing 190-250 g by collagenase perfusion. The survival rate of the hepatocytes at the time of release was 80% or more by a trypan blue exclusion test. 1.2 to 1.6 × 10 ⁸ cells were obtained per animal.

As the basic medium, Williams' E
In the medium, dexamethasone (0.1 μM), insulin (0.1 μM), aprotinin (5,000 KIU /
L), penicillin G (20,000 IU / L), streptomycin (20 mg / L), amphotericin B (5
(0 μg / L) and 10% fetal calf serum.

1-2. Culture method In monolayer culture, 1 × 10 ⁶ viable cells were suspended in 2 ml of medium and seeded on a dish. The medium was oxygenated by surface aeration. Oxygen was dissolved in the medium by changing the oxygen partial pressure in the gas phase of the incubator to 75 to 375 mmHg. The dissolved oxygen concentration in this medium is calculated as 1
Corresponding to 02-510 μM. For example, for surface ventilation:
5% CO ₂ -50% air-45% N ₂ (10% O ₂ , 7
5 mmHg O ₂ partial pressure, 102 μM O ₂ ), 5% CO
₂ -95% air (20% O _2, 15 2 mmHg of partial pressure of O _2, O ₂ of _{204μM), 5% CO 2 -55} % air -4
0% O ₂ (50% O ₂ , O ₂ partial pressure of 375 mmHg, 5
Conditions such as 10 μM O ₂ ) were used.

In the perfusion culture, a packed-bed reactor was used. FIG. 3 is a schematic diagram of a culture device according to the present experimental example. This apparatus 17 includes a packed bed reactor 18, a reservoir 19, and a roller pump 20. The packed bed reactor 18 and the reservoir 19 are connected by lines 21 and 22. In the middle of the line 21, an oxygenator 23 is provided,
Oxygen was introduced into the medium. Further, by disposing an oxygen electrode 24 in the middle of the line 22, the dissolved oxygen concentration of the medium was measured. This device 17 was maintained at 37 ° C. using a water jacket.

In order to seed the hepatocytes into the packed-bed reactor 18, hepatocytes were suspended in 3 mL of a medium to prepare a hepatocyte suspension, and this was added to the suspension at a rate of 8 to 10 × 1 per module.
As will be 0 ^7, it was injected from the top of the reactor 18. This culture device 17 is placed in a clean bench, and a total amount of 50 mL of the medium is passed through the surface ventilation in the reservoir 19,
It was oxygenated by an oxygenator 23 and perfused. In the oxygenator 23, a silicon tube 16 as shown in FIG. 1 was used. Change the oxygen concentration in the medium (60-470 μM)
Alternatively, by changing the perfusion rate of the medium (17-50 mL / min),
Hepatocytes were cultured for 5 days. The oxygen concentration in the medium was adjusted using the same gas phase as in monolayer culture, and the perfusion rate was adjusted with a pump. In addition, in the perfusion culture, even if oxygen is introduced into the medium through an oxygenator with 5% CO ₂ -95% air (20% O ₂ ), which is a normal culture condition, the result of oxygen consumption by the cultured cells The oxygen concentration of the medium upon exiting the reactor ranges from 200 μM to 150 μM calculated in monolayer culture.
It decreases around M. Therefore, the condition of the oxygen concentration of 60 to 470 μM used in this experiment is the oxygen concentration of the medium at the outlet of the reactor, and 102 to 510 at the inlet of the reactor.
Corresponds to an oxygen concentration of μM.

The experimental protocol will be described with reference to FIG. FIG. 4 is an explanatory diagram showing a culture time and a medium exchange time. Straight lines extending to the left and right indicate the passage of time. The numbers scaled on this straight line represent the culture time. At each of these times, the medium was changed. The black or white arrow below this straight line indicates the type of medium. Black arrows indicate fetal bovine serum in Williams'E medium,
This is a basal medium supplemented with hormones and antibiotics, and the white arrow is a basal medium supplemented with 1 mM ammonium chloride.

As shown in FIG. 4, at time 0, hepatocytes were seeded in a plate-type dish or a packed-bed reactor and culture was started. To remove floating cells, the medium was changed 3 hours after the start of the culture, and thereafter the medium was changed daily. In order to evaluate the ability of hepatocytes to metabolize ammonia, 1 mM ammonium chloride was added to the basal medium 20 hours (day 2) and 68 hours (day 4) after the start of culture, and the ammonia concentration was measured over time. did.

The culture experiment was completed 92 hours (day 5) after the start of the culture, and the number of hepatocytes immobilized in the reactor or dish was measured. The number of hepatocytes was determined by crushing the cells with an ultrasonic crusher, measuring the amount of DNA by the fluorescent dye method,
It was determined by converting the NA amount to the number of cells.

1-3. Evaluation of ammonia metabolism Ammonia is a waste product resulting from the metabolism of amino acids, and converting it to urea is one of the important functions of the liver. The 1 mM ammonia concentration is the highest level commonly found in patients with liver failure.

In order to quantitatively evaluate the ability of hepatocytes to metabolize ammonia under each culture condition, a kinetic analysis was attempted. That is, when ammonia concentration in the medium is measured over time after loading with ammonia, the change in concentration can be approximated by the following equation (1) [N. Ohshima, M. Shiot
a, H. Kusano, G. Wada, T. Tsunetsugu, K. Ookawa an
dK.Yanagi, (1994), Kinetic analysis of the perfo
rmance of a hybrid-typeartificial liver support sy
Stem utilizing isolated hepatocytes. Mater.Sci. En
g., C1: 79-85]. dC / dt = -KmNC (1) where C: ammonia concentration (mM), Km: ammonia metabolic rate constant (L / cell / hr), and N: hepatocyte concentration (cell / L).

FIG. 5 is a graph showing the relationship between the ammonia concentration and the culture time. This graph shows the time course of the ammonia concentration in the culture solution on the second day (◆) and the fourth day (▲) of the culture. The slope of this graph corresponds to KmN,
In this experimental example, the value of KmN was mainly used as an index of metabolic ability.

1-4. In this experiment, the concentration of alanine transaminase (ALT) was measured every time the medium was changed. ALT, also called glutamate-pyruvate transaminase (GPT), is an enzyme released when hepatocytes are destroyed. When 1 g of hepatocytes and 1 × 10 ⁸ cells are destroyed, about 40
The ALT of the IU is released. The lower this value, the less hepatocytes have been damaged in the medium.

2. Results and discussion in monolayer culture 2-1. FIG. 6 is a graph showing the cell immobilization rate and the dissolved oxygen concentration. In this graph, the effect of the dissolved oxygen concentration on the cell immobilization rate can be known. The vertical axis of this graph represents the immobilization rate (%), and the seeded living cells (retained cell densit
y) shows what percentage was fixed after 4 days of culture. As shown in FIG. 6, the cell immobilization rate tended to be low at low oxygen concentrations, and at a oxygen concentration of 200 to 510 μM, the immobilization rate was almost constant at 50 to 70%.

2-2. FIG. 7 is a graph showing the ammonia metabolizing ability. FIG.
In the graph, ● indicates the metabolic capacity on the second day of culture, and Δ indicates the metabolic capacity on the fourth day of culture. The metabolic ability on the second day of culture is significantly improved depending on the dissolved oxygen concentration. In particular, 300-400 μM
Excellent metabolic ability was obtained at an oxygen concentration of. Since the immobilization rate of the cells was almost the same at any oxygen concentration, it was considered that the metabolic capacity per cell was improved under the high oxygen concentration.
On the other hand, the metabolic ability on the fourth day of culture is slightly improved at an oxygen concentration of 200 μM, and the metabolic ability is slightly reduced in both high oxygen and low oxygen concentrations.

2-3. FIG. 8 is a graph showing the amount of ALT released. In this graph, AL released into the medium after 4 days of culture
Shows the total amount of T. As shown in FIG. 8, the release of ALT is
It can be seen that the oxygen concentration tends to be slightly higher at low oxygen concentration, but is hardly affected by the dissolved oxygen concentration.

3. Results and consideration in packed bed reactor 3-1. FIG. 9 is a graph showing the cell immobilization rate and the dissolved oxygen concentration. This immobilization rate was obtained by examining the effect of the dissolved oxygen concentration, and shows, as in the case of monolayer culture (FIG. 5), what percentage of the inoculated living cells were immobilized after 4 days of culture. However,
In this experiment, the medium was perfused (17 mL / min) using a packed bed reactor as shown in FIG. As shown in this figure, the immobilization rate of the cells was improved by increasing the dissolved oxygen concentration. Since the cell immobilization rate at the time of one day of culture is about 30%, the cells after four days of culture
An immobilization rate of ４０40% is a significant improvement.

3-2. FIG. 10 is a graph showing the ammonia metabolizing ability. The symbols in this figure are also the same as those in the monolayer culture of FIG.
In the perfusion culture, the metabolic capacity increases due to an increase in the oxygen concentration on both the second day of culture (●) and the fourth day of culture (△). However,
Even if it exceeds 300 μM, there is almost no difference.

3-3. Released ALT Amount and Dissolved Oxygen Concentration FIG. 11 is a graph showing the amount of released ALT.
As shown in FIG. 11, up to an oxygen concentration of 300 μM, A
The amount of LT released decreases with an increase in oxygen concentration.
It slightly increases where it exceeds 0 μM.

3-4. FIG. 12 is a graph showing the effect of the perfusion rate on the cell immobilization rate. The immobilization rate was obtained by examining the effect of the perfusion rate using a gas phase of 5% CO ₂ -95% air (20% O ₂ ) in an oxygenator with an oxygen concentration of 5%. In this figure,
As in the case of examining the effect of oxygen concentration (FIG. 9), it shows what percentage of the seeded cells was fixed after 4 days of culture. As shown in FIG. 11, the immobilization rate of the cells was around 20% at any perfusion rate and slightly improved to the right, but there was almost no difference.

3-5. Ammonia metabolism ability and perfusion rate FIG. 13 is a graph showing ammonia metabolism ability. The symbols in the figure are the same as those in FIG. 10 showing the effect of the oxygen concentration. As shown in FIG. 13, the perfusion rate was slightly low at a low perfusion rate, and hardly affected by the perfusion rate.

3-6. FIG. 14 is a graph showing the amount of ALT released.
As shown in this figure, up to a perfusion rate of 30 mL / min,
With the perfusion rate, the release of ALT tends to decrease slightly. In this packed bed reactor, 17-34 mL
Per minute, preferably 20-30 mL / min, was considered appropriate.

3-7. Ammonia metabolic capacity and dissolved oxygen concentration per cell Instead of using the KmN value representing the metabolic capacity of the entire apparatus, the dissolved oxygen concentration is calculated using the Km value representing the metabolic capacity per immobilized cell. The effect on metabolic capacity was evaluated. FIG. 15 is a graph showing this ammonia metabolism ability. As the cell number, the number of cells after the completion of the culture for 4 days was used, and the result of metabolic ability on the second day of the culture was obtained. In the monolayer culture using a dish (デ ィ), the metabolic capacity per cell increased as the oxygen concentration increased. On the other hand, in the perfusion culture using a packed-bed reactor (perfusion rate: 17 mL / min), the metabolic ability was poor at low oxygen concentration, but reached almost constant at oxygen concentration of 200 μM or more. The metabolic capacity of this packed-bed reactor is better than that of a dish culture under 20% oxygen, which is considered to be almost comparable to the metabolic capacity of hepatocytes in vivo. Sex was shown.

Experimental Example 2 1. Experimental Method In this experimental example, under the same conditions as in Experimental Example 1, the influence of the oxygen concentration in the medium and the effect of the perfusion rate of the medium were examined. However, the oxygenation of the culture medium was 5% CO ₂ -50% air-45%
N ₂ (10% O ₂ ), 5% CO ₂ -95% air (20%
O ₂ ) and 5% CO ₂ -55% air-40% O ₂ (50
% O ₂ ), and oxygenation of the medium in perfusion culture is performed by surface aeration into the reservoir, and the perfusion rate of the medium is set to 8, 17, 34 mL / min. did.

In this experimental example, in order to evaluate the ability of metabolizing ammonia, 1 mM ammonium chloride was added to the medium on the first and third days of the culture, and the culture experiment was terminated on the fourth day of the culture. In this experimental example, the value of Km in the approximate expression (1) was calculated by the least squares method, and was used as an index of the ammonia metabolizing ability per cell.

[0061] 2. Results and discussion in monolayer culture 2-1. FIG. 16 is a graph showing the ammonia metabolizing ability. Columns with diagonal lines indicate ammonia metabolizing ability on the first day of culture, and columns without diagonal lines indicate ammonia metabolizing ability on the third day of culture. As shown in FIG.
The metabolic capacity of hepatocytes is the best under the condition of% oxygen concentration,
Metabolic capacity decreased under high and low oxygen conditions. In particular, it was considered that high oxygen concentration conditions had little effect on metabolic ability in the initial stage of culture, but had a bad influence on maintenance of cell activity.

3. Results and consideration in packed bed reactor 3-1. Effects of Perfusion Rate and Dissolved Oxygen Concentration on Cell Immobilization Rate Five perfusion culture experiments were performed under the conditions shown in Table 1 for the perfusion flow rate of the medium and the oxygen concentration during culture. As shown in Table 1, after 4 days of culture, 15 to 20% of the living cells seeded in the reactor were immobilized in the substrate. The cell immobilization rate was slightly lower under extreme conditions of high oxygen concentration and low oxygen concentration (Experiment Nos. 4 and 5), suggesting that there was a problem in maintaining the cells under these conditions. On the other hand, at an oxygen concentration of 20%, the perfusion rate of the medium did not affect the immobilization rate of the cells. In the study of the present inventors using the same packed-bed reactor, the immobilization rate of cells in the substrate was about 30% on the first day of perfusion culture and about 15% on the seventh day. It was considered that the number of hepatocytes immobilized in the PVF resin gradually decreased during the period of the perfusion culture.

[0063]

[Table 1]

3-2. FIG. 17 is a graph showing the relationship between the ammonia concentration and the culture time. This graph shows the time-dependent change in the ammonia concentration in the culture solution on the first day (■) and the third day (●) of the culture. This figure shows the time-dependent change of the ammonia concentration in the medium in the perfusion culture. The solid line and the broken line are the results of the simulation by the equation (1). The metabolic capacity was evaluated by calculating the value of Km · N from the fitting curve, dividing the value by the cell density (N) at the end of culture, and
An index of ammonia metabolic capacity per unit, Km, was obtained. In the perfusion culture, the immobilized cell density on the first day of culture is estimated to be higher than the value at the end of the culture. Therefore, the Km value on the first day of culture in the perfusion culture group shown below is overestimated. it is conceivable that.

3-3. FIG. 18 is a graph showing the ammonia metabolizing ability. Columns with diagonal lines indicate ammonia metabolizing ability on the first day of culture, and columns without diagonal lines indicate ammonia metabolizing ability on the third day of culture. As shown in this figure, in perfusion culture using a packed-bed reactor, the metabolic ability of hepatocytes showed a favorable value depending on the oxygen concentration during aeration. In this experimental example, oxygenation of the medium for perfusion culture was performed only by surface aeration in the reservoir. Therefore, 50
Even with the aeration of% oxygen, the concentration of dissolved oxygen in the medium may not be as high as in a dish with the same conditions.

3-4. FIG. 19 is a graph showing the ammonia metabolizing ability. Also in this figure, the first day of culture and the third day of culture were compared. This figure shows the effect of the perfusion rate of the medium on the metabolic capacity of ammonia. In the experiment at the high perfusion rate (34 mL / min; Experiment No. 3) shown in Table 1, the ammonia metabolizing ability showed a slightly better tendency. When discussing the perfusion rate of the medium in a packed-bed reactor, the perfusion rate is more related to the linear velocity in the reactor than the volume flow rate.
8 cm / min. Even at this rate, no outflow of hepatocytes outside the reactor was observed except immediately after the start of culture.

As described above in Experimental Examples 1 and 2, in a high-density culturing system for hepatocytes using a monolayer culture and a packed-bed reactor, the oxygen concentration in the liquid medium was reduced to 204-200.
969 μM, and the liquid medium was 5.4 μm.
By perfusing at a linear velocity of １10.8 cm / min,
It was found that the metabolic ability of cultured hepatocytes could be further improved. In particular, the function of cultured hepatocytes tended to be maintained well by controlling the dissolved oxygen in the liquid medium to a predetermined concentration, and to be maintained slightly better by further increasing the perfusion rate of the liquid medium.

[0068]

According to the method for culturing hepatocytes of the present invention at high density, the metabolic ability of the cultured hepatocytes can be maintained in a more appropriate state. Further, according to the high-density hepatocyte culture device of the present invention, it is possible to provide a hybrid artificial liver capable of maintaining the metabolic ability of the cultured hepatocytes in a more appropriate state.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an example of a culture device.

FIG. 2 is a schematic view of an example of an artificial liver.

FIG. 3 is a schematic diagram of a culture device according to an experimental example.

FIG. 4 is an explanatory diagram showing a culture time and a medium exchange time.

FIG. 5 is a graph showing a relationship between an ammonia concentration and a culture time.

FIG. 6 is a graph showing the immobilization rate of hepatocytes.

FIG. 7 is a graph showing ammonia metabolism ability.

FIG. 8 is a graph showing the amount of ALT released.

FIG. 9 is a graph showing the immobilization rate of hepatocytes.

FIG. 10 is a graph showing ammonia metabolism ability.

FIG. 11 is a graph showing the amount of ALT released.

FIG. 12 is a graph showing the immobilization rate of hepatocytes.

FIG. 13 is a graph showing ammonia metabolism ability.

FIG. 14 is a graph showing the amount of ALT released.

FIG. 15 is a graph showing ammonia metabolism ability.

FIG. 16 is a graph showing ammonia metabolism ability.

FIG. 17 is a graph showing the relationship between ammonia concentration and culture time.

FIG. 18 is a graph showing ammonia metabolism ability.

FIG. 19 is a graph showing ammonia metabolism ability.

[Explanation of symbols]

 1, 17 Culture device 2, 18 Packed bed reactor 3 Reactor 3a Lower port 3b Upper port 4 Resin porous carrier 5 Liquid medium 6 Teflon sphere 7 Partition plate 8 Hepatocyte 9 Cock 10 Water jacket 11 Warm water 12, 20, 27 Pump 13, 19 Reservoir 13a Upper port 14, 23 Oxygenator 14a Upper port 15 Oxygen 16, 21, 22, 28, 29, 30, 31 Line 24 Oxygen electrode 25 Artificial liver 26 Plasma separator 27 Pump

──────────────────────────────────────────────────続 き Continuation of the front page Preliminary examination (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 1/00-5/28 C12M 1/00-3/10

Claims (7)

(57) [Claims] Claims: 1. A hepatocyte is immobilized on the surface and in pores of a resin porous carrier, and the resin porous carrier is arranged in a reactor, wherein the reactor has an inlet and an outlet for a liquid medium. Introducing the liquid medium from the inlet of the reactor,
The resin porous carrier is immersed in the liquid medium, the liquid medium is brought into direct contact with the hepatocytes, the liquid medium is taken out from the outlet of the reactor, and the liquid medium is inserted into the inlet of the reactor. When culturing the hepatocytes by returning and perfusing the liquid medium, the oxygen concentration of the liquid medium at the outlet of the reactor is adjusted to 2
A high-density culture method for hepatocytes, characterized by maintaining the concentration at 04 to 969 μM. 2. The high-density culture method for hepatocytes according to claim 1, wherein the liquid medium taken out from the outlet is exposed to a gas having an oxygen partial pressure of 152 to 722 mmHg. 3. The method according to claim 1, wherein the liquid medium in the reactor is 5.4.
3. The method for culturing hepatocytes at a high density according to claim 1 or 2, wherein the cells are allowed to flow at a linear velocity of 10.8 cm / min. 4. The method according to claim 3, wherein the liquid medium is divided so that the flow of the liquid medium in the reactor is not biased. 5. The flow of the liquid medium in the reactor is controlled by a baffle plate.
A method for high-density culture of hepatocytes as described. 6. Immobilizing hepatocytes on a porous resin carrier, immersing the porous resin carrier in a liquid medium, and culturing the hepatocytes, wherein the oxygen concentration of the liquid medium is adjusted to 300 to 400 μM. A high-density culture method for hepatocytes, characterized by maintaining. 7. A hepatocyte, a resin porous carrier on which the hepatocytes are immobilized, a reactor containing the resin porous carrier, and oxygen in the liquid medium in which the resin porous carrier is immersed. An oxygenator to be introduced, wherein the reactor has an inlet and an outlet for a liquid medium, and the liquid medium is introduced from the inlet of the reactor, and the resin porous carrier is filled with the liquid. Immersed in a culture medium, bringing the liquid culture medium into direct contact with the hepatocytes, removing the liquid culture medium from the outlet of the reactor, returning the liquid culture medium to the inlet of the reactor, and perfusing the liquid culture medium A high-density hepatocyte culture device for culturing the hepatocytes, wherein the oxygen concentration of the liquid medium at the outlet of the reactor is 2%.
A high-density culture apparatus for hepatocytes, which is maintained at 04 to 969 μM.