JPWO2006090933A1 - Hepatitis C virus particles and methods of propagation - Google Patents

Abstract

Provided is a proliferating HCV capable of efficiently performing HCV gene replication, HCV particle formation and release, and a method for proliferating the same. A method for growing HCV particles using a hydrogel-forming polymer in which the aqueous solution is in a fluid sol state at a temperature lower than the sol-gel transition temperature and reversibly in a hydrogel state at a temperature higher than the sol-gel transition temperature. The cells are cultured in a state in which a mixture of HCV-containing cells and an aqueous solution of the hydrogel-forming polymer is gelled at a temperature higher than the sol-gel transition temperature.

Description

  The present invention relates to a proliferated hepatitis C virus (hereinafter referred to as “HCV”) particle and a method for proliferating the same.

HCV is a virus whose host is human hepatocytes, and the HCV gene was cloned in 1989. As a result of clarifying the properties of each viral antigen using various gene expression systems, a very effective diagnostic system has been developed, which enables accurate diagnosis of hepatitis C and the occurrence of post-transfusion hepatitis. Was almost suppressed.
On the other hand, a cultured cell line that efficiently replicates and proliferates HCV has not yet been established. In recent years, self-replicating (replicon) cells of the HCV gene have been produced, but virus particle formation has not been successful. Although significant progress has been made in the study of gene replication mechanisms by using replicon cells, there are still many unclear points regarding HCV infection, invasion, particle formation mechanisms, and the like. For this reason, research and development of therapeutic drugs and vaccines are not sufficiently advanced. As an animal experiment system, an HCV infection model using chimpanzees has been established, but it is very expensive, lacks reproducibility due to individual differences, and has a large use restriction from the viewpoint of animal welfare, There are problems such as.
For the culture system of human hepatocytes, fetal hepatocytes, primary cultured cells using surgically excised liver tissue or the like, or cell lines derived from primary hepatocellular carcinoma are used. However, the former is problematic in that it is not easy to obtain and poor in proliferation, and the latter is disadvantageous in that dedifferentiation proceeds and the original hepatocyte function is reduced. In epithelial cells such as liver parenchymal cells, it is known that when cells are three-dimensionally cultured, the original cell polarity is formed and a differentiation function is expressed. It has been reported that when human hepatocytes are three-dimensionally cultured with an artificial liver device such as a bioreactor, production of liver proteins such as albumin and drug metabolism function are enhanced (Patent Document 1).
International Publication WO 01/14517

An object of the present invention is to provide a proliferating HCV and a proliferating method thereof in which the above-mentioned drawbacks of the prior art are eliminated.
Another object of the present invention is to provide a proliferating HCV capable of efficiently performing HCV gene replication, HCV particle formation and HCV particle release from cells, and a method for proliferating the same.
As a result of earnest research, the inventor has obtained a gel using a hydrogel-forming polymer that becomes a fluid sol state at a temperature lower than the sol-gel transition temperature and reversibly enters a hydrogel state at a temperature higher than the sol-gel transition temperature. It has been found that culturing cells containing HCV particles using the system in a state is extremely effective for achieving the above object.
The cultured HCV particles of the present invention are based on the above findings. More specifically, both HCV (hepatitis C virus) -RNA and HCV-core protein are present in the same fraction formed by centrifugation under a concentration gradient. It is characterized by doing.
According to the present invention, there is further provided a hydrogel-forming polymer whose aqueous solution is in a fluid sol state at a temperature lower than the sol-gel transition temperature and reversibly in a hydrogel state at a temperature higher than the sol-gel transition temperature. Use; mixing an aqueous solution of the gel-forming material in a fluid sol state at a temperature lower than the sol-gel transition temperature and cells containing the grown HCV particles; at a temperature higher than the sol-gel transition temperature, In a state where the mixture is gelled, the cells are cultured (HCV particles are released from the cells), the mixture is solated at a temperature lower than the sol-gel transition temperature, and the HCV particles released from the cells are There is provided a method for growing HCV particles, characterized in that they are collected or detected.
Furthermore, according to the present invention, there is provided a method for evaluating an antiviral agent using HCV particles produced by the above proliferation method.
As described above, according to the present invention, a simple and highly versatile tissue culture matrix, TGP (Thermoversable gelation polymer; hydrogel-forming polymer used in the present invention) is used to easily and efficiently produce HCV. Can be cultured.
A TGP gel is a matrix using a temperature-sensitive polymer material, and becomes an aqueous solution at a low temperature (15 ° C. or lower) and a gel at a high temperature (25 ° C. or higher), contrary to an agar gel or gelatin gel. Since it is easy to set the hardness of the gel within a range in which cells and tissues can grow freely, three-dimensional culture of cells and the like is possible.
In addition, since the TGP gel is easily melted by lowering the temperature, it can be recovered in a form that minimizes damage to cells and the like. Furthermore, unlike an artificial liver device, the TGP gel can arbitrarily select a culture scale from a small scale of about 0.1 mL to 100 mL or more, and the apparatus used for culture is for ordinary cell culture. No special equipment such as a bioreactor is required. Therefore, it can be used for various applications from high-throughput antiviral agent screening to large-scale preparation of virus particles.
In the present invention, as a culture system, for example, a multiwell plate (for example, 6-well, 12-well, 24-well-, 96-well plate; usually made of plastic) used for clinical examination can be used.

FIG. 1 is a phase contrast micrograph of HCV-expressing cells (RCYM1) cultured three-dimensionally with TGP (A, B and C).
FIG. 2 is an electron micrograph of an HCV-expressing cell (RCYM1) cultured three-dimensionally with TGP (D).
FIG. 3 is an electron micrograph of HCV-expressing cells (RCYM1) three-dimensionally cultured with TGP (E).
FIG. 4 is a graph showing the presence of HCV-RNA in the culture supernatant.
FIG. 5 is a graph showing the presence of HCV core protein in the culture supernatant.
FIG. 6 is an electron micrograph of HCV particles released out of cultured cells (A 1.04. G / ml; B 1.18 g / ml).
FIG. 7 is a TEM photograph (A anti-E1, E2 antibody) showing a 50-60 nm particle-like structure to which a colloidal gold-labeled anti-HCV antibody is bound, and a negative control (using mouse serum as the primary antibody). It is a TEM photograph (B normal mouse serum) which shows the state at the time of binding | bonding of a gold colloid labeled anti- HCV antibody with respect to FIG.
FIG. 8 is a graph (A, B, C) showing the results of a sucrose density gradient analysis of the culture supernatant of RCYM1 cells.
FIG. 9 is a photograph showing an example of an embodiment in which Huh-7 and RCYM1 cells form spheroids in TGP (A, a1). FIG. 9B is an enlarged photograph of a part (a1) in FIG. 9A.
FIG. 10 is a photograph showing an example of an embodiment in which Huh-7 and RCYM1 cells form spheroids in TGP (B, b1). FIG. 10B is an enlarged photograph of the part (b1) of FIG. 10A.
FIG. 11 is a photograph showing an example of an embodiment in which Huh-7 and RCYM1 cells form spheroids in TGP (C, D, E, F, G, H).
FIG. 12 shows HCV protein expression in RCYM1 cells and secretion of viral molecules in TGP culture (A, B, C, D).
FIG. 13 is a diagram showing expression of HCV protein in RCYM1 cells and secretion of viral molecules in TGP culture (E, F, G, H).
FIG. 14 is an electron micrograph of ultrathin sections of RCYM1 cells grown in TGP (A, B, C).
FIG. 15 is an immunoelectron micrograph of ultrathin sections of RCYM1 cells cultured in TGP (A, B, C).
FIG. 16 is a diagram showing infectivity of HCV particles secreted from RCYM1 cells cultured in TGP and neutralization of the infectivity (A, B).
FIG. 17 shows inhibition of HCV particle production by IFN and RBV (A, B).

Hereinafter, the present invention will be described more specifically with reference to the drawings as necessary. In the following description, “parts” and “%” representing the quantity ratio are based on mass unless otherwise specified.
(Proliferated HCV particles)
The proliferating HCV particles of the present invention are characterized in that both HCV (hepatitis C virus) -RNA and HCV-core protein are present in the same fraction formed by centrifugation under a concentration gradient. In the present invention, “both HCV-RNA and HCV-core protein are present” in “the same fraction formed by centrifugation under a concentration gradient” can be easily determined under the experimental conditions of Examples 1 to 3 described later. Can be confirmed.
(Confirmation method of HCV particles)
In the present invention, for example, the presence of HCV particles can be confirmed by the following method.
(1) As described later, it is confirmed by PCR that a new HCV gene has been formed in the culture supernatant of the HCV-containing cell culture system. (2) Thereby, it is estimated that "HCV" exists under the supernatant. (3) Furthermore, as shown in the Example mentioned later, it confirms that both HCV-RNA and HCV-core exist in the same density fraction. (4) In addition, the presence of HCV particles can be confirmed by TEM (transmission electron microscope) observation of the density fraction.
(Confirmation using colloidal gold labeled antibody)
The presence of the HCV particles of the present invention can be reliably verified by an antigen-antibody reaction with an anti-HCV antibody labeled with colloidal gold. Such a reaction with the anti-HCV antibody can be performed, for example, under the conditions of “Example 4” described later.
(HCV propagation method)
The HCV growth method of the present invention comprises a hydrogel-forming polymer whose aqueous solution is in a fluid sol state at a temperature lower than the sol-gel transition temperature and reversibly in a hydrogel state at a temperature higher than the sol-gel transition temperature. It is characteristic to use. More specifically, in the present invention, an aqueous solution of the gel-forming material in a fluid sol state at a temperature lower than the sol-gel transition temperature is mixed with cells containing the grown HCV particles. Culturing the cells and releasing HCV particles from the cells in a gelled state at a temperature above the sol-gel transition temperature; and solating the mixture at a temperature below the sol-gel transition temperature; HCV particles released from the cells are collected.
(HCV-containing cells)
The “HCV-containing cells” to be used in the present invention are not particularly limited as long as HCV can be included in the cells during the cell culture / proliferation. As the HCV-containing cells, for example, the following can be used (for details of cells that can include such HCV, see, for example, the literature Pietschmann, T. et al., Journal of Virology 76; 4008. -21 (2002) Aizaki, H. et al., Virology 314; 16-25 (2003)).
(Specific examples of HCV-containing cells)
Huh7 (Pietschmann, T. et al., Journal of Virology 76; 4008-21 (2002). Aizaki, H. et al. Virology 314; 16-25 (2003))
(Suitable HCV-containing cells)
From the viewpoint of the replication efficiency of the HCV gene, Huh7 cells (for example, commercially available from the Human Science Research Resource Bank) can be suitably used as the HCV-containing cells.
(One aspect of HCV proliferation method)
In the present invention, for example, HCV can be propagated as follows. (Introduction of HCV gene into HCV-containing cells)
An HCV gene is introduced into HCV-containing cells (for example, Huh7 cells) by a conventional method. Can refer to).
(Culture of HCV-containing cells)
(1) HCV-containing cells (for example, Huh cells) are cultured in a flask using a normal medium (Dulbecco medium).
(2) The grown cells are detached from the flask wall by trypsinization (2 to 3 minutes) and resuspended in Dulbecco's medium.
(3) Count the cells using a blood cell counter to adjust the concentration (ie, the number of cells in a certain volume).
(4) In an appropriate container (for example, a test tube having a capacity of 10 cc) on ice, an aqueous solution of a sol-form hydrogel-forming polymer (for example, commercially available from Meviol Co., Ltd. (Hiratsuka City, Kanagawa)) Meviol gel ") and the above suspension are mixed and suspended uniformly by pipetting.
(5) The suspension is dispensed into a 6-well plate and cultured at 37 ° C. (in about 5 minutes, the suspension containing cells hardens in a gel state).
(6) The Dulbecco medium warmed to 37 ° C. is overlaid on the gel.
(7) Incubate in a carbon dioxide gas incubator for 7 to 10 days.
(8) The “37 ° C. Dulbecco medium” is changed every 4 days (by decantation).
(HCV recovery)
(1) After the above culture, the lower layer is a gel and the upper layer is a supernatant (if it is a large container, the cooled medium is directly poured into this container, and the gel + supernatant is collected. May be recovered). In this system, since the container is a test tube and is small, the supernatant and the gel are collected separately as described below.
(2) First, the supernatant is collected. The remaining gel is placed in a refrigerator to make a sol, added to a previously cooled medium and suspended. This suspension is combined with the above supernatant.
(3) Separate cells / supernatant by centrifugation.
(4) Concentrate the supernatant by pelleting (i.e., remove solids by centrifugation; however, if necessary, it may be by "ultrafiltration" which is milder than pelleting).
(5) The pellet derived from the supernatant is suspended and centrifuged under a concentration gradient.
(6) As shown in the examples described later, both HCV-RNA and core protein are present in the fraction with a specific density (that is, it is determined that HCV particles are present).
(7) HCV particles are confirmed by TEM observation of the same sample as necessary, as shown in the examples described later.
(Details of each material)
Hereinafter, each material to be used in the present invention will be described in detail.
(Hydrogel-forming polymer)
The hydrogel-forming polymer used in the present invention has a thermoreversible sol-gel transition in which the aqueous solution becomes a sol state at a temperature lower than the sol-gel transition temperature and becomes a gel state at a temperature higher than the sol-gel transition temperature. Indicates.
In the present invention, the “hydrogel-forming polymer” constituting the hydrogel has a crosslinking structure or a network structure, and based on the structure, a hydrolyzed liquid such as water is retained therein. A polymer having the property of forming a gel. The “hydrogel” refers to a gel containing at least a crosslinked or network structure containing a polymer and water supported or held in the structure (dispersed liquid).
The “dispersed liquid” retained in the crosslinked or network structure is not particularly limited as long as it is a liquid containing water as a main component. More specifically, the dispersion liquid may be water itself, and may be either an aqueous solution and / or a water-containing liquid. The water-containing liquid preferably contains 80 parts or more, more preferably 90 parts or more of water with respect to 100 parts of the whole water-containing liquid.
Unless it is contrary to the object of the present invention, the dispersion liquid may contain an organic solvent (for example, a hydrophilic solvent such as ethanol having compatibility with water) or a contrast agent in a predetermined content.
(Sol-gel transition temperature)
In the present invention, the definition and measurement of “sol state”, “gel state”, and “sol-gel transition temperature” are described in the literature (H. Yoshioka et al., Journal of macromolecular Science, A31 (1), 113 (1994)). That is, the dynamic elastic modulus of the sample at an observation frequency of 1 Hz is measured by gradually changing the temperature from the low temperature side to the high temperature side (1 ° C./1 min), and the storage elastic modulus ( The temperature at which G ′, the elasticity term) exceeds the loss modulus (G ″, viscosity term) is defined as the sol-gel transition temperature. In general, the state of G ″> G ′ is defined as sol, and the state of G ″ <G ′ is defined as gel. In measuring the sol-gel transition temperature, the following measurement conditions can be preferably used.
<Dynamic / loss elastic modulus measurement conditions>
Measuring device (trade name): Stress-controlled rheometer AR500, manufactured by TA Instruments Inc. Sample solution (or dispersion) concentration (however, as the concentration of “hydrogel-forming polymer having sol-gel transition temperature”) : 10 (weight)% Amount of sample solution: about 0.8 g Shape and dimensions of measurement cell: Acrylic parallel disk (diameter: 4.0 cm), gap: 600 μm Measurement frequency: 1 Hz Applicable stress: in the linear region.
In the present invention, from the viewpoint of preventing thermal damage of cells or cultured HCV particles, the sol-gel transition temperature is preferably higher than 0 ° C, preferably not higher than 37 ° C, and more preferably higher than 5 ° C. It is preferably 35 ° C. or lower (particularly 10 ° C. or higher and 33 ° C. or lower).
Such a hydrogel-forming polymer having a suitable sol-gel transition temperature is easily selected from the specific compounds described below according to the screening method (sol-gel transition temperature measurement method) described above. be able to. In the operation of gelling an aqueous solution of a hydrogel-forming polymer in the present invention and culturing HCV-containing cells, the sol-gel transition temperature (a ° C.) described above is injected with the temperature during culture (b ° C.). It is preferable to set the temperature between the cooling temperature (c ° C.) for the sol formation in an operation such as recovery. That is, it is preferable that there is a relationship of b>a> c between the above three temperatures a ° C., b ° C., and c ° C. More specifically, (b-a) is preferably 1 to 36 ° C, more preferably 2 to 30 ° C, and (ac) is 1 to 35 ° C, more preferably 2 to 30 ° C. Is preferred.
(Followability of the hydrogel-forming polymer aqueous solution)
In the present invention, a hydrogel based on an aqueous solution of a hydrogel-forming polymer exhibits a solid behavior at a higher frequency from the viewpoint of the balance of conformity to morphological changes for cells and the like, It is preferable to exhibit liquid behavior for low frequencies. More specifically, the followability to the operation of the hydrogel can be suitably measured by the following method.
(Measuring method for tracking performance)
In the present invention containing a hydrogel-forming polymer, an aqueous solution of hydrogel-forming polymer (1 mL as a hydrogel) is put in a test tube having an inner diameter of 1 cm in a sol state (temperature lower than the sol-gel transition temperature). The test tube is held for 12 hours in a water bath at a temperature sufficiently higher than the sol-gel transition temperature of the aqueous solution of the gel-forming polymer (for example, about 10 ° C. higher than the sol-gel transition temperature). Gel the gel.
Next, the time (T) until the solution / air interface (meniscus) is deformed by its own weight when the test tube is turned upside down is measured. Where 1 / T (sec ^-1 ) For lower frequency operation, the hydrogel behaves as a liquid, 1 / T (sec ^-1 For higher frequency operation, the hydrogel will behave as a solid. In the present invention, in the case of a hydrogel, T is 1 minute to 24 hours, preferably 5 minutes to 10 hours.
(Steady flow viscosity)
In the present invention, the gel-like properties of a hydrogel based on an aqueous solution of a hydrogel-forming polymer can be suitably measured by measuring the steady flow viscosity. The steady flow viscosity η (eta) can be measured, for example, by a creep experiment. In the creep experiment, a constant shear stress is applied to the sample and the temporal change of shear strain is observed. Generally, in the creep behavior of a viscoelastic body, the shear rate initially changes with time, but thereafter the shear rate becomes constant. The ratio between the shear stress and the shear rate at this time is defined as the steady flow viscosity η. This steady flow viscosity is sometimes referred to as Newtonian viscosity. Here, however, the steady flow viscosity must be determined within a linear region that is largely independent of shear stress.
Specifically, a stress-controlled viscoelasticity measuring device (AR500, manufactured by TA Instruments) is used as a measuring device, an acrylic disk (diameter 4 cm) is used as a measuring device, and a sample thickness is 600 μm for at least 5 minutes. Observe the above measurement time creep behavior (delay curve). Sampling time is once per second for the first 100 seconds and then once every 10 seconds. In determining the applied shear stress (stress), the displacement angle is 2 × 10 by applying the shear stress for 10 seconds. ^-3 Set to the lowest value detected above rad. The analysis employs at least 20 measured values after 5 minutes. In the present invention, a hydrogel based on an aqueous solution of a hydrogel-forming polymer has a η of 5 × 10 5 at 37 ° C. ³ ~ 1x10 ⁷ It is preferably Pa · sec, and more preferably 8 × 10. ³ ~ 8x10 ⁶ Pa · sec, especially 1 × 10 ⁴ Pa · sec or more, 7 × 10 ⁶ It is preferably Pa · sec or less.
The above η is 5 × 10 ³ If it is less than Pa · sec, the fluidity is relatively high even in short-time observation, and it becomes easy to move from within the cell culture system. On the other hand, η is 1 × 10 ⁷ When Pa · sec is exceeded, the gel tends to hardly exhibit fluidity even when observed for a long time, and the followability of the aqueous solution of the hydrogel-forming polymer to the deformation of the cell culture system becomes insufficient. Also, η is 1 × 10 ⁷ If Pa · sec is exceeded, the possibility of the gel becoming brittle becomes strong, and after a slight pure elastic deformation, the tendency to easily cause brittle fracture that breaks at once increases.
(Dynamic elastic modulus)
In the present invention, the gel-like properties of a hydrogel based on an aqueous solution of a hydrogel-forming polymer can be suitably measured by a dynamic elastic modulus. The gel has an amplitude γ ₀ , Strain γ (t) = γ with frequency ω / 2π ₀ When cos ωt (t is time) is given, the constant stress is σ ₀ , Σ (t) = σ, where δ is the phase difference ₀ Assume that cos (ωt + δ) is obtained. | G | = σ ₀ / Γ ₀ Then, the ratio (G ″ / G ′) between the dynamic elastic modulus G ′ (ω) = | G | cos δ and the loss elastic modulus G ″ (ω) = | G | sin δ is an index representing gel-like properties. It becomes.
In the present invention, a hydrogel based on an aqueous solution of a hydrogel-forming polymer behaves as a solid with respect to a strain of ω / 2π = 1 Hz (corresponding to a fast operation), and ω / 2π = 10. ^-4 It behaves as a fluid to distortions of Hz (corresponding to slow motion). More specifically, in the present invention, a hydrogel based on an aqueous solution of a hydrogel-forming polymer preferably exhibits the following properties (for details of such elastic modulus measurement, see, for example, literature: Ryohei Oda Edit, Modern Industrial Chemistry 19, page 359, Asakura Shoten, 1985).
When ω / 2π = 1 Hz (frequency at which the gel behaves as a solid), (G ″ / G ′) s = (tan δ) s is preferably less than 1 (more preferably 0.8 or less, Particularly preferably 0.5 or less).
ω / 2π = 10 ^-4 In the case of Hz (frequency at which the gel behaves as a liquid), (G ″ / G ′) L = (tan δ) L is preferably 1 or more (more preferably 1.5 or more, particularly preferably 2 more than).
It is preferable that the ratio {(tan δ) s / (tan δ) L} of (tan δ) s to (tan δ) L is less than 1 (more preferably 0.8 or less, particularly preferably 0). .5 or less).
<Measurement conditions>
Concentration of hydrogel-forming polymer in aqueous solution of hydrogel-forming polymer: Approximately 8% by mass Temperature: 37 ° C. Measuring instrument: Stress-controlled rheometer (model name: AR500, manufactured by TA Instruments)
Shape and dimensions of measurement cell: Stainless steel parallel disk (diameter: 4.0 cm), gap: 600 μm Applicable stress: in the linear region.
(Fluidity at low temperature)
In the present invention, since the aqueous solution of the hydrogel-forming polymer is injected into the cell culture system at a low temperature, it preferably has an appropriate viscosity at a low temperature. Viscosity can be determined by normal static viscosity measurement.
In the present invention, the aqueous hydrogel-forming polymer solution preferably has a viscosity at 10 ° C. of 0.005 to 100 Pa · sec, more preferably 0.01 to 10 Pa · sec, particularly 0.1 Pa · sec or more. It is preferably 1 Pa · sec or less.
<Measurement conditions>
Concentration of hydrogel-forming polymer in aqueous solution of hydrogel-forming polymer: about 8% by mass Temperature: 10 ° C. Measuring instrument: Stress-controlled rheometer (model name: AR500, manufactured by TA Instruments)
Shape and dimensions of measurement cell: Stainless steel parallel disk (diameter: 4.0 cm), gap: 600 μm Applicable stress: in the linear region.
(Hydrogel-forming polymer)
As long as it exhibits a thermoreversible sol-gel transition as described above (that is, has a sol-gel transition temperature), the hydrogel-forming polymer that can be used in the aqueous solution of the hydrogel-forming polymer in the present invention is There is no particular limitation.
Specific examples of the polymer in which the aqueous solution has a sol-gel transition temperature and reversibly shows a sol state at a temperature lower than the transition temperature include, for example, a block copolymer of polypropylene oxide and polyethylene oxide. Known polyalkylene oxide block copolymers; etherified celluloses such as methyl cellulose and hydroxypropyl cellulose; chitosan derivatives (KR Holme et al., Macromolecules, 24, 3828 (1991)) and the like are known.
As a polyalkylene oxide block copolymer, a pluronic F-127 (trade name, manufactured by BASF Wyandotte Chemicals Co.) gel in which polyethylene oxide is bonded to both ends of polypropylene oxide has been developed. It is known that this high-concentration aqueous solution of Pluronic F-127 becomes a hydrogel at about 20 ° C. or higher and becomes an aqueous solution at a lower temperature. However, in the case of this material, it becomes a gel state only at a high concentration of about 20% by mass or more, and even if it is kept at a high concentration of about 20% by mass or more and higher than the gelation temperature, water is further added. The gel will dissolve. Pluronic F-127 has a relatively small molecular weight, and not only exhibits a very high osmotic pressure in a high gel state of about 20% by mass or more, but also easily penetrates the cell membrane, and thus may adversely affect cell culture. There is sex.
On the other hand, in the case of etherified cellulose represented by methyl cellulose, hydroxypropyl cellulose and the like, usually, the sol-gel transition temperature is high and is about 45 ° C. or higher (N. Sarkar, J. Appl. Polym. Science, 24, 1073, 1979). In contrast, since the temperature during cell culture is usually around 37 ° C., the etherified cellulose is in a sol state, and it is a fact that the etherified cellulose is used as an aqueous solution of a hydrogel-forming polymer. It is difficult.
As described above, the problems of conventional polymers that have a sol-gel transition point in the aqueous solution and reversibly show a sol state at a temperature lower than the transition temperature are as follows: 1) From the sol-gel transition temperature Once gelled at a high temperature, the gel dissolves when water is further added. 2) The sol-gel transition temperature is higher than the temperature during cell culture (near 37 ° C), and the body temperature is in a sol state. 3) In order to make it gel, it is necessary to make the polymer concentration of the aqueous solution very high.
On the other hand, according to the study by the present inventors, a hydrogel-forming polymer having a sol-gel transition temperature that is preferably higher than 0 ° C. and not higher than 37 ° C. (eg, a plurality of blocks having cloud points) And a hydrophilic block, and the aqueous solution has a sol-gel transition temperature and a polymer that reversibly shows a sol state at a temperature lower than the sol-gel transition temperature) to form a hydrogel It has been found that the above problems can be solved when an aqueous solution of a conductive polymer is constructed.
(Suitable hydrogel-forming polymer)
In the present invention, the hydrogel-forming polymer utilizing a hydrophobic bond that can be suitably used as an aqueous solution of the hydrogel-forming polymer is preferably formed by combining a plurality of blocks having a cloud point and a hydrophilic block. . The hydrophilic block is preferably present because the hydrogel becomes water-soluble at a temperature lower than the sol-gel transition temperature, and the plurality of blocks having a cloud point have a hydrogel having a sol-gel transition temperature. It is preferably present to change to a gel state at a higher temperature. In other words, a block having a cloud point dissolves in water at a temperature lower than the cloud point and changes to insoluble in water at a temperature higher than the cloud point, so that the block forms a gel at a temperature higher than the cloud point. It serves as a cross-linking point including a hydrophobic bond. That is, the cloud point derived from the hydrophobic bond corresponds to the sol-gel transition temperature of the hydrogel.
However, the cloud point and the sol-gel transition temperature do not necessarily coincide. This is because the cloud point of the “block having a cloud point” described above is generally affected by the bond between the block and the hydrophilic block.
The hydrogel used in the present invention utilizes the property that not only the hydrophobic bond becomes stronger as the temperature increases, but also the change is reversible with respect to temperature. From the viewpoint that a plurality of crosslinking points are formed in one molecule and a gel having excellent stability is formed, the hydrogel-forming polymer preferably has a plurality of “blocks having cloud points”.
On the other hand, the hydrophilic block in the hydrogel-forming polymer has a function of changing the water-soluble polymer to water-soluble at a temperature lower than the sol-gel transition temperature, as described above. It has a function of forming a hydrous gel state while preventing the hydrogel from aggregating and precipitating due to excessive increase in hydrophobic binding force at a temperature higher than the transition temperature.
Furthermore, it is desirable that the hydrogel used in the present invention is decomposed and absorbed in cell culture. That is, in the present invention, it is preferable that the hydrogel-forming polymer is decomposed in a cell culture by a hydrolysis reaction or an enzyme reaction to be absorbed and excreted as a low molecular weight body that is harmless to cell culture.
In the present invention, when the hydrogel-forming polymer is formed by combining a plurality of blocks having a cloud point and a hydrophilic block, at least one of a block having a cloud point and a hydrophilic block is preferable. Both are preferably those that are degraded and absorbed within the cell culture.
(Multiple blocks with cloud points)
The block having a cloud point is preferably a polymer block having a negative solubility-temperature coefficient in water, and more specifically, polypropylene oxide, a copolymer of propylene oxide and another alkylene oxide, Polymers selected from the group comprising poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, copolymers of N-substituted acrylamide derivatives and N-substituted methacrylamide derivatives, polyvinyl methyl ether, polyvinyl alcohol partially acetylated products Can be preferably used.
In order to make a block having a cloud point decomposed and absorbed in cell culture, it is effective to make the block having a cloud point a polypeptide composed of a hydrophobic amino acid and a hydrophilic amino acid. Alternatively, polyester-type biodegradable polymers such as polylactic acid and polyglycolic acid can be used as blocks having cloud points that are decomposed and absorbed in cell culture.
The above polymer (block having a cloud point) has a cloud point higher than 4 ° C. and not higher than 40 ° C., and the polymer used in the present invention (a compound in which a plurality of blocks having a cloud point and a hydrophilic block are combined) The sol-gel transition temperature is preferably higher than 0 ° C and not higher than 37 ° C.
Here, the cloud point is measured by, for example, cooling an aqueous solution of about 1% by mass of the above polymer (block having a cloud point) to form a transparent uniform solution, and then gradually increasing the temperature (temperature increase rate of about 1). C./min), and the point at which the solution becomes cloudy for the first time is taken as the cloud point.
Specific examples of poly N-substituted acrylamide derivatives and poly N-substituted methacrylamide derivatives that can be used in the present invention are listed below.
Poly-N-acroylpiperidine; poly-Nn-propylmethacrylamide; poly-N-isopropylacrylamide; poly-N, N-diethylacrylamide; poly-N-isopropylmethacrylamide; poly-N-cyclopropylacrylamide; Poly-N-acryloylpyrrolidine; poly-N, N-ethylmethylacrylamide; poly-N-cyclopropylmethacrylamide; poly-N-ethylacrylamide.
The polymer may be a homopolymer or a copolymer of a monomer constituting the polymer and another monomer. As another monomer constituting such a copolymer, either a hydrophilic monomer or a hydrophobic monomer can be used. In general, copolymerization with a hydrophilic monomer raises the cloud point of the product and copolymerization with a hydrophobic monomer lowers the cloud point of the product. Therefore, a polymer having a desired cloud point (for example, a cloud point higher than 4 ° C. and lower than 40 ° C.) can be obtained also by selecting the monomer to be copolymerized.
(Hydrophilic monomer)
Examples of the hydrophilic monomer include N-vinyl pyrrolidone, vinyl pyridine, acrylamide, methacrylamide, N-methyl acrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, and acrylic having an acid group. Acid, methacrylic acid and salts thereof, vinyl sulfonic acid, styrene sulfonic acid and the like, and N, N-dimethylaminoethyl methacrylate, N, N-diethylaminoethyl methacrylate, N, N-dimethylaminopropyl having a basic group Examples include, but are not limited to, acrylamide and salts thereof.
(Hydrophobic monomer)
On the other hand, examples of the hydrophobic monomer include acrylate derivatives and methacrylate derivatives such as ethyl acrylate, methyl methacrylate and glycidyl methacrylate, N-substituted alkylmethacrylamide derivatives such as Nn-butylmethacrylamide, vinyl chloride, acrylonitrile and styrene. And vinyl acetate and the like, but are not limited thereto.
(Hydrophilic block)
On the other hand, as the hydrophilic block to be combined with the above-described block having a cloud point, specifically, methyl cellulose, dextran, polyethylene oxide, polyvinyl alcohol, poly N-vinyl pyrrolidone, polyvinyl pyridine, polyacrylamide, polymethacrylamide , Poly N-methylacrylamide, polyhydroxymethyl acrylate, polyacrylic acid, polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid and their salts; poly N, N-dimethylaminoethyl methacrylate, poly N, N-diethylaminoethyl methacrylate , Poly N, N-dimethylaminopropylacrylamide and salts thereof.
In addition, hydrophilic blocks are desirably decomposed, metabolized and excreted in cell culture, and culture polymers of hydrophilic cells such as proteins such as albumin and gelatin, polysaccharides such as hyaluronic acid, heparin, chitin and chitosan. Is preferably used.
The method for bonding the block having a cloud point and the hydrophilic block is not particularly limited. For example, a polymerizable functional group (for example, acryloyl group) is introduced into one of the blocks to give the other block. This can be done by copolymerizing monomers. Further, the combined product of a block having a cloud point and the hydrophilic block may be obtained by block copolymerization of a monomer that gives a block having a cloud point and a monomer that gives a hydrophilic block. Is possible. In addition, the block having a cloud point and the hydrophilic block are bonded to each other by introducing a reactive functional group (for example, a hydroxyl group, an amino group, a carboxyl group, an isocyanate group, etc.) in advance and bonding them together by a chemical reaction. Can also be done. At this time, usually a plurality of reactive functional groups are introduced into the hydrophilic block. In addition, the bond between the polypropylene oxide having a cloud point and the hydrophilic block is repeated, for example, by anionic polymerization or cationic polymerization by repeatedly repeating propylene oxide and a monomer (for example, ethylene oxide) constituting “another hydrophilic block”. By polymerizing, a block copolymer in which polypropylene oxide and a “hydrophilic block” (for example, polyethylene oxide) are bonded can be obtained. Such a block copolymer can also be obtained by copolymerizing monomers constituting a hydrophilic block after introducing a polymerizable group (for example, acryloyl group) to the terminal of polypropylene oxide. Furthermore, the polymer used in the present invention can also be obtained by introducing a functional group capable of binding reaction with a functional group (for example, hydroxyl group) at the end of polypropylene oxide into the hydrophilic block and reacting both. . Further, the hydrogel-forming polymer used in the present invention can also be obtained by linking materials such as Pluronic F-127 (trade name, manufactured by Asahi Denka Kogyo Co., Ltd.) in which polyethylene glycol is bonded to both ends of polypropylene glycol. Can be obtained.
The polymer of the present invention in a mode including a block having a cloud point is water-soluble together with the hydrophilic block at the temperature lower than the cloud point, because the above-mentioned “block having a cloud point” is present in the molecule. It is completely dissolved in water and shows a sol state. However, when the temperature of the aqueous solution of the polymer is raised to a temperature higher than the cloud point, the “block having a cloud point” existing in the molecule becomes hydrophobic and associates between different molecules by hydrophobic interaction. .
On the other hand, since the hydrophilic block is still water-soluble at this time (when heated to a temperature higher than the cloud point), the polymer of the present invention has a hydrophobic association between the blocks having a cloud point in water. A hydrogel having a three-dimensional network structure as a crosslinking point is generated. When the temperature of this hydrogel is cooled again to a temperature lower than the cloud point of the “block having cloud point” existing in the molecule, the block having the cloud point becomes water-soluble and the crosslinking point due to hydrophobic association is released. The hydrogel structure disappears and the polymer of the present invention becomes a complete aqueous solution again. Thus, the sol-gel transition of the polymer of the present invention in a preferred embodiment is based on reversible changes in hydrophilicity and hydrophobicity at the cloud point of the block having the cloud point present in the molecule. Therefore, it has complete reversibility in response to temperature changes.
(Gel solubility)
As described above, in the present invention including at least a polymer having a sol-gel transition temperature in an aqueous solution, the hydrogel-forming polymer is substantially insoluble in water at a temperature higher than the sol-gel transition temperature (d ° C.). It is reversibly water soluble at a temperature (e ° C.) lower than the sol-gel transition temperature.
The high temperature (d ° C.) is preferably a temperature that is 1 ° C. or more higher than the sol-gel transition temperature, and more preferably 2 ° C. or more (particularly 5 ° C. or more). The “substantially water-insoluble” means that the amount of the polymer dissolved in 100 mL of water at the temperature (d ° C.) is 5.0 g or less (more preferably 0.5 g or less, particularly 0.1 g or less). ) Is preferable.
On the other hand, the above-mentioned low temperature (e ° C.) is preferably 1 ° C. or more lower than the sol-gel transition temperature (in absolute value), more preferably 2 ° C. or higher (particularly 5 ° C. or higher). preferable. The “water-soluble” means that the amount of the polymer dissolved in 100 mL of water at the temperature (e ° C.) is preferably 0.5 g or more (more preferably 1.0 g or more). Further, “reversibly water-soluble” means that the aqueous solution of the hydrogel-forming polymer is once gelled (at a temperature higher than the sol-gel transition temperature), even after the sol-gel transition temperature. It means that it exhibits the above-mentioned water solubility at a low temperature.
The polymer preferably has a 10% aqueous solution at 5 ° C. and a viscosity of 10 to 3,000 centipoise (more preferably 50 to 1,000 centipoise). Such viscosity is preferably measured under the following measurement conditions, for example.
Viscometer: Stress-controlled rheometer (Model name: AR500, manufactured by TA Instruments)
Rotor diameter: 60 mm Rotor shape: parallel flat plate In the present invention, the hydrogel-forming polymer aqueous solution is gelled at a temperature higher than the sol-gel transition temperature and then immersed in a large amount of water. Does not dissolve. The characteristics of the aqueous solution of the hydrogel-forming polymer can be confirmed, for example, as follows.
That is, in the present invention, 0.15 g of the hydrogel-forming polymer is dissolved in 1.35 g of distilled water at a temperature lower than the sol-gel transition temperature (for example, under ice cooling) to prepare a 10 wt% aqueous solution. The aqueous solution was poured into a plastic petri dish having a diameter of 35 mm and heated to 37 ° C. to form a gel having a thickness of about 1.5 mm in the petri dish, and then the weight of the whole petri dish containing the gel (F gram) is measured. Subsequently, after the whole petri dish containing the gel was allowed to stand at 37 ° C. for 10 hours in 250 ml of water, the weight (g gram) of the whole petri dish containing the gel was measured, and the dissolution of the gel from the gel surface was measured. Evaluate presence or absence. At this time, in the hydrogel-forming polymer in the present invention, the weight reduction rate of the gel, that is, (f−g) / f is preferably 5.0% or less, and more preferably 1.0%. Or less (particularly 0.1% or less).
In the present invention, the aqueous solution of the hydrogel-forming polymer is gelled at a temperature higher than the sol-gel transition temperature and then immersed in a large amount of water (by volume, about 0.1 to 100 times the gel). However, the gel does not dissolve over a long period of time. Such a property of the polymer used in the present invention is achieved by, for example, the presence of two or more (plural) blocks having a cloud point in the polymer.
On the other hand, when a similar gel was prepared using the aforementioned Pluronic F-127 in which polyethylene oxide was bonded to both ends of polypropylene oxide, the gel was completely dissolved in water after standing for several hours. The inventors have found that this is the case.
From the standpoint of suppressing the non-gelling cytotoxicity to the lowest possible level, the concentration in water, that is, {(polymer) / (polymer + water)} × 100 (%), 20% or less (and 15 It is preferable to use a hydrogel-forming polymer that can be gelled at a concentration of not more than%, particularly not more than 10%.
The molecular weight of the hydrogel-forming polymer used in the present invention is preferably from 30,000 to 30 million, more preferably from 100,000 to 10 million, and still more preferably from 500,000 to 5 million.
(Confirmation of HCV particle infectivity)
In the present invention, as described later, the fact that the HCV particles secreted into the culture medium of 3D culture are infectious means that, for example, the HCV particles against Huh-7.5.1 cells that highly allow HCV replication. This can be confirmed by the behavior of Infections are neutralized by anti-E2 antibodies or by patient sera that prevent E2 protein binding to human cells. As will be described later, the usefulness of the TGP-3D culture system for evaluation of antiviral drugs can also be demonstrated. It can be confirmed that the 3D culture system induced by Huh-7 gives the production of infectious HCV particles. This system can be a valuable tool for further studying HCV morphogenesis in the natural host cell environment.
The present invention will be specifically described with reference to the following examples and drawings. However, the technical scope of the present invention is not limited to the following examples.

Production Example 1
  10 g of a polypropylene oxide-polyethylene oxide copolymer (propylene oxide / ethylene oxide average degree of polymerization of about 60/180, manufactured by Asahi Denka Kogyo Co., Ltd .: Pluronic F-127) was dissolved in 30 ml of dry chloroform, and in the presence of phosphorus pentoxide, Hexamethylene diisocyanate (0.13 g) was added, and the reaction was allowed to proceed for 6 hours under reflux at the boiling point. After distilling off the solvent under reduced pressure, the residue was dissolved in distilled water, and ultrafiltration was performed using an ultrafiltration membrane (Amicon PM-30) with a molecular weight cut off of 30,000 to obtain a high molecular weight polymer and a low molecular weight polymer. Fractionated. The obtained aqueous solution was frozen to obtain F-127 high polymer and F-127 low polymer.
  The F-127 high polymer obtained above (in the present invention, hydrogel-forming polymer, TGP-1) was dissolved in distilled water at a concentration of 8% by mass under ice cooling. When this aqueous solution was gently heated, the viscosity gradually increased from 21 ° C. and solidified at about 27 ° C. to form a hydrogel. When this hydrogel was cooled, it returned to an aqueous solution at 21 ° C. This change was repeatedly observed reversibly. On the other hand, the F-127 low polymer dissolved in distilled water at a concentration of 8% by mass below freezing did not gel at all even when heated to 60 ° C. or higher.
  Production Example 2
  160 mol of ethylene oxide was added by cationic polymerization to 1 mol of trimethylolpropane to obtain a polyethylene oxide triol having an average molecular weight of about 7000.
  After dissolving 100 g of the polyethylene oxide triol obtained above in 1000 ml of distilled water, 12 g of potassium permanganate was gradually added at room temperature, and the reaction was allowed to proceed for about 1 hour. After removing the solid matter by filtration, the product was extracted with chloroform, and the solvent (chloroform) was distilled off under reduced pressure to obtain 90 g of a polyethylene oxide tricarboxylate.
  10 g of the polyethylene oxide tricarboxylate obtained above and 10 g of polypropylene oxide diamino (propylene oxide average polymerization degree: about 65, manufactured by Jefferson Chemical Co., USA, trade name: Jeffamine D-4000, cloud point: about 9 ° C.) After dissolving in 1000 ml of carbon tetrachloride and adding 1.2 g of dicyclohexylcarbodiimide, the mixture was reacted for 6 hours under reflux at the boiling point. After cooling the reaction solution and removing the solid matter by filtration, the solvent (carbon tetrachloride) was distilled off under reduced pressure, and the residue was vacuum dried to produce a hydrogel in which a plurality of polypropylene oxides and polyethylene oxides were combined. A forming polymer (TGP-2) was obtained. This was dissolved in distilled water at a concentration of 5% by mass under ice-cooling, and its sol-gel transition temperature was measured and found to be about 16 ° C.
  Production Example 3
  96 g of N-isopropylacrylamide (manufactured by Eastman Kodak), 17 g of N-acryloxysuccinimide (manufactured by Kokusan Chemical Co., Ltd.) and 7 g of n-butyl methacrylate (manufactured by Kanto Chemical Co., Ltd.) are dissolved in 4000 ml of chloroform, After the substitution, 1.5 g of N, N′-azobisisobutyronitrile was added and polymerized at 60 ° C. for 6 hours. After the reaction solution was concentrated, it was reprecipitated (reprecipitated) in diethyl ether. The solid was collected by filtration and then vacuum dried to obtain 78 g of poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate).
  To the poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate) obtained above, an excess of isopropylamine was added to obtain poly (N-isopropylacrylamide-co-n-butyl methacrylate). Obtained. The cloud point of this aqueous solution of poly (N-isopropylacrylamide-co-n-butyl methacrylate) was 19 ° C.
  Chloroform 10 g of the above poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate) and 5 g of both ends aminated polyethylene oxide (molecular weight 6,000, manufactured by Kawaken Fine Chemical Co., Ltd.) It melt | dissolved in 1000 ml and made it react at 50 degreeC for 3 hours. After cooling to room temperature, 1 g of isopropylamine was added and allowed to stand for 1 hour, and then the reaction solution was concentrated, and the residue was precipitated in diethyl ether. After the solid matter was collected by filtration, it was vacuum-dried to form a hydrogel-forming polymer (TGP-3) in the present invention in which a plurality of poly (N-isopropylacrylamide-co-n-butyl methacrylate) and polyethylene oxide were combined. )
  The TGP-3 thus obtained was dissolved in distilled water at a concentration of 5% by mass under ice cooling, and the sol-gel transition temperature was measured to be about 21 ° C.
  Production Example 4
  (Sterilization method)
  In the present invention, 2.0 g of the hydrogel-forming polymer (TGP-3) is put into an EOG (ethylene oxide gas) sterilization bag (trade name: hybrid sterilization bag, manufactured by Hogi Medical Co., Ltd.), and an EOG sterilizer ( The bag was filled with EOG with Easy Pack (manufactured by Inoue Seieido) and left at room temperature all day and night. Further, after standing at 40 ° C. for half a day, the EOG was removed from the bag and aerated. The bag was sterilized by placing it in a vacuum dryer (40 ° C.) and leaving it for half a day with occasional aeration.
  It was separately confirmed that the sol-gel transition temperature of the polymer was not changed by this sterilization operation.
  Production Example 5
  After dissolving 37 g of N-isopropylacrylamide, 3 g of n-butyl methacrylate and 28 g of polyethylene oxide monoacrylate (molecular weight 4,000, manufactured by NOF Corporation: PME-4000) in 340 ml of benzene, 2,2 ′ -0.8 g of azobisisobutyronitrile was added and reacted at 60 ° C for 6 hours. The obtained reaction product was dissolved by adding 600 ml of chloroform, and the solution was added dropwise to 20 L (liter) of ether to cause precipitation. The obtained precipitate was recovered by filtration, and the precipitate was vacuum-dried at about 40 ° C. for 24 hours, and then dissolved again in 6 L of distilled water, and a hollow fiber ultrafiltration membrane (H1P100 manufactured by Amicon Co., Ltd.) having a molecular weight cut-off of 100,000. -43) and concentrated to 2 liters at 10 ° C. The concentrated solution was diluted by adding 4 l of distilled water, and the above dilution operation was repeated. The above dilution and ultrafiltration concentration operations were further repeated 5 times to remove those having a molecular weight of 100,000 or less. What was not filtered by this ultrafiltration (remaining in the ultrafiltration membrane) was recovered and freeze-dried, and in the present invention having a molecular weight of 100,000 or more, 60 g of a hydrogel-forming polymer (TGP-4) was added. Obtained.
  In the present invention obtained as described above, 1 g of a hydrogel-forming polymer (TGP-4) was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 25 ° C.
  Production Example 6
  In the present invention of Production Example 3, the hydrogel-forming polymer (TGP-3) was dissolved in distilled water at a concentration of 10% by mass, and η at 37 ° C. was measured.⁵  Pa · sec. On the other hand, after agar was dissolved in distilled water at a concentration of 2% by mass at 90 ° C. and gelled at 10 ° C. for 1 hour, η at 37 ° C. was measured. 10⁷Pa · sec).
  Production Example 7
  71.0 g of N-isopropylacrylamide and 4.4 g of n-butyl methacrylate were dissolved in 1117 g of ethanol. An aqueous solution obtained by dissolving 22.6 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF Corporation) in 773 g of water was added thereto, and the mixture was heated to 70 ° C. under a nitrogen stream. While maintaining 70 ° C. under a nitrogen stream, 0.8 mL of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 8 mL of 10% ammonium persulfate (APS) aqueous solution were added and stirred for 30 minutes. Further, 0.8 mL of TEMED and 8 mL of 10% APS aqueous solution were added four times at 30-minute intervals to complete the polymerization reaction. After cooling the reaction solution to 10 ° C. or lower, 5 L of 10 ° C. cold distilled water was added for dilution, and the mixture was concentrated to 2 L at 10 ° C. using an ultrafiltration membrane having a fractional molecular weight of 100,000.
  The concentrated solution was diluted by adding 4 L of cold distilled water, and the above ultrafiltration concentration operation was performed again. The above dilution and ultrafiltration concentration operations were further repeated 5 times to remove those having a molecular weight of 100,000 or less. What was not filtered by this ultrafiltration (remaining in the ultrafiltration membrane) was recovered and freeze-dried, and in the present invention having a molecular weight of 100,000 or more, 72 g of a hydrogel-forming polymer (TGP-5) was added. Obtained.
  In the present invention obtained as described above, 1 g of the hydrogel-forming polymer (TGP-5) was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 20 ° C.
  Production Example 8
  42.0 g of N-isopropylacrylamide and 4.0 g of n-butyl methacrylate were dissolved in 592 g of ethanol. An aqueous solution in which 11.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF Corporation) was dissolved in 65.1 g of water was added thereto, and the mixture was heated to 70 ° C. under a nitrogen stream. While maintaining 70 ° C. under a nitrogen stream, 0.4 mL of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 4 mL of 10% ammonium persulfate (APS) aqueous solution were added and stirred for 30 minutes. Furthermore, 0.4 mL of TEMED and 4 mL of 10% APS aqueous solution were added four times at 30 minute intervals to complete the polymerization reaction. The reaction solution was cooled to 5 ° C. or lower, diluted by adding 5 L of 5 ° C. cold distilled water, and concentrated to 2 L at 5 ° C. using an ultrafiltration membrane having a fractional molecular weight of 100,000.
  The concentrated solution was diluted by adding 4 L of cold distilled water, and the above ultrafiltration concentration operation was performed again. The above dilution and ultrafiltration concentration operations were further repeated 5 times to remove those having a molecular weight of 100,000 or less. What was not filtered by this ultrafiltration (remaining in the ultrafiltration membrane) was recovered and freeze-dried. In the present invention having a molecular weight of 100,000 or more, 40 g of the hydrogel-forming polymer (TGP-6) was added. Obtained.
  In the present invention obtained as described above, 1 g of the hydrogel-forming polymer (TGP-6) was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 7 ° C.
  Production Example 9
  45.5 g of N-isopropylacrylamide and 0.56 g of n-butyl methacrylate were dissolved in 592 g of ethanol. An aqueous solution in which 11.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF Corporation) was dissolved in 65.1 g of water was added thereto, and the mixture was heated to 70 ° C. under a nitrogen stream. While maintaining 70 ° C. under a nitrogen stream, 0.4 mL of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 4 mL of 10% ammonium persulfate (APS) aqueous solution were added and stirred for 30 minutes. Furthermore, 0.4 mL of TEMED and 4 mL of 10% APS aqueous solution were added four times at 30 minute intervals to complete the polymerization reaction. After cooling the reaction solution to 10 ° C. or lower, 5 L of 10 ° C. cold distilled water was added for dilution, and the mixture was concentrated to 2 L at 10 ° C. using an ultrafiltration membrane having a fractional molecular weight of 100,000.
  The concentrated solution was diluted by adding 4 L of cold distilled water, and the above ultrafiltration concentration operation was performed again. The above dilution and ultrafiltration concentration operations were further repeated 5 times to remove those having a molecular weight of 100,000 or less. What was not filtered by this ultrafiltration (remaining in the ultrafiltration membrane) was recovered and freeze-dried, and in the present invention having a molecular weight of 100,000 or more, 22 g of the hydrogel-forming polymer (TGP-7) was added. Obtained.
  In the present invention obtained as described above, 1 g of the hydrogel-forming polymer (TGP-7) was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 37 ° C.
  Production Example 10
  44.0 g of N-isopropylacrylamide and 1.68 g of n-butyl methacrylate were dissolved in 592 g of ethanol. An aqueous solution in which 11.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by NOF Corporation) was dissolved in 65.1 g of water was added thereto, and the mixture was heated to 70 ° C. under a nitrogen stream. While maintaining 70 ° C. under a nitrogen stream, 0.4 mL of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 4 mL of 10% ammonium persulfate (APS) aqueous solution were added and stirred for 30 minutes. Furthermore, 0.4 mL of TEMED and 4 mL of 10% APS aqueous solution were added four times at 30 minute intervals to complete the polymerization reaction. After cooling the reaction solution to 10 ° C. or lower, 5 L of 10 ° C. cold distilled water was added for dilution, and the mixture was concentrated to 2 L at 10 ° C. using an ultrafiltration membrane having a fractional molecular weight of 100,000.
  The concentrated solution was diluted by adding 4 L of cold distilled water, and the above ultrafiltration concentration operation was performed again. The above dilution and ultrafiltration concentration operations were further repeated 5 times to remove those having a molecular weight of 100,000 or less. What was not filtered by this ultrafiltration (remaining in the ultrafiltration membrane) was recovered and freeze-dried, and in the present invention having a molecular weight of 100,000 or more, 22 g of the hydrogel-forming polymer (TGP-8) was added. Obtained.
  In the present invention obtained above, 1 g of the hydrogel-forming polymer (TGP-8) was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 26 ° C.
Example 1
(Detection of HCV-RNA in TGP cultured cell culture supernatant)
  Dicistronic HCV gene clone (consisting of hepatitis C virus (HCV) 5 'untranslated region gene-neomycin resistance gene-encephalomyocarditis virus 5' untranslated region gene-HCV total protein gene-HCV 5 'untranslated region gene) Was used as a template, and the gene was introduced into Huh7 cells by electroporation (for details of such HCV gene introduction methods, see, for example, the literature Pietschmann, T. et al., Journal of Virology 76: 4008-21 (2002). ).
  The cells obtained above were cultured for 3 weeks in ASF104 medium (AJINOMOTO) containing geneticin 0.5 mg / ml, D-glucose 4 g / L, and fetal bovine serum 2%, and a drug resistant cell line (RCYM1) was selected. The retention of HCV genomic RNA in RCYM1 cells was confirmed by real-time reverse transcription (RT) -PCR, and the production of HCV protein by Western blotting. 5 × 10 this cell⁶Subculture was performed in a flask up to a single cell.
  To 1 g of TGP (TGP prepared in Production Example 3) 10 mL of the culture solution (described above) and cooled to 4 ° C., the cells cultured in the flask were added, and 800 μL / well was poured into a 6-well plate at 37 ° C. And TGP was gelled. The gel seeded with the cells was overlaid with 3 ml of a culture solution warmed to 37 ° C., and cultured at 37 ° C. for 10 days. During the culture, the culture medium that was overlaid every 4 days was collected and replaced with a new culture medium.
  After completion of the culture, the gel was cooled to 4 ° C. to be sol, diluted by adding a culture solution, and RCYM1 cells that were three-dimensionally cultured by centrifugation were collected. From the culture supernatant, cell disruptions and the like were removed by centrifugation at 8000 g for 50 min. The supernatant was centrifuged at 25,000 rpm for 4 hours. The pellet was dissolved in 1 ml of TNE buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA) and fractionated by 10-60% sucrose density gradient centrifugation. After measuring the specific gravity of each fraction, it was diluted 3-fold with TNE buffer, centrifuged at 50,000 rpm for 2 hours, and the precipitate was redissolved in 100 μL of TNE buffer. RNA was extracted from this solution, and the amount of HCV-RNA in each fraction was quantified by a real-time RT-PCR method. As a control, the culture supernatant obtained by monolayer culture of the same number of RCYM1 cells for 10 days was analyzed by the same method.
  The base sequences of real-time RT-PCR primers and probes used for the analysis are shown below.
  5′-GAGTGTCGTGGCAGCCTCCA-3 ′ 5′-CACTCGCAAGCACCCTATCA-3 ′ 5 ′-(6-carbo × yfluorescine) Applied Biosystems) was used. The total amount of the reaction solution was 50 μL, and the temperature and the like were performed under the following reaction conditions.
  After the reaction at 60 ° C. for 1 min, 50 ° C. for 60 min, and 95 ° C. for 5 min, 50 cycles of 94 ° C. for 15 sec, 55 ° C. for 10 sec, 69 ° C. for 1 min were performed. As standard RNA, HCV-RNA synthesized in vitro was diluted and used as a standard.
  The result is shown in FIG. The black circle (●) in FIG. 4 is the TGP culture supernatant, and the white circle (◯) in the figure is the amount of HCV-RNA in the monolayer culture supernatant. The vertical axis represents the amount of HCV-RNA (10⁴  copy / fraction), the horizontal axis is the specific gravity (g / ml) of each fraction. As shown in the figure, HCV-RNA was detected from the TGP culture supernatant of RCYM1 cells with a specific gravity of 1.18 g / ml as a peak. This density was close to the value reported as the density of HCV particles in a study using sera of HCV-infected patients, and it was confirmed that HCV-like particles were present in the culture supernatant.
Example 2
(Detection of HCV-core protein in TGP cultured cell culture supernatant)
  5 × 10 3 Huh7 cells (RCYM1 cells) expressing the entire protein of hepatitis C virus and carrying the autonomously replicating dicistronic HCV genome⁶Subculture was performed in a flask up to a single cell. Cells cultured in the flask were added to the culture solution added to TGP and cooled to 4 ° C., 800 μL / well was dispensed into a 6-well plate, and the TGP was gelated by heating to 37 ° C. The gel was overlaid with 3 ml of the culture and cultured at 37 ° C. for 10 days. During the culture, the culture medium that was overlaid every 4 days was collected and replaced with a new culture medium. The culture solution used was the same as in Example 1.
  After completion of the culture, the gel was cooled to 4 ° C. to be sol, diluted by adding a culture solution, and RCYM1 cells that were three-dimensionally cultured by centrifugation were collected. From the culture supernatant, cell disruptions and the like were removed by centrifugation at 8000 g for 50 min. The supernatant was centrifuged at 25,000 rpm for 4 hours. The pellet was dissolved in 1 ml of TNE buffer and fractionated by 10-60% sucrose density gradient centrifugation. After measuring the specific gravity of each fraction, it was diluted 3-fold with TNE buffer, centrifuged at 50,000 rpm for 2 hours, and the precipitate was redissolved in 100 μL of TNE buffer. Using this solution as a sample, the amount of HCV-core protein in each fraction was quantified by ELISA.
  As a control, the culture supernatant obtained by monolayer culture of the same number of RCYM1 cells for 10 days was analyzed by the same method. The result is shown in FIG.
  The black circle (●) in FIG. 5 is the TGP culture supernatant, and the white circle (◯) in the figure is the amount of HCV-core protein in the monolayer culture supernatant. The vertical axis represents the amount of HCV-core protein (fmol / fraction), and the horizontal axis represents the specific gravity (g / ml) of each fraction. As shown in the figure, HCV-core protein was detected from the TGP culture supernatant of RCYM1 cells with a specific gravity of 1.18 g / ml as a peak. This density is close to the value reported for the density of HCV particles in studies using HCV-infected patient sera. Further, according to Example 1, the presence of HCV-RNA was also confirmed at the same density, and it was confirmed that HCV-like particles were present in the culture supernatant.
Example 3
(Detection of HCV particles in TGP cultured cell culture supernatant)
  Huh7 cells (RCYM1 cells) expressing the entire protein of hepatitis C virus and carrying the dicistronic HCV genome that replicates autonomously were prepared. 5 × 10 this cell⁶Subculture was performed in a flask up to a single cell. Cells cultured in a flask were added to a culture solution added to TGP and cooled to 4 ° C., 800 μL / well was dispensed into a 6-well plate, and the mixture was heated to 37 ° C. to gel TGP. After adding 3 ml of the culture solution warmed to 37 ° C., the gel was cultured at 37 ° C. for 10 days.
  After completion of the culture, the gel was cooled to 4 ° C. to be sol, diluted by adding a culture solution, and RCYM1 cells that were three-dimensionally cultured by centrifugation were collected. From the culture supernatant, cell disruptions and the like were removed by centrifugation at 8000 g for 50 min. The supernatant was centrifuged at 25,000 rpm for 4 hours. The pellet was dissolved in 1 ml of TNE buffer and fractionated by 10-60% sucrose density gradient centrifugation. A fraction with a specific gravity of 1.04 g / ml and a fraction of 1.18 g / ml which was the peak of HCV-RNA and HCV-core protein were diluted three-fold with TNE buffer and centrifuged at 50,000 rpm for 2 hr. This precipitate was redissolved in 20 μL of TNE buffer. After staining with 4% uranium acetate, observation was performed with a transmission electron microscope. The result is shown in FIG. In the 1.18 g / ml fraction, a virus particle-like structure having a membrane structure and a complicated internal structure was observed at a diameter of 50-60 nm, whereas in the 1.04 g / ml fraction, such a structure was observed. No structure was observed. This particle-like structure was identical in size and density to that reported as a particle in a study using HCV-infected serum, and was confirmed to be an HCV particle.
  FIG. 6 shows electron microscope (TEM) photographs of the 1.18 g / ml fraction (A) and the 1.04 g / ml fraction (B) obtained above. As shown in FIG. 6, HCV particles were confirmed in the 1.18 g / ml fraction.
Example 4
(Immunostaining of HCV particles in TGP cultured cell culture supernatant)
  Huh7 cells (RCYM1 cells) that express the entire protein of hepatitis C virus and maintain a dicistronic HCV genome that replicates autonomously were prepared. 5 × 10 this cell⁶Subculture was performed in a flask up to a single cell. Cells cultured in a flask were added to a culture solution added to TGP and cooled to 4 ° C., 800 μL / well was dispensed into a 6-well plate, and the mixture was heated to 37 ° C. to gel TGP. After adding 3 ml of the culture solution warmed to 37 ° C., the gel was cultured at 37 ° C. for 10 days. After completion of the culture, the gel was cooled to 4 ° C. to be sol, diluted by adding a culture solution, and RCYM1 cells that were three-dimensionally cultured by centrifugation were collected. From the culture supernatant, cell disruptions and the like were removed by centrifugation at 8000 g for 50 min. The supernatant was centrifuged at 25,000 rpm for 4 hours. The pellet was dissolved in 1 ml of TNE buffer and fractionated by 10-60% sucrose density gradient centrifugation. 1 ml of a fraction having a specific gravity of 1.18 g / ml, which was the peak of HCV-RNA and HCV-core protein, was diluted three-fold with TNE buffer and centrifuged at 50,000 rpm for 2 hours. This precipitate was redissolved in 20 μL of TNE buffer. This solution was reacted with an anti-HCV envelope mouse antibody as a primary antibody and an anti-mouse antibody labeled with a 10 nm gold colloid as a secondary antibody, washed, and then observed with a transmission electron microscope. The results are shown in FIG. 7A (Anti-E1, E2 Ab). As shown in FIG. 7A, it was observed that gold colloid was bound to the particle-like structure of 50-60 nm by the antigen-antibody reaction.
  On the other hand, when the same treatment was performed using mouse serum as the primary antibody as a negative control, a particle image in which colloidal gold was bound as shown in FIG. 7B (Normalmouse serum) could not be observed. From these results, it was reconfirmed that E1 and E2 which are HCV structural proteins are present on the surface of the particle-like structure and are HCV particles.
Example 5
(Radial flow type bioreactor (RFB)) Secretion of HCV particles from RCYM1 carrying genomic length dicistonic HCV-RNA cultured in culture)
  The replicable capacity of selectable genome-length HCV-RNA in FLC4 cells was first evaluated. However, no G418 resistant colonies were observed, indicating that FLC4 cells do not support replication of these HCV-RNAs. Therefore, subsequent experiments were performed with a stable Huh-7 cell line (RCYM1). And Con1 clone l389neo / core-37NK 5.1 (genotype 1b) (Pietschmann, T., V. Lohmann, A. Kaul, N. Krieger, G. Rink, G. Rutter, D. Strand, and R. Bartenschlager.2002. Persistently expressed by RNA of the transfection of RNA from the persistent and transient replication of full-length hepatitis C virus genes in cell culture, J. Virol. 76: 4008-4021). Supports full-length HCV-RNA replication did. HCV-RNA levels in RCYM1 cells were approximately 5 × 10 6 determined by real-time RT-PCR.⁶Copy / μg total RNA. HCV protein expression and subcellular localization was confirmed by Western blotting and immunofluorescence analysis. To develop a 3D RFB culture, RCYM1 cells were first loaded onto an RFB column by flowing a cell suspension, and the cells were then attached to carrier beads. The cells grew within the 3D matrix and the culture medium was circulated in a radial direction through the column. To determine if HCV particles were secreted from RCYM1 cells in the RFB culture system, culture fluid was collected after 5-10 days of culture and fractionated by continuous 10-60% (wt / vol) sucrose density gradient centrifugation. It was done. HCV-RNA and core protein were found primarily at 1.15 to 1.20 g / ml, maximizing at the 1.18 g / ml fraction (FIGS. 8A and B).
  FIG. 8 is a graph showing the results of sucrose density gradient analysis of the culture supernatant of RCYM1 cells. Culture media collected from RFB cultured RCYM1 (black circles), monolayer cultured RCYM1 (white squares) and RFB cultured 5-15 cells (white triangles) as described in “Materials and Methods” Was separated.
  FIG. 8A: HCV-RNA in individual fractions was measured by real-time RT-PCR. The average of duplicate measurements was plotted against the corresponding fractional density.
  FIG. 8B: HCV core protein in individual fractions was determined by ELISA. The average of duplicate measurements was plotted against density.
  FIG. 8C: The culture broth of RCM1 cells cultured in RFB was treated with 0.2% NP40 (white circle) and then centrifuged with a sucrose gradient. Each fraction was examined for HCV-RNA by real-time RT-PCR.
  In the same experiment using 5-15 cells (the subgenomic HCV replicon was replicated), a similar peak corresponding to HCV-RNA observed in RCYM1 cells was not detected. Substantial amounts of HCV-RNA were detected in the 1.03-1.07 g / ml fraction in both 5-15 and RCYM1 cells in the RFB culture system (FIG. 8A). Previous report by Pietschmann et al. (Pietschmann, T., V. Lohmann, A. Kaul, N. Krieger, G. Rink, G. Rutter, D. Strand, and R. Bartenschlanger. 2002. Persistent and trandent trandent and trandent. These RNAs released from the cells in the subgenomic replicon did not match the virus pieces, consistent with length hepatitis C virus genes in cell culture. J. Virol. 76: 4008-4021). When an equivalent number of RCYM1 cells were cultured in a monolayer culture system, only limited amounts of HCV-RNA and core protein were detected in the culture supernatant (FIGS. 8A and B).
  Mature HCV virions are believed to have a nucleocapsid and an outer envelope composed of a lipid membrane and a viral envelope glycoprotein. The culture fluid was treated with NP40 to solubilize lipids and then subjected to sucrose density gradient centrifugation. HCV (FIG. 8C) aggregated to a density of 1.22 g / ml rather than 1.18 g / ml) (FIG. 8C) indicates that the density of HCV particles has been shifted to a higher density due to de-envelopment. Show. Transmission electron microscope (TEM) of 1.18 g / ml fraction (subjected to negative staining after concentration) showed a particle structure with a diameter of 30-60 nm and a main particle size of 50 nm. . Similar particle-like structures were not observed in the same density fraction of RCYM1-monolayer culture or in the 1.07 g / ml fraction of RCYM1-RFB culture. These results indicate that in the RFB system, the production and secretion of HCV particles was possible with a selectable bistatic HCV genome.
Example 6
Production and secretion of HCV particles from spheroid cultures of RCYM1 cells using thermoreversible gelling polymer (TGP)
  RCYM1 cells seeded in TGP as in Examples 1 and 2 formed spheroids after 3 days of culture, and numerous spheroids having a diameter of about 1 mm were observed after 7-10 days of culture. After 8 days of culture, spheroids were fixed and examined by scanning electron microscopy (FIGS. 9A and 9B (a1)) and ultrathin sections by TEM (FIGS. 10A (B) and 10B (b1)). ).
  FIGS. 9 to 11 are photographs showing examples of embodiments in which Huh-7 and RCYM1 cells form spheroids in TGP. Scanning electron micrographs (FIGS. 9A and 9B (a1)) and transmission electron micrographs (FIGS. 10A (B) and 10B (b1)) of RCYM1 cells cultured in TGP for 8 days are shown.
    Black arrow: villiform process, white arrow: bile tubule-like structure.
  11A-11F (C-H): intracellular station of connexin32 in Huh-7 cells cultured in TGP (FIGS. 11A-C (CE)) and monolayers (FIGS. 11D-11F (F-H)) Presence.
  FIG. 11A (C) and 11D (F): immunostaining of connexin 32
  11B (D) and 11E (G): phase contrast microscope
  FIG. 11C (E): Overlay of 11A (C) and 11B (D)
  The nuclei were colored by post-staining with propidium iodide.
  Well developed villus-like processes (polarizing epithelial features) were observed on the cell surface (FIGS. 9A and 9B (a1)). In the space between cells, bile tubule-like structures were also observed, which appeared to be connected via tight junctions (FIGS. 10A (B) and 10B (b1)). This cell morphology is similar to that observed in RFB culture (Garoff, H., R. Hewson, and D. J. Opstelten. 1998. Virus measurement by budging. Microbiol. Mol. Biol. Rev. 62). 1177-1190. Grakoui, A., D.W. McCourt, C. Wychowski, S. M. Feinstone, and C. M. Rice. 1993. Characteristic of the hepatitis C-development of serine dependent polyprotein cleavage sites.J Virol. 67: 2832-2843), well correlated with characteristics of mature liver tissue. Observation of connexin32, which plays an important role in the formation of gap junctions between cells by immunostaining, localizes connexin32 on the surface of cell adhesion in TGP cultures (FIGS. 11A-11C (CE)), whereas monolayers In cultured tissues, it was found that it is mainly distributed in the cytoplasm (FIGS. 11D-11F (FH)). This indicates that Huh-7 cells gained cell polarity in the 3D culture system.
  It is known that replication of HCV replicon in Huh-7 cells depends on host cell growth. Here, it was found that the growth of RCYM1 cells in the TGP culture system was significantly slower than that of the cells in the monolayer culture (FIG. 12A). Therefore, the expression of HCV protein (FIG. 12B) and viral RNA copy number in RCYM1 spheroids were apparently lower than those observed in monolayer cells. Results from sucrose density gradient analysis of the culture supernatant showed co-precipitation of HCV-RNA and core protein at a density of 1.15 to 1.20 g / ml and a peak at 18 g / ml. (FIGS. 12C (C) and 12D (D)). This distribution was consistent with the pattern obtained in RFB culture (FIGS. 8A and B). It should be noted that due to the slower growth of cells, fewer cells were used in 3D cultures in these experiments compared to monolayer cultures.
  12 to 13 are diagrams showing expression of HCV protein in RCYM1 cells and secretion of viral molecules in TGP culture.
  FIG. 12A: Cell growth curves of TGP culture (black circle) and monolayer (white circle) of RCYM1 cells. Cells were harvested on days 0, 3, 6, and 9 after inoculation and cell numbers were determined.
  FIG. 12B: Western blotting of HCV core and NS5A protein in RCYM1 cells and control Huh-7 cells.
  12C and 12D: Sucrose density gradient analysis of culture supernatants of RCYM1 cells. HCV core protein (C) and viral RNA (D) in individual fractions were determined by ELISA and real-time RT-PCR, respectively. Representative data from three independent experiments are shown. Black circle: HCV-RNA in TGP culture, white circle: HCV-RNA in monolayer culture; black triangle: HCV core protein in TGP culture, white triangle: core protein in monolayer culture.
  Figures 13A-13D (E-H): Electron micrographs of HCV particles in the supernatant of TGP cultured RCYMl cells.
  FIG. 13A (E): negative staining of HCV particles in a 1.18 g / ml density fraction
  As shown in FIG. 13D (H), the spherical structure was absent in the 1.05 g / ml density fraction.
  FIG. 13B (F): Ultrathin section of HCV particles. Precipitated HCV particle samples were prepared from the 1.18 g / ml fraction.
  FIG. 13C (G): Immunogold labeling of HCV particles with anti-E2 antibody in a 1.18 g / ml density fraction. Gold particles: 5 nm, Bars, 50 nm.
  TEM analysis of the 1.18 g / ml fraction after negative staining showed a particle structure with a diameter of 50-60 nm and spike-like projections (FIG. 13A (E)). Observation of the ultrathin section showed a lipid bilayer-like membrane structure having a width of about 5 nm (FIG. 13B (F)). Immunoelectron microscopy using anti-E2 antibody revealed HCV envelope protein on the particle surface (FIG. 13C (G)). Although significant amounts of HCV-RNA were detected in the supernatant 1.03-1.05 g / ml fractions (FIG. 12A), no virus-like particle structure was observed in these fractions (FIG. 13D (H )). These results were consistent with the results from the RFB system shown above. Thus, the effectiveness of the 3D cell culture system in the virus particle structure was shown in the TGP culture system and the RFB system using cells derived from human liver.
Example 7
Ultrastructural localization of HCV particles in TGP-cultured spheroids of RCYM1 cells
  The subcellular localization of HCV particles generated in RCYM1-TGP culture at the ultrastructural level was then determined by EM analysis ultrathin sections. Spherical particles with a membrane-like structure with protrusions on the short surface (diameter 50-60 nm) were observed mainly in the ER membrane (FIG. 14A) as in the ER wide sac (FIG. 14B). .
  FIG. 14 is an electron micrograph of an ultrathin section of RCYM1 cells grown in TGP. HCV particles in RGPM1 cells cultured in TGP. Globular virus-like particles (arrows) with a diameter of 50-60 nm were observed in the ER membrane (FIGS. 14A and B), and cytoplasmic vesicles.
  In follicles, these virus-like particles were often accompanied by amorphous material. Previously, Shimizu et al. Reported that virus-like particles similar in morphology and size were observed in human B cells infected with HCV (Shimizu, YK, SM Feinstone, M. Kohara). , R.H.Pur cells, and H.Yoshikura.1996.Hepatitis Cvirus: detection of intra cells, ultra virus particles by electron microscopy.Hepatology 23: 205-209). Similar particle-like structures were not observed in RCYM1 cells in monolayer culture or in subgenomic replicon 5-15 cells in TGP culture.
  To determine whether the virus-like particles observed using conventional TEM in this experiment were HCV particles, immunoelectron microscopic analysis was performed using anti-core and anti-E1 antibodies. Double labeling experiments showed that virus-like particles that cooperate with the ER membrane were immunoreactive with both HCV proteins and that the E1 protein surrounded the core protein (FIG. 15A). . According to the findings of the present inventors, this is the first report clearly showing that the virus envelope protein surrounds the core protein in HCV particle formation. As a negative control, slices prepared from 5-15 cells containing subgenomic RNA were stained with these antibodies and they showed negligible levels of background in immunostaining.
  Due to the low resolution and contrast of micrographs, it is generally difficult to visualize intracellular microstructure and perform antigenic protein localization using an immunogold electron microscope. To overcome this difficulty, a silver sensitized immunogold labeling method was applied in this experiment (FIGS. 15B and 15C). Using this method, approximately 20 nm antigen-reactive immune gold particles were observed.
  FIG. 15 is an immunoelectron micrograph of an ultrathin section of RCYM1 cells cultured in TGP. FIG. 15A (A): Double immunostaining with anti-E1 and anti-core monoclonal antibodies. Core protein-specific gold particles (10 nm diameter) and E1 protein-specific gold particles (5 nm diameter) formed rosettes on the surface of the ER membrane.
  Figures 15B (B) and 15C (C)): Silver-sensitized immunity gold with anti-core (B) and anti-E1 (C) antibodies. A second antibody conjugated with 1.4 nm diameter gold particles was applied, and then the particles were enlarged with a silver sensitizer. Arrows indicate that virus-like particles react with anti-core and anti-E1 antibodies.
  Specific immunolabeling of the core and E1 proteins was mainly detected within the ER and on the ER membrane. Strong immunopositive reactions were detected in cytoplasmic vesicles and on virus-like particles observed on the ER membrane. Such immune labeling was not observed when normal mouse serum was used as the first antibody. These results confirm conventional TEM ultrastructural observations and suggest that the formation of HCV particles is achieved by the putative core particle bundling in the ER membrane.
Example 8
Dependence of infectivity of HCV particles on E2 glycoprotein
  To determine whether HCV particles released from RCYM1 cells cultured in the TGP system are infectious, naive Huh-7.5.1 cells (HCV-negative Huh-7.5 (7) Cells were inoculated with the culture supernatant of RCYM1 spheroids and analyzed by immunofluorescence staining for NS5A protein 4 days after inoculation (FIG. 16A).
  FIG. 16 shows the infectivity of HCV particles secreted from RGPM1 cells cultured in TGP and neutralization of the infectivity.
  FIG. 16A (A): Huh7.5.1 cells infected with HCV particles (upper panel: infection (+)) or uninfected (lower panel: infection (−)) were cultured for 4 days, Immunostained with NS5A antibody. Nuclei were colored by post-staining with DAPI.
  FIG. 16B (B): Huh7.5. 1 cells post-treated with anti-E2 antibody (AP33), NOB antibody, or anti-FLAG antibody were infected with HCV particles and incubated for 4 days. HCV-RNA in the cells was determined by real-time RT-PCR. Average values in triplicate samples are shown along with standard deviations.
  About 1% Huh-7.5.1 cells were observed to be NS5A positive. In contrast, NS5A-positive cells were not detected when Huh-7.5.1 cells were inoculated using cell supernatant samples obtained from 5-15 cells cultured in TGP. To further determine whether viral envelope proteins mediate infection with HCV particles, serum from patients with high titers of anti-E2 and NOB antibodies (1 / 3500-1 / 4000) (Ishii, K. et al.). , D. Rosa, Y. Watanabe, T. Katayama, H. Harada, C. Wyatt, K. Kiyosawa, H. Aizaki, Y. Matsuura, M. Hughtoni, S. Abrigani, and T. Miyah. of antibodies inhibiting the binding of envelope to human cells s correlated with natural resolution of chronic hepati is C.Hepatology 28: 1117-1120), or with anti-FLAG antibody (FIG. 16B), HCV particles were pre-incubated. Intracellular HCV-RNA levels decreased to 43%, 28%, and 26%, respectively, in the presence of anti-E2 antibodies, NOB3, and NOB4. No reduction of viral RNA in infected cells was observed after treatment with anti-FLAG antibody. Taken together, these results indicate that HCV particles produced by RCYM1 cells cultured in TGP are infectious and that the viral envelope protein plays an important role in the infection of Huh-7.5.1 cells. Strongly suggest.
Example 9
Potential use of TGP culture system for HCV production and evaluation of antiviral agents
  Lindenbach et al. Recently reported that a cell culture system that supports complete replication of the HCV genotype 2a clone is useful for the evaluation of antiviral drugs (Lindenbach, BD, MJ Evans, A. et al. J. Syder, B. Walk, TL Tellinghuisen, CC Liu, T. Maruyama, R. O. Hynes, D. R. Burton, JA McKeating, and C. R. Rice. Complete replication of hepatitis C virus in cell culture. Science 309: 623-626). To date, however, this complete HCV culture system has not been extended to genotype 1, which is most often detected and most difficult to treat in patients with hepatitis C.
  Here we show the potential utility of TGP cultures of RCYM1 cells for evaluating anti-HCV drugs (FIG. 17).
  FIG. 17 shows inhibition of HCV particle production by IFN and RBV. RGPM1 cells cultured in TGP were treated with 100 IU / ml IFN-α or 100 μm RBV, and then HCV-RNA in the cells (FIG. 17A) and HCV-RNA in the medium (FIG. 17B) were determined. It was. Media from individual samples was fractionated by sucrose gradient centrifugation and HCV particle positive (1.18 g / ml) and negative (1.04 g / ml) fractions were analyzed. Mean values and standard deviations in triplicate samples are shown. Black bar: control without treatment, shade bar: IFN-α, white bar; RBV.
  Intracellular HCV-RNA levels in TGP cultured RCYM1 cell spheroids were reduced to 90% after 3 days of culture with 100 IU / ml IFN-α (FIG. 17B). Similarly, the extracellular HCV particle concentration calculated using the HCV-RNA copy number of the 1.18 g / ml supernatant fraction was 89% with IFN-α treatment (FIG. 17A). Furthermore, HCV particle production was inhibited to the same extent (85%) as intracellular HCV-RNA by treatment with 100 μM RBV (FIG. 17A). The level of HCV-RNA detected in the 1.04 g / ml fraction of the culture supernatant of the untreated group is about 14 times that in the 1.18 g / ml fraction, which is equal to that of IFN-α or RBV. (FIG. 17A). Although the mechanism underlying this increase is not known, a similar phenomenon was observed when highly cytotoxic drugs were evaluated using TGP-RCYM1 cultures. Thus, as a result of cell death due to the mild cytotoxic effects of IFN and RBV, some cellular proteins that cooperate with HCV-RNA may be released into the culture supernatant.
  In summary, these results demonstrate that the HCV production model based on TGP culture is useful for assessing HCV particle production and the inhibitory action of anti-HCV drugs.
[Sequence Listing]

Claims (7)

A cultured HCV particle, wherein both HCV (hepatitis C virus) -RNA and HCV-core protein are present in the same fraction formed by centrifugation under a concentration gradient. A cultured HCV particle characterized by being capable of binding to an anti-HCV antibody labeled with gold colloid. Using a hydrogel-forming polymer whose aqueous solution becomes a fluid sol state at a temperature lower than the sol-gel transition temperature and reversibly becomes a hydrogel state at a temperature higher than the sol-gel transition temperature,
Mixing an aqueous solution of the gel-forming material in a fluid sol state at a temperature lower than the sol-gel transition temperature with cells containing the grown HCV particles,
Culturing the cells with the mixture gelled at a temperature higher than the sol-gel transition temperature;
A method for growing HCV particles, wherein the mixture is made into a sol at a temperature lower than a sol-gel transition temperature to recover HCV particles released from the cells. The method for growing HCV particles according to claim 3, wherein the cells are collected in addition to the collection of the released HCV particles. The method for growing HCV particles according to claim 3 or 4, wherein the sol-gel transition temperature is in the range of 0 ° C to 37 ° C. The method for growing HCV particles according to any one of claims 3 to 5, wherein the aqueous solution of the hydrogel-forming polymer is substantially water-insoluble in a gel state. Using a hydrogel-forming polymer whose aqueous solution becomes a fluid sol state at a temperature lower than the sol-gel transition temperature and reversibly becomes a hydrogel state at a temperature higher than the sol-gel transition temperature,
Mixing an aqueous solution of the gel-forming material in a fluid sol state at a temperature lower than the sol-gel transition temperature with cells containing the grown HCV particles,
Culturing the cells in the presence of the drug to be evaluated in a gelled state of the mixture at a temperature above the sol-gel transition temperature;
A method for evaluating a drug, wherein the mixture is made into a sol at a temperature lower than a sol-gel transition temperature, and HCV particles released from the cells are collected or detected.