JP2019156804A - Cataract inhibitor - Google Patents

Abstract

To provide novel effective agents for cataract treatment.SOLUTION: Provided is a cataract inhibitor or therapeutic agent that contains a culture supernatant component obtained by culturing mesenchymal stem cells. Preferably, provided is an inhibitor or therapeutic agent that comprises the culture supernatant component of bone marrow mesenchymal stem cells or adipose tissue-derived mesenchymal stem cells. Preferably, provided is an inhibitor or therapeutic agent that comprises an exosome. Preferably, provided is an inhibitor or therapeutic agent that is an eye drop or an ophthalmic ointment.SELECTED DRAWING: None

Description

  The present invention relates to a substance that suppresses white turbidity of a lens. More specifically, the present invention relates to an agent in the form of eye drops or eye ointment containing at least a part of the culture supernatant of mesenchymal stem cells.

  Cataract is a disease in which the eye lens becomes cloudy. The direct cause occurs when crystallin, which is a protein constituting the lens, aggregates and the transparency of the lens is lost. As a cause of cataract, aging, diabetes, ultraviolet rays, etc. have been reported, but in any case, the root cause has not been elucidated, and once the transparency is lost, it is not possible to restore the original lens I can't. For this reason, drug treatment is used to delay the progression of cataracts as much as possible, or treatment is carried out by crushing the turbid lens with ultrasonic waves (ultrasonic lens emulsification) and inserting an intraocular lens . In recent years, research on regenerative medicine has become active, and it has become clear that various diseases can be treated by cell transplantation treatment in which stem cells are transplanted.

  Mesenchymal stem cells are a type of somatic stem cell and have the ability to differentiate into mesenchymal cells, i.e., bone cells, cardiomyocytes, chondrocytes, fat cells, etc. Application to regenerative medicine such as construction is expected. In addition to this, mesenchymal stem cells have anti-inflammatory action, immunomodulation action, and the like, and thus have already been used for the treatment of various autoimmune diseases and graft-versus-host disease. Furthermore, it has been reported that liver cirrhosis, which is a chronic liver disease, is also effective in suppressing fibrosis of liver tissue.

  Under such circumstances, it has been found that mesenchymal stem cells transplanted in vivo in cell transplantation treatment not only regenerate the tissue by the proliferation and differentiation of the cells themselves. That is, it has become clear that various properties possessed by physiologically active substances such as various cytokines secreted from cells contribute to tissue regeneration and disease site healing.

  When mesenchymal stem cells are cultured in vitro, physiologically active substances are released into the culture medium. Thus, an example has been reported in which a culture solution used for culturing mesenchymal stem cells is recovered and tissue is regenerated using this culture solution containing a large amount of substances released from the cells. Ueda et al. Showed in an experiment using rats that the culture supernatant of bone marrow mesenchymal stem cells had bone regeneration ability (Non-patent Document 1). Among these, the culture supernatant of bone marrow mesenchymal stem cells contains a lot of insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), etc., and these factors are used for tissue regeneration. It is suggested to be involved.

  In addition, Arimura et al. Have reported that the culture supernatant of bone marrow mesenchymal stem cells has an anti-inflammatory action and exhibits preventive and therapeutic effects on enteritis (Patent Document 1).

  Recently, vesicles called exosomes secreted from mesenchymal stem cells contain various proteins and RNAs, and this has been reported to have the same therapeutic effect as mesenchymal stem cells (Non-patent Document 2). ).

Japanese Patent No. 6132459

Tissue EngineeringPart A. 2012; 18: 1479-1489 Drug Delivery System. 2014; 29-2: 141-151

  Cataract is a disease in which the lens of the eye becomes cloudy, and the direct cause is caused by aggregation of crystallin, which is a protein constituting the lens, and loss of transparency of the lens. The root cause of cataracts has not been elucidated, and one of the treatment methods is pharmacotherapy to delay the progression of cataracts as much as possible, but this is not always effective and is a new effect. Drug development is required.

As a result of intensive studies on the above problems, the present inventors have found that the above problems can be solved, and have completed the present invention.
That is, the outline of the present invention is as follows.
1. An inhibitor or therapeutic agent for cataract comprising a culture supernatant component obtained by culturing mesenchymal stem cells.
2. 2. The inhibitor or therapeutic agent for cataracts according to 1, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells or adipose tissue-derived mesenchymal stem cells.
3. 3. The inhibitor or therapeutic agent for cataract according to 1 or 2, wherein the culture supernatant component contains exosomes.
4). The inhibitor or therapeutic agent for cataract according to any one of 1 to 3, which is an eye drop or an eye ointment.

  According to the present invention, the opacity of the lens can be prevented, so that cataract can be effectively prevented or treated.

It is a schematic diagram which shows an example of the cell culture container used for preparation of the culture supernatant of a mesenchymal stem cell. It is a schematic diagram which shows an example of the cell culture apparatus used for preparation of the culture supernatant of a mesenchymal stem cell. It is the schedule of culture supernatant preparation in an Example. It is a graph which shows the score transition of the rat cataract using the culture supernatant obtained in the Example. It is a graph which shows the score transition of the rat cataract using the exosome solution prepared from the culture supernatant obtained in the Example.

(Mesenchymal stem cells)
In the present invention, the mesenchymal stem cells are not particularly limited, but bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and the like are preferable. Further, not only primary cells but also mesenchymal stem cells established / immortalized by genetic modification or the like can be used. The animal species is not particularly limited, and any animal species such as human, mouse and rat can be used.

(Culture supernatant of mesenchymal stem cells)
In the present invention, the culture supernatant of mesenchymal stem cells refers to a culture solution that has been in direct contact with cells or indirectly through a semipermeable membrane when cells are cultured for a certain period (several hours to several days). Is obtained by separating cells from cells. Consent with culture medium conditioned medium, Conditioned medium, etc.

(Culture medium)
In the present invention, the composition of the culture solution used for production of the culture supernatant is not particularly limited. For example, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium Eagle, Alpha Modulation (αMEM), Rowell Park Memorial Institute medium, RPMI What was prepared by adding etc. can be used.

  In the present invention, it may be preferable that the culture medium to be used does not contain animal serum in some cases. This is because animal serum contains abundant physiologically active substances such as cell growth factors, and the presence of these physiologically active substances sometimes interferes with the purpose of using the culture supernatant or acts negatively. This is because there is a possibility.

  In the present invention, in order to culture a mesenchymal stem cell and obtain a culture supernatant, it is preferable to use a cell culture container in which a semipermeable membrane is housed as a culture substrate. Such a cell culture container not only can save space because it can increase volumetric efficiency, but also can efficiently recover the culture supernatant by using a semipermeable membrane having a specific configuration. it can.

(Semipermeable membrane)
In the present invention, the semipermeable membrane used as a culture substrate is preferably one having a structure that can hold cells on the surface of the semipermeable membrane and permeate a solution or a low-molecular substance. More specifically, the culture supernatant component does not permeate the semipermeable membrane, but the culture fluid component preferably has a structure (pore diameter) that permeates the semipermeable membrane. Specifically, a semipermeable membrane having a characteristic that exosomes (about 30 nm to 150 nm) in the culture supernatant do not permeate the membrane and γ-globulin (about 8.4 nm) as a culture solution component permeates is preferable. . Then, it is preferable to use an ultrafiltration membrane having a pore radius of about 5 nm to 20 nm as the semipermeable membrane.

  In the present invention, the material of the semipermeable membrane is not particularly limited, and examples thereof include acrylic resins such as 2-hydroxyethyl methacrylate and polymethyl methacrylate, cellulose resins such as cellulose acetate and regenerated cellulose, polysulfone and polyethersulfone, and the like. Polysulfone resin, polyester resin such as polylactic acid and polyhydroxyalkanoate, polyolefin resin such as polyethylene and polypropylene, polyvinyl alcohol, epoxy resin, polyacrylonitrile, polyvinylidene fluoride, polystyrene, polyamide and the like can be suitably used. These derivatives may be the main component.

  In the present invention, the semipermeable membrane may be a material obtained by chemically modifying the material. For example, it may be hydrophilized. By using a semipermeable membrane subjected to a hydrophilic treatment, it becomes easy to supply a liquid component such as a culture solution to cultured cells. Examples of the method for hydrophilizing the semipermeable membrane include a method of treating the semipermeable membrane with a hydrophilic polymer such as ethylene-vinyl alcohol copolymer, glycerin, and ethanol. Depending on the cells to be used, a coating such as collagen or fibronectin may be applied to improve adhesion to the semipermeable membrane.

  In the present invention, the semipermeable membrane may be a flat membrane or a hollow fiber membrane, but it is preferable to use a hollow fiber membrane from the viewpoint of volume efficiency. In the case of the hollow fiber membrane, if the inner diameter is too small, the culture volume cannot be secured, or stress is applied to the cultured cells. On the other hand, if the inner diameter is too large, the volumetric efficiency is lowered and the merit of the hollow fiber membrane culture vessel is impaired. Further, the thickness of the semipermeable membrane is preferably about 10 μm to 200 μm in consideration of the permeability of the culture solution components and the membrane strength.

  In the present invention, the method for producing a culture supernatant of mesenchymal stem cells is not particularly limited. For example, the hollow fiber membrane is used as a cell culture substrate, and the mesenchymal system is used on the lumen side or the outer lumen side of the hollow fiber membrane. What is necessary is just to culture a stem cell. For example, a cell suspension in which mesenchymal stem cells are suspended in a culture solution or the like is filled in the lumen of the hollow fiber membrane, and the culture solution is continuously or intermittently provided on the lumen side and the outer lumen side of the hollow fiber membrane. Mesenchymal stem cells are cultured by perfusion or the like. In addition, intermittent perfusion refers to repeating the process of temporarily stopping or advancing the flow of the culture solution. Here, the interval at which the flow is stopped or advanced is not particularly limited, and may be equal or irregular. The culture solution has a role of supplying nutrients and oxygen necessary for the cells, and conversely discharging waste products. In this way, a culture supernatant of mesenchymal stem cells can be obtained by collecting the culture medium on the hollow fiber membrane lumen side after culturing for a certain period.

(Cell culture vessel)
In the present invention, a cell culture container used for producing a culture supernatant of mesenchymal stem cells contains several to tens of thousands of hollow fiber membranes in a cylindrical container having four openings, and both ends of the hollow fiber membranes. Can be prepared by liquid-tightly adhering and fixing to a cylindrical container. Such a cell culture vessel can greatly increase the culture area per unit volume, and can simplify the culture operation, so that cell culture can be carried out efficiently.

  The configuration of such a cell culture container is not particularly limited. For example, as shown in FIG. 1, a necessary number of hollow fiber membranes are bundled in a cylindrical container having four openings (end ports and side ports) as needed. The form accommodated is mentioned. Specifically, in the cell culture container 1, the plurality of hollow fiber membranes 3 are sealed in a state where the inner and outer cavities of each hollow fiber membrane are separated at both ends, and so as not to block the hollow portion of the hollow fiber membrane. A material (for example, polyurethane potting agent) 8 is bonded and fixed to the end of the cylindrical container 2. That is, of the four openings, the two end ports 6 a and 6 b communicate with the lumen (hollow part) 5 of the hollow fiber membrane 3. On the other hand, of the opening, the two side ports 7a and 7b communicate with a space (external side) 4 that is inside the cylindrical container 2 and outside the hollow fiber membrane, The culture medium or the like introduced from one of the side ports 7a or 7b is led out from the other side port 7b or 7a that passes through the outer cavity side 4.

(Cell culture device)
FIG. 2 shows an example of a cell culture apparatus using a hollow fiber membrane cell culture vessel. The end port 6a communicating with the hollow fiber membrane lumen 5 of the cell culture vessel 1 introduces and sends a cell suspension containing mesenchymal stem cells from the introduction port 40 and the culture solution storage vessel 9 A flow path for feeding the cell culture solution from is connected. A valve 20 is provided in the middle of the flow path so that the flow paths of the cell suspension and the cell culture solution can be switched. The end port 6b communicating with the hollow fiber membrane lumen 5 of the cell culture vessel 1 is connected to a flow path for discharging the culture supernatant after culturing, and the flow rate is adjusted in the middle of the flow path. The valve 21 for switching the flow path to the valve 21 and the liquid feeding pump 31, the culture supernatant collection container 11 or the discharge port 50 are provided. On the other hand, to the side port 7 a communicating with the hollow fiber membrane outer space 4 of the cell culture container 1, a flow path for sending the culture solution from the culture solution storage container 8 to the hollow fiber membrane outer space 4 is connected. . The side port 7b communicating with the hollow fiber membrane outer space 4 is connected to a flow path for discharging the culture solution, and a liquid feed pump 30 is provided in the middle of the flow path. It is connected to the collection container 10 for collecting the culture medium. In the present invention, it is preferable that at least the culture solution storage containers 8 and 9, the flow path, and the cell culture container 1 are installed in a CO ₂ incubator.

(Manufacture of culture supernatant)
When culturing mesenchymal stem cells, a cell suspension is introduced into the hollow fiber membrane lumen of the cell culture container, and the mesenchymal stem cells are seeded on the surface of the hollow fiber membrane. Mesenchymal stem cells are cultured while adjusting the culture environment by flowing a cell culture medium through both. Then, since the mesenchymal stem cells release various secretions (proteins, cytokines, exosomes) into the culture solution, these secretions are collected together with the cell culture solution.

The production of the culture supernatant will be described with reference to FIG. The cell suspension is fed from the introduction port 40, and the hollow fiber membrane lumen 5 is filled with the cell suspension. After the cell suspension is filled, the valve 20 is closed. After filling the hollow fiber membrane lumen 5 with the cell suspension, the cells are allowed to adhere to the surface of the hollow fiber membrane by standing for a certain time. After standing for a certain period of time, the valves 20, 21, and 22 are switched so that the culture medium storage container 9, the hollow fiber membrane lumen 5, and the discharge port 50 communicate with each other, and the pumps 30 and 31 are activated to hollow the cell culture container. A cell culture fluid is passed through both the membrane lumen 5 and the hollow fiber membrane outer lumen 4. At this time, the flow rate of the culture solution is preferably adjusted according to the degree of cell growth and the environment. At least the cell culture container, the culture solution storage container, and the flow path connecting them are installed in an incubator equipped with a temperature and CO ₂ concentration control mechanism. After culturing for several days, the culture solution in the culture solution storage container 9 is replaced with a culture solution for collecting the culture supernatant. After exchanging the culture solution for collecting the culture supernatant, the valve 22 is switched and the culture supernatant is recovered in the culture supernatant recovery container 11.

  The flow rate of the culture solution, particularly the culture solution flowing through the hollow fiber membrane lumen 5, is preferably adjusted according to the degree of cell growth and the environment. The method for examining the degree of cell proliferation is not particularly limited, but can be performed based on the measurement results such as the concentration of glucose and lactate in the culture solution.

  In the present invention, the culture supernatant means a product obtained by removing mesenchymal stem cells from the collected culture solution. For example, a residual medium component (of the culture solution component before culture, The mesenchymal system in which at least a part of components that do not contribute to prevention or treatment of lens tissue hardening in the present invention, such as components remaining in the subsequent culture solution) and moisture in the culture solution, is further removed. It is contained in the culture supernatant of stem cells. A suspension containing exosomes extracted from the collected culture fluid (culture supernatant) is also within the scope of the present invention.

  In addition, in the culture supernatant, other active ingredients such as antiallergic or antihistamine ingredients, decongestant ingredients, local anesthetic ingredients, vitamin ingredients (vitamins A and B groups) unless an undesirable interaction is caused by the formulation. C), amino acid components (eg, valine, leucine, isoleucine, serine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, lysine, histidine, citrulline, ornithine, cystine, taurine, glycine) Furthermore, you may contain. As such other active ingredients, various known drugs can be appropriately used. In addition, other active ingredients may be formulated separately from the agent of the present invention and administered to the same subject at the same time or at a time difference, or by the same route or different routes.

(Measurement of average pore radius of semipermeable membrane)
Cut dozens of hollow fiber membranes that have been sufficiently wetted with pure water to about 5 mm, remove excess water with filter paper, pack them in a sealed pan, DSC (DSC-7, manufactured by Perkin-Elmer, differential scanning calorimeter) Measure the melting curve at. The measurement is performed in the range of −45 ° C. to 15 ° C. at a temperature increase rate of 2.5 ° C./min. The water present in the pores is affected by the base material, lowers its freezing point, and exhibits a peak at a place different from free water (melted at around 0 ° C.) (temperature range lower than that of free water). The amount of heat of fusion (ΔHp) of the region surrounded by the peak of the portion where the freezing point is lowered and the baseline is obtained, and the amount of pore water (Wp) is calculated from the amount of heat of fusion (ΔHm) per unit weight of water. The sample measured by DSC is completely dried, and the weight of evaporated water (total water content Wt) is determined. From these values, Vp (pore volume porosity) is calculated by the following equation.
Wp = ΔHp / ΔHm
Vp (%) = Wp / (Wt + Mp / ρp) × 100
Mp: Polymer weight ρp: Polymer specific gravity From the melting curve obtained as described above, the peak top of the peak where the freezing point is lowered is read, and the freezing point (freezing point) lowering degree due to capillary condensation of water in the pore is expressed by the following equation. The pore radius (r) can be simply calculated using
r (nm) = Degree of freezing point (° C.) / 164 × 10

(Measurement of inner diameter, outer diameter, film thickness)
A sample holder in which a cross section of the hollow fiber membrane was exposed was prepared by using a 2 mm-thick SUS platelet having a 3 mmφ hole and filling the hole with an appropriate amount of wet hollow fiber membrane and cutting it. This was placed on the stage of a Nikon microscope (ECLIPSE LV100), and then a Nikon image processing apparatus (DIGITAL SIGN DS-U2) and a CCD camera (DS-Ri1) were activated. Using NIS Element D3.00 SP6 as image analysis software, calculate the outer diameter and inner diameter of the hollow fiber membrane by measuring the outer diameter and inner diameter of the cross section of the hollow fiber membrane displayed on the screen using the measurement function of the analysis software. did.

(Preparation of hollow fiber membrane 1)
26.5 wt% of polyethersulfone (Ultrason (registered trademark) 6020P manufactured by BASF), 1 wt% of vinylpyrrolidone / vinyl acetate copolymer (Luvitec (registered trademark) VA64 manufactured by BASF), N-methyl-2-pyrrolidone (NMP) 39.9 wt% of Mitsubishi Chemical Co., Ltd.) and 32.6 wt% of triethylene glycol (TEG, Mitsui Chemical Co., Ltd.) were mixed and dissolved at 55 ° C. to obtain a uniform solution. From the annular part of the double-tube nozzle, the obtained film-forming solution was discharged from the center part as a core liquid with a mixture of NMP 42.75 wt%, TEG 52.25 wt%, and RO water 5 wt%, and through the air gap, NMP 27 wt% , TEG was introduced into a coagulation bath filled with an external coagulation liquid consisting of 33 wt% and RO water 40 wt%. At this time, the nozzle temperature was set to 50 ° C., and the temperature of the external coagulation liquid was set to 30 ° C. After drawing out from the coagulation bath, washing was carried out by running a water washing tank at 55 ° C., and wound up by a winder. The wound hollow fiber membrane was made into a hollow fiber membrane bundle having a number of 100 and a length of 30 cm, and was washed by dipping in 85 ° C. RO water in an upright state. Then, it was immersed in a high-pressure steam sterilizer containing hot water of 40 ° C., and high-pressure hot water treatment was performed under conditions of 140 ° C. × 20 min. Thereafter, microwave drying was performed at an internal temperature of 35 ° C. The high-pressure hot water treatment and microwave drying were repeated three times to produce a hollow fiber membrane 1. The resulting hollow fiber membrane 1 had an inner diameter of 230 μm, an outer diameter of 310 μm, and a film thickness of 40 μm. The average pore radius was 5 nm.

(Preparation of hollow fiber membrane 2)
Cellulose triacetate (manufactured by Daicel Chemical Industries) 18 wt%, NMP 57.4 wt%, and TEG 24.6 wt% were mixed and dissolved to obtain a film forming solution. The obtained film-forming solution is discharged from the annular part of the double-tube nozzle from the central part of liquid paraffin as the core liquid, and through the air gap, external coagulation consisting of a mixed liquid of NMP 14 wt%, TEG 6 wt%, RO water 80 wt% It was led to a coagulation bath filled with liquid. At this time, the nozzle temperature was set to 105 ° C., and the temperature of the external coagulating liquid was set to 40 ° C. After drawing out from the coagulation bath, washing was carried out by running a 30 ° C. water-washing tank, passing through a glycerin bath at 50 ° C. and 60 wt%, dried and wound up on a winder. The obtained hollow fiber membrane 2 had an inner diameter of 240 μm, an outer diameter of 276 μm, and a film thickness of 18 μm. Moreover, the average pore diameter was 18 nm.

(Preparation of hollow fiber membrane 3)
Cellulose triacetate (manufactured by Daicel Chemical Industries) 19.5 wt%, NMP 57.75 wt%, and TEG 24.75 wt% were mixed and dissolved to obtain a film forming solution. The obtained film-forming solution is discharged from the annular part of the double-tube nozzle from the central part of liquid paraffin as the core liquid, and through the air gap, external coagulation consisting of a mixed liquid of NMP 14 wt%, TEG 6 wt%, RO water 80 wt% It was led to a coagulation bath filled with liquid. At this time, the nozzle temperature was set to 105 ° C., and the temperature of the external coagulating liquid was set to 40 ° C. After drawing out from the coagulation bath, washing was carried out by running a 30 ° C. water-washing tank, passing through a glycerin bath at 50 ° C. and 60 wt%, dried and wound up on a winder. The resulting hollow fiber membrane 3 had an inner diameter of 250 μm, an outer diameter of 300 μm, and a film thickness of 25 μm. Moreover, the average pore diameter was 11 nm.

[Example 1]
A cell culture container shown in FIG. 1 was prepared using a hollow fiber membrane 1 whose inner surface was previously coated with collagen (Nitta gelatin). Furthermore, using the resulting cell culture vessel constitutes a cell culture apparatus shown in FIG. 2, was placed in a CO ₂ incubator were conducted this experiment. A solution of human bone marrow mesenchymal stem cells (CELL APPLICATIONS Inc.) suspended in a culture solution was injected into the hollow fiber membrane lumen (the number of seeded cells was 5.0 × 10 5 cells / module). At this time, since the total culture area in the cell culture vessel (membrane area based on the inner diameter of the hollow fiber membrane) was 108 cm ² , the cell seeding density was calculated to be about 4630 cells / cm ² . The culture solution is DMEMGlutaMAX (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) from the start of culture (cell seeding) to 96 hours later, and after 96 hours of collecting the culture supernatant, MF- medium (mesenchymal stem cell growth medium, Toyobo) was used.

  FIG. 3 shows a schedule for preparing the culture supernatant. Cultivation was carried out for 7 days (after 168 hours) after cell seeding (culture start). During this time, the flow rate (linear velocity) of the culture fluid flowing through the hollow fiber lumen averaged 0.066 mm / min from 96 hours after cell seeding, and averaged from 0.6 hours to 144 hours. From 20 mm / min, 144 hours to 168 hours, the average was 0.33 mm / min. On the other hand, the speed of the culture solution flowing through the hollow fiber outer space was set to 3.4 mm / min from the start to the end of the culture. The cell culture supernatant was collected for 72 hours from 96 hours to 168 hours after the start of culture. A hollow fiber module culture similar to this was further carried out for one system to obtain a culture supernatant. The total amount of the obtained culture supernatant was about 18 ml. The culture supernatant was dispensed immediately after collection and stored frozen at −80 ° C. until use. The flow rate was calculated based on the hollow fiber membrane lumen volume and the hollow fiber membrane lumen volume by measuring the flow rates flowing out from the hollow fiber membrane lumen and the outer lumen with a flow meter installed. After 168 hours from the culture, the cells were digested with trypsin, detached and recovered, and the number of cells was counted. As a result, 1.3 × 10 7 cells / module of cells were recovered, and the proliferation rate was 26 times.

[Example 2]
A cell culture experiment was conducted in the same manner as in Example 1 except that the hollow fiber membrane 2 was used. Since the total culture area in the cell culture vessel (membrane area based on the inner diameter of the hollow fiber membrane) was 113 cm ² , the cell seeding density was calculated to be about 4425 cells / cm ² .
After 168 hours of culture, the cells were digested with trypsin, detached and recovered, and the number of cells was counted. As a result, 1.1 × 10 7 cells were recovered, and the proliferation rate was 22 times.

[Example 3]
A cell culture experiment was conducted in the same manner as in Example 1 except that the hollow fiber membrane 3 was used. Since the total culture area in the cell culture vessel (membrane area based on the inner diameter of the hollow fiber membrane) was 118 cm ² , the cell seeding density was calculated to be about 4237 cells / cm ² .
After 168 hours of culture, the cells were digested with trypsin, detached and recovered, and the number of cells was counted. As a result, 1.5 × 10 ^ 7 cells were recovered, and the proliferation rate was 30 times.

[Example 4]
Four collagen-coated petri dishes (culture area 55 cm ² , Asahi Techno Glass) were seeded with human bone marrow mesenchymal stem cells (CELLAPPLICATIONS Inc.) at a cell seeding density of about 5100 cells / cm ² . As in Example 1, DMEMGlutaMAX (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) was used up to 96 hours after seeding the cells, as in Example 1, and MF-medium (Life Technologies) was used after 96 hours. The medium was changed to a mesenchymal stem cell growth medium, Toyobo), and cultured.

  FIG. 3 shows a schedule for preparing the culture supernatant. Culture medium exchange was performed 48 hours after the start of culture and 96 hours after. Thereafter, the culture medium was not changed, and the culture was terminated when it reached 100% confluence in 168 hours from the start of the culture. The culture solution which was cultured for 72 hours from the last medium exchange to the end of the culture was collected as a culture supernatant. The amount of the culture supernatant was 20.0 mL in total. The culture supernatant was dispensed immediately after collection and stored frozen at −80 ° C. until use. After 168 hours from the culture, the cells were digested with trypsin, detached and recovered, and the number of cells was counted. As a result, 2.8 × 10 6 cells / dish cells were recovered, and the growth rate was about 10 times.

[Experiment 1]
(Preparation of cataract model rat)
A male model rat, SDJ / Jcl rat (CLEA Japan), who develops cataract due to type 2 diabetes, was obtained at 15 weeks of age, and after habituation, it was subjected to the experiment from 20 weeks of age. The experiment was performed in 3 groups, with 5 animals in each group.

(Administration of culture supernatant to rats)
The culture supernatant collected in each Example and a new culture solution not in contact with the cells (MF-medium, Toyobo) were instilled into both eyes of SDJ / Jcl rats (5 mice in each group). That is, each culture supernatant and culture solution was instilled (administered) by 10 μL per rat eye using a micropipette. Instillation was performed once a day, and was performed every day for 20 weeks from the age of 20 to 40 weeks.

[Experiment 2]
(Exosome extraction from culture supernatant)
Exosomes were extracted from each culture supernatant obtained in the examples. MagCapture TM Exosome Isolation Kit PS (Wako Pure Chemical Industries, model number: 293-77601) was used for extraction of exosomes. In each case, the obtained exosomes were suspended in the same amount of phosphate buffered saline (PBS) as the original culture supernatant. Moreover, what performed extraction operation of the exosome similarly from the new culture solution (MF-medium, Toyobo) which is not made to contact with the cell was prepared for the administration to a control group.

(Administration of extracted exosome components to rats)
The prepared exosome-containing PBS (three types including the control group) solution was instilled (administered) in an amount of 10 μL per rat eye using a micropipette. Instillation was performed once a day, every day for 20 weeks from the age of 20 to 40 weeks.

[Experiment 3]
(Observation and scoring of lens turbidity)
In Experiments 1 and 2, during the administration of the culture supernatant or exosome, the lens was observed once a week with 1% atropine sulfate instilled into both eyes of the rat in the mydriatic state. At the time of observation, inhalation anesthesia was performed with diethyl ether, and the turbidity and the turbidity generation site were observed in detail with an ophthalmoscope (manufactured by Welch Allen) and scored according to the classification shown in Table 1.

  The transition of the score obtained by observation is shown in FIG. 4 and FIG. FIG. 4 shows the results of instilling (administering) the culture supernatant obtained in Experiment 1 to rats. On the other hand, FIG. 5 shows the result of instilling (administering) the exosome extract obtained in Experiment 2 to rats. It has been shown that the mesenchymal stem cell culture supernatant of the present invention or a solution containing exosomes extracted from the culture supernatant has an effect of suppressing turbidity of the lens in an experiment using a cataract model rat.

  Although cataract develops due to the turbidity of the lens, the turbidity of the lens is effectively suppressed by instilling an eye drop comprising a mesenchymal stem cell culture supernatant or a solution containing exosomes of the present invention. Can be prevented or treated.

1 Cell culture container 2 Container 3 Semipermeable membrane (hollow fiber membrane)
4 Hollow fiber membrane outer space 5 Hollow fiber membrane lumens 6a, 6b End ports 7a, 7b Side ports 8, 9 Culture solution storage container 10 Recovery container 11 Culture supernatant recovery container 20, 21, 22 Valve 30, 31 Feed pump 40 Inlet 50 Outlet

Claims (4)

  An inhibitor or therapeutic agent for cataract comprising a culture supernatant component obtained by culturing mesenchymal stem cells.   The cataract suppressor or therapeutic agent according to claim 1, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells or adipose tissue-derived mesenchymal stem cells.   The cataract suppressant or therapeutic agent according to claim 1 or 2, wherein the culture supernatant component comprises exosomes.   The inhibitor or therapeutic agent for cataract according to any one of claims 1 to 3, which is an eye drop or an eye ointment.