JP2019156742A - Lens hardening inhibitor or therapeutic agent - Google Patents

Abstract

To solve the problem that presbyopia occurs because the lens becomes hardened, loses elasticity, and makes it difficult to adjust nearby focus, and that there is no effective treatment or prevention for presbyopia.SOLUTION: The present invention is a hardening-preventing agent or therapeutic agent for lens tissue comprising a culture supernatant component obtained by culturing mesenchymal stem cells.SELECTED DRAWING: None

Description

  The present invention relates to a substance that suppresses hardening of a crystalline lens.

  Presbyopia refers to a situation that occurs when the ability to adjust the focus of the eye declines and causes difficulties when looking at nearby objects, and is commonly called "presbyopia". The lens of the eye is a tissue that refracts light.When looking into the distance, the muscles around the lens reduce its thickness by pulling the lens, and conversely, The muscles loosen and focus is brought about by the thickness returning to the original due to the elasticity of the crystalline lens. However, as the lens hardens and loses its elasticity with aging, it becomes difficult to adjust the focus, especially for presbyopia.

  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, immunoregulatory action and the like, and thus have already been used for the treatment of various autoimmune diseases and graft-versus-host diseases. 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 been clarified 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, such 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 (for example, 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. Moreover, 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 (for example, Patent Document 1).

  More recently, vesicles called exosomes secreted from mesenchymal stem cells have been reported to contain various proteins and RNA, which have the same therapeutic effects as mesenchymal stem cells (for example, non- Patent Document 2).

JP 2013-18756 A

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

  As described above, presbyopia occurs because the lens is cured and loses its elasticity, making it difficult to adjust the focus nearby. However, there is no effective treatment or prevention method for presbyopia until now, and a new 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. A lens anti-sclerosing agent or therapeutic agent comprising a culture supernatant component obtained by culturing mesenchymal stem cells.
2. 2. The lens hardening inhibitor or therapeutic agent according to 1, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells or adipose tissue-derived mesenchymal stem cells.
3. 3. The lens hardening inhibitor or therapeutic agent according to 1 or 2, wherein the culture supernatant component comprises exosomes.

  According to the present invention, it is possible to prevent the onset of presbyopia because it is possible to suppress the decrease in elasticity of the lens and the progression of the decrease.

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. 2 is a schedule for preparing a culture supernatant in Example 1. FIG. It is an example of the load curve which shows the result of the compression test of a rat crystalline lens. It is a graph which shows the compression recovery property of the rat lens which instilled the culture supernatant. It is a graph which shows the compression recovery property of the rat lens which instilled the exosome suspension.

(Mesenchymal stem cells)
In the present invention, the mesenchymal stem cells are not particularly limited, but bone marrow-derived mesenchymal stem cells or adipose tissue-derived mesenchymal stem cells 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.

(Cell 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 Modification (αMEM), Rosell Park Memorial Institute medium (RPMI), etc. Can be used.

  In the present invention, it may be preferable that the cell culture medium 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, exosomes (approximately 30 nm to 150 nm) in the culture supernatant do not permeate the membrane, and γ-globulin (approximately 8.4 nm), which is a culture solution component, is preferably a semipermeable membrane having a property of permeating the membrane. . Then, it is preferable to use an ultrafiltration membrane having a pore diameter of about 10 nm to 30 nm as the semipermeable membrane.

  The organic polymer material includes acrylic resins such as 2-hydroxyethyl methacrylate and polymethyl methacrylate, cellulose resins such as cellulose acetate and regenerated cellulose, polysulfone resins such as polysulfone and polyethersulfone, polylactic acid and polyhydroxyalkanoic acid. Polyester resins such as acrylate, polyolefin resins such as polyethylene and polypropylene, polyvinyl alcohol, epoxy resins, 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. The thickness of the semipermeable membrane is preferably about 10 to 200 μm in consideration of the permeability of the culture medium 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, mesenchymal stem cells release various secretions (proteins, cytokines, exosomes) into the culture solution, and 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 drugs known per se 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.

(Mean pore diameter of membrane)
Measurement of the average pore diameter of the produced membrane was performed using a Palm Materials (PPM, CFP-1200AEX) apparatus manufactured by Porous Materials. The test type was Capillary Flow Porometry Wet Up / DryUp, and GalWick (surface tension 15.7 dyne / cm) was used as a test solution. The hollow fiber membrane used for the measurement was processed into a dedicated small element so that the measurement by the PPM was possible. A hollow acrylic sleeve having an outer diameter of 8.5 mm, a thickness of 1 mm, and a length of 4 cm was prepared so as to fit the hollow fiber membrane measurement sample holder (sample insertion port opening diameter 8.5 mm) attached to the apparatus. After passing the hollow fiber membrane through the sleeve, the inside of the sleeve was filled with a curable resin and cured. As for the holder insertion side of the sleeve, the hollow fiber membrane that protrudes from the sleeve end surface is cut with the cured resin at the sleeve end surface so that the cross section of the hollow fiber membrane is taken out, and the side opposite to the insertion side (membrane sample measurement side) For the above, the remaining hollow fiber membrane was cut off, leaving a length slightly exceeding 3 cm from the sleeve end surface (exactly, the interface between the cured resin and the hollow fiber membrane). A small element for measurement was completed by applying and sealing a curable resin to the tip of the hollow fiber membrane so that the effective length of the hollow fiber membrane was 3 cm. After inputting the following measurement parameters (automatic test parameter values) into the measurement software attached to the PPM, the well-dried small element was inserted and fixed in the sample holder, and the holder was set in the PPM. The measurement was first performed under Dry, and then the membrane sample was immersed in GalWick for 10 minutes, and then measurement was performed under Wet.
<Automatic test parameter values for pore diameter distribution measurement test>
(0) Minimum pressure 0 (KPA), Maximum pressure 300 (KPA), 200000 maxflow (cc / m)
(1) Bubble point test / integrity test: 15.0 bubbleflow (cc / m), 100 F / PT (old bubbletime), 0.00 minbppres (KPA), 1.0 zerotime (sec)
(2) Motor valve control; 3 v2incr (cts * 3)
(3) Regulator control; 1 preginc, 2 pulse delay
(4) Lohm calibration; 1330.683346 maxpress (KPA), 0.070 pulsewidth (sec)
(5) Data determination routine; 30 mineqtime (sec), 50 presslew (cts * 3), 50 flowslew (cts * 3), 50 equiter (0.1 sec), 5 aveiter (0.1 sec), 0.69 maxpdif ( KPA), 30.0 maxfdif (cc / m)
Note that cts is a machine constant and represents “count number”, and cts * 3 means that cts is tripled.

(Preparation of hollow fiber membrane 1)
26 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, Mitsubishi (Chemical Co., Ltd.) 32.85 wt% and triethylene glycol (TEG, Mitsui Chemical Co., Ltd.) 40.15 wt% 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 obtained hollow fiber membrane 1 had an inner diameter of 200 μm, an outer diameter of 260 μm, and a film thickness of 30 μm. Moreover, the average pore diameter was 12 nm.

(Preparation of hollow fiber membrane 2)
Cellulose triacetate (manufactured by Daicel Chemical Industries) 19 wt%, NMP 56.7 wt%, and TEG 24.3 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 200 μm, an outer diameter of 230 μm, and a film thickness of 15 μm. Moreover, the average pore diameter was 30 nm.

(Preparation of hollow fiber membrane 3)
Cellulose triacetate (manufactured by Daicel Chemical Industries) 17.5 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 obtained hollow fiber membrane 3 had an inner diameter of 200 μm, an outer diameter of 250 μm, and a film thickness of 25 μm. The average pore diameter was 92 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 98 cm ² , the cell seeding density was calculated to be about 5100 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) from cell seeding (culture start). During this time, the flow rate (linear velocity) of the culture fluid flowing through the hollow fiber membrane lumen was 0.066 mm / min on average for 96 hours after cell seeding, and 0.20 mm on average for 96 hours to 144 hours after cell seeding. / Min From 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 outer space of the hollow fiber membrane was 3.4 mm / min from the start to the end of the culture (168 hours). The cell culture supernatant was collected for 72 hours from 96 hours after the start of the culture to 168 hours. The total amount of the culture supernatant was 7.9 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 of culture, the cells were digested with trypsin, detached and recovered, and the number of cells was counted. As a result, 1.2 × 10 ^ 7 cells were recovered, and the proliferation rate was 24 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. In addition, since the total culture area in the cell culture vessel (membrane area based on the inner diameter of the hollow fiber membrane) was 99 cm ² , the cell seeding density was calculated to be about 5050 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, 1.4 × 10 7 cells were recovered, and the proliferation rate was 28 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 container (membrane area based on the inner diameter of the hollow fiber membrane) was 98 cm ² , the cell seeding density was calculated to be about 5100 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, 0.9 × 10 ^ 7 cells were recovered, and the proliferation rate was 18 times.

[Example 4]
Human bone marrow mesenchymal stem cells (CELL APPLICATIONS Inc.) were seeded on ^two collagen-coated petri dishes (culture area 55 cm ² , Asahi Techno Glass) so that the cell seeding density was about 5100 cells / cm ² . As in Example 1, the culture solution was DMEM GlutaMAX (Life Technologies) supplemented with 10% fetal calf serum (Life Technologies) until 96 hours after seeding the cells, and MF-medium after 96 hours. The medium was changed to (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 total amount of the culture supernatant was 10.0 ml. 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, 2.6 × 10 ^ 6 cells were recovered, and the proliferation rate was 9.3 times.

[Experiment 1]
(Induction of rat lens hardening)
Rats were exposed to cigarette smoke to induce lens hardening. That is, ten 9-week-old male SD rats were placed in a sealable case (width 40 cm × depth 40 cm × height 20 cm). Tobacco (Seven Star (registered trademark)) was attached to a 50 mL syringe, mainstream smoke was sucked into the syringe and collected, and this was blown into a case containing a rat. Suction of mainstream smoke and blowing into the case were repeated 12 times, and the mainstream smoke was blown into a total 600 mL chamber, and then allowed to stand for 30 minutes. The same procedure was repeated 4 times every hour to expose the rats to mainstream smoke. This mainstream smoke exposure was carried out daily for 3 weeks.

(Administration of culture supernatant to rats)
Each culture supernatant collected in the Examples and a new culture solution (MF-medium, Toyobo) not in contact with the cells were administered by eye drops to rats exposed to tobacco smoke. That is, each culture supernatant or culture solution was instilled using a micropipette at 10 μL per rat eye. Instillation was performed once a day prior to exposure to tobacco smoke, which was performed daily for 3 weeks.

[Experiment 2]
(Exosome extraction from culture supernatant)
Exosomes were extracted from each culture supernatant obtained in the examples. For the extraction of exosomes, MagCapture ^™ Exosome Isolation Kit PS (Wako Pure Chemical Industries, model number: 293-77601) was used. Each of the obtained exosomes was used by suspending in 1/10 amount of phosphate buffered saline (PBS) of 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 (3 types including the control group) solution was administered (instilled) using a micropipette at 10 μL per eye of each rat. Instillation was performed once a day prior to exposure to tobacco smoke, which was performed daily for 3 weeks.

[Experiment 3]
(Measurement of lens hardness)
After completion of Experiments 1 and 2, the rats were euthanized and the lens was removed from both eyes. Next, the hardness of the crystalline lens was measured using a compression tester (Kato Tech, model number: KES-G5). For the measurement, a load of 50 g was applied using a ² cm ² compressor, and the load at the same indentation distance and the repulsion therefrom were measured. The measurement was performed under the conditions of 20 ° C. and 65% Rh. The compression recovery property was evaluated from the compression load curve obtained by the measurement. That is, Ba / (Aa + Ba) × 100 was obtained from the compression load curve shown in FIG. It can be said that the closer this value is to 100, the greater the recovery from compression, that is, the elasticity.

  The measurement results of the compression recovery property are shown in FIGS. FIG. 5 shows the results of measuring the compression recovery of the rat lens instilled with the culture supernatant obtained in Experiment 1. Moreover, FIG. 6 shows the result of measuring the compression recovery property of the rat lens instilled with a suspension containing exosomes extracted from the culture supernatant. It was shown that the solution containing the culture supernatant of the mesenchymal stem cells of the present invention has an effect of preventing the induced elasticity reduction of the lens.

  Conventionally, presbyopia has developed due to the hardening of the lens, but by applying a solution containing the culture supernatant of the mesenchymal stem cells of the present invention, hardening of the eye lens tissue is effectively prevented. It becomes possible to prevent or treat presbyopia.

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 (3)

  An anti-sclerosing agent or therapeutic agent for lens tissue, comprising a culture supernatant component obtained by culturing mesenchymal stem cells.   The lens tissue sclerosis preventive agent 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 lens tissue hardening inhibitor or therapeutic agent according to claim 1 or 2, wherein the culture supernatant component comprises exosomes.