Compositions and methods for preventing or treating eye diseases

Abstract

The present disclosure provides a composition and method for preventing or treating eye diseases. Specifically, a method for preparing mesenchymal stem cell conditioned medium is provided, which includes isolating mesenchymal stem cells from an individual, maintaining the mesenchymal stem cells in a mesenchymal stem cell maintenance medium, collecting the mesenchymal stem cells at passages 2 to 5, culturing the mesenchymal stem cells for several hours in a medium supplemented with fetal bovine serum and a mesenchymal stem cell medium adjuvant to obtain the mesenchymal stem cell conditioned medium, collecting the conditioned medium, and filtering it after centrifugation. The present disclosure also provides a method for preventing or treating an ocular epithelial tissue disorder such as dry eye disease, which includes administering the conditioned medium to the eye of an individual. The present disclosure further provides a pharmaceutical composition comprising the conditioned medium.

Description

Technical Field

[0001] The present disclosure relates to a conditioned medium of adipose-derived stem cells, and more particularly to a conditioned medium of adipose-derived stem cells for preventing or treating ocular disorders. The present disclosure also relates to a method for preparing a conditioned medium of adipose-derived stem cells. Background Art

[0002] The incidence of dry eye syndrome (DES) or dry eye has been increasing. Dry eye causes ocular discomfort and impairs visual quality (1). Current treatment strategies for dry eye syndrome include providing lubricants, anti-inflammatory drugs such as corticosteroids or cyclosporine, punctal occlusion, or even autologous serum. Punctal occlusion reduces tear film loss but increases cytokines or enzymes in the tear film (2). Topical use of autologous serum drops provides lubrication mimicking natural tears and certain biochemical components. However, the preparation and storage of autologous serum are inconvenient, and its effects on the symptoms and signs of dry eye are inconsistent or lacking (3). Since traditional treatment methods are not ideal in daily practice, further exploration is needed.

[0003] It is believed that stem cells have excellent functions in degenerative diseases, and it has been reported that paracrine factors produced by stem cells can enhance tissue regeneration and healing effects (4-6). Their repair ability and anti-inflammatory effects in epithelial damage have also been demonstrated (7, 8). Since corneal and conjunctival epithelial damage and inflammation are two important causes of dry eye, the effects of stem cells and paracrine factors on dry eye are worthy of further study. Although stem cells themselves may have tumorigenic and antigenic properties, an urgent problem to be solved in the related field is to develop a pharmaceutical composition for effectively preventing or treating dry eye. Summary of the Invention

[0004] The present disclosure provides a method for treating an ocular epithelial tissue disorder in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a bioactive agent, the bioactive agent comprising a composition prepared by: obtaining mesenchymal stem cells; culturing the mesenchymal stem cells in a culture medium; collecting the culture medium; and obtaining a fraction less than 30 kD from the collected culture medium. In at least one embodiment, the culture medium is supplemented with serum and a mesenchymal stem cell culture adjuvant (MCA). In certain embodiments, the serum is fetal bovine serum (FBS) or human serum, and its concentration in the culture medium ranges from 5% to 15%. In certain embodiments, the culture medium is supplemented with 10% serum and a mesenchymal stem cell culture adjuvant. In certain embodiments, the serum (e.g., FBS) is added at a concentration of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% for 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours or 132 hours.

[0005] In at least one embodiment, the mesenchymal stem cell culture adjuvant comprises at least one of fibroblast growth factor 2 (FGF-2), N-acetyl-L-cysteine (NAC), and L-ascorbic acid-2-phosphate (AsA2P). In certain embodiments, the concentration of fibroblast growth factor-2 in the mesenchymal stem cell culture adjuvant ranges from 5 ng / mL to 15 ng / mL. In certain embodiments, the concentration of fibroblast growth factor-2 in the mesenchymal stem cell culture adjuvant is 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL or 15 ng / mL.

[0006] In at least one embodiment, the concentration of N-acetyl-L-cysteine in the mesenchymal stem cell culture adjuvant ranges from 1 mM to 5 mM. In certain embodiments, the concentration of N-acetyl-L-cysteine in the mesenchymal stem cell culture adjuvant is 1 mM, 2 mM, 3 mM, 4 mM or 5 mM.

[0007] In at least one embodiment, the concentration range of L-ascorbic acid-2-phosphate in the mesenchymal stem cell culture medium adjuvant is from 0.1 mM to 0.5 mM. In certain embodiments, the concentration of the L-ascorbic acid-2-phosphate in the mesenchymal stem cell culture medium adjuvant is 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM or 0.5 mM.

[0008] In certain embodiments, the mesenchymal stem cell culture medium contains about 10 ng / mL fibroblast growth factor 2 (FGF-2), about 2 mM N-acetyl-L-cysteine (NAC), and 0.2 mM L-ascorbic acid-2-phosphate (AsA2P).

[0009] In at least one embodiment, the mesenchymal stem cells are cultured in the medium for at least 2 passages, such as 3 passages, 4 passages or 5 passages.

[0010] In at least one embodiment, the mesenchymal stem cells are obtained from an individual, wherein the individual is another individual. In certain embodiments, the individual is a mammal. In certain embodiments, the individual is a human. In certain embodiments, the mesenchymal stem cells are obtained from the adipose tissue of an individual.

[0011] In at least one embodiment of the present disclosure, the present disclosure also provides a composition comprising a fraction less than 30 kDa prepared in the following manner: obtaining mesenchymal stem cells from an individual; culturing the mesenchymal stem cells in a medium; collecting the medium; and obtaining a fraction less than 30 kDa from the collected medium.

[0012] In at least one embodiment, a fraction less than 10 kDa (such as less than 8 kDa, less than 5 kDa, less than 3 kDa, less than 2 kDa or less than 1 kDa) is obtained from the collected medium.

[0013] In at least one embodiment of the present disclosure, the present disclosure also provides the use of a composition comprising a fraction less than 30 kDa for external use in the treatment of ocular epithelial tissue disorders. In certain embodiments, the ocular epithelial tissue disorder is dry eye. In certain embodiments, the dry eye is caused by at least one of the following reasons: tear deficiency, dry air or aging.

[0014] The present disclosure provides a method for treating dry eye syndrome in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a bioactive agent, the bioactive agent comprising a composition prepared by: obtaining adipose-derived stem cells (ADSCs); maintaining the adipose-derived stem cells in a first culture medium; culturing the adipose-derived stem cells in a second culture medium; collecting the second culture medium; and obtaining a fraction less than 30 kDa (<30 kDa) from the collected second culture medium.

[0015] In certain embodiments, the present disclosure provides a method for preparing adipose-derived stem cell-conditioned medium (ADSC-CM), comprising isolating adipose-derived stem cells (ADSCs) from an individual; maintaining the adipose-derived stem cells in a mesenchymal stem cell maintenance medium; collecting the adipose-derived stem cells of passages 2 to 5; culturing the adipose-derived stem cells in phenol red-free Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 1 mM to 5 mM glutamine, 5% to 15% fetal bovine serum (FBS), and a mesenchymal stem cell culture adjuvant (MCA) for 36 hours to 132 hours to obtain adipose-derived stem cell-conditioned medium (ADSC-CM); collecting the adipose-derived stem cell-conditioned medium; and centrifuging, followed by filtration. In certain embodiments, glutamine is supplemented in an amount of 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, and FBS is supplemented in an amount of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% for 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, or 132 hours.

[0016] In at least one embodiment of the present disclosure, the ADSC-CM comprises active components having a molecular weight less than 30 kDa, for example, less than 20 kDa, less than 10 kDa, less than 8 kDa, less than 5 kDa, less than 3 kDa, less than 2 kDa, less than 1 kDa.

[0017] The present disclosure also provides a conditioned medium of adipose-derived stem cells (ADSC-CM) for treating or preventing dry eye, wherein the conditioned medium of the adipose-derived stem cells is obtained by the foregoing method, and the conditioned medium of the adipose-derived stem cells contains active ingredients with a molecular weight of less than 30 kDa, for example, less than 20 kDa, less than 10 kDa, less than 8 kDa, less than 5 kDa, less than 3 kDa, less than 2 kDa, less than 1 kDa.

[0018] In at least one embodiment of the present disclosure, the conditioned medium of adipose-derived stem cells contains active ingredients with a molecular weight of less than 3 kDa.

[0019] The present disclosure also provides a conditioned medium of adipose-derived stem cells (ADSC-CM) for treating or preventing dry eye, wherein the conditioned medium of the adipose-derived stem cells is obtained by the foregoing method, and the conditioned medium of the adipose-derived stem cells contains active ingredients with a molecular weight of less than 3 kDa.

[0020] In at least one embodiment of the present disclosure, the conditioned medium of adipose-derived stem cells contains active ingredients with a molecular weight of less than 1 kDa.

[0021] The present disclosure also provides a conditioned medium of adipose-derived stem cells (ADSC-CM) for treating or preventing dry eye, wherein the conditioned medium of the adipose-derived stem cells is obtained by the foregoing method, and the conditioned medium of the adipose-derived stem cells contains active ingredients with a molecular weight of less than 1 kDa.

[0022] The present disclosure also provides a method for treating or preventing dry eye, including administering the conditioned medium of adipose-derived stem cells obtained by the foregoing method to the eye of an individual, wherein the conditioned medium of the adipose-derived stem cells contains active ingredients with a molecular weight of less than 30 kDa.

[0023] The present disclosure also provides a method for treating or preventing dry eye, including administering the conditioned medium of adipose-derived stem cells obtained by the foregoing method to the eye of an individual, wherein the conditioned medium of the adipose-derived stem cells contains active ingredients with a molecular weight of less than 3 kDa.

[0024] The present disclosure also provides a method for treating or preventing dry eye, including administering the conditioned medium of adipose-derived stem cells obtained by the foregoing method to the eye of an individual, wherein the conditioned medium of the adipose-derived stem cells contains active ingredients with a molecular weight of less than 1 kDa.

[0025] The present disclosure further provides a composition containing the conditioned medium of adipose-derived stem cells obtained by the foregoing method, wherein the conditioned medium of the adipose-derived stem cells contains active ingredients with a molecular weight of less than 30 kDa.

[0026] The present disclosure also provides a composition comprising a conditioned medium of adipose-derived stem cells obtained by the foregoing method, wherein the conditioned medium of the adipose-derived stem cells comprises active ingredients with a molecular weight of less than 3 kDa.

[0027] The present disclosure also provides a composition comprising a conditioned medium of adipose-derived stem cells obtained by the foregoing method, wherein the conditioned medium of the adipose-derived stem cells comprises active ingredients with a molecular weight of less than 1 kDa. Brief Description of the Drawings

[0028] The present disclosure can be more fully understood by reading the description of the following embodiments and referring to the accompanying drawings, wherein:

[0029] Figure 1 It shows the effects of different culture medium formulations on the viability of human corneal epithelial cells (HCECs) measured by Cell Counting Kit-8 (CCK-8 assay) in a dry stress study. The HCECs were grown to approximately 80% confluence and then air-dried for 10 minutes. After drying, the cells were transferred to different culture media. After incubation for 2 hours, the cells were counted using the CCK-8 assay. The control group represents HCECs without air-drying treatment. R represents Refresh Plus Lubricant eye drops. IM represents IMDM supplemented with 10% fetal bovine serum and glutamine. IMMCA represents IM supplemented with 10 ng / mL fibroblast growth factor 2 (FGF-2), 2 mM N-acetyl-L-cysteine, and 0.2 mM L-ascorbic acid-2-phosphate. ADSC-CM represents IMMCA conditioned by culturing adipose-derived mesenchymal stem cells. CEM represents corneal epithelial cell basal medium supplemented with components of the corneal epithelial cell growth kit. The values are presented as the mean ± SEM of three repeated experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group. #P < 0.05, P < 0.001, compared with the CEM group;

[0030] Figure 2 It shows the results of immunoblot analysis of JUK, P38, and Erk1 / 2 gene expression in HCECs from the dry stress study, where P represents "phosphorylated" and t represents "total";

[0031] Figure 3Shows the tear volume of BALB / c mice housed in a controlled environment chamber (CEC) treated with different eye drops. Tear volume was measured using a phenol red thread, in millimeters. Mean tear volume for the non-dry control, dry control, Refresh eye drop group, IMMCA group, and ADSC-CM group are shown. * represents comparison with non-dry control group, *p < 0.05, **p < 0.01, ***p < 0.001. # represents comparison with dry control group, #p < 0.05, ##p < 0.01, p < 0.001. & represents comparison with IMMCA group, &p < 0.05, &&p < 0.01, &&&p < 0.001. % represents comparison with Refresh eye drop group, %p < 0.05, %%p < 0.01, %%%p < 0.001. Mean ± SEM, N = 4. Once daily, 0.2% benzalkonium chloride (BAK) was administered to each eye;

[0032] Figure 4 Shows fluorescein staining and rose bengal staining of BALB / c mice. Corneal staining increased in mice from CEC. Topical administration of R, IMMCA, or ADSC-CM reversed rose bengal staining. Fluorescein staining scores were as follows: 0 = no staining; 1 = slight punctate staining (<30 dots); 2 = punctate staining (>30 dots) but no diffusion; 3 = severe diffuse staining but no positive plaques; and, 4 = positive fluorescein plaques. Rose bengal staining scores of the cornea were as follows: 1 = few isolated dots; 2 = numerous isolated dots; and, 3 = fused dots (maximum score of 9). *p < 0.05, compared with non-dry control group. +p < 0.05, compared with dry control group. ++p < 0.01, compared with dry control group. N = 5;

[0033] Figure 5A and 5B Shows confocal microscopy of corneal epithelium and corneal epithelial tight junction barrier integrity in a CEC-induced dry eye model in BALB / c mice. BF: bright field;

[0034] Figures 6A to 6E Shows the results of periodic acid-Schiff (PAS) staining of conjunctival goblet cells in CEC-induced dry eyes of different groups. Figure 6A : Non-dry control group. Figures 6B to 6E : Mice from CEC as dry control group ( Figure 6B ), and mice topically administered with R ( Figure 6C ), IMMCA ( Figure 6D ), and ADSC-CM ( Figure 6E ). Magnification: 400X. Scale bar: 20μm, 5μm sections;

[0035] Figure 6F Show the statistical comparison of conjunctival goblet cell density between dry eye induced by different groups of CEC. * represents comparison with non-dry control group, *p<0.05, ***p<0.001. + represents comparison with non-dry control group, +p<0.05, +++p<0.001;

[0036] Figure 7 Show the effect of different eye drops on the expression of membrane-associated mucin MUC16 in the conjunctival epithelium of BALB / c mice in CEC by immunohistochemical analysis. Arrows in the ADSC-CM group indicate the conjunctival epithelium. Magnification: 400X. Scale bar: 50μm, 5μm sections;

[0037] Figures 8A to 8E Show the results of transmission electron microscopy of the corneas from BALB / c mice caged in CEC treated with different eye drops. Figure 8A : Non-dry control group; Figure 8B : Dry control group; Figure 8C : Group R; Figure 8D : IMMCA group; Figure 8E : ADSC-CM group. Magnification: 40,000X. Scale bar: 500nm;

[0038] Figures 9A to 9E Show the results of scanning electron microscopy of the corneas from BALB / c mice caged in CEC treated with different eye drops. Figure 9A : Non-dry control group; Figure 9B : Dry control group; Figure 9C : Group R; Figure 9D : IMMCA group; Figure 9E : ADSC-CM group. Magnification: 25,000X;

[0039] Figures 10A to 10D Show the effect of different culture medium preparations and ADSC-CM fractions on the viability of HCEC measured by CCK-8 in dry stress studies and hyperosmotic stress studies. Values are presented as the mean ± SEM of three repeated experiments. *P<0.05, **P<0.01, ***P<0.001, compared with the control group. #P<0.05, ##P<0.01, P<0.001, compared with the CEM group;

[0040] Figure 11Shown are the effects of different culture medium formulations and fractions of ADSC-CM on the viability of HCECs measured by CCK-8 in dry stress studies. Values are presented as the mean ± SEM of three replicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group. #P < 0.05, ##P < 0.01, P < 0.001, compared with the CEM group;

[0041] Figure 12 Shown are the effects of different culture medium formulations and the 0-1 kDa fraction of ADSC-CM on the viability of HCECs measured by CCK-8 in dry stress studies. Values are presented as the mean ± SEM of three replicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group. #P < 0.05, ##P < 0.01, P < 0.001, compared with the CEM group;

[0042] Figures 13A to 13C Shown are the tear volumes of mice housed in the CEC chamber treated with different fractions of ADSC-CM. Mean tear volumes for each group are shown. *Compared with the non-dry control group, *p < 0.05, **p < 0.01, ***p < 0.001. #Compared with the dry control group, #p < 0.05, ##p < 0.01, p < 0.001. Mean ± SEM, N = 4;

[0043] Figures 14A to 14B 、 Figures 15A to 15B 、and Figures 16A to 16B Shown are the confocal microscopy results of corneal epithelium and corneal epithelial tight junction barrier integrity in BALB / c mice in a CEC-induced dry eye model treated with different fractions of ADSC-CM. BF: bright field;

[0044] Figures 17A to 17E Shown are the PAS staining results of conjunctival goblet cells in CEC-induced dry eyes of different groups. Figure 17A : Non-dry control group. Figures 17B to 17E : Mice from CECs as the dry control group ( Figure 17B ), and mice treated with ADSC-CM ( Figure 17C ), ADSC-CM 30-100 kDa ( Figure 17D ), and ADSC-CM 0-30 kDa ( Figure 17E ). Magnification: 400X. Scale bar: 20 μm, 3 μm sections;

[0045] Figure 17F Shown is the statistical comparison of conjunctival goblet cell density between CEC-induced dry eyes of different groups. #Compared with the dry control group, #p < 0.05;

[0046] Figures 18A to 18G Shows the PAS staining results of conjunctival goblet cells in dry eyes induced by different groups of CEC. Figure 18A : Non-dry control group. Figures 18B to 18G : Mice from CEC as dry control group ( Figure 18B ), and mice treated with ADSC-CM ( Figure 18C ), ADSC-CM > 10 kDa ( Figure 18D ), ADSC-CM < 10 kDa ( Figure 18E ), ADSC-CM > 3 kDa ( Figure 18F ), and ADSC-CM < 3 kDa ( Figure 18G ). Magnification: 400X. Scale bar: 20 μm, 3 μm sections;

[0047] Figure 18H Shows the statistical comparison of conjunctival goblet cell density among dry eyes induced by different groups of CEC. *Compared with the non-dry control group, *p < 0.05, **p < 0.01, ***p < 0.001. #Compared with the dry control group, #p < 0.05, ##p < 0.01, p < 0.001;

[0048] Figures 19A to 19F Shows the PAS staining results of conjunctival goblet cells in dry eyes induced by different groups of CEC. Figure 19A : Non-dry control group. Figures 19B to 19F : Mice from CEC as dry control group ( Figure 19B ), and mice treated with ADSC-CM ( Figure 19C ), ADSC-CM 0 - 3 kDa ( Figure 19D ), ADSC-CM 0 - 1 kDa ( Figure 19E ), and IMDM ( Figure 19F ). Magnification: 400X. Scale bar: 50 μm, 3 μm sections;

[0049] Figure 19G Shows the statistical comparison of conjunctival goblet cell density among dry eyes induced by different groups of CEC. *Compared with the non-dry control group, *p < 0.05, **p < 0.01, ***p < 0.001. #Compared with the dry control group, #p < 0.05, ##p < 0.01, p < 0.001. &Compared with the IMDM group, &p < 0.05, &&p < 0.01, &&&p < 0.001;

[0050] Figures 20A to 20E Shows the results of immunohistochemical analysis of MUC16 expression in mouse conjunctival epithelium. Figure 20A : Non-dry control group;Figure 20B : Dry control group; Figure 20C : ADSC-CM group; Figure 20D : 30-100 kDa fraction of ADSC-CM group; Figure 20E : 0-30 kDa fraction of ADSC-CM group. Magnification: 400X. Scale bar: 20 μm, 3 μm sections. Data show that compared with those treated with other eye drops and the dry control group, conjunctival MUC16 expression is higher in those treated with ADSC-CM and the 0-30 kDa fraction of ADSC-CM. Arrows indicate MUC16 expression on the surface of the conjunctival epithelium;

[0051] Figures 21A to 21G Shows the results of immunohistochemical analysis of MUC16 expression in the mouse conjunctival epithelium. Figure 21A : Non-dry control group; Figure 21B : Dry control group; Figure 21C : ADSC-CM group; Figure 21D : >10 kDa fraction of ADSC-CM group; Figure 21E : <10 kDa fraction of ADSC-CM group; Figure 21F : >3 kDa fraction of ADSC-CM group; Figure 21G : <3 kDa fraction of ADSC-CM group. Magnification: 400X. Scale bar: 20 μm, 3 μm sections. Data show that compared with those treated with other eye drops and the dry control group, conjunctival MUC16 expression is higher in those treated with ADSC-CM and the <10 kDa fraction and <3 kDa fraction of ADSC-CM. Arrows indicate MUC16 expression on the surface of the conjunctival epithelium;

[0052] Figures 22A to 22F Shows the results of immunohistochemical analysis of MUC16 expression in the mouse conjunctival epithelium. Figure 22A : Non-dry control group; Figure 22B : Dry control group; Figure 22C : ADSC-CM group; Figure 22D : 0-3 kDa fraction of ADSC-CM group; Figure 22E : 0-1 kDa fraction of ADSC-CM group; Figure 22F : IMDM group. Magnification: 200X. Scale bar: 50 μm, 3 μm sections. Data show that compared with those treated with IMDM and the dry control group, conjunctival MUC16 expression is higher in those treated with ADSC-CM and the 0-3 kDa fraction and 0-1 kDa fraction of ADSC-CM;

[0053] Figure 23AImmunohistochemical analysis of MUC4 in the corneal epithelium of mice treated with the 0 - 30 kDa fraction of ADSC-CM or the 30 - 100 kDa fraction of ADSC-CM. Magnification: 400X. Scale bar: 20 μm, 3 μm sections;

[0054] Figure 23B Immunohistochemical analysis of MUC4 in the corneal epithelium of mice treated with the >10 kDa fraction of ADSC-CM, the <10 kDa fraction of ADSC-CM, the >3 kDa fraction of ADSC-CM, or the <3 kDa fraction of ADSC-CM. Magnification: 400X. Scale bar: 20 μm, 3 μm sections;

[0055] Figure 24 Immunohistochemical analysis of MUC4 in the corneal epithelium of mice treated with the 0 - 3 kDa fraction of ADSC-CM or the 0 - 1 kDa fraction of ADSC-CM. Magnification: 400X. Scale bar: 20 μm, 3 μm sections;

[0056] Figures 25A to 25E Scanning electron microscopy results of the corneas from BALB / c mice housed in CEC and treated with different eye drops. Figure 25A : Non-dry control group; Figure 25B : Dry control group; Figure 25C : ADSC-CM group; Figure 25D : 30 - 100 kDa fraction of ADSC-CM group; Figure 25E : 0 - 30 kDa fraction of ADSC-CM group. Magnification: 25,000X;

[0057] Figures 26A to 26G Scanning electron microscopy results of the corneas from BALB / c mice housed in CEC and treated with different eye drops. Figure 26A : Non-dry control group; Figure 26B : Dry control group; Figure 26C : ADSC-CM group; Figure 26D : >10 kDa fraction of ADSC-CM group; Figure 26E : <10 kDa fraction of ADSC-CM group; Figure 26F : >3 kDa fraction of ADSC-CM group; Figure 26G : <3 kDa fraction of ADSC-CM group. Magnification: 25,000X; and

[0058] Figures 27A to 27F Scanning electron microscopy results of the corneas from BALB / c mice housed in CEC and treated with different eye drops. Figure 27A : Non-dry control group;Figure 27B : Dry control group; Figure 27C : ADSC-CM group; Figure 27D : 0-3 kDa fraction of ADSC-CM group; Figure 27E : 0-1 kDa fraction of ADSC-CM group; Figure 27F : IMDM group. Magnification: 25,000X. Detailed implementation manners

[0059] The following examples are used to illustrate the present disclosure. Based on the description of the present disclosure, those skilled in the art can conceive other aspects of the present disclosure. The present disclosure can also be implemented or applied as disclosed in different examples. For different aspects and applications, the examples can be modified and / or changed to implement the present disclosure without departing from its scope.

[0060] It should be further noted that, as used in this specification, unless clearly stated and indubitably limited to one reference, the singular forms "a", "an" and "the" include plural references. Unless explicitly excluded in the context, the term "or" and the term "and / or" can be used interchangeably.

[0061] The present disclosure provides a method for treating dry eye syndrome in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a bioactive agent, the bioactive agent comprising a composition prepared by: obtaining adipose-derived stem cells (ADSCs); maintaining the adipose-derived stem cells in a first culture medium; culturing the adipose-derived stem cells in a second culture medium; collecting the second culture medium; obtaining a fraction less than 30 kDa from the collected second culture medium.

[0062] For maintaining and culturing adipose-derived stem cells, different types of culture media can be used and selected by those skilled in the art. In at least one embodiment, the culture medium is selected from the group consisting of alpha minimum essential medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute (RPMI) medium, Opti-MEM, improved minimum essential medium (IMEM), Iscove's Modified Dulbecco's Medium (IMDM), and AIM-V medium. The cells can be cultured in various media containing fetal bovine serum or other growth factors for expansion. In at least one embodiment, the cells are transferred to a medium substantially lacking serum, and the conditioned medium preparation before administration can be performed by various means; for example, the conditioned medium can be aseptically filtered or concentrated under certain conditions. In certain embodiments, the conditioned medium is further prepared to obtain different fractions containing different molecules, and the molecules of the different fractions have the following size ranges: greater than 100 kDa, greater than 30 kDa, greater than 3 kDa, greater than 1 kDa, less than 100 kDa, less than 50 kDa, less than 40 kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa, less than 5 kDa, less than 3 kDa, less than 1 kDa, between greater than 0 kDa and 100 kDa, between greater than 0 kDa and 30 kDa, between greater than 0 kDa and 3 kDa, or between greater than 0 kDa and 1 kDa.

[0063] In certain embodiments, conditioned media are used to manufacture the active ingredient of a pharmaceutical preparation. In at least one embodiment, stem cell conditioned media can be administered alone as a therapeutic agent. In certain embodiments, administration can include known pharmaceutical preparation modalities, including tablets, capsules, or elixirs for oral administration, suppositories for rectal administration, sterile solutions for parenteral or intramuscular administration, liposomal or encapsulated preparations. In such preparations, the therapeutic agent is used alone or conjugated to, for example, a delivery agent or vehicle. It should be appreciated that the therapeutic entities of the present disclosure will be administered together with suitable carriers, excipients, and / or other agents incorporated into the preparation to provide improved transfer, delivery, tolerance, etc. Suitable bulk preparations can be found in formularies known to all pharmaceutical chemists. Such preparations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipids (cationic or anionic) containing vehicles such as Lipofectin, DNA conjugates, anhydrous absorption pastes, water-in-oil emulsions and oil-in-water emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowaxes. Any of the foregoing mixtures can be suitable for treatment and therapy according to the present disclosure, provided that the active ingredient in the preparation is not inactivated by the preparation and the preparation and its route of administration are physiologically compatible and tolerable.

[0064] In some embodiments, the compositions prepared by the present disclosure are administered by topical formulations. Topical formulations can be used to treat conditions related to skin diseases. For example, formulations in topical form can consist of, for example, aqueous and non-aqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, sprays, skin patches, hydrocarbon bases, and powders, and can contain excipients such as solubilizers, penetration enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols, and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrrolidone). In at least one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. The topical formulations of the present disclosure can include dermatologically acceptable carriers, for example, substances capable of delivering other components of the formulation to the skin, and those components can be acceptably administered or absorbed by the skin. Typical carriers include solvents to dissolve or disperse the therapeutic agent, and optionally one or more excipients or other vehicle components. Carriers that can be used for the topical formulations according to the present disclosure can include, by way of non-limiting examples, water, acetone, ethanol, ethylene glycol, propylene glycol, 1,3-butanediol, acrylate copolymers, isopropyl myristate, isopropyl palmitate, mineral oil, butter, aloe vera gel, talc, vegetable oils, vegetable juices, plant extracts, plant powders, other plant derivatives, lanolin, urea, petroleum products, tar products, plant or animal fats, plant or animal oils, soaps, triglycerides, and keratin. The topical formulations of the present disclosure are prepared by mixing the compositions of the present disclosure with topical carriers according to methods well known in the art, for example, the standard reference Remington: The Science and Practice of Pharmacy, 1577-1591, 1672-1673, 866-885 (Alfonso R. Gennaro ed. 19th ed. 1995) and Ghosh et al., Transdermal and Topical Drug Delivery Systems (1997). In certain embodiments, if desired, humectants or wetting agents, sunscreens, fragrances, dyes, and / or thickeners such as paraffin, jojoba oil, PABA, waxes, surfactants, occlusives, humectants, emulsifiers, emollients, lipid-free cleansers, antioxidants, and lipophilic agents can be added to the topical formulations of the present disclosure. The topical formulations of the present disclosure can be designed to remain on the skin and not be washed off in the short term after application. Alternatively, the topical formulations can be designed to be rinsed off within a specific time after use.

[0065] The present disclosure provides a method for preparing a conditioned medium (ADSC-CM) of adipose-derived stem cells, comprising isolating adipose-derived stem cells (ADSCs) from an individual; maintaining the adipose-derived stem cells in a mesenchymal stem cell maintenance medium; collecting the adipose-derived stem cells that have been passaged 2 to 5 times; culturing the adipose-derived stem cells in phenol red-free Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 1 mM to 5 mM glutamine, 5% to 15% fetal bovine serum (FBS), and a mesenchymal stem cell medium adjuvant (MCA) for 36 hours to 132 hours to obtain a conditioned medium (ADSC-CM) of adipose-derived stem cells, wherein the MCA comprises 5 ng / mL to 15 ng / mL fibroblast growth factor 2 (FGF-2), 1 mM to 5 mM N-acetyl-L-cysteine (NAC), and 0.1 mM to 0.5 mM L-ascorbic acid-2-phosphate (AsA2P); collecting the conditioned medium of the adipose-derived stem cells; and centrifuging, followed by filtration.

[0066] In one embodiment of the present disclosure, the IMDM is supplemented with 2 mM glutamine, 10% FBS, and MCA for 72 hours.

[0067] In one embodiment of the present disclosure, the MCA comprises about 5 ng / mL to 15 ng / mL fibroblast growth factor 2 (FGF-2), 1 mM to 5 mM N-acetyl-L-cysteine (NAC), and 0.1 mM to 0.5 mM L-ascorbic acid-2-phosphate (AsA2P). In certain embodiments of the present disclosure, the MCA comprises about 10 ng / mL FGF-2, about 2 mM N-acetyl-L-cysteine, and about 0.2 mM L-ascorbic acid-2-phosphate (AsA2P).

[0068] In at least one embodiment of the present disclosure, the method further comprises a step of freezing after filtration.

[0069] In at least one embodiment of the present disclosure, the individual is a mammal. In certain embodiments, the individual is a human, a rat, a mouse, a pig, a rabbit, a sheep, a goat, a cat, a dog, a calf, or a baboon.

[0070] In at least one embodiment of the present disclosure, the conditioned medium of adipose-derived stem cells comprises active ingredients with a molecular weight less than 30 kDa.

[0071] In certain embodiments of the present disclosure, the conditioned medium of adipose-derived stem cells comprises active ingredients with a molecular weight less than 3 kDa.

[0072] In yet another embodiment of the present disclosure, the conditioned medium of adipose-derived stem cells comprises active ingredients with a molecular weight less than 1 kDa.

[0073] The present disclosure also provides a conditioned medium of adipose-derived stem cells (ADSC-CM) for preventing or treating dry eye. In at least one embodiment of the present disclosure, the conditioned medium of adipose-derived stem cells contains active ingredients with a molecular weight less than 30 kDa. In certain embodiments of the present disclosure, the conditioned medium of adipose-derived stem cells contains active ingredients with a molecular weight less than 3 kDa. In certain embodiments of the present disclosure, the conditioned medium of adipose-derived stem cells contains active ingredients with a molecular weight less than 1 kDa.

[0074] As used herein, the term "dry eye" refers to a disorder in which the tear film is caused by insufficient tear fluid or excessive evaporation, which causes damage to the ocular surface between the eyelids and is associated with a syndrome of ocular discomfort (37).

[0075] The present disclosure also provides a method for treating or preventing dry eye, including administering the conditioned medium of adipose-derived stem cells obtained by the foregoing method to the eye of an individual. In one embodiment, the conditioned medium of adipose-derived stem cells contains active ingredients with a molecular weight less than 30 kDa. In certain embodiments of the present disclosure, the conditioned medium of adipose-derived stem cells contains active ingredients with a molecular weight less than 3 kDa. In yet another embodiment of the present disclosure, the conditioned medium of adipose-derived stem cells contains active ingredients with a molecular weight less than 1 kDa. The present disclosure also provides a pharmaceutical composition comprising the conditioned medium of adipose-derived stem cells obtained by the foregoing method. In at least one embodiment, the conditioned medium of adipose-derived stem cells contains active ingredients with a molecular weight less than 30 kDa. In certain embodiments of the present disclosure, the conditioned medium of adipose-derived stem cells contains active ingredients with a molecular weight less than 3 kDa. In certain embodiments of the present disclosure, the conditioned medium of adipose-derived stem cells contains active ingredients with a molecular weight less than 1 kDa.

[0076] The following are embodiments further demonstrating the efficacy of the present disclosure, but do not attempt to limit the scope of the present disclosure.

[0077] Examples

[0078] Preparation Example 1: Isolation and Maintenance of Conditioned Medium of Adipose-Derived Stem Cells (ADSC-CM)

[0079] This study was approved by the Institutional Review Board of Fooyin University Hospital (IRB102-130), and informed consent was obtained from all study participants. Human adipose tissue was obtained from the subcutaneous abdominal fat during cosmetic liposuction of three women aged 23, 28, and 30 years, respectively. Stromal-vascular fraction cells were isolated using a method similar to that provided by Griesche et al. (9). Type I collagenase (Sigma) was added to a final concentration of 0.4 mg / mL, and the enzymatic digestion reaction was carried out in a hybridization oven at 37°C, at a 30° angle, and at 15 rpm for 45 minutes. The digested adipose tissue was centrifuged at 400×g for 10 minutes to yield stromal-vascular fraction (SVF) pellets for subsequent ADSC culture. To maintain and expand the ADSC population, the cells were cultured in mesenchymal stem cell maintenance medium containing Iscove's Modified Dulbecco's Medium (IMDM, Gibco), 10% fetal bovine serum (FBS, Gibco), and 10 ng / mL FGF-2 (R&D Systems), as previously described (10, 11). ADSCs at passages 2 to 5 (P2 to P5) were used in the experiments.

[0080] Preparation Example 2: Preparation of ADSC-CM

[0081] ADSCs were seeded at a density of 1×10 6 cells per flask into 150 cm 2 tissue culture flasks (BD Falcon, 355001, Durham, NC) and cultured in phenol red-free Iscove's Modified Dulbecco's Medium (IMDM) (Gibco) supplemented with 2 mM glutamine (Gibco), 10% FBS (HyClone), and mesenchymal stem cell culture adjuvant (MCA) containing 10 ng / mL fibroblast growth factor 2 (FGF-2, R&D Systems), 2 mM N-acetyl-L-cysteine (NAC, Sigma), and 0.2 mM L-ascorbic acid-2-phosphate (AsA2P, Sigma). After 72 hours of culture, the ADSC-conditioned medium (ADSC-CM) was collected, centrifuged at 300×g for 5 minutes, and filtered through a 0.22-μm syringe filter. ADSC-CM from P2 to P5 ADSCs was collected and mixed, and then aliquoted and frozen for further use.

[0082] Preparation Example 3: Fractions of ADSC-CM with Different Molecular Weight Sizes

[0083] Prepare different sized fractions of ADSC-CM using Millipore Amicon Ultra-15 centrifugal tubes or Spectrum Labs hollow fiber filters. Specifically, pool the protein-containing fractions (as evaluated by SDS-PAGE), and concentrate to a final concentration of 1 mg / mL using an Amicon centrifugal concentrator (molecular weight cut-off (MWCO) = 30 kDa, 3 kDa, or 1 kDa). Collect the permeate flow-through and concentrate to a final concentration of 1.5 mg / mL. Quickly freeze the samples and store at -20 °C for later use.

[0084] The concentrated conditioned medium was diluted in IMDM and added to the cells to quantify the number of viable cells by Cell Counting Kit-8.

[0085] For the lyophilized conditioned medium test, prepare equal aliquots (1 mL) of the dialyzed sample in 5 mL lyophilization tubes and then lyophilize in a programmable lyophilizer.

[0086] For the heat stability test, incubate the conditioned medium at 56 °C for 30 minutes or at 100 °C for 3 minutes.

[0087] For lipid extraction, treat the conditioned medium with hexane at a 1:1 ratio for 3 minutes and collect the lower aqueous phase.

[0088] For the charge test, first dialyze the conditioned medium into 20 mM Tris (pH 8.0). After dialysis, apply the sample to an SP-Sepharose cation exchange column. Elute the column with 20 mM Tris, 1 M NaCl (pH 8.0) at 6 column volumes.

[0089] Example 1: In vitro human corneal epithelial cell (HCEC) dry stress study using ADSC-CM

[0090] Maintain normal primary HCEC from the American Type Culture Collection (ATCC, Manassas, VA, USA) according to the instructions. Let HCEC grow in corneal epithelial cell basal medium (CEM, ATCC) supplemented with the components of the corneal epithelial cell growth kit. Culture the cells at 37 °C in a humid atmosphere with 5% carbon dioxide. Change the medium every 2 or 3 days. In this example, only sub-confluent HCEC at passage 4 were used.

[0091] Modified in vitro dry stress (12 - 14) was used on HCECs. Briefly, HCECs were grown to approximately 80% confluence. The medium was aspirated, and the culture dishes were air-dried at 37°C for 10 minutes. After dry stress, the cells were transferred to different media, which included CEM (Refresh Plus Lubricant eye drops, abbreviated as R; Allergan, Westport, Ireland), IMDM supplemented with 10% fetal bovine serum and glutamine (abbreviated as IM), IMDM supplemented with 10% FBS, glutamine, and MCA (abbreviated as IMMCA, used as a control medium that had not been conditioned by ADSCs), and ADSC-CM. After incubation for 2 hours, the cells were counted using a cell counting kit 8 (CCK-8 assay, Enzo Life Sciences, Farmingdale, NY, USA), or lysed in radio-immunoprecipitation assay (RIPA) buffer for immunoblot analysis.

[0092] For the cell viability assay, 10 μL of CCK-8 reagent was added to the cells grown in a 96-well culture plate containing 100 μL of different media as described above. The cells were incubated at 37°C for 3 hours. The absorbance at 450 nm was measured using a microplate reader (MicroQuant, BioTek Instruments, Inc., Winooski, VT, USA). The results were plotted as the mean ± standard error of the mean of three replicate experiments. As Figure 1 shown, the in vitro dry stress results of HCECs showed that drying for 10 minutes caused a significant decrease in cell viability or proliferation. However, this decrease was reversible, and ADSC-CM showed the best protective effect against the dry stress of HCECs.

[0093] For immunoblot analysis, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed on ice for 20 minutes using RIPA buffer (Millipore, Billerica, MA, USA) containing Halt protease and phosphatase inhibitor cocktail (Pierce, Thermo Fisher Scientific, Rockford, IL, USA). The cell extracts were centrifuged at 13,200 rpm for 10 minutes at 4°C, and the supernatant was collected for the experiment. The protein concentration of the cell extracts was determined by the Bradford method using a Bradford protein assay kit (Amresco, Ohio, USA) with bovine serum albumin of known concentration as the standard.

[0094] Separate 50 μg of protein samples on a 10% SDS-polyacrylamide gel, then perform electrophoresis and blot onto a polyvinylidene fluoride (PVDF) membrane (Sigma). Block the membrane with PBS containing 0.1% Tween-20 (PBS-T) and 5% non-fat milk at room temperature for 1 hour, then incubate overnight at 4°C with appropriate primary antibodies, which are rabbit monoclonal anti-Erk1 / 2 (extracellular signal-regulated kinase 1 / 2) antibody, rabbit monoclonal anti-phospho-Erk1 / 2 (Thr202 / Tyr204) antibody, rabbit monoclonal anti-P38 mitogen-activated protein kinase (MAPK) antibody, rabbit monoclonal anti-phospho-P38MAPK (Thr180 / Tyr182) antibody, rabbit monoclonal anti-SAPK / JNK (stress-activated protein kinase / c-Jun NH2-terminal kinase) antibody, rabbit monoclonal anti-phospho-SAPK / JNK (Thr183 / Tyr185) antibody diluted 1:1000 with Seppro removal buffer (Sigma) (Cell Signaling Technology, Beverly, MA, USA), or rabbit monoclonal anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody diluted 1:5000 (Cell Signaling Technology). After washing with PBS-T, incubate the membrane with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (GeneTex, Irvine, CA, USA) at room temperature for 1 hour. Develop the signal using VisGlow chemiluminescent substrate, horseradish peroxidase system (Visual Protein, Taipei, Taiwan, China). Acquire images using a Wealtec KETA imaging system.

[0095] Figure 2 The results shown in indicate that ADSC-CM increased the expression of phosphorylated JUK (P-JUK), phosphorylated P38 (P-P38), and phosphorylated Erk1 / 2 (P-Erk1 / 2) in HCEC.

[0096] Example 2: Study of dry eye in live animals using ADSC-CM

[0097] 1. Establishment of a murine dry eye model:

[0098] All experimental procedures were approved by the Institutional Animal Care and Use Committee of Tzu Chi University. As previously described, dry eye-related ocular surface signs were induced in BALB / c mice in a controlled environment chamber (CEC) (15). Briefly, 13-week-old BALB / c mice were exposed to a CEC with a relative humidity of 10 ± 3% and a temperature of 21–25 °C, and an air flow of 10–15 L / min was monitored and maintained. The mice were divided into five groups. Each experimental group and control group consisted of five mice. Four groups were kept in the CEC as dry eye groups, and one group was kept in a chamber with a humidity of 75 ± 3% as a normal non-dry control group. Among the four CEC dry eye groups, one group served as a dry eye control group and received no eye drops, while the other three groups received the following eye drops, respectively: R, IMMCA (used as a control medium not conditioned by ADSCs), and ADSC-CM medium, twice a day for a total of 28 days. Approximately 50 μL of a single drop was administered each time. Tear secretion measurements were performed weekly.

[0099] At the end of the experiment (i.e., on day 28 at the endpoint), the ocular surface was evaluated by fluorescein staining and rose bengal staining, and corneal thickness was estimated by optical coherence tomography. The mice were then sacrificed, and the eyeballs were preserved for immunohistochemical studies and electron microscopy.

[0100] In addition, statistical analysis of the data is expressed as the mean ± standard error of the mean. One-way analysis of variance (ANOVA) and two-sample t-tests were used to compare the CCK-8 assay, fluorescein and rose bengal staining, and conjunctival goblet cell density. p < 0.05 was considered statistically significant.

[0101] 2. Tear secretion assay

[0102] Tear secretion was estimated by the length of the discolored area of the tear-absorbing Zone-Quick phenol red thread (Showa Pharmaceutical Co., Ltd., Japan). Briefly, excess tears were removed at a standard time of 4 seconds, and then the Zone-Quick phenol red thread was clamped with small forceps and placed in the lateral fornix for 30 seconds. The left eye was measured first, and then the right eye. The average of the two eyes was calculated for analysis. In this study, each experimental group and control group consisted of 10 eyes (n = 5 mice / group).

[0103] As Figure 3As shown, compared with other groups in the tear secretion assay, the volume of tears secreted by mice in the ADSC-CM group was significantly different. In the first and second weeks of treatment, the mice in the ADSC-CM group had a tear volume comparable to that of the non-dry control group mice, indicating that ADSC-CM could maintain the same tear volume level as the non-dry control group. In the third and fourth weeks, the volume of tears secreted by mice in the ADSC-CM group was still significantly higher than that of the dry control group and the mice treated with IMMCA.

[0104] 3. Fluorescein staining and rose bengal staining assays

[0105] After a 1 μL drop of 1% fluorescein was applied to the conjunctival sac for 90 seconds, the cornea was independently evaluated using the following fluorescein staining scoring system: 0 = no staining; 1 = slight punctate staining (<30 dots); 2 = punctate staining (>30 dots) but no diffusion; 3 = severe diffuse staining but no positive plaques; and, 4 = positive fluorescein plaques (16).

[0106] After a 1 μL drop of 1% rose bengal was slowly instilled into the conjunctival sac for 15 seconds, the rose bengal staining of the cornea was scored using the following Van Bijsterveld system: 1 = a few isolated dots; 2 = numerous isolated dots; and, 3 = fused dots (maximum score of 9 points) (17).

[0107] As Figure 4 shown, the results showed that the dry control group had the highest staining score, which was reduced by the administration of R, IMMCA, or ADSC-CM. It was noted that the staining in the ADSC-CM group was reduced the most and was reversed to a level similar to that of the non-dry control group.

[0108] 4. Immunofluorescence double staining

[0109] The eyes were fixed in 10% formaldehyde. After paraffin embedding, 3-μm sections were deparaffinized in xylene, rehydrated in a series of ethanol solutions, and washed twice with distilled water. Antigen retrieval was performed for 15 minutes at 90 to 95 °C using DAKO target retrieval solution (DAKO, Glostrup, Denmark) with pH = 9. The sections were blocked in PBS with 0.3% Triton X-100 and 1% BSA at room temperature for at least 1 hour. The slides were incubated overnight at 4 °C with rabbit anti-ZO-1 (Mid) (1:100; Invitrogen, Camarillo, CA, USA), mouse anti-occludin (1:50; Thermo Scientific, Rockford, IL, USA), or goat anti-cytokeratin 12 (1:50; Santa Cruz, Santa Cruz, CA, USA), and then incubated for 1 hour at room temperature with Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (1:800; Jackson ImmunoResearch, West Grove, PA, USA), Dylight 550-conjugated goat anti-mouse IgG (H+L), or Dylight 550-conjugated donkey anti-goat IgG (H+L) (1:500; Bethyl, Montgomery, TX, USA). The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR, USA). The slides were mounted and examined using a Zeiss LSM 510 META confocal microscope. In negative controls, the primary antibodies were replaced with blocking buffer.

[0110] The results are shown in Figure 5A and 5B . Figure 5A showed that the expression of zonula occludens-1 (ZO-1) and occludin in BALB / c mice in CECs was inhibited. Although external administration of R or IMMCA alleviated the inhibition of expression, ADSC-CM showed the best remedy. In another experiment, Figure 5B showed that the expression of ZO-1 and keratin 12 (K12) in BALB / c mice in CECs was also inhibited. Mice receiving external administration of ADSC-CM showed the best expression levels. Thus, the results demonstrated a decrease in the expression of ZO-1, occludin, and K12 induced by CECs in dry eye. This decrease was partially reversed by R and IMMCA, but the best expression was still seen in the ADSC-CM group.

[0111] 5. Histological analysis and immunohistochemical (IHC) assay

[0112] For histological examination, eyes were fixed in 10% formaldehyde and embedded in paraffin. Central vertical sections of 3 μm thickness were stained with hematoxylin-eosin or periodic acid Schiff (PAS). The corneal epithelium morphology and the thickness of the epithelium and stroma at the central cornea were measured, and the mean conjunctival goblet cell density was calculated by ImageJ examination.

[0113] As Figures 6A to 6E shown, the PAS staining results showed that CEC-induced dry eye reduced conjunctival goblet cells. R and IMMCA partially reversed this reduction, while ADSC-CM best maintained the goblet cell density. Figure 6F Statistical comparisons between the mean conjunctival goblet cell densities observed in each group were provided. As Figure 6F shown, among all treatment groups, ADSC-CM had the highest mean conjunctival goblet cell density and was comparable to the non-dry control group.

[0114] For MUC16 immunohistochemistry, eyes were fixed in 10% formaldehyde. After paraffin embedding, sections of 8 μm thickness were deparaffinized in xylene, rehydrated in a series of ethanol solutions, and washed twice with distilled water. Antigen retrieval was performed for 15 minutes at 90 to 95 °C with DAKO target retrieval solution (DAKO, Glostrup, Denmark) at pH = 9. MUC16 staining was performed on 8-μm thick sections using Histofine mouse staining kit (Nichirei, Tokyo, Japan). Sections were incubated overnight at 4 °C with anti-MUC16 (1:50; Santa Cruz, Santa Cruz, CA, USA) and finally stained with Histofine simple stain MAX PO for 10 minutes. The horseradish peroxidase reaction was developed using 3,3'-diaminobenzidine tetrahydrochloride and cobalt (D-0426, Sigma, Saint Louise, Missouri, USA). Experiments for negative control groups were also performed without using the primary antibody. After dehydration in graded ethanol and xylene, sections were mounted in Histokit (Hecht Assistent, Sondheim, Germany) and analyzed.

[0115] As Figure 7 shown, immunohistochemical analysis of MUC16 showed that MUC16 expression in the conjunctiva of the non-dry group was continuous, while MUC16 expression was discontinuous in the dry, R, and IMMCA groups. However, in CEC dry eye mice, topical application of ADSC-CM helped maintain continuous MUC16 expression. In addition, the results also showed that the ADSC-CM group had the best MUC16 expression. Arrows in the ADSC-CM group indicate conjunctival epithelial cells.

[0116] 6. Transmission electron microscopy (TEM) analysis

[0117] First, fresh corneal samples were fixed in 2% paraformaldehyde for 24 hours, then fixed in 2.5% glutaraldehyde solution in 0.2 M cacodylate buffer, then fixed with 1% tannic acid at pH 7.0 - 7.3 for 24 hours, and post-fixed in 1% osmium tetroxide solution in 0.2 M cacodylate buffer for 1 hour. After further fixation, the samples were en bloc stained with 0.2% uranyl acetate for 2 hours, then dehydrated through ethanol / acetone, and embedded in pure Spurr resin at room temperature for 8 hours. Finally, the samples were polymerized at 62 °C for 48 hours, and then photographed under a transmission electron microscope (Hitachi H-7500, Hitachi Ltd., Japan).

[0118] As Figures 8A to 8E shown, TEM studies showed that in CEC-induced dry eye mice, the tight junctions and interdigitation between corneal epithelial cells were reduced. Treatment with ADSC-CM increased the interdigitation and tight junction formation between adjacent corneal epithelial cells.

[0119] 7. Scanning electron microscopy (SEM) analysis

[0120] First, fresh corneas were fixed in 2% paraformaldehyde for 24 hours, then fixed in 2.5% glutaraldehyde solution and 1% tannic acid in 0.2 M cacodylate buffer at pH 7.0 to 7.3 for 24 hours, and post-fixed with 1% osmium tetroxide solution in 0.2 M cacodylate buffer for 1 hour. Then, the samples were dehydrated by a critical point dryer (Hitachi Ltd., Japan) and coated with palladium using an ion sputter coater (Hitachi Ltd., Japan). Finally, the samples were observed and photographed using a scanning electron microscope (Hitachi Ltd., Japan).

[0121] As Figures 9A to 9E shown, SEM studies demonstrated that microvilli loss occurred in the corneal epithelium of CEC-induced dry eye mice. External application of R and IMMCA partially preserved the microvilli of the corneal epithelium, while ADSC-CM had the best effect in protecting microvilli from dry damage.

[0122] Example 3: In vitro human corneal epithelial cell (HCEC) dry stress study using different molecular weight fractions of ADSC-CM

[0123] As described in Preparation Example 3 above, portions of ADSC-CM with different molecular weight sizes were prepared. ADSC-CM portions containing >750 kDa, 0-750 kDa, 300-750 kDa, 0-300 kDa, 100-300 kDa, 0-100 kDa, 30-100 kDa, and 0-30 kDa were obtained and subjected to dry stress and hyperosmotic stress, and then evaluated by CCK-8 assay. The dry stress study by CCK-8 assay was as described in Example 2 above. For the hyperosmotic stress study, HCECs were grown to approximately 60% confluence and then treated with fresh medium (311 milliosmoles (mOsM)) or medium containing 90 mM NaCl (480 mOsM) for 24 hours. After hyperosmotic treatment, the cells were cultured in different media. After incubation for 2 hours, the cells were assayed by CCK-8.

[0124] The results are shown in Figures 10A to 10D . It is shown that the ADSC-CM portions containing 0 to 750 kDa, 0 to 300 kDa, 0 to 100 kDa, and 0 to 30 kDa have the effect of protecting cells of ADSC-CM, and this protective effect protects the cells from being damaged by dry stress or hyperosmotic stress.

[0125] ADSC-CM portions with other molecular sizes were prepared to analyze the molecular size of the active ingredient in ADSC-CM. ADSC-CM portions containing 0 to 30 kDa, 0 to 10 kDa, 0 to 3 kDa, >30 kDa, >10 kDa, and >3 kDa were obtained, and the dry stress study as described above was performed on them and evaluated by CCK-8 assay. The results are shown in Figure 11 . It indicates that, in terms of the effect of maintaining the protection of cells of ADSC-CM from being damaged by dry stress, the ADSC-CM portions containing 0 to 30 kDa, 0 to 10 kDa, and 0 to 3 kDa have a better effect than the ADSC-CM portions containing >30 kDa, >10 kDa, and >3 kDa.

[0126] The ADSC-CM portion containing 0-1 kDa was used and the same dry stress study was repeated and evaluated by CCK-8 assay. The results are shown in Figure 12 . The ADSC-CM portion containing 0-1 kDa is still effective in protecting cells from being damaged by dry stress, and the percentage of cells maintained is similar to those treated with ADSC-CM.

[0127] Example 4: In vivo animal study of dry eye using different molecular weight size portions of ADSC-CM

[0128] The use of live animal studies was further carried out to evaluate the therapeutic effect of the ADSC-CM portion prepared above on dry eye. As described above, tear secretion assays, immunofluorescence double staining, histological analysis, immunohistochemistry (IHC) assays, transmission electron microscopy (TEM) analysis, and scanning electron microscopy (SEM) analysis were performed.

[0129] Figures 13A to 13C Results of tear secretion assays using different ADSC-CM portions with 30 to 100 kDa, 0 to 30 kDa, >10 kDa, <10 kDa, >3 kDa, <3 kDa, 0 to 3 kDa, and 0 to 1 kDa are shown. The results show that the ADSC-CM portions with 0 to 30 kDa, <10 kDa, <3 kDa, 0 to 3 kDa, and 0 to 1 kDa were able to stimulate the treated mice in the dry chamber to secrete a tear volume as much as or even more than that of the non-dry control group.

[0130] Furthermore, Figures 14A to 14B 、 Figures 15A to 15B and Figures 16A to 16B show the immunofluorescence double staining results of the ADSC-CM portions with 30 to 100 kDa, 0 to 30 kDa ( Figures 14A to 14B ), >10 kDa, <10 kDa, >3 kDa, <3 kDa ( Figures 15A to 15B ), 0 to 3 kDa, and 0 to 1 kDa ( Figures 16A to 16B ), respectively, to analyze the effect of different eye drops on the expression of corneal epithelial specific protein K12 and on the integrity of the tight junction barrier through the expression of ZO-1 and occludin. From the results, it can be seen that the expression of corneal epithelial specific protein K12 did not change with the treatment of different eye drops, while a decrease in the expression levels of tight junction related proteins ZO-1 and occludin in the corneal epithelium of mice was observed in the dry-treated mice, but it could be successfully prevented by ADSC-CM and the ADSC-CM portions with 0 to 30 kDa, <10 kDa, <3 kDa, 0 to 3 kDa, and 0 to 1 kDa.

[0131] Histological analysis was performed as described above to compare the effects of ADSC-CM portions of different molecular sizes on corneal epithelial morphology, the thickness of the epithelium and stroma at the central cornea, and the average conjunctival goblet cell density. As Figures 17A to 17F shown, ADSC-CM and the ADSC-CM portion with 0 to 30 kDa reversed the decrease in conjunctival goblet cells observed in CEC-induced dry eye, while the ADSC-CM portion with 30 to 100 kDa did not show the same effect.

[0132] Figures 18A to 18HAmong them, the ADSC-CM fractions with <10 kDa and <3 kDa partially reversed the conjunctival goblet cell reduction observed in CEC-induced dry eye, while the ADSC-CM fractions with >10 kDa and >3 kDa did not show the same effect. Figures 19A to 19G Among them, the ADSC-CM fractions with 0 to 3 kDa and 0 to 1 kDa partially reversed the conjunctival goblet cell reduction observed in CEC-induced dry eye, while IMDM treatment did not show the same effect.

[0133] In addition, IHC assays were performed to examine MUC4 expression and MUC16 expression in mice treated with different molecular size fractions of ADSC-CM. Figures 20A to 20E Shows the MUC16 expression results of mice caged in CEC and treated with ADSC-CM and different ADSC-CM fractions. It shows that ADSC-CM ( Figure 20C ) and the ADSC-CM fraction with 0 to 30 kDa ( Figure 20D ) induced the suppressed MUC16 expression observed in CEC-induced dry eye, while the ADSC-CM fraction with 30 to 100 kDa ( Figure 20E ) did not show the same effect. Figure 20E The arrow in [X] indicates conjunctival epithelial cells.

[0134] Figures 21A to 21G Shows the MUC16 expression results of mice caged in CEC and treated with ADSC-CM and different ADSC-CM fractions. Dry eyes treated with ADSC-CM ( Figure 21C ), the ADSC-CM fraction with <10 kDa ( Figure 21D ), and the ADSC-CM fraction with <3 kDa ( Figure 21E ) had higher MUC16 expression than those treated with the ADSC-CM fraction with >10 kDa ( Figure 21F ) and the ADSC-CM fraction with >3 kDa ( Figure 21G ).

[0135] Figures 22A to 22F Shows the MUC16 expression results of mice caged in CEC and treated with ADSC-CM and the ADSC-CM fractions of 0 to 3 kDa and 0 to 1 kDa. Dry eyes treated with ADSC-CM ( Figure 22C ), the ADSC-CM fraction with 0 to 3 kDa ( Figure 22D ), and the ADSC-CM fraction with 0 to 1 kDa ( Figure 22E ) had MUC16 expression similar to that of the non-dry control group and those treated with ADSC-CM, while those treated with IMDM ( Figure 22F ) had lower MUC16 expression similar to that of the dry control group.

[0136] Similarly, the MUC4 expression levels of animals housed in the CEC and treated with different ADSC-CM fractions were also examined. Figure 23A The MUC4 expression results of those treated with ADSC-CM and the 30 to 100 kDa and 0 to 30 kDa fractions of ADSC-CM are shown, while Figure 23B the MUC4 expression results of those treated with ADSC-CM and the >10 kDa, <10 kDa, >3 kDa, and <3 kDa fractions of ADSC-CM are shown. It shows that the 0 to 30 kDa, <10 kDa, and <3 kDa fractions of ADSC-CM show MUC4 expression levels similar to those of the non-dry control group and those treated with ADSC-CM, while a decrease in MUC4 was observed in the dry control group and those treated with the >10 kDa and >3 kDa fractions of ADSC-CM.

[0137] Figure 24 The MUC4 expression results of those treated with ADSC-CM and the 0 to 3 kDa and 0 to 1 kDa fractions of ADSC-CM are shown. Both the 0 to 3 kDa and 0 to 1 kDa fractions of ADSC-CM show MUC4 expression levels similar to those of the non-dry control group and those treated with ADSC-CM, while the mice treated with IMDM show a decreased MUC4 expression level similar to that seen in the dry control group.

[0138] In addition, as described above, SEM studies were performed on mice housed in the CEC and treated with different ADSC-CM fractions. As Figures 25A to 25E shown, CEC induced the degeneration and loss of microvilli of the corneal epithelium in dry eye mice ( Figure 25B ). The topical application of the <30 kDa fraction of ADSC-CM ( Figure 25E ) maintained the microvilli of the corneal epithelium and had an effect similar to that of those treated with ADSC-CM ( Figure 25C ) and the non-dry control group. However, the 30 to 100 kDa fraction of ADSC-CM ( Figure 25D ) did not show the same effect.

[0139] Similarly, Figures 26A to 26G the SEM results of those treated with the >10 kDa, <10 kDa, >3 kDa, and <3 kDa fractions of ADSC-CM are shown. The topical application of the <10 kDa fraction of ADSC-CM ( Figure 26E ) and the <3 kDa fraction of ADSC-CM ( Figure 26G ) maintained the microvilli of the corneal epithelium and had an effect similar to that of those treated with ADSC-CM ( Figure 26C ) and the non-dry control group ( Figure 26A) Similar effects. However, dry eyes treated with the >10 kDa fraction of ADSC-CM ( Figure 26D ) and the >3 kDa fraction of ADSC-CM ( Figure 26F ) could not maintain the microvilli of the corneal epithelium induced in dry eyes.

[0140] Furthermore, Figures 27A to 27F SEM results of dry eyes treated with the 0 to 3 kDa and 0 to 1 kDa fractions of ADSC-CM are shown. The 0 to 3 kDa fraction of ADSC-CM ( Figure 27D ) and the 0 to 1 kDa fraction of ADSC-CM ( Figure 27E ) both showed a similar maintenance of corneal epithelial cell microvilli as observed in the non-dry control group ( Figure 27A ) and those treated with ADSC-CM ( Figure 27C ), while mice treated with IMDM ( Figure 27F ) showed corneal epithelial microvilli loss and degradation similar to that seen in the dry control group ( Figure 27B ).

[0141] Examples 1 to 4 above clearly show that ADSC-CM containing active ingredients with molecular weights less than 30 kDa, less than 3 kDa, and less than 1 kDa effectively treats dry eyes in an individual.

[0142] In the in vitro HCEC dry stress study, drying for ten minutes caused a decrease in HCEC viability, which could not be alleviated by Refresh, IMDM, or IMMCA, but the decrease in viability was significantly compensated for by ADSC-CM and the fractions of ADSC-CM with active ingredients having molecular weights less than 30 kDa, less than 3 kDa, and less than 1 kDa. Western blot analysis performed simultaneously also showed an increase in the expression of P38 or p-Erk1 / 2 in HCECs cultured in ADSC-CM.

[0143] Mice in the dry CEC had more evaporation and thus more corneal fluorescein staining and rose bengal staining. This staining was the mildest in the ADSC-CM group. Immunohistochemical studies of the cornea also showed that mice in the CEC had significantly reduced expression levels of ZO-1 and occludin, which are markers of tight junctions. By administering ADSC-CM and its fractions with molecular weights less than 30 kDa, less than 3 kDa, and less than 1 kDa, the decrease in ZO-1 and occludin expression levels caused by dry injury was almost reversed or even better. The corneal epithelium in the ADSC-CM group also showed the best K12 expression, indicating the maintenance of corneal epithelial characteristics. The beneficial effects of ADSC-CM and its fractions on the tight junctions of the cornea were demonstrated not only by confocal immunohistochemical studies but also in TEM studies. The tight junctions maintained by ADSC-CM help with the housekeeping function of the corneal epithelium, which is to establish a boundary with the external environment and provide a barrier against fluid loss, toxin stimulation, and pathogen entry.

[0144] ADSC-CM and its fractions not only protect the tight junctions of corneal epithelial cells but also protect conjunctival goblet cells and the expression of membrane-associated mucin MUC16. The density of conjunctival goblet cells in CEC mice decreased significantly, and this phenomenon was alleviated by Refresh, IMMCA, or ADSC-CM. ADSC-CM and its fractions increased the goblet cell density to the highest degree, even higher than that in non-dry mice. The expression of MUC16 in the non-dry control group was continuous, while the expression of MUC16 in the dry control group was discontinuous. ADSC-CM and its fractions helped maintain the integrity of MUC16 expression.

[0145] Dry injury caused a loss of corneal epithelial microvilli. Although lubrication with Refresh or IMMCA alleviated the microvilli damage caused by dry stress, ADSC-CM and its fractions maintained the microvilli best.

[0146] In summary, this disclosure demonstrated the effect of ADSC-CM on dry-induced ocular surface injury, possibly achieved through the activation of Erk1 / 2 and P38.

[0147] The present disclosure has been described using exemplary embodiments. However, it should be understood that the scope of the present disclosure is not limited to the disclosed embodiments. On the contrary, it is intended to cover various modifications and similar rearrangements. Therefore, the scope of the claims should be given the broadest interpretation to cover all such modifications and similar arrangements.

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Claims

1. Use of a pharmaceutical composition for the preparation of a medicament for preventing or treating an ocular epithelial tissue disorder in an individual in need thereof, said pharmaceutical composition comprising a therapeutically effective amount of a bioactive agent, said bioactive agent comprising a composition prepared as follows: Obtain mesenchymal stem cells; Maintain the mesenchymal stem cells in a culture medium; Collect the culture medium; and Obtain a fraction containing active ingredients with a molecular weight less than 10 kDa from the collected culture medium; wherein the culture medium is supplemented with serum and a mesenchymal stem cell culture medium adjuvant; wherein the mesenchymal stem cells are obtained from adipose tissue; wherein the ocular epithelial tissue disorder is dry eye; and wherein the mesenchymal stem cell culture medium adjuvant comprises at least one of fibroblast growth factor-2, N-acetyl-L-cysteine, and L-ascorbic acid-2-phosphate.

2. The use according to claim 1, wherein the serum is fetal bovine serum or human serum, and its concentration in the culture medium ranges from 5% to 15%.

3. The use according to claim 1, wherein the concentration range of fibroblast growth factor-2 in the mesenchymal stem cell culture medium adjuvant is 5 ng / mL to 15 ng / mL.

4. The use according to claim 1, wherein the concentration range of N-acetyl-L-cysteine in the mesenchymal stem cell culture medium adjuvant is 1 mM to 5 mM.

5. The use according to claim 1, wherein the concentration range of L-ascorbic acid-2-phosphate in the mesenchymal stem cell culture medium adjuvant is 0.1 mM to 0.5 mM.

6. The use according to claim 1, wherein the fraction containing the active ingredient has a molecular weight less than 3 kDa.

7. The use according to claim 6, wherein the fraction containing the active ingredient has a molecular weight less than 1 kDa.

8. The use according to claim 1, wherein the mesenchymal stem cells are cultured in the culture medium for at least 2 passages.

9. The use according to claim 1, wherein the mesenchymal stem cells are obtained from another individual.

10. The use according to claim 9, wherein the other individual is a mammal.

11. The use according to claim 10, wherein the other individual is a human.

12. A composition comprising the fraction less than 10 kDa obtained according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

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