Corneal tissue exosomes, methods of making, uses, and pharmaceutical compositions thereof

Abstract

A kind of corneal tissue exosome of corneoscleral tissue after corneal transplantation is directly cultured quickly, the whole manufacturing process is simple, the procedures of thawing, separating and culturing cells in traditional cell culture process are omitted, and has high production and homogeneity, and can reduce the generation of biomedical waste.The corneal tissue exosome obtained from corneoscleral tissue has specific protein expression, and has the potential to treat wound repair, eye diseases, cancer and immune-related diseases.

Description

Technical Field

[0001] This invention relates to an exosome, specifically to a corneal tissue exosome generated by corneal and scleral tissue culture, its preparation method, its application, and its pharmaceutical composition. Background Technology

[0002] Current technologies exist that utilize exosomes to encapsulate drugs, proteins, or nucleotides, achieving transport, biocompatibility, and drug delivery effects. However, the production of these exosomes often requires stringent conditions, resulting in low production quantities and difficulty in preservation. This limits their application in the medical industry, and currently, exosomes are typically considered carriers without direct therapeutic efficacy. Therefore, developing exosomes that are easy to produce, have high preservation efficiency, and possess therapeutic effects is a pressing goal in related fields. Summary of the Invention

[0003] In order to develop exosomes that are easy to produce, have high preservation efficiency, and have therapeutic effects, this invention provides a corneal tissue exosome obtained by culturing a corneal scleral tissue.

[0004] The corneal and scleral tissue in question is a tissue explant of mammalian origin.

[0005] Furthermore, the corneal scleral tissue is corneal scleral tissue left after corneal transplantation, which is rich in limbal tissue and does not contain conjunctiva, endothelial cell, or iris tissue.

[0006] The corneal and scleral tissue comprises a sclera, a cornea, and a stromal cell.

[0007] The corneal tissue exosomes contain Integrin α9 protein and / or CD59 protein expression.

[0008] The present invention further provides a method for preparing corneal tissue exosomes, the steps of which include: The corneal and scleral tissue was collected and cultured in a culture environment comprising a serum-free medium (SFM) and a culture time between 12 and 72 hours; and Collect the serum-free culture medium from the culture dish in which the corneal and scleral tissue has been cultured, centrifuge and take a supernatant and / or take a concentrated solution after purification and filter it through a 0.22 μm membrane to obtain the corneal tissue exosomes.

[0009] The present invention further provides a method for collecting the corneal and scleral tissue, the steps of which include: Under sterile conditions, a conjunctival tissue, an endothelial cell, and an iris tissue were scraped away from the sclera in the corneal region using a No. 15 blunt scalpel.

[0010] The corneal and scleral tissue was cultured in this environment 10 to 17 days after being excised.

[0011] Among them, corneal tissue exosomes can be used for wound repair, cancer or immune disease treatment.

[0012] The method for obtaining corneal tissue exosomes and the corneal tissue exosomes provided by this invention have the following advantages: Exosomes from the corneal tissue can be collected directly after corneal transplantation, resulting in the reuse of biological resources and reducing the generation of biomedical waste. The overall process for obtaining corneal exosomes from this corneal and scleral tissue is simple, eliminating the thawing, separation, and cell culture steps required in traditional cell culture procedures. This significantly reduces time costs and results in a high total production volume of corneal exosomes, making mass production highly feasible. Exosomes obtained from corneal tissue exhibit specific protein expression and have the potential to treat wound healing, eye diseases, cancer, and immune-related diseases. Attached Figure Description

[0013] Figure 1 This is a flowchart of a preferred embodiment of the present invention; Figure 2 This is an electronic image of a preferred embodiment of the present invention; Figures 3A to 3B This is a comparison diagram of the size and concentration of corneal exosomes in a preferred embodiment of the present invention; Figures 4A to 4B This is a comparison diagram of the source, size, and concentration of corneal exosome cells in a preferred embodiment of the present invention; Figure 5 as well as Figure 6This is a protein expression diagram of corneal tissue exosomes, representing a preferred embodiment of the present invention. Figures 7A to 7B This is a comparison chart of growth factor performance in Experiment 3 of the preferred embodiment of the present invention; Figures 8A to 8B This is a comparison diagram of the crawling ability of cells in Experiment 6, representing a preferred embodiment of the present invention. Figure 9 This is a comparison of the cell crawling ability of corneal tissue exosomes after TFF purification, according to a preferred embodiment of the present invention. Figures 10A to 10B This is a protein performance comparison diagram from Experiment 2 of the preferred embodiment of the present invention; Figure 11 This is an analysis diagram of the affinity reaction between corneal tissue exosomes and osteopontin, representing a preferred embodiment of the present invention. Detailed Implementation

[0014] Please refer to Figure 1 This invention provides a corneal tissue exosome with high expression of integrin α9, directly generated from corneoscleral tissue culture. The steps for collecting the corneal tissue exosome from this corneoscleral tissue include: Step S1: Collect the corneal tissue.

[0015] The keratoscleral tissue is a mammalian-source tissue explant.

[0016] The corneal tissue is at least a portion of the cornea, a transparent, thin membrane covering the anterior end of the eyeball.

[0017] In this embodiment, the corneoscleral tissue is rich in limbaltissue, which is located around the cornea and connects to the sclera.

[0018] Preferably, the corneal and scleral tissue is a ring-shaped tissue left after corneal transplantation following ex vivo transplantation.

[0019] In this study, the keratoscleral tissue was preferably cultured in this environment for 10 to 17 days after ex vivo, ensuring the consistency and homogeneity of the exosomes produced. Referring to Table 1, samples 1 to 5 represent groups where the keratoscleral tissue was cultured for 10 to 17 days after ex vivo; samples 6 to 9 represent groups where the keratoscleral tissue was cultured for more than 17 days after ex vivo. The keratoscleral tissues from samples 1 to 3 and 6 to 7 were from Caucasians (United States), while those from samples 4 to 5 and 8 to 9 were from Asians (Taiwan). ZetaView nanoparticle tracking analysis revealed that the exosomes collected from samples 1 to 5, with a culture time of 10 to 17 days after ex vivo ex vivo, exhibited homogeneous particle diameters (peak values ​​between 100-125 nm). Samples 6 to 9, whose corneal and scleral tissues were cultured for 19 days or more after ex vivo, showed high heterogeneity in the particle size of the collected exosomes (peak values ​​ranging from 49.4 to 91.1 nm), with a tendency for the peak value to shift to the left. Subsequent data further demonstrate that exosomes generated from corneal and scleral tissues cultured for 10 to 17 days not only exhibited high homogeneity but also demonstrated significant therapeutic effects in wound healing experiments, showing a dose-dependent therapeutic effect.

[0020] Table 1

[0021] Preferably, the corneal annulus tissue is a hollow ring, and the ratio of the minimum diameter formed by its inner circumference to the maximum diameter formed by its inner circumference is between 7:20 and 8.5:15.

[0022] Preferably, the corneal tissue undergoes a pretreatment process to remove a conjunctiva, an endothelial cell, and an iris tissue before culturing to generate corneal tissue exosomes.

[0023] Step S2: Disperse the keratoscleral tissue into multiple culture dishes for culture.

[0024] The corneal and scleral tissue can be dispersed into culture dishes of 2, 6, 15 or 24 depending on its size or the requirements of subsequent operations, and this invention does not limit the scope of the invention.

[0025] In a first embodiment, the corneal sclera tissue is cut into multiple corneal flaps, and the multiple corneal flaps are dispersed into multiple culture dishes. In this embodiment, the annular corneal sclera tissue is cut into six equal parts to form six corneal flaps, and the six corneal flaps are placed in the culture tanks of a six-well culture dish for culture.

[0026] In a second embodiment, the corneal tissue is first cut into six equal parts to form six corneal flaps, and then each corneal flap is cut into small fragments by scissors and placed in each culture tank of the six-well culture dish for culture; or the six corneal flaps are first homogenized to form a corneal homogenate, and then placed in each culture tank of the six-well culture dish for culture.

[0027] In this second embodiment, the corneal homogenate is prepared by first placing the corneal and scleral tissue in the serum-free culture medium and then homogenizing it using a homogenizer. The homogenizer is used at a temperature of 2-8°C, a frequency of 50-80Hz, and a duration of 100-140 seconds per homogenization, which can be repeated several times as needed, with intervals of 30 seconds to 2 minutes between each homogenization, and the homogenate is placed on ice.

[0028] Step S3: Provide a culture environment to generate corneal tissue exosomes.

[0029] The culture environment includes a serum-free medium (SFM) and a culture time between 12 and 72 hours.

[0030] The serum-free medium (SFM) can be selected from MEM (minimalessential medium), α-MEM (alpha minimal essential medium), DMEM (Dulbecco's modified minimal essential medium), IMDM (Iscove's modified dulbecco's medium), MSCM (Mesenchymal stem cell medium), SF-1 (SF1hMSC medium), MSC NutriStem® XF Medium, CellCor™ EXO CD, MesenCult™-ACF Plus Culture Kit, or HEK GM medium.

[0031] Furthermore, the appropriate amount of serum-free culture medium is determined based on the type of culture dish and the size of the corneal flap in each dish. Preferably, 0.5 to 3 ml of serum-free culture medium is added to each culture dish.

[0032] In this embodiment, the serum-free medium (SFM) is α-MEM (alphaminimal essential medium), and 1 ml of the serum-free medium is added to each culture dish, and the culture time is 24 hours.

[0033] Step S4: Collect the corneal tissue exosomes.

[0034] The serum-free culture medium from which the corneal and scleral tissue was cultured was collected in the culture dish. The supernatant was then filtered through a 0.22 μm membrane using a batch centrifugation technique to obtain the filtered corneal tissue exosomes, which can then be preserved.

[0035] The corneal tissue exosomes are stored in a cryogenic environment at -80 degrees Celsius for at least 4 months.

[0036] The centrifugation of this batch was carried out in the following order: 200-500 centrifugal force (g) for 10-20 minutes; 1000-2000 centrifugal force (g) for 15-30 minutes; and 9000-14000 centrifugal force (g) for 30-60 minutes.

[0037] In one embodiment, the serum-free culture medium is subjected to batch centrifugation, and the corneal tissue exosomes are obtained by filtering the supernatant. The production yield of the corneal tissue exosomes can still be as high as 1.1-5.2 x 10⁻⁶ per milliliter. 11 The particle count, after conversion, is approximately 1.8-6.7 x 10⁻⁶ per 24 hours. 12 Exosomes.

[0038] Step S5 (optional): Purify the corneal tissue exosomes.

[0039] The supernatant was filtered through a 0.22 μm membrane and then reconstituted. Tangential flow filtration (TFF) was then used to further purify and concentrate the corneal exosomes by adjusting osmotic pressure and selecting the appropriate filter membrane. TFF purification of the corneal exosomes removed approximately 90% of the impurities.

[0040] In addition, in other embodiments, the method for collecting corneal tissue exosomes through the limbal tissue described above can also be combined with other existing methods for separating, purifying or extracting the exosomes, such as particle size separation chromatography (SEC), ultrafiltration (UF), precipitation, or immunoaffinity chromatography (IA).

[0041] Experiment 1 The present invention further analyzes the exosomes produced by the aforementioned method, wherein the exosomes produced from the keratoscleral tissue used in Experiment 1 were all obtained through the aforementioned first embodiment. Figure 2 The results showed that the corneal tissue exosomes produced by directly culturing the corneal and scleral tissue were not significantly different in morphology or size from general exosomes obtained from mesenchymal stem cells (MSCs). Similarly, ZetaView nanoparticle tracking analysis revealed that the peak diameter of the exosomes produced by the above method was between 75 and 125 nm, indicating that the corneal tissue exosomes conformed to the general exosome diameter standard of 30 to 200 nm.

[0042] Further analysis is needed to determine whether corneal tissue exosomes produced from corneal tissue under different conditions differ.

[0043] Figures 3A-3B Corneal and scleral tissues were obtained from Caucasian (USA U) and Asian (Taiwan, China T) individuals, respectively. The corneal and scleral tissues were then cultured using the aforementioned first embodiment, and corneal tissue exosomes were obtained from each tissue. The results showed that there was no difference in size or concentration between the Caucasian (USA U) group and the Asian (Taiwan, China T) group, indicating that the corneal tissue exosomes obtained according to this invention have high homogeneity.

[0044] Figures 4A-4B The study compared the differences in corneal tissue exosomes cultured from corneal and scleral tissues of different ages. The results showed that the age of origin did not affect the particle size or production concentration of the corneal tissue exosomes, nor did it affect the preservation effect, indicating that the corneal tissue exosomes obtained according to this invention have high homogeneity.

[0045] Further comparisons were made between the corneal tissue exosomes (U1 to U9 and T1 to T4, T8 and T10) obtained by the method provided in this invention and general exosomes obtained conventionally through mesenchymal stem cells (MSCs) and retinal pigment epithelial cells (RPEs).

[0046] Please refer to Table 2. ZetaView nanoparticle tracking analysis revealed that the production concentration of corneal tissue exosomes produced using the above method ranged from 1.1 to 5.2 x 10⁻⁶ per milliliter. 11The presence of particles indicates that the corneal tissue exosome yield obtained according to the present invention is higher per unit time than that obtained by traditional cell culture methods.

[0047] It is worth noting that the method for obtaining corneal tissue exosomes provided by this invention is not only simple in procedure but also low in time cost. After obtaining the tissue, only 24 hours of rapid culture are required to stably produce exosomes of up to 1.8-6.7 x 10⁻⁶. 12 The method provides a quantity of corneal tissue exosomes. However, obtaining general exosomes through conventional culture methods usually requires the use of cell lines or the isolation of target cells from tissues for cell culture. The process of culturing cells to the point where suitable conditions for harvesting general exosomes often takes more than two weeks, or even several months, and the resulting quantity of general exosomes cannot reach the quantity obtained in just 24 hours of culture as described in this invention. Furthermore, the corneal tissue exosomes obtained through the method provided by this invention can be stored for a long time, helping to reduce the number and frequency of repeated exosome culture procedures required by an operator, and possessing significant potential to accelerate scientific research or drug development in the medical industry.

[0048] Table 2 compares the yield of the circumferential corneal tissue obtained by the present invention with that of the ARPE19 cell line (mesenchymal stem cell, MSC) in 175T culture dishes. Even after continuous culture for 72 hours, the ARPE19 cell line failed to achieve the same yield as the ARPE19 cell line obtained 24 hours after culturing in 175T dishes. This demonstrates the feasibility of mass production of the corneal tissue exosomes obtained by the present invention.

[0049] Table 2

[0050] Furthermore, this invention compares the expression levels of common marker proteins found in exosomes. For example... Figure 5 As shown, the corneal tissue exosomes expressed CD63, CD9, CD81, and Alix proteins in the Western blot results. This indicates that the corneal tissue exosomes obtained by the method provided in this invention have great potential for genetic engineering.

[0051] This invention also tested the expression of common marker proteins for limbal stem cells (P63-α and Integrin α9) and common marker proteins for stem cells (ABCG2 and Notch1) in corneal exosomes. The experiments were also conducted using Western blot. Figure 6 It can be observed that the corneal tissue exosomes (U1 to U9, T1 to T4, T8, and T10) obtained by the method provided by the present invention exhibit high expression of Notch1 and Integrin α9 proteins. This indicates that the corneal tissue exosomes obtained by the method provided by the present invention reflect the characteristics of the corneal limbal tissue and limbal stem cells.

[0052] Experiment 2 This invention further compares the corneal tissue exosomes obtained by the method provided by this invention with exosomes obtained from other ocular tissues or cells. The corneal tissue exosomes used in Experiment 2 were all obtained through the aforementioned second embodiment. Each group in Experiment 2 included: Experimental group 1: The corneal tissue exosomes produced by the aforementioned second embodiment; Experimental Group 2: A central corneal tissue exosome produced from the central cornea (i.e., the corneal tissue from which the corneal rim tissue has been removed) of one eye via the aforementioned second embodiment; Experimental Group 3: The corneal and scleral tissue taken from the cornea in Experimental Group 2, processed using the corneal tissue exosomes produced in the aforementioned second embodiment; and Control group 1: General exosomes obtained from the culture medium of ARPE19, a retinal pigment epithelial cell line.

[0053] Please refer to Figure 10AIn the Western blot results, the corneal tissue exosomes obtained from experimental groups 1-3, the central corneal tissue exosomes, and the general exosomes obtained from control group 1 all showed a certain degree of expression for common exosome marker proteins. Notably, compared to other groups, the corneal tissue exosomes produced from the limbal tissue-rich scleral tissue in experimental groups 1 and 3 highly expressed CD9 and CD81 proteins.

[0054] Then you can see Figure 10B In the study, experimental groups 1 through 3 all showed expression of P63-α, Notch1, and Integrin α9 proteins (especially in the corneal tissue exosomes obtained from experimental groups 1 and 3, where the expression was most significant), while the exosomes obtained from control group 1 showed almost no expression of these proteins. Notably, compared to other groups, experimental groups 1 and 3 showed high expression of CD59 protein through the corneal tissue exosomes. This demonstrates the uniqueness of corneal tissue exosomes secreted by the cornea and sclera, and their CD59-rich nature makes them a promising candidate platform for drug delivery.

[0055] Experiment 3 The aforementioned Experiments 1 and 2 and Figure 6 , 10A Furthermore, 10B confirmed that the corneal tissue exosomes were rich in Integrin α9. This aligns with a previous report (doi: 10.3390 / cancers12113379) which described Integrin α9 as a receptor for the SVVYGLB domain. The SVVYGLB domain is specific and commonly found in high levels and overexpressed osteopontin (OPN) in various cancer cells, including but not limited to breast cancer, prostate cancer, colorectal cancer, head and neck cancer, liver cancer, and lung cancer. This invention further detects the presence of Integrin α9 in the corneal tissue exosomes. + Whether EVs can produce a labeling effect on osteopontin. Experiment 3 included the following groups: Experimental Group 1: The corneal tissue exosomes produced by the aforementioned second embodiment were dissolved in 1 ml of Dulbecco's Phosphate Buffered Saline (DPBS); Control group 1: Common exosomes obtained from the culture medium of ARPE19 retinal pigment epithelial cell line were dissolved in 1 mL of Dulbecco's Phosphate Buffered Saline (DPBS); and Control group 2: Phosphate-buffered saline (PBS).

[0056] In this study, the exosome content in experimental group 1 and control group 1 was determined by Nanoparticle Tracking Analysis (NTA). The concentration in experimental group 1 was 1.6 mg / mL. 10 11 Particles, control group 1 concentration was 1.1 g / mL. 10 11 Particles.

[0057] The affinity of each of the above groups for this osteopontin will be further tested using fluorescence staining. The overall procedure is as follows: 1. Add 100 μl of coating buffer containing osteopontin (200 ng) to each well of a 96-well plate and allow it to react at 4°C for 8-12 hours. 2. Take 100 μl of fluorescent dye (PKH67) and 1-5 μl of fluorescent dye (PKH67) from experimental group 1 and control group 1 respectively, mix them evenly, and then add 1 ml of diluent C (Diluent C) and 200 μl of 10% BSA dissolved in PBS in sequence. Mix evenly and let it stand at room temperature for 5-40 minutes. 4. Centrifuge at ultra-high speed (100,000×g) for 1 hour to collect the corneal tissue exosomes that have been deposited and which are fluorescent, as well as the general exosomes, and then rehydrate them with 100 μl of PBS; 5. Add each group to the respective well of the reaction pan containing the coating solution, and react at room temperature for 20-30 minutes; 6. After the reaction, discard the solution, rinse with PBS, and observe the fluorescence reaction under a fluorescence microscope.

[0058] Please refer to Figure 11 Among them, experimental group 1 and control group 1 were respectively treated with exosome content of 5×10 8 / μl, 1×10 9 / μl and 2×10 9 / μl was used to conduct the affinity reaction of osteopontin. The results showed that the corneal tissue exosomes provided by experimental group 1 had a better affinity reaction than the general exosomes provided by control group 1, and the binding effect of experimental group 1 to osteopontin was significantly improved with the higher content of exosomes.

[0059] The corneal tissue exosome (Integrin α9) + EVs can bind to osteopontin (OPN) overexpressing cancer cells (such as breast cancer, prostate cancer, colorectal cancer, head and neck cancer, liver cancer, and lung cancer), improving tumor-targeting specificity to the SVVYGLB domain and avoiding the non-specific tissue toxicity commonly seen when binding to the RGD domain on OPN. Furthermore, preliminary verification shows that these corneal tissue exosomes (Integrin α9+ EVs) have a higher binding affinity to OPN proteins compared to general exosomes with low Integrin α9 protein expression, suggesting a potential future development of corneal tissue exosomes (Integrin α9+ EVs). + EVs have emerged as a candidate platform for drug delivery in cancer treatment.

[0060] In addition, when the corneal tissue exosomes are rich in CD59, the corneal tissue exosomes (CD59) show increased expression. + EVs can inhibit the formation of the membrane attack complex (MACAssembly) after the complement protein in the immune response is activated, thus preventing the corneal exosomes (CD59) from forming. + EVs have emerged as a candidate platform for drug delivery in the treatment of complement activation diseases, inflammatory diseases, and autoimmune diseases.

[0061] Based on the above, corneal exosomes expressing specific proteins (such as Integrin α9) can be selected according to requirements. + EV or CD59 + Exosomes (EVs) are used to deliver drugs or treatments specifically targeting a particular disease, cell, or tissue. Among these methods, selecting exosomes exhibiting specific protein expression can be achieved using tangential flow filtration (TFF) technology. Further purification of the exosomes is then performed simultaneously with affinity chromatography using antibodies (such as CD59 or Integrin α9 antibodies) to classify the exosomes based on whether or not they express the specific protein.

[0062] Further reference Figure 7AIn addition to 7B, when detecting the expression levels of multiple growth factors in the corneal tissue exosomes and general exosomes, the corneal tissue exosomes obtained by the method provided by this invention are also found to be rich in various growth factors, especially bFGF, EGFR, VEGF, VEGFR3, TGF-bata3, PDFG AA, PDFG BB, SCF, NT4, and IGFBP1. This demonstrates the potential of the corneal tissue exosomes for cell or tissue repair.

[0063] Experiment 4 Furthermore, the present invention also compared the production concentration of corneal tissue exosomes in the first embodiment and the second embodiment, and compared whether the production concentration of corneal tissue exosomes was affected by different ethnicities, different tissue fragmentation methods, and whether or not the tissue was filtered through a 0.22 μm membrane (as shown in Tables 3 to 5).

[0064] As can be seen from Table 3 of the first embodiment, the supernatant derived from corneal and scleral tissues of different ethnicities does not affect the final particle size and production concentration of the corneal tissue exosomes. This shows that the corneal tissue exosomes obtained by the method provided by the present invention are homogeneous, not easy to aggregate and precipitate, and have batch-to-batch stability. The corresponding results can be obtained from Tables 3 and 4 below.

[0065] Table 4 shows that the production concentration of corneal tissue exosomes obtained by cutting the corneal tissue into fine fragments with scissors or by homogenizing it in the second embodiment is approximately one power higher than that in the first embodiment. Furthermore, filtration through a 0.22 μm membrane does not affect the production concentration of corneal tissue exosomes.

[0066] Table 3, First Embodiment

[0067] Table 4, Second Embodiment

[0068] Experiment 5 Further comparison of the technology provided by this invention with existing techniques for culturing ex vivo tissue using a collagenase. Specifically, the groups in Experiment Five included: Experimental Group 1: The corneal and scleral tissue was cut into small fragments using the scissors according to the method provided in the second embodiment of the present invention and then cultured.

[0069] Comparative Example 1: The corneal and scleral tissue was cut into small fragments with scissors, and then a collagenase was added and reacted for 30 minutes to decompose the fragmented corneal and scleral tissue.

[0070] Comparative Example 2: The corneal and scleral tissue was cut into small fragments by scissors, and then the collagenase was added and reacted for 60 minutes to decompose the fragmented corneal and scleral tissue.

[0071] In Comparative Examples 1 and 2, after the corneal and scleral tissue reacted with the collagenase, the collagenase was washed away using a buffer solution (such as phosphate-buffered saline, PBS), and then the corneal and scleral tissue was placed in a culture tank for culture.

[0072] The results in Table 5 show that, without the addition of collagenase, experimental group 1 significantly outperformed comparative examples 1 and 2, which included collagenase. Further investigation suggests that this may be because the rigidity of the corneal and scleral tissue is higher than that of general tissues, falling between the cornea and sclera, with a Young's coefficient of approximately 100-5000 kPa (the retina is approximately 0.1-10 kPa; visceral tissues are approximately 0.5-30 kPa). This makes it difficult for the corneal and scleral tissue to be broken down by collagenase, and may even reduce the efficiency of corneal exosome production.

[0073] As can be seen from the results of comparisons in Tables 2 to 4 above, in the second embodiment, the corneal tissue flap can be homogenized and cultured under appropriate conditions to obtain more corneal tissue exosomes, which is about 4 times the yield in the first embodiment. In addition, the present invention omits the collagenase treatment, which is a significant breakthrough from the concept of the old technology and shows the mass production potential of increased yield after the method is optimized.

[0074] Table 5

[0075] Experiment Six Further analysis was conducted on the repair capacity of the corneal exosome cells provided by this invention. Human corneal epithelial cells (HCE-T) were cultured on cell discs and tested using a wound healing assay (scratch assay). A wounding area was scraped out of the cell disc contaminated with HCE-T cells, and control groups were given PBS, while general exosomes derived from mesenchymal stem cells (2 x 10⁶ cells / year) were administered. 8 Particles and corneal tissue exosomes (randomly selected U1, U9, U30, T15, and T17) obtained by the method provided by the present invention, 2 x 10 8Particles were used to assess the impact of each group on cell crawling ability. This could be detected after 24 hours. Figure 8A In the group treated with corneal tissue exosomes obtained by the method provided by this invention, the wounding area in the cell disc was significantly reduced, confirming that the corneal tissue exosomes obtained by the method provided by this invention have similar therapeutic effects on wound repair and are better than conventional exosomes derived from mesenchymal stem cells. The differences are even more clearly visible in the charts analyzing the area of ​​the wounding area in each group at 0 hours, 2 hours, 5 hours, 8 hours, and 24 hours after treatment. Figure 8B ).

[0076] Figure 9 The next step compared whether corneal tissue exosomes purified via TFF in step S5 affected their ability to repair cells. Similarly, a wound healing assay (Scratch assay) was performed, with the control group receiving PBS and the group receiving unpurified exosomes at a concentration of 2 x 10⁻⁶. 8 The corneal tissue exosomes (pre-TFF) and those purified via TFF at concentrations of 2 x 10⁻⁶ were used. 8 1.0 x 10 8 0.5 x 10 8 0.25 x 10 8 and 0.125 x 10 8 The corneal tissue exosomes (post-TFF) were used to assess the impact of each group on cell crawling ability.

[0077] The results show that corneal tissue exosomes purified via TFF (post-TFF) have the same cell repair efficacy as those not purified via TFF (pre-TFF), while the efficacy of post-TFF at 2 x 10⁻⁶ mmol / L is significantly higher than that of 2 x 10⁻⁶ mmol / L. 8 Up to 0.125 x 10 8 The corneal exosomes exhibit dose-dependent potency, even at a concentration of 0.125 x 10⁻⁶. 8 Under these conditions, the effect on the corneal cell crawling ability of HCE-T cells remained significantly different within 24 hours, unaffected by the TFF purification process. This demonstrates the ability of corneal tissue exosomes to repair corneal wounds and suggests their potential as a candidate drug for treating eye diseases, including but not limited to dry eye, corneal erosion, and corneal ulcers.

[0078] The method for obtaining corneal tissue exosomes and the corneal tissue exosomes provided by this invention have the following advantages: Exosomes from corneal tissue can be quickly collected directly from the corneal and scleral tissue after corneal transplantation, resulting in the reuse of biological resources and reducing the generation of biomedical waste. The overall process for obtaining corneal tissue exosomes from corneal and scleral tissue is simple, eliminating the thawing, separation, and cell culture steps required in traditional cell culture processes. This significantly reduces time costs and results in a high total production volume of corneal tissue exosomes, making mass production highly feasible. Exosomes obtained from corneal tissue exhibit specific protein expression and have the potential to treat wound healing, eye diseases, cancer, and immune-related diseases.

Claims

1. A corneal exosome, characterized in that, Obtained by culturing a corner of scleral tissue, the steps include: Collect a corner scleral tissue and break it up, wherein the corner scleral tissue contains an annular corneal rim tissue; The fragmented keratoscleral tissue was dispersed into multiple culture dishes and cultured in a culture environment comprising a serum-free medium and a culture time between 12 and 72 hours, wherein no collagenase was added to the keratoscleral tissue before or during culture; and The serum-free culture medium from which the corneal and scleral tissue was cultured was collected in the culture dish. After centrifugation, a supernatant was filtered through a 0.22 μm membrane to obtain the corneal tissue exosomes.

2. The corneal tissue exosomes as provided in claim 1, characterized in that, The keratoscleral tissue was a mammalian-source tissue explant, and was cultured in this culture environment for 10 to 17 days after ex vivo.

3. The corneal tissue exosomes as provided in claim 2, characterized in that, It includes corneal concentric ring tissue, and the ratio of the minimum diameter formed by its inner periphery to the maximum diameter formed by its inner periphery is between 7:20 and 8.5:

15.

4. The corneal tissue exosomes as provided in claim 3, characterized in that, This corneal tissue does not contain a conjunctiva, an endothelial cell, or an iris.

5. The corneal tissue exosomes as provided in claim 1, characterized in that, Includes Integrin α9 protein expression.

6. The corneal tissue exosomes as provided in claim 1, characterized in that, Includes CD59 protein expression.

7. A method for preparing corneal tissue exosomes, characterized in that, The steps include: Collect a corneal sclera tissue and break it up, wherein the corneal sclera tissue is a corneal helix tissue containing an annular shape; The fragmented keratoscleral tissue was dispersed into multiple culture dishes and cultured in a culture environment comprising a serum-free medium and a culture time between 12 and 72 hours, wherein no collagenase was added to the keratoscleral tissue before or during culture; and The serum-free culture medium from which the corneal and scleral tissue was cultured was collected in the culture dish. After centrifugation, a supernatant was filtered through a 0.22 μm membrane to obtain the corneal tissue exosomes.

8. The method as provided in claim 7, characterized in that, The keratoscleral tissue was an ammammalian-derived tissue explant and was cultured in this environment after 10 to 17 days.

9. The method as provided in claim 8, characterized in that, The ratio of the minimum diameter formed by the inner periphery of this corneal tissue to the maximum diameter formed by the inner periphery is between 7:20 and 8.5:

15.

10. The method as provided in claim 9, characterized in that, The corneal and scleral tissue was dispersed into multiple culture dishes after being fragmented or homogenized.

11. The method as claimed in claim 10, characterized in that, The serum-free medium (SFM) may include MEM (minimal essential medium), α-MEM (alpha minimal essential medium), or DMEM (Dulbecco's modified minimal essential medium).

12. The method as claimed in claim 7, characterized in that, The centrifugation was performed in sequence at 200-500 g for 10-20 minutes; 1000-2000 g for 15-30 minutes; and 9000-14000 g for 30-60 minutes.

13. The method as claimed in claim 12, characterized in that, The supernatant was filtered through a 0.22 μm membrane to obtain corneal tissue exosomes.

14. The method as claimed in claim 13, characterized in that, The supernatant was purified and filtered through a 0.22 μm membrane to obtain corneal tissue exosomes. The purification process included tangential flow filtration (TFF), particle size chromatography (SEC), ultrafiltration (UF), precipitation, or immunoaffinity (IA) techniques.

15. The use of corneal tissue exosomes as described in claim 1 for cell repair or complement activation-related immune diseases.

16. The use of corneal tissue exosomes as described in claim 5 for targeting cancer cells expressing osteopontin.

17. A composition, characterized in that, It includes a corneal tissue exosome as provided in claim 1.

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