Human corneal limbus microenvironment cell exosome eye drops as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical engineering, and particularly relates to human corneal limbus microenvironment cell exosome eye drops as well as a preparation method and application thereof. The invention discloses human corneal limbus microenvironment cell exosome eye drops. The unique active ingredient in the eye drops is human corneal limbus microenvironment cell exosome. The invention aims to provide eye drops containing human corneal limbus microenvironment cell exosomes. The eye drops are suitable for repairing corneal injuries caused by various factors clinically and relieving eye diseases such as visual fatigue, xerophthalmia, corneal limbus stem cell deficiency and immune lesions caused by graft-versus-host diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering technology, and in particular relates to human limbal microenvironment cell exosome eye drops, a preparation method and applications thereof. Background Art

[0002] Limbal stem cells (LSCs) are a group of stem cells located at the Vogt palisade at the base of the cornea. Limbal stem cells continuously proliferate and differentiate to maintain the stability and renewal of the corneal epithelium. Limbal stem cells (LSCs) play a vital role in maintaining corneal transparency and health. Factors such as chemical damage or thermal burns to the ocular surface, dry eyes, and prolonged wear of contact lenses can cause decompensation in the number and function of limbal stem cells, leading to limbal stem cell deficiency (LSCD). Limbal stem cell deficiency causes the corneal epithelium to lose its ability to regenerate and repair, causing conjunctivalization of the corneal epithelium, neovascularization, corneal stromal scarring, and even corneal autolysis and ulcers.

[0003] Currently, commonly used treatments for limbal stem cell deficiency include autologous / allogeneic limbal transplantation, autologous / allogeneic limbal stem cell transplantation, in vitro cultured oral mucosal cell transplantation, and amniotic membrane transplantation. While these approaches have achieved some clinical efficacy, they still present numerous challenges, including limbal transplant rejection, difficulty in in vitro expansion and differentiation of limbal stem cells, poor biological efficacy of oral mucosal cell transplantation, and the inability of amniotic membrane transplantation to repair the corneal epithelium.

[0004] Mesenchymal stem cells (MSCs) are a type of stromal multipotent stem cell originally isolated and characterized from bone marrow (BMMSCs). Multiple studies have demonstrated that BMMSCs have the potential to differentiate into epithelial cells. Therefore, BMMSCs may be used to treat LSCD.

[0005] Limbal microenvironment cells (LNCs) are a group of primitive cells isolated and cultured from the microenvironment of limbal stem cells (LSCs) that have the functions of endothelial progenitor cells and mesenchymal stem cells (MSCs). They can support corneal epithelial progenitor cells (LEPCs) under in vitro 3D culture conditions, as shown by increased expression of P63α and decreased CK12 in LEPCs. Therefore, they are named limbal microenvironment cells.

[0006] LNCs express multiple embryonic stem cell, neural crest cell, and MSC markers, and possess stronger stem cell properties than BMMSCs. LNCs are more effective than BMMSCs in treating LSCD. Therefore, as corneal MSCs, LNCs are a more ideal stem cell for corneal disease treatment than MSCs from other sources.

[0007] The importance of stem cell exosomes in the clinical treatment of diseases is increasingly recognized. Exosomes are small, nanoscale extracellular vesicles secreted by cells, carrying bioactive substances such as nucleic acids, proteins, and lipids, with diameters ranging from 30 to 150 nm. Exosomes have been found to be secreted by most cell types, including immune cells, neurons, epithelial cells, endothelial cells, embryonic cells, cancer cells, and mesenchymal stem cells (MSCs). Exosomes can play a role in physiological and pathological processes, acting as mediators of intercellular communication and substance exchange, and have important inflammatory and immunomodulatory effects. Furthermore, exosomes can deliver a variety of bioactive substances and easily inactivated or degraded components through multiple pathways and sites, safely transferring them to target cells to participate in regulatory processes such as tissue repair, tumor diagnosis, and treatment. The endogenous and heterogeneous nature of exosomes gives them broad and unique advantages in the fields of disease diagnosis and treatment.

[0008] Currently, there are a few reports on the use of exosome components extracted from stem cell culture media for corneal damage repair, relief of visual fatigue, and dry eye syndrome. However, there are no reports on the use of exosomes from human limbal microenvironment cells (LNC) to prepare eye drops suitable for corneal damage repair, relief of visual fatigue, dry eye syndrome, limbal stem cell deficiency, and eye lesions caused by graft-versus-host disease. Summary of the Invention

[0009] The purpose of the present invention is to provide an eye drop containing exosomes from human limbal microenvironment cells, which is suitable for repairing corneal damage caused by various clinical factors, relieving visual fatigue, dry eyes, limbal stem cell deficiency, and eye diseases caused by graft-versus-host disease.

[0010] The present invention provides human limbal microenvironment cell exosome eye drops, wherein the only active ingredient in the eye drops is human limbal microenvironment cell exosomes.

[0011] Furthermore, the eye drops are composed of human corneal limbal microenvironment cell exosomes and a balanced salt solution.

[0012] The present invention provides a method for preparing exosomes from human limbal microenvironment cells, comprising the following steps:

[0013] (1) Culture dishes covered with 5% human acellular amniotic membrane gel were placed in a 37°C constant temperature cell culture incubator with a volume concentration of 5% CO2 to culture human corneal limbal microenvironment cells;

[0014] (2) Cultivate cells until they are 80-90% confluent and continue culturing for 48 h;

[0015] (3) After centrifugation, the precipitate was collected to obtain exosomes;

[0016] (4) Washing the precipitate and resuspending it, followed by secondary centrifugation, and resuspending the precipitate to prepare exosomes from human limbal microenvironment cells.

[0017] Furthermore, the human corneal limbal microenvironment cells in step (1) are 4th generation human corneal limbal microenvironment cells.

[0018] Furthermore, the specific operation of the centrifugation in step (3) is:

[0019] At 4°C, first centrifuge at 300g for 10 minutes, aspirate the supernatant, then centrifuge at 2000g for 10 minutes, aspirate the supernatant, high-speed centrifuge at 10000g for 30 minutes, aspirate the supernatant, ultracentrifuge at 140000g for 90 minutes, remove the supernatant, and the obtained precipitate is the exosome.

[0020] The present invention provides a method for preparing the human limbal microenvironment cell exosome eye drops according to claim 1, comprising the following steps:

[0021] The prepared human corneal limbal microenvironment cell exosomes were mixed with a balanced salt solution at a volume ratio of 1:200-400 to obtain human corneal limbal microenvironment cell exosome eye drops.

[0022] Furthermore, the concentration of exosomes from human limbal microenvironment cells is 1×10 7 P / mL.

[0023] The present invention provides an application of human limbal microenvironment cell exosomes in the preparation of eye drops for treating eye diseases.

[0024] Furthermore, the eye diseases include corneal damage repair, relief of visual fatigue, dry eye or limbal stem cell deficiency.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The eye drops described in this invention are effective for treating corneal damage repair, alleviating visual fatigue, dry eye, limbal stem cell deficiency, and other eye diseases caused by graft-versus-host disease. The stem cell exosome eye drops have excellent stability and bioactivity, facilitating safe and effective localized ocular treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are slit lamp photographs of the mouse cornea on the 3rd and 5th days after treatment with the eye drops prepared in Example and Comparative Examples 1 and 2, as well as fluorescent staining images on the 0th, 1st, 3rd, 4th and 5th days. DETAILED DESCRIPTION

[0028] Example

[0029] Preparation of exosomes from human limbal microenvironment cells

[0030] (1) The 4th generation human corneal limbal microenvironment cells were selected and cultured in a culture dish covered with 5% human decellularized amniotic membrane gel using MESCM culture medium in a 37°C constant temperature cell culture incubator with a volume concentration of 5% CO2. The cells were cultured until 80-90% confluence and then cultured for 48 hours.

[0031] (2) Extracting exosomes from the cells obtained in step (1) by ultraspeed gradient centrifugation:

[0032] Centrifuge at 300g for 10 minutes at 4°C and aspirate the supernatant.

[0033] Then centrifuge at 2000g for 10 min and aspirate the supernatant.

[0034] Centrifuge at 10000g for 30 minutes, and draw the supernatant.

[0035] Ultracentrifugation was performed at 140000g for 90 min, and the supernatant was removed. The obtained precipitate was the exosome.

[0036] The precipitate was washed once with balanced salt solution and resuspended, then centrifuged again at 140,000 g for 90 min, and the precipitate was resuspended in 500-1000 μL balanced salt solution to obtain exosomes from human corneal limbal microenvironment cells.

[0037] (3) The recovery rate of exosomes from human limbal microenvironment cells can reach 80-90%, and the particle concentration after sterilization and filtration is approximately 2×10 10 P / mL.

[0038] Among them, exosomes can be stably stored at 2-8°C for 3 days and at -20°C for 1 month.

[0039] Long-term storage requires a low temperature of -80°C.

[0040] Preparation of eye drops containing exosomes from human limbal microenvironment cells

[0041] (1) The human corneal limbal microenvironment cell exosomes obtained above were used as the therapeutic active ingredient, wherein the concentration of LNC exosomes was 2.1x10 10 P / mL;

[0042] (2) Balanced salt solution: a medical compound sodium chloride injection that is less irritating to the human ocular surface; used to prepare human limbal microenvironment cell exosome eye drops;

[0043] (3) The exosomes from step (1) and the balanced salt solution from step (2) were mixed at a volume ratio of 1:300 (20 μL of exosome suspension was added to 6 mL of balanced salt solution and mixed) at 4°C in a sterile environment to obtain a particle concentration of 1x10 7 P / mL of human limbal microenvironment cell exosome eye drops.

[0044] (4) The amount of eye drops containing exosomes from human limbal microenvironment cells is prepared in the form of 5 vials, each 1 mL.

[0045] Comparative Example 1

[0046] The difference from the embodiment is that the LNC exosomes in the eye drop components are replaced by 0.05% recombinant bovine basic fibroblast growth factor (0.05% rh-bFGF), and the rest are the same.

[0047] Comparative Example 2

[0048] The difference from the embodiment is that the eye drops do not contain LNC, and the other components are the same.

[0049] Animal experimental methods

[0050] The mouse corneal epithelial injury model was established using the Algerbrush II method.

[0051] After modeling, 6-8 week old C57BL / 6 male mice were selected and the eye drops of the experimental example and comparative examples 1 and 2 were used for eye drops 3 times a day at 9:00 AM, 13:00 AM, and 17:00 PM, respectively, with a dosage of 5 μL / eye / time. The treatment time was 5 days.

[0052] After treatment with the eye drops prepared in the experimental example and comparative examples 1 and 2, the eyeballs of the mice were photographed with a slit lamp and corneal fluorescence staining was performed on d0, d1, d3, d4, and d5, respectively.

[0053] The experimental results are as follows Figure 1 As shown:

[0054] As can be seen, the corneal epithelium of the mice in Example 1 began to heal on day 1 after treatment and was substantially healed by day 3. In contrast, the corneal epithelial defect area of ​​the mice in Comparative Examples 1 and 2 did not change significantly on day 1 after treatment. By day 3, a small area of ​​corneal epithelial defect still remained in the cornea of ​​the mice in Comparative Example 1, while the area of ​​corneal epithelial defect in the mice in Comparative Example 2 was relatively larger. The corneas of the mice in Comparative Examples 1 and 2 were substantially healed on days 4 and 5 after treatment, respectively.

[0055] The above results show that after modeling of corneal epithelial damage in mice, compared with rh-bFGF (Comparative Example 1) and balanced salt solution (Comparative Example 2), the eye drops containing human LNC exosomes (Example) according to the present invention can repair the corneal epithelium and stem cell damage in mice faster after treatment, indicating that the composition of the eye drops according to the present invention has certain specificity and can effectively treat corneal damage repair, relieve visual fatigue, dry eye, limbal stem cell deficiency, and eye diseases such as graft-versus-host disease.

[0056] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A human limbal microenvironment cell exosome eye drops, characterized in that: The only active ingredient in the eye drops is exosomes from human limbal microenvironment cells.

2. The eye drops according to claim 1, characterized in that The eye drops are composed of human corneal limbal microenvironment cell exosomes and a balanced salt solution.

3. A method for preparing exosomes from human limbal microenvironment cells, characterized in that: The steps include: (1) Culture dishes covered with 5% human acellular amniotic membrane gel were placed in a 37°C constant temperature cell culture incubator with a volume concentration of 5% CO2 to culture human corneal limbal microenvironment cells; (2) Cultivate cells until they are 80-90% confluent and continue culturing for 48 h; (3) After centrifugation, the precipitate was collected to obtain exosomes; (4) Washing the precipitate and resuspending it, followed by secondary centrifugation, and resuspending the precipitate to prepare exosomes from human corneal limbal microenvironment cells.

4. The preparation method according to claim 3, characterized in that The human corneal limbal microenvironment cells in step (1) are 4th generation human corneal limbal microenvironment cells.

5. The preparation method according to claim 3, characterized in that The specific operation of the centrifugation in step (3) is: At 4°C, first centrifuge at 300g for 10 minutes, aspirate the supernatant, then centrifuge at 2000g for 10 minutes, aspirate the supernatant, high-speed centrifuge at 10000g for 30 minutes, aspirate the supernatant, ultracentrifuge at 140000g for 90 minutes, remove the supernatant, and the obtained precipitate is the exosome.

6. A method for preparing the human corneal limbal microenvironment cell exosome eye drops according to claim 1, characterized in that: The steps include: The human corneal limbal microenvironment cell exosomes prepared according to claim 3 are mixed with a balanced salt solution at a volume ratio of 1:200-400 to obtain human corneal limbal microenvironment cell exosome eye drops.

7. The preparation method according to claim 6, characterized in that The concentration of exosomes from human corneal limbal microenvironment cells is 1×10 7 P / mL.

8. Use of human limbal microenvironment cell exosomes obtained by the preparation method according to claim 3 in the preparation of eye drops for treating eye diseases.

9. The use according to claim 8, characterized in that The eye diseases include corneal damage repair, relief of visual fatigue, dry eye, limbal stem cell deficiency, and eye lesions caused by graft-versus-host disease.

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