Bioengineering corneal cell culture solution for corneal ulcer repair and preparation process of bioengineering corneal cell culture solution

Abstract

The invention discloses a bioengineering corneal cell culture solution for repairing corneal ulcer and a preparation process of the bioengineering corneal cell culture solution. According to the invention, the addition of various growth factors not only promotes the proliferation and differentiation of corneal cells, but also enhances the signal transduction between cells, thereby accelerating the repair process of corneal ulcer. The epidermal growth factor and the basic fibroblast growth factor mainly promote cell proliferation and migration and provide a new cell source for an ulcer surface; the transforming growth factor-beta regulates the synthesis and remodeling of an extracellular matrix, and is helpful for recovering the normal structure of the cornea; and the corneal cell growth factor specifically promotes the growth of corneal epithelial cells. Through the multi-factor synergistic effect, the ulcer repairing speed is increased, the repairing quality is improved, scar formation is reduced, and a clearer and healthier vision recovery effect is brought to a patient.

Description

Technical Field

[0001] The present invention belongs to the technical field of corneal ulcer repair, and specifically relates to a bioengineered corneal cell culture medium for corneal ulcer repair and its preparation process. Background Art

[0002] Corneal ulcer is a common ophthalmic disease, referring to a localized inflammation and ulcer on the corneal surface. It is usually caused by bacterial, viral, or fungal infections or trauma, and can also be caused by factors such as chronic eye diseases, immune system problems, or malnutrition. Patients mainly present symptoms such as eye pain, tearing, blurred vision, foreign body sensation, and photophobia. The treatment of corneal ulcer requires different measures according to the cause, including local antibiotics, antiviral or antifungal drugs, and severe cases may require surgical treatment. If not treated in time, corneal ulcer may lead to vision impairment or even blindness. Therefore, once relevant symptoms appear, medical treatment should be sought in a timely manner for professional diagnosis and treatment. Corneal ulcer not only seriously affects the patient's vision but may even lead to blindness. In the process of corneal ulcer repair, the proliferation, differentiation, and matrix remodeling of corneal cells play a crucial role. Therefore, the development of a culture medium that can effectively promote the growth and repair of corneal cells has become a research hotspot.

[0003] However, the existing corneal cell culture media only contain one or a few antibacterial components, which cannot comprehensively inhibit the growth of bacteria and fungi, increasing the risk of infection. At the same time, the anti-inflammatory components in the existing culture media may have limited effects and cannot effectively reduce the inflammatory reaction at the ulcer site, resulting in the persistence of pain and edema and affecting the healing process. Summary of the Invention

[0004] The purpose of the present invention is to provide a bioengineered corneal cell culture medium for corneal ulcer repair and its preparation process to solve the above-mentioned problems.

[0005] The technical solution adopted by the present invention is as follows: A bioengineered corneal cell culture medium for corneal ulcer repair, the cell culture medium comprising:

[0006] Advanced DMEM / F12 medium: 50 parts by weight;

[0007] Fetal bovine serum: 10 parts by weight;

[0008] Human serum albumin: 2 parts by weight;

[0009] Epidermal growth factor: 0.00015 - 0.00020 parts by weight;

[0010] Fibroblast growth factor: 0.00015 - 0.00020 parts by weight;

[0011] Transforming growth factor-β: 0.0001 to 0.0005 parts by weight;

[0012] Keratocyte growth factor: 0.00005 to 0.0008 parts by weight;

[0013] Penicillin / streptomycin: 0.1 to 0.5 parts by weight;

[0014] Amphotericin B: 0.0025 to 0.0050 parts by weight;

[0015] Vitamin C: 1 part by weight;

[0016] Calcium ions: 0.4 part by weight

[0017] N-acetyl-L-cysteine: 1.14 parts by weight;

[0018] Dexamethasone: 0.0068 part by weight;

[0019] Prednisolone: 0.0022 part by weight;

[0020] Collagen I: 0.5 part by weight;

[0021] Hyaluronic acid: 0.1 part by weight;

[0022] Heparin: 0.15 part by weight;

[0023] Polyvinylpyrrolidone: 1.5 parts by weight.

[0024] In a preferred embodiment, the preparation process comprises the following steps:

[0025] Comprising the following steps:

[0026] S1: Take an appropriate amount of Advanced DMEM / F12 medium and place it in a sterile container as the basis of the culture solution; S2: Add fetal bovine serum to the basic medium to provide necessary growth factors and nutrients;

[0027] S3: Add human serum albumin to enhance the nutritional components of the culture solution and promote cell growth;

[0028] S4: Sequentially add epidermal growth factor, fibroblast growth factor, transforming growth factor-β and keratocyte growth factor to promote cell proliferation, differentiation and matrix remodeling;

[0029] S5: Add penicillin / streptomycin and amphotericin B to prevent bacterial and fungal contamination and protect the cell growth environment;

[0030] S6: Add vitamin C and calcium ions to provide necessary vitamins and minerals and support the normal physiological functions of cells;

[0031] S7: Add N-acetyl-L-cysteine, dexamethasone and prednisolone to regulate the cell growth environment and reduce the inflammatory response;

[0032] S8: Finally, add collagen I, hyaluronic acid, heparin, and polyvinylpyrrolidone to provide extracellular matrix components to support cell attachment and growth;

[0033] S9: Preserve the prepared culture medium to obtain the bioengineered corneal cell culture medium for corneal ulcer repair, thus completing the entire preparation process.

[0034] In a preferred embodiment, in step S1, ensure that the temperature of the culture medium is room temperature of 20 - 25 °C to avoid the influence of too low or too high temperature on the stability and solubility of the culture medium; before use, check the expiration date and appearance of the culture medium to ensure no precipitation and no color change, meeting the usage standards.

[0035] In a preferred embodiment, in step S2, the serum needs to be taken out from the low-temperature storage condition and slowly thawed at room temperature before use to avoid repeated freezing and thawing; when adding the serum, use a sterile pipette and slowly add it along the wall of the container to reduce the generation of bubbles, and gently stir until completely mixed.

[0036] In a preferred embodiment, in step S3, human serum albumin is pre-dissolved in sterile water to prepare a solution with an appropriate concentration; during dissolution, gently stir in a 37 °C water bath to accelerate dissolution; the dissolved HSA solution is filtered and sterilized through a 0.22-micron sterile filter membrane, and then slowly added to the culture medium while stirring to ensure uniform distribution.

[0037] In a preferred embodiment, in step S4, the growth factor is pre-dissolved in sterile phosphate buffer to prepare a high-concentration stock solution, which is aliquoted and stored at -80 °C; when in use, take an appropriate amount of the stock solution and dilute it to the working concentration, filter it through a sterile filter membrane, and then add it to the culture medium in sequence. Gently stir after adding each growth factor to ensure thorough mixing.

[0038] In a preferred embodiment, in step S5, penicillin / streptomycin is provided in the form of a 100-fold concentrated solution and needs to be diluted proportionally before use; amphotericin B is dissolved in sterile distilled water to prepare a stock solution, which is aliquoted and stored at 2 - 8 °C; when adding, use a sterile pipette and slowly add it to the culture medium while stirring to ensure uniform distribution.

[0039] In a preferred embodiment, in step S6, vitamin C is pre-dissolved in sterile water to prepare a solution to avoid prolonged exposure to air to prevent oxidation; calcium ions are provided in the form of CaCl2 and directly added to the culture medium and stirred until completely dissolved; ensure that vitamin C and CaCl2 are at room temperature before addition to avoid temperature changes affecting the stability of the culture medium.

[0040] In a preferred embodiment, in step S7, N-acetyl-L-cysteine ​​is dissolved in sterile water to prepare a solution, which is filtered through a sterile filter membrane before being added; ensuring that all additives are at room temperature before being added to maintain the homogeneity of the culture medium.

[0041] In a preferred embodiment, in step S8, collagen I is pre-dissolved in a sterile acetic acid solution, hyaluronic acid and heparin are dissolved in sterile water, and PVP can be directly added to the culture medium; after each component is added, it is necessary to fully stir to ensure complete dissolution and uniform distribution; the final culture medium is filtered and sterilized through a 0.22-micron sterile filter membrane, and stored at 2-8°C after packaging to avoid light and repeated freezing and thawing.

[0042] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0043] 1. In the present invention, the addition of multiple growth factors not only promotes the proliferation and differentiation of corneal cells, but also enhances the signal transduction between cells, thereby accelerating the repair process of corneal ulcers. Epidermal growth factor and basic fibroblast growth factor mainly promote cell proliferation and migration, providing a new source of cells for the ulcer surface; transforming growth factor-β regulates the synthesis and remodeling of the extracellular matrix, which helps to restore the normal structure of the cornea; and corneal cell growth factor specifically promotes the growth of corneal epithelial cells. This multi-factor synergistic effect not only increases the speed of ulcer repair, but also improves the quality of repair, reduces scar formation, and brings patients a clearer and healthier vision recovery effect.

[0044] 2. In the present invention, the antibacterial and anti-inflammatory ingredients penicillin / streptomycin, amphotericin B, dexamethasone and prednisolone are combined. The effective combination of these ingredients not only provides a sterile growth environment for corneal cells, but also reduces the inflammatory response at the ulcer site through anti-inflammatory effects, reducing the risk of infection. Penicillin / streptomycin and amphotericin B effectively inhibit the growth of bacteria and fungi and prevent infection complications; while dexamethasone and prednisolone, as powerful anti-inflammatory drugs, can reduce inflammatory edema at the ulcer site, relieve pain, and promote healing. Therefore, the culture fluid not only improves the safety of the culture fluid, but also significantly improves the therapeutic effect of corneal ulcers, shortens the recovery time, and reduces the pain and economic burden of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the flowchart of the method of the present invention. Detailed implementation mode

[0046] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0047] Example 1:

[0048] Refer to Figure 1 ,

[0049] A bioengineered corneal cell culture medium for corneal ulcer repair, which includes:

[0050] Advanced DMEM / F12 medium: 50 parts by weight;

[0051] Fetal bovine serum (FBS, charcoal stripped): 10 parts by weight;

[0052] Human serum albumin (HSA): 2 parts by weight;

[0053] Epidermal growth factor (EGF): 0.00015 parts by weight;

[0054] Fibroblast growth factor (bFGF): 0.00015 parts by weight;

[0055] Transforming growth factor-β (TGF-β): 0.0001 parts by weight;

[0056] Keratinocyte growth factor (KGF): 0.00005 parts by weight;

[0057] Penicillin / streptomycin: 0.1 parts by weight;

[0058] Amphotericin B: 0.0025 parts by weight;

[0059] Vitamin C (ascorbic acid phosphate): 1 part by weight;

[0060] Calcium ions: 0.4 parts by weight (in the form of CaCl2)

[0061] N-acetyl-L-cysteine (NAC): 1.14 parts by weight;

[0062] Dexamethasone: 0.0068 parts by weight;

[0063] Prednisolone: 0.0022 parts by weight;

[0064] Collagen I: 0.5 parts by weight;

[0065] Hyaluronic acid: 0.1 part by weight;

[0066] Heparin: 0.15 part by weight;

[0067] Polyvinylpyrrolidone (PVP): 1.5 parts by weight.

[0068] The preparation process includes the following steps:

[0069] Including the following steps:

[0070] S1: Take an appropriate amount of Advanced DMEM / F12 medium and place it in a sterile container as the basis of the culture solution;

[0071] S2: Add fetal bovine serum (FBS, charcoal stripped) to the basic medium to provide necessary growth factors and nutrients;

[0072] S3: Add human serum albumin (HSA) to enhance the nutritional components of the culture solution and promote cell growth

[0073] S4: Sequentially add epidermal growth factor (EGF), fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), and keratinocyte growth factor (KGF) to promote cell proliferation, differentiation, and matrix remodeling

[0074] S5: Add penicillin / streptomycin and amphotericin B to prevent bacterial and fungal contamination and protect the cell growth environment

[0075] S6: Add vitamin C (ascorbic acid phosphate) and calcium ions (in the form of CaCl2) to provide necessary vitamins and minerals and support the normal physiological functions of cells

[0076] S7: Add N-acetyl-L-cysteine (NAC), dexamethasone, and prednisolone to regulate the cell growth environment and reduce inflammatory reactions

[0077] S8: Finally, add collagen I, hyaluronic acid, heparin, and polyvinylpyrrolidone (PVP) to provide extracellular matrix components and support cell attachment and growth

[0078] S9: Preserve the prepared culture solution to obtain the bioengineered corneal cell culture solution for corneal ulcer repair, and complete the entire preparation process.

[0079] In step S1, ensure that the temperature of the medium is room temperature 20 - 25 °C to avoid the influence of too low or too high temperature on the stability and solubility of the medium. Before use, check the expiration date and appearance of the medium to ensure no precipitation and no color change, meeting the usage standards.

[0080] In step S2, before use, the serum needs to be taken out from the low-temperature storage conditions (such as -20°C or -80°C), placed at room temperature and slowly thawed to avoid repeated freezing and thawing. When adding the serum, use a sterile pipette and slowly add it along the wall of the container to reduce the generation of bubbles, and gently stir until completely mixed.

[0081] In step S3, human serum albumin should be pre-dissolved in sterile water to prepare a solution with an appropriate concentration. During dissolution, gently stir in a 37°C water bath to accelerate dissolution. The dissolved HSA solution should be filtered through a 0.22-micron sterile filter membrane to remove bacteria, and then slowly added to the medium while stirring to ensure uniform distribution.

[0082] In step S4, growth factors should be pre-dissolved in sterile phosphate-buffered saline (PBS) to prepare a high-concentration stock solution, which is aliquoted and stored at -80°C. When in use, take an appropriate amount of the stock solution and dilute it to the working concentration. After filtering through a sterile filter membrane, add it to the medium in sequence. Gently stir after adding each growth factor to ensure thorough mixing.

[0083] In step S5, penicillin / streptomycin is usually provided in a 100-fold concentrated solution and needs to be diluted proportionally before use. Amphotericin B should be dissolved in sterile distilled water to prepare a stock solution, which is aliquoted and stored at 2 - 8°C. When adding, use a sterile pipette and slowly add it to the medium while stirring to ensure uniform distribution.

[0084] In step S6, vitamin C should be pre-dissolved in sterile water to prepare a solution, and avoid long-term exposure to air to prevent oxidation. Calcium ions are provided in the form of CaCl2 and are directly added to the medium and stirred until completely dissolved. Ensure that both vitamin C and CaCl2 are at room temperature before adding to avoid temperature changes affecting the stability of the medium.

[0085] In step S7, N-acetyl-L-cysteine is dissolved in sterile water to prepare a solution, which is added after filtering through a sterile filter membrane. Ensure that all additives are at room temperature before adding to maintain the homogeneity of the medium.

[0086] In step S8, collagen I should be pre-dissolved in sterile acetic acid solution, hyaluronic acid and heparin should be dissolved in sterile water, and PVP can be directly added to the medium. After adding each component, it is necessary to stir thoroughly to ensure complete dissolution and uniform distribution. The final culture medium should be filtered through a 0.22-micron sterile filter membrane to remove bacteria, aliquoted and stored at 2 - 8°C, avoiding light and repeated freezing and thawing.

[0087] As can be seen from the above: In the present invention, the addition of multiple growth factors not only promotes the proliferation and differentiation of corneal cells, but also enhances the signal transduction between cells, thereby accelerating the repair process of corneal ulcers. Epidermal growth factor and basic fibroblast growth factor mainly promote cell proliferation and migration, providing a new cell source for the ulcer surface; transforming growth factor-β regulates the synthesis and remodeling of the extracellular matrix, contributing to the restoration of the normal structure of the cornea; corneal cell growth factor specifically promotes the growth of corneal epithelial cells. This synergistic effect of multiple factors not only increases the speed of ulcer repair, but also improves the repair quality, reduces scar formation, and brings clearer and healthier vision recovery for patients.

[0088] In the present invention, antibacterial and anti-inflammatory components are combined, such as penicillin / streptomycin, amphotericin B, dexamethasone, and prednisolone. The effective combination of these components not only provides a sterile growth environment for corneal cells, but also reduces the inflammatory reaction at the ulcer site through anti-inflammatory effects, reducing the risk of infection. Penicillin / streptomycin and amphotericin B effectively inhibit the growth of bacteria and fungi, preventing infection complications; while dexamethasone and prednisolone, as potent anti-inflammatory drugs, can reduce the inflammatory edema at the ulcer site, relieve pain, and promote healing. Thus, this culture medium not only improves the safety of the culture medium, but also significantly enhances the treatment effect of corneal ulcers, shortens the recovery time, and reduces the pain and economic burden of patients.

[0089] Example Two:

[0090] Refer to Figure 1 ,

[0091] A bioengineered corneal cell culture medium for corneal ulcer repair, comprising:

[0092] Advanced DMEM / F12 medium: 50 parts by weight;

[0093] Fetal bovine serum (FBS, charcoal stripped): 10 parts by weight;

[0094] Human serum albumin (HSA): 2 parts by weight;

[0095] Epidermal growth factor (EGF): 0.00020 parts by weight;

[0096] Fibroblast growth factor (bFGF): 0.00020 parts by weight;

[0097] Transforming growth factor-β (TGF-β): 0.0005 parts by weight;

[0098] Corneal cell growth factor (KGF): 0.0008 parts by weight;

[0099] Penicillin / streptomycin: 0.5 parts by weight;

[0100] Amphotericin B: 0.0050 parts by weight;

[0101] Vitamin C (ascorbic acid phosphate): 1 part by weight;

[0102] Calcium ions: 0.4 parts by weight (in the form of CaCl2)

[0103] N-acetyl-L-cysteine (NAC): 1.14 parts by weight;

[0104] Dexamethasone: 0.0068 parts by weight;

[0105] Prednisolone: 0.0022 parts by weight;

[0106] Collagen I: 0.5 parts by weight;

[0107] Hyaluronic acid: 0.1 parts by weight;

[0108] Heparin: 0.15 parts by weight;

[0109] Polyvinylpyrrolidone (PVP): 1.5 parts by weight.

[0110] The preparation process includes the following steps:

[0111] Including the following steps:

[0112] S1: Take an appropriate amount of Advanced DMEM / F12 medium and place it in a sterile container as the basis of the culture solution;

[0113] S2: Add fetal bovine serum (FBS, charcoal stripped) to the basic medium to provide necessary growth factors and nutrients;

[0114] S3: Add human serum albumin (HSA) to enhance the nutritional components of the culture solution and promote cell growth

[0115] S4: Sequentially add epidermal growth factor (EGF), fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β) and keratinocyte growth factor (KGF) to promote cell proliferation, differentiation and matrix remodeling

[0116] S5: Add penicillin / streptomycin and amphotericin B to prevent bacterial and fungal contamination and protect the cell growth environment

[0117] S6: Add vitamin C (ascorbic acid phosphate) and calcium ions (in the form of CaCl2) to provide necessary vitamins and minerals to support the normal physiological functions of cells

[0118] S7: Add N-acetyl-L-cysteine (NAC), dexamethasone and prednisolone to regulate the cell growth environment and reduce the inflammatory response.

[0119] S8: Finally, add collagen I, hyaluronic acid and heparin, as well as polyvinylpyrrolidone (PVP) to provide extracellular matrix components and support cell attachment and growth.

[0120] S9: Preserve the prepared culture medium to obtain the bioengineered corneal cell culture medium for corneal ulcer repair, thus completing the entire preparation process.

[0121] In step S1, ensure that the temperature of the culture medium is at room temperature of 20 - 25 °C to avoid the influence of too low or too high temperature on the stability and solubility of the culture medium. Before use, check the expiration date and appearance of the culture medium to ensure no precipitation and no color change, meeting the usage standards.

[0122] In step S2, the serum needs to be taken out from the low-temperature storage conditions (such as -20 °C or -80 °C) before use and slowly thawed at room temperature to avoid repeated freezing and thawing. When adding the serum, use a sterile pipette and slowly add it along the wall of the container to reduce the generation of bubbles, and gently stir until completely mixed.

[0123] In step S3, human serum albumin should be pre-dissolved in sterile water to prepare a solution with an appropriate concentration. During dissolution, gently stir in a 37 °C water bath to accelerate dissolution. The dissolved HSA solution should be filtered and sterilized through a 0.22-micron sterile filter membrane, and then slowly added to the culture medium while stirring to ensure uniform distribution.

[0124] In step S4, the growth factor should be pre-dissolved in sterile phosphate buffer (PBS) to prepare a high-concentration stock solution, which is aliquoted and stored at -80 °C. When in use, take an appropriate amount of the stock solution and dilute it to the working concentration, filter it through a sterile filter membrane, and then add it to the culture medium in sequence. Gently stir after adding each growth factor to ensure thorough mixing.

[0125] In step S5, penicillin / streptomycin is usually provided in a 100-fold concentrated solution and needs to be diluted proportionally before use. Amphotericin B should be dissolved in sterile distilled water to prepare a stock solution, which is aliquoted and stored at 2 - 8 °C. When adding, use a sterile pipette and slowly add it to the culture medium while stirring to ensure uniform distribution.

[0126] In step S6, vitamin C should be dissolved in sterile water in advance and prepared into a solution to avoid long-term exposure to air to prevent oxidation. Calcium ions are provided in the form of CaCl2, directly added to the culture medium, and stirred until completely dissolved. Ensure that vitamin C and CaCl2 are both at room temperature before addition to avoid temperature changes affecting the stability of the culture medium.

[0127] In step S7, N-acetyl-L-cysteine ​​is dissolved in sterile water to prepare a solution, which is filtered through a sterile filter membrane before being added. Ensure that all additives are at room temperature before addition to maintain the homogeneity of the culture medium.

[0128] In step S8, collagen I should be pre-dissolved in a sterile acetic acid solution, hyaluronic acid and heparin should be dissolved in sterile water, and PVP can be directly added to the culture medium. After each component is added, it must be fully stirred to ensure complete dissolution and uniform distribution. The final culture solution should be sterilized by filtering through a 0.22 micron sterile filter membrane, and stored at 2-8°C after aliquoting to avoid light and repeated freezing and thawing.

[0129] From the above, it can be known that in the present invention, the addition of multiple growth factors not only promotes the proliferation and differentiation of corneal cells, but also enhances the signal transduction between cells, thereby accelerating the repair process of corneal ulcers. Epidermal growth factor and basic fibroblast growth factor mainly promote cell proliferation and migration, providing a new source of cells for the ulcer surface; transforming growth factor-β regulates the synthesis and remodeling of the extracellular matrix, which helps to restore the normal structure of the cornea; and corneal cell growth factor specifically promotes the growth of corneal epithelial cells. This multi-factor synergistic effect not only increases the speed of ulcer repair, but also improves the quality of repair, reduces scar formation, and brings patients a clearer and healthier vision recovery effect.

[0130] In the present invention, antibacterial and anti-inflammatory ingredients such as penicillin / streptomycin, amphotericin B, dexamethasone and prednisolone are combined. The effective combination of these ingredients not only provides a sterile growth environment for corneal cells, but also reduces the inflammatory response at the ulcer site through anti-inflammatory effects, thereby reducing the risk of infection. Penicillin / streptomycin and amphotericin B effectively inhibit the growth of bacteria and fungi and prevent infection complications; while dexamethasone and prednisolone, as potent anti-inflammatory drugs, can reduce inflammatory edema at the ulcer site, relieve pain, and promote healing. Thus, the culture fluid not only improves the safety of the culture fluid, but also significantly improves the therapeutic effect of corneal ulcers, shortens the recovery time, and reduces the pain and economic burden of patients.

[0131] It should be noted that in this text, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article, or device comprising the said element.

[0132] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A bioengineered corneal cell culture solution for corneal ulcer repair, characterized in that: The cell culture fluid comprises: Advanced DMEM / F12 medium: 50 parts by weight; Fetal bovine serum: 10 parts by weight; Human serum albumin: 2 parts by weight; Epidermal growth factor: 0.00015-0.00020 parts by weight; Fibroblast growth factor: 0.00015-0.00020 parts by weight; Transforming growth factor-β: 0.0001-0.0005 parts by weight; Corneal cell growth factor: 0.00005-0.0008 parts by weight; Penicillin / streptomycin: 0.1-0.5 parts by weight; Amphotericin B: 0.0025-0.0050 parts by weight; Vitamin C: 1 part by weight; Calcium ion: 0.4 parts by weight N-acetyl-L-cysteine: 1.14 parts by weight; Dexamethasone: 0.0068 parts by weight; Prednisolone: ​​0.0022 parts by weight; Collagen I: 0.5 parts by weight; Hyaluronic acid: 0.1 parts by weight; Heparin: 0.15 parts by weight; Polyvinyl pyrrolidone: 1.5 parts by weight.

2. The process for preparing a bioengineered corneal cell culture solution for corneal ulcer repair according to claim 1, characterized in that: The preparation process The following steps are involved: The following steps are involved: S1: Take an appropriate amount of Advanced DMEM / F12 medium and place it in a sterile container as the basis of the culture medium; S2: adding fetal bovine serum to the basal medium to provide necessary growth factors and nutrients; S3: adding human serum albumin to enhance the nutrient content of the culture medium and promote cell growth; S4: epidermal growth factor, fibroblast growth factor, transforming growth factor-β, and keratocyte growth factor were added in sequence to promote cell proliferation, differentiation, and matrix remodeling; S5: Add penicillin / streptomycin and amphotericin B to prevent bacterial and fungal contamination and protect the cell growth environment; S6: Add vitamin C and calcium ions to provide necessary vitamins and minerals to support normal physiological functions of cells; S7: adding N-acetyl-L-cysteine, dexamethasone and prednisolone to adjust the cell growth environment and reduce inflammatory response; S8: Finally, collagen I, hyaluronic acid and heparin, as well as polyvinylpyrrolidone, are added to provide extracellular matrix components to support cell attachment and growth; S9: The prepared culture medium is preserved to obtain the bioengineered corneal cell culture medium for corneal ulcer repair, thus completing the entire preparation process.

3. The process for preparing a bioengineered corneal cell culture solution for corneal ulcer repair according to claim 1, characterized in that: In step S1, ensure that the temperature of the culture medium is room temperature 20-25°C to avoid the influence of too low or too high temperature on the stability and solubility of the culture medium; before use, check the validity period and appearance of the culture medium to ensure that there is no precipitation, no color change, and it meets the use standards.

4. The process for preparing a bioengineered corneal cell culture solution for corneal ulcer repair according to claim 1, characterized in that: In step S2, the serum needs to be taken out from the low-temperature storage condition before use and slowly thawed at room temperature to avoid repeated freezing and thawing; when adding the serum, use a sterile pipette to slowly add it along the wall of the container to reduce the generation of bubbles, and gently stir until completely mixed.

5. The process for preparing a bioengineered corneal cell culture solution for corneal ulcer repair according to claim 1, characterized in that: In step S3, human serum albumin is pre-dissolved in sterile water to prepare a solution of appropriate concentration; during dissolution, it is gently stirred in a 37° C. water bath to accelerate dissolution; the dissolved HSA solution is sterilized by filtering through a 0.22 μm sterile filter membrane, and then slowly added to the culture medium while stirring to ensure uniform distribution.

6. The process for preparing a bioengineered corneal cell culture solution for corneal ulcer repair according to claim 1, characterized in that: In step S4, the growth factors are pre-dissolved in sterile phosphate buffer to prepare a high-concentration storage solution, which is stored at -80°C after being packaged; when used, an appropriate amount of the storage solution is diluted to a working concentration, filtered through a sterile filter membrane, and added to the culture medium in sequence, and gently stirred after each growth factor is added to ensure sufficient mixing.

7. The process for preparing a bioengineered corneal cell culture solution for corneal ulcer repair according to claim 1, characterized in that: In step S5, penicillin / streptomycin is provided in the form of a 100-fold concentrated solution, which needs to be diluted proportionally before use; amphotericin B is dissolved in sterile distilled water to prepare a storage solution, which is stored at 2-8° C. after being divided and packaged; when adding, use a sterile pipette to slowly add it to the culture medium, stirring while adding to ensure uniform distribution.

8. The process for preparing a bioengineered corneal cell culture solution for corneal ulcer repair according to claim 1, characterized in that: In step S6, vitamin C is pre-dissolved in sterile water to prepare a solution to avoid long-term exposure to air to prevent oxidation; calcium ions are provided in the form of CaCl2 and directly added to the culture medium and stirred until completely dissolved; it is ensured that vitamin C and CaCl2 are both at room temperature before addition to avoid temperature changes affecting the stability of the culture medium.

9. The process for preparing a bioengineered corneal cell culture solution for corneal ulcer repair according to claim 1, characterized in that: In step S7, N-acetyl-L-cysteine ​​is dissolved in sterile water to prepare a solution, which is filtered through a sterile filter membrane before being added; ensuring that all additives are at room temperature before being added to maintain the homogeneity of the culture medium.

10. The process for preparing a bioengineered corneal cell culture solution for corneal ulcer repair according to claim 1, characterized in that: In step S8, collagen I is pre-dissolved in a sterile acetic acid solution, hyaluronic acid and heparin are dissolved in sterile water, and PVP can be directly added to the culture medium; after each component is added, it needs to be fully stirred to ensure complete dissolution and uniform distribution; the final culture medium is filtered and sterilized through a 0.22-micron sterile filter membrane, and stored at 2-8°C after packaging to avoid light and repeated freezing and thawing.

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