Construction method of tissue engineering corneal epithelial cells

Abstract

The invention relates to a construction method of tissue engineering corneal epithelial cells. The construction method comprises the following steps: obtaining corneal limbus tissue blocks; digesting the tissue block, and separating to obtain epithelial cells of the tissue block; the method comprises the following steps: attaching epithelial cells to ultrathin amniotic membrane-umbilical cord derived mesenchymal stem cells, culturing in an SHEM culture medium, changing the SHEM culture medium once every two days, culturing for a period of time, and culturing the epithelial cells for a period of time by using a gas-liquid interface culture method to promote the multi-layer growth of the epithelial cells, thereby obtaining the tissue engineering corneal epithelial cells. Through a controllable preparation scheme, the amniotic membrane carrier with a certain thickness is obtained so as to increase the transparency of the amniotic membrane carrier, meanwhile, the matrix surface of the amniotic membrane carrier is inoculated with MSC, the healthy corneal limbus matrix microenvironment of a human body is simulated, damaged corneal limbus tissue / corneal epithelium is better repaired or regenerated, and the ocular surface microenvironment is improved. The produced ultrathin amnion combined mesenchymal stem cell carrier can obviously improve the effect of a tissue engineering corneal epithelium transplantation operation.

Description

Technical Field

[0001] The present invention relates to the technical field of corneal cell culture, and in particular to a method for constructing tissue-engineered corneal epithelial cells. Background Art

[0002] Limbal stem cells renew and replenish corneal epithelial cells. A certain number of these cells plays a crucial role in maintaining corneal epithelial homeostasis and transparency. Limbal stem cell dysfunction (LSCD) is caused by a deficiency in stem cell numbers or loss of their differentiation potential. It often manifests as extensive destruction of the ocular surface tissue, leading to persistent corneal epithelial defects, chronic inflammation, conjunctivalization of the corneal epithelium, and neovascularization. This leads to decreased corneal transparency, resulting in severe visual impairment and even blindness. The incidence of LSCD is increasing due to ocular chemical injuries, thermal burns, ocular cicatricial pemphigoid, and severe dry eye. Currently, corneal stem cell deficiency is primarily treated by transplanting healthy limbal stem cells from the unaffected eye and transplanting tissue-engineered corneal epithelium. However, patients with complete bilateral corneal stem cell deficiency can only undergo allogeneic transplantation, which may result in a moderate immune response and ultimately leads to surgical failure. Therefore, transplantation of tissue-engineered corneal epithelial stem cells is an effective treatment for corneal epithelial stem cell deficiency.

[0003] The choice of carrier is a key factor in the construction of tissue-engineered corneal epithelium, as the maintenance of stem cell phenotype, the determination of differentiation direction, and the proliferation and apoptosis of stem cells are closely related to the microenvironment in which the stem cells reside. However, to date, there is no ideal tissue-engineered corneal epithelial carrier that can mimic the limbal microenvironment, resulting in low stem cell expansion efficiency and a high risk of failure after transplantation. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a method for constructing tissue-engineered corneal epithelial cells.

[0005] To achieve the above-mentioned object of the invention, the present invention provides a method for constructing tissue-engineered corneal epithelial cells, comprising:

[0006] Obtain limbal tissue blocks;

[0007] The tissue blocks were digested in SHEM medium containing Dispase II to isolate and obtain epithelial cells from the tissue blocks;

[0008] Epithelial cells are attached to ultra-thin amniotic membrane-umbilical cord-derived mesenchymal stem cells and cultured in SHEM medium. The SHEM medium is changed every two days. After a period of culture, the epithelial cells are cultured for a period of time using the air-liquid interface culture method to promote the growth of epithelial cells in multiple layers, thereby obtaining tissue-engineered corneal epithelial cells.

[0009] In one embodiment, the limbal tissue is cut into 2 mm×2 mm tissue blocks, and then the tissue blocks are incubated in SHEM medium containing 2 mg / ml Dispase II at 4° C. for 14 h to separate and obtain epithelial cells from the tissue blocks.

[0010] In one embodiment, a method for preparing ultrathin amniotic membrane-umbilical cord-derived mesenchymal stem cells comprises:

[0011] Umbilical cord-derived mesenchymal stem cells are inoculated on the reverse side of the ultra-thin amniotic membrane to form ultra-thin amniotic membrane-umbilical cord-derived mesenchymal stem cells.

[0012] In one embodiment, a method for preparing umbilical cord-derived mesenchymal stem cells comprises:

[0013] The umbilical cord was cleaned under sterile conditions, the arteries and veins were stripped, the Warburg jelly was separated, and the tissue blocks were cut into 1×1 mm pieces. After digestion with type IV collagenase overnight, the cells were cultured in DMEM medium to obtain umbilical cord-derived mesenchymal stem cells.

[0014] In one embodiment, a method for preparing an ultrathin amniotic membrane comprises:

[0015] The amniotic membrane was separated from the placenta and rinsed with HBSS buffer until there was no blood stain;

[0016] The amniotic membrane is spread flat on the amniotic ring, and after digestion and removal of the amniotic epithelium, the de-epithelialized amniotic membrane is obtained; then a sterilized cylindrical nanosponge is immersed in 1.5 mg / mL type IV collagenase and placed under the amniotic ring. The circular surface of the nanosponge is tightly attached to the matrix surface of the de-epithelialized amniotic membrane. After digestion, the ultra-thin amniotic membrane is obtained.

[0017] In one embodiment, the amniotic epithelium is removed using 0.02% EDTA digestion.

[0018] In one embodiment, the circular surface of the nanosponge is closely attached to the stromal surface of the de-epithelialized amniotic membrane, and after digestion at 37° C. for 75-90 minutes, the ultra-thin amniotic membrane is obtained.

[0019] In one embodiment, the cells are cultured in SHEM medium, which is changed every two days. After 8-10 days of culture, the epithelial cells are cultured using an air-liquid interface method for 3 days to promote the multilayer growth of the epithelial cells and obtain tissue-engineered corneal epithelial cells.

[0020] The advantages and beneficial effects of the present invention over the prior art are:

[0021] 1. Amniotic membrane (AM) is widely used in reconstructive surgery as a carrier of corneal epithelial tissue. Ultrathin amniotic membrane (UAM) is made by using collagenase IV to peel off some stroma from the de-epithelialized amniotic membrane (DAM), thinning the matrix to about 30 μm, and its wet and dry forms become cell-free and optically transparent, and its collagen skeleton becomes dense and regular. In order to better simulate the limbal stem cell microenvironment, the present invention, based on UAM, inoculates umbilical cord-derived mesenchymal stem cells (MSCs) on the reverse side of UAM (UAM-MSCs). The engineered rabbit corneal epithelial cell (RCEC) sheet generated by amplifying corneal epithelial cells on UAM-MSCs has good transparency and thickness. Moreover, the expression of progenitor cell (K14, PAX6, ABCG2) genes in tissue-engineered cell sheets amplified by UAM-MSCs is higher than that in tissue-engineered cell sheets amplified by UAM and DAM. Furthermore, two weeks after surgery, corneas implanted with UAM-MSCs and UAM-derived cell sheets exhibited greater transparency and more stratified epithelium than corneas implanted with DAM-derived cell sheets. Connexin 43 gene expression was higher in corneas implanted with UAM-MSCs-derived cell sheets than in corneas implanted with UAM- and DAM-derived cell sheets. Electron microscopy revealed tighter junctions between corneal epithelial cells in corneas implanted with UAM-MSCs-derived cell sheets. Furthermore, expression of anti-angiogenic proteins (TIMP-1 and thrombospondin-1) was higher in corneas implanted with UAM-MSCs-derived cell sheets than in corneas implanted with UAM- and DAM-derived cell sheets. In summary, tissue-engineered corneal epithelium cultured with UAM-MSCs exhibits superior efficacy because the transplanted tissue is more transparent and more similar to the phenotype of native tissue than tissue obtained with DAM. MSCs possess potent immunomodulatory, proliferation, and paracrine functions, as well as the ability to differentiate into mesodermal and non-mesodermal cell lineages, better simulating the ocular surface microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Schematic diagram of the process of in vitro expansion of limbal stem cells on the surface of DAM, UAM and UAM-MSCs; Figure 1 Figure A in the middle shows the expansion process of limbal stem cells on the epithelial surface of three carriers. Figure 1Middle Figure B shows the expansion process of mesenchymal stem cells on the UAM-MSCs matrix surface. Figure 1 Figure C in the middle shows the transparency comparison of three carriers;

[0024] Figure 2 The H&E staining results of tissue-engineered corneal epithelial sheets constructed on the surfaces of DAM, UAM, and UAM-MSCs are shown. The scale bar in the figure is 25 μm.

[0025] Figure 3 To observe the cell phenotype of tissue-engineered corneal epithelial sheets constructed on the surface of DAM, UAM and UAM-MSCs; Figure 3 Middle panel A shows the immunofluorescence staining results of corneal epithelial cell proliferation and differentiation-related markers K12, K14, and PAX6; Figure 3 Middle panel B shows the protein expression levels of K12, K14, and PAX6; Figure 3 Middle panel C shows the RNA expression levels of K12, K14, PAX6, and ABCG2;

[0026] Figure 4 Results of three tissue-engineered corneal epithelial sheet transplantations in the treatment of limbal stem cell injury models; Figure 4 Middle Figure A shows slit lamp photography of three tissue-engineered corneal epithelial cells transplanted into animal models for the treatment of limbal stem cell deficiency. Figure 3 Middle Figure B is the corneal epithelial healing curve;

[0027] Figure 5 The CFDA SE traced corneal epithelial stem cells before and two weeks after three types of tissue-engineered corneal epithelial sheet transplantation.

[0028] Figure 6 The results of H&E staining of frozen sections of rabbit cornea after three types of tissue-engineered corneal epithelial sheet transplantation to treat the corneal limbal stem cell injury model;

[0029] Figure 7 The cell phenotypes of rabbit corneal epithelial cells after three types of tissue-engineered corneal epithelial sheet transplantation; DAPI blue nuclear staining. Figure 7 Figure A shows the immunofluorescence staining results of corneal epithelial cell proliferation and differentiation-related markers K12, K14, and PAX6; Figure 7 Figure B shows the RNA expression levels of K3, ABCG2, and PAX6;

[0030] Figure 8 To investigate the connection between corneal epithelial cells after transplantation of three tissue-engineered corneal epithelial sheets to treat LSCD models; Figure 8 Middle Figure A shows the results of transmission electron microscopy; Figure 8 Middle panel B shows the RNA expression level of Connexin 43, a cell junction-related marker;

[0031] Figure 9 The RNA expression levels of anti-angiogenic factors TIMP-1 and Thrombospondin-1 in the cornea after three tissue-engineered corneal epithelial sheet transplantations in the treatment of LSCD models were compared. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] Materials preparation:

[0034] Animals: Male New Zealand white rabbits, weighing 2–2.5 kg and aged 3–4 months, were provided by the Animal Center of Guizhou Medical University (Guizhou, China). All animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. This study was approved by the Animal Ethics Committee of Guizhou Medical University (NO.2000928).

[0035] Human placentas were obtained from healthy donors at the time of cesarean section in accordance with the tenets of the Declaration of Helsinki for use in human research and were approved by the Institutional Review Board of the Affiliated Hospital of Guizhou Medical University (Guiyang, China). Informed consent was obtained from the donors.

[0036] Human umbilical cords were obtained from healthy donors at the time of cesarean section in accordance with the tenets of the Declaration of Helsinki for use in human research and were approved by the Institutional Review Board of the Affiliated Hospital of Guizhou Medical University (Guiyang, China). Informed consent was obtained from the donors.

[0037] Example 1

[0038] A method for constructing tissue-engineered corneal epithelial cells, comprising:

[0039] The amniotic membrane was separated from the placenta and rinsed with HBSS buffer until no blood stains were found; the amniotic membrane was spread flat on an amniotic ring and digested with 0.02% EDTA to remove the amniotic epithelium, thereby obtaining de-epithelialized amniotic membrane;

[0040] New Zealand white rabbits were sacrificed, the central corneal tissue was removed, and the remaining limbal tissue was cut into 2 mm × 2 mm tissue blocks. The tissue blocks were then incubated in SHEM medium containing 2 mg / ml Dispase II at 4°C for 14 h, and the epithelial cells were isolated and obtained from the tissue blocks. The epithelial cells were attached to de-epithelialized amniotic membrane (DAM) and cultured in SHEM medium. The SHEM medium was changed every two days. After 10 days of culture, the epithelial cells were cultured using the air-liquid interface method for 3 days to promote the growth of epithelial cells in multiple layers, thereby obtaining tissue-engineered corneal epithelial cells.

[0041] All the above steps are carried out under sterile conditions to prevent microbial contamination during the preparation process.

[0042] Example 2

[0043] A method for constructing tissue-engineered corneal epithelial cells, comprising:

[0044] The amniotic membrane was separated from the placenta and rinsed with HBSS buffer until free of blood. The amniotic membrane was spread flat on an amniotic ring and digested with 0.02% EDTA to remove the amniotic epithelium, thereby obtaining de-epithelialized amniotic membrane. A sterilized cylindrical nanosponge soaked in 1.5 mg / mL type IV collagenase was then placed under the amniotic ring, with the circular surface of the nanosponge pressed against the matrix surface of the de-epithelialized amniotic membrane. The membrane was digested at 37°C for 75 minutes to remove the loose collagen components in the amniotic matrix while retaining the dense portion, thereby obtaining ultra-thin amniotic membrane.

[0045] New Zealand white rabbits were sacrificed, the central corneal tissue was removed, and the remaining limbal tissue was cut into 2 mm × 2 mm tissue blocks. The tissue blocks were then incubated in SHEM medium containing 2 mg / ml Dispase II at 4°C for 14 hours to isolate and obtain epithelial cells from the tissue blocks. The epithelial cells were attached to an ultrathin amniotic membrane (UAM) and cultured in SHEM medium, which was changed every two days. After 10 days of culture, the epithelial cells were cultured using the air-liquid interface method for 3 days to promote the growth of epithelial cells in multiple layers, thereby obtaining tissue-engineered corneal epithelial cells.

[0046] All the above steps are carried out under sterile conditions to prevent microbial contamination during the preparation process.

[0047] Example 3

[0048] A method for constructing tissue-engineered corneal epithelial cells, comprising:

[0049] The amniotic membrane was separated from the placenta and rinsed with HBSS buffer until free of blood. The amniotic membrane was spread flat on an amniotic ring and digested with 0.02% EDTA to remove the amniotic epithelium, thereby obtaining de-epithelialized amniotic membrane. A sterilized cylindrical nanosponge soaked in 1.5 mg / mL type IV collagenase was then placed under the amniotic ring, with the circular surface of the nanosponge pressed against the matrix surface of the de-epithelialized amniotic membrane. The membrane was digested at 37°C for 75 minutes to remove the loose collagen components in the amniotic matrix while retaining the dense portion, thereby obtaining ultra-thin amniotic membrane.

[0050] Under sterile conditions, the umbilical cord was cleaned, the arteries and veins were stripped, and Warburg's jelly was separated. The cord was then cut into 1×1 mm pieces. After overnight digestion with type IV collagenase, the cells were cultured in DMEM medium to obtain umbilical cord-derived mesenchymal stem cells. The umbilical cord-derived mesenchymal stem cells were then inoculated onto the reverse side of the ultrathin amniotic membrane to form an ultrathin amniotic-umbilical cord-derived mesenchymal stem cell system.

[0051] New Zealand white rabbits were sacrificed, the central corneal tissue was removed, and the remaining limbal tissue was cut into 2 mm × 2 mm tissue blocks. The tissue blocks were then incubated in SHEM medium containing 2 mg / ml Dispase II at 4°C for 14 h, and epithelial cells were isolated from the tissue blocks. The epithelial cells were attached to ultrathin amniotic membrane-umbilical cord-derived mesenchymal stem cells (UAM-MSCs) and cultured in SHEM medium. The SHEM medium was changed every two days. After 10 days of culture, the epithelial cells were cultured using the air-liquid interface method for 3 days to promote the growth of epithelial cells in multiple layers, thereby obtaining tissue-engineered corneal epithelial cells.

[0052] All the above steps are carried out under sterile conditions to prevent microbial contamination during the preparation process.

[0053] Detection method:

[0054] Optical transmittance characteristics of AM:

[0055] To estimate the optical transmittance of de-epithelialized amniotic membrane (DAM), ultrathin amniotic membrane (UAM), and UAM seeded with umbilical cord mesenchymal stem cells (MSCs) on the stromal side (UAM-MSCs), scanning images were acquired. Then, after limbal stem cells were cultured on the epithelial side of AM tissue, the transparency was photographed with a digital camera and compared.

[0056] Histology of AM:

[0057] AM tissues were fixed with 4% paraformaldehyde at 4°C for 30 min, then embedded in OCT compound (Sakura, Tablet-Tek, Torrance, USA) and stored at −80°C. 6-μm-thick frozen sections were stained with hematoxylin-eosin (Shanghai Sanggong Biotechnology Co., Ltd., China) and observed under a light microscope (Nikon Eclipse 50i, Tokyo, Japan).

[0058] Immunofluorescence staining:

[0059] Immunofluorescence staining: 6-μm-thick frozen sections were fixed with 4% paraformaldehyde for 15 min at room temperature and incubated in 0.2% Triton X-100 (Solarbio, Beijing, China) for 10 min. Sections were rinsed three times with PBS for 5 min each and preincubated with 2% bovine serum albumin (BSA) (Servicebio, China). Sections were then incubated with primary antibodies (anti-K12 (1:50, Abcam, UK), anti-K14 (1:50, Abcam, UK), and anti-PAX6 (1:50, Abcam, UK)) for 14–16 h overnight at 4°C. Sections were washed four times with PBS for 10 min each and incubated with AlexaFluor 594-labeled immunoglobulins (1:300, Invitgen, Carlsbad, USA) for 1 h at room temperature. The sections were then rinsed with PBS four times for 10 min each time, and then incubated with DAPI (Solarbio, China) at room temperature for 15 min, rinsed with PBS three times for 5 min each time, and photographed using a laser confocal microscope.

[0060] Western blot analysis:

[0061] After the incubation period, epithelial cell sheets were harvested with a cell scraper and extracted with cold lysis buffer consisting of a high-efficiency RIPA tissue / cell rapid lysis buffer and protease inhibitors (Solarbio, China). Protein concentration was determined using a BAC protein assay kit (Solarbio, China). Western blot analysis was performed using anti-K12 (1:1000, Abcam, UK), anti-K14 (1:1000, Abcam, UK), and anti-PAX6 (1:500; Abcam, Cambridge Science Park, UK) antibodies. Results were visualized using enhanced chemiluminescence reagent (Xiamen Lulong Company, China) and recorded using a transilluminator (ChemiDoc XRS, Bio-Rad, Hercules, CA, USA).

[0062] RNA extraction and RT-PCR:

[0063] DAM, UAM and UAM-MSCs epithelial cell sheets were collected with a cell scraper and RNA was extracted from Invitgen, CA, and equal amounts of RNA were reverse transcribed into cDNA using a high-efficiency reverse transcription kit (EZBioscience, USA). Real-time polymerase chain reaction (qPCR) was performed using the CFX96™ Real-Time PCR Detection System with a 2× SYBR Green qPCR Master Mix Kit (EZBioscience, USA) according to the manufacturer's instructions. A template-minus control, a reaction system without template, was included for each experiment to provide a negative control for subsequent PCR reactions. The amplification protocol consisted of 5-min denaturation at 95°C, followed by 40 cycles of denaturation at 95°C for 10 s and annealing and extension at 60°C for 30 s. SYBR Green fluorescence was measured after each extension step, and amplification specificity was assessed using melting curve analysis. Relative quantitative real-time PCR was analyzed using the comparative CT method with GAPDH as an internal control. The primer sequence pair used was: PAX6, forward primer 5'-ATAACCTGCCTATGCAACCCC-3', and back primer 5'-CTGCATATGTGGGGGAGTGT-3'. For P63, the forward primer was 5'-CGCCCCTTTCGTCA GAACAC-3' and the rear primer was 5'-GTGCTGAGGAAGGTACTGCAT-3'. For ABCG2, the forward primer was 5'-ACTACCCATGCGGATGTTGC-3' and the rear primer was 5'-GCCACGGACACTA CACTCTG-3'. For K12, the forward primer was 5'-ACTGAAGAGCTGGCCTACAT-3' and the rear primer was 5'-GGTGAGGTCCACTCCTGGT-3'. For K14, the forward primer was 5'-GACCATTGAGGACCTGAGGA-3' and the rear primer was 5'-GGCTCTCAATCTGCATCTCC-3'. For PCNA, the forward primer was 5'-GGGTGAAGTTTTCCGCCAGT-3' and the rear primer was 5'-CTGTAGGAGAAAGCGGAGTGG-3'. For TIMP-1, the forward primer was 5'-CTTCTGCAACTCCGACCTTG-3' and the back primer was 5'-GTACCCGCAGACACTTTCCAT-3'. For thrombospondin-1, the forward primer was 5'-TGTGTGGCCAACGCAACTTA-3' and the back primer was 5'-GCTGGGTTGTAATGGAACGG-3'. For connexin43, the forward primer was 5'-CCTG AGTGCCGTTTACACCT-3' and the back primer was 5'-AGAGACACCAACGACACCAC-3'.

[0064] Transplantation of tissue-engineered corneal epithelial cells in a rabbit full LSCD model:

[0065] Transplantation of the tissue-engineered corneal epithelial cells cultured in Examples 1 to 3 was performed: Fifteen New Zealand white rabbits were anesthetized by intramuscular injection of 1% sodium pentobarbital (3 ml / kg). One eye of each animal underwent a 2 mm wide, 0.2 mm deep limbal lamellar keratectomy and central corneal epithelial scraping to establish a LSCD model. The animals were then randomly divided into four groups: the first group served as the control group, which did not undergo amniotic membrane transplantation after successful model establishment; the second group received tissue-engineered corneal epithelium transplanted with DAM as a carrier; the third group received tissue-engineered corneal epithelium transplanted with UAM as a carrier; and the fourth group received tissue-engineered corneal epithelium transplanted with UAM-MSCs as a carrier. The tissue-engineered corneal epithelium was secured to the recipient's limbus with 10-0 sutures. Subsequently, another intact layer of AM was sutured to the bulbar conjunctiva, covering the entire cornea to protect the underlying transplanted tissue-engineered corneal epithelium, as is standard practice in ocular surface reconstruction surgery. Postoperatively, deproteinized calf blood ophthalmic gel (Sugaojie, Shenyang, China) was administered three times daily, tobramycin-dexamethasone eye drops (Alcon, USA) three times daily, and tobramycin-dexamethasone eye ointment (Alcon, Spain) once daily. The animals were observed and photographed under a slit lamp microscope (Chongqing Kanghua Technology, China) every two days. One week after surgery, the AM patch was removed. Two weeks after surgery, the animals were sacrificed, and corneal and limbal tissues were obtained for histological examination.

[0066] Test results:

[0067] (1) Acquisition of new UAM-MSCs carrier and its transparency: In order to obtain a more transparent and ideal carrier, the frozen-thawed amniotic membrane was treated with trypsin and type IV collagenase to obtain ultra-thin amniotic membrane (UAM), and mesenchymal stem cells (MSCs) extracted from the umbilical cord were inoculated into the matrix surface of the ultra-thin amniotic membrane. MSCs can adhere to and proliferate on the matrix surface of the ultra-thin amniotic membrane, thus obtaining a new UAM-MSCs carrier. Figure 1 As shown in part A, the process of in vitro expansion of corneal limbal stem cells on the surface of DAM, UAM and UAM-MSCs was observed under a microscope. After the epithelial sheet was inoculated, it was found that the corneal limbal epithelial sheets on the surfaces of the two groups of carriers were of the same size and attached relatively flatly. Figure 1 As shown in D3, the epithelial sheets of group 3 were still closely attached to the amniotic membrane carrier underneath, and cells gradually migrated to the periphery of the epithelial sheet, showing an oval epithelial-like morphology. Figure 1As shown in Figure A, D7, the surface cells of the epithelial sheet gradually begin to shed, while the underlying adherent cells gradually expand outward, forming a sheet-like pattern of typical cobblestone-like epithelial cells on the amniotic membrane. Generally, by around day 8-10, the epithelial cells reach 100% confluence. At this point, the cells are cultured using an air-liquid interface for approximately three days to allow for stratified growth. After completion of the culture, the formation of stratified epithelial tissue can be observed under a phase-contrast microscope, at which point stem cell transplantation can be performed. Figure 1 Figure B in the middle shows the expansion of limbal stem cells on the epithelial side and mesenchymal stem cells on the stromal side in the UAM-MSCs group. Generally, around the 8th to 10th day, the stromal cells can reach 100% fusion. Figure 1 Figure C in the middle shows a comparison of the optical transparency of three tissue-engineered corneal epithelial sheets. UAM-MSCs demonstrated superior transparency compared to DAM. Although UAM-MSCs have an additional cell layer, their transparency is very similar to that of UAM. Observation of in vitro expansion of limbal stem cells revealed no significant difference in the growth rate of epithelial cells when expanded on the three carrier surfaces. Compared with the DAM and UAM groups, the morphological changes of limbal stem cells on the epithelial surface in the UAM-MSCs group were slower, with a more oval shape retained. Furthermore, the stromal mesenchymal stem cells in the UAM-MSCs group exhibited a well-developed morphology. Therefore, UAM-MSCs can serve as a novel carrier for constructing tissue-engineered corneal epithelial cells.

[0068] (2) Research on the construction of tissue-engineered corneal epithelial cells using UAM-MSCs: Based on the novel UAM-MSCs carrier, the present invention transplanted rabbit limbal stem cells into the epithelial surface. Figure 2 As shown, the present invention performed HE staining on three types of tissue-engineered corneal epithelial sections, namely DAM, UAM, and UAM-MSCs. The tissue-engineered corneal epithelial sheet constructed based on DAM has about 3 to 5 layers of cells, and the underlying amniotic membrane matrix is thinner than before culture, but still clearly visible. The attachment between DAM and the epithelial sheet is relatively loose, and there is obvious separation at the edge. The tissue-engineered corneal epithelial sheet constructed based on UAM has about 5-7 cells, with good cell polarity, the surface cells are arranged in a flat shape, and the bottom cells are arranged in a columnar shape. The tissue-engineered corneal epithelial sheet constructed based on UAM-MSCs has a thicker epithelial cell stratification than the UAM group, the surface cells are arranged in a flat shape, and the bottom cells are arranged in a columnar shape.

[0069] (3) In order to further observe the cell phenotype of the constructed tissue-engineered corneal epithelial sheet, epithelial sheet specimens, proteins and total RNA were collected, and immunofluorescence staining, Western blot and real-time PCR were performed to detect the expression of cell differentiation (K12), progenitor cells (K14, PAX6, ABCG2), and related markers. Figure 3As shown in the immunofluorescence results in Figure A, the expression of corneal epithelial progenitor cell markers K14 and PAX6 in the epithelial sheets of the UAM-MSCs group was stronger than that in the other two groups. Figure 3 Chinese Library B and Figure 3 Western blot and real-time PCR results in Figure C further demonstrate that the corneal epithelial progenitor cell markers K14 and PAX6 are more strongly expressed in the epithelial sheets of the UAM-MSCs group compared to the other two groups. Furthermore, real-time PCR results revealed that ABCG2 expression was higher in the UAM-MSCs group than in the other two groups. K12, a marker for terminal corneal epithelial cell differentiation, was least expressed in the UAM group. Expression in the UAM-MSCs group was slightly higher than in the UAM group, but significantly lower than in the DAM group. This higher K12 result may be due to the thicker tissue-engineered corneal epithelial sheets in the UAM-MSCs group compared to the UAM group.

[0070] (4) Treatment of the corneal limbal stem cell injury model with the new tissue-engineered corneal epithelium of UAM-MSCs: First, the present invention scraped the stem cells and central epithelial cells of the rabbit cornea to construct a stem cell injury model. Then, three tissue-engineered corneal epithelia of DAM, UAM, and UAM-MSCs were transplanted into the animal injury model. Then, sodium fluorescein was used to take photos to record the growth of the corneal epithelium, and the pictures were processed with Image J. Figure 4 As shown in Figure A in the middle, photos taken one week after transplantation revealed that the amniotic membrane dressing covering the ocular surface had not dissolved, and there was no significant difference between the three groups. Two weeks after surgery, the amniotic membrane dressing covering the ocular surface was removed, and observation under the slit lamp revealed that the corneal stroma of the rabbits in the DAM transplantation group was still significantly turbid, the corneal epithelium was not smooth, and sodium fluorescein staining revealed that the cornea below the cornea had flaky staining and the epithelium was incomplete. The corneal stroma of the rabbits in the UAM transplantation group was still slightly turbid, the corneal epithelium was not smooth, and sodium fluorescein staining revealed scattered flaky staining in the center of the cornea and incomplete epithelium. However, the cornea of the rabbits in the UAM-MSCs transplantation group had recovered its transparency, the graft was well attached to the corneal stroma, the corneal epithelium was smooth, and sodium fluorescein staining was negative. In the figure Figure 4 B. The corneal epithelial healing rate curve also showed that the corneal epithelial healing in the UAM-MSCs group was better than that in the other two groups.

[0071] (5) In order to further prove that the rabbit corneal epithelium is the tissue-engineered corneal epithelial sheet transplanted by the present invention and to observe whether the corneal epithelium can survive on the ocular surface, the present invention used CFDA SE to trace the cells before transplantation. Figure 5As shown, two weeks after transplantation, the animals were sacrificed and their eyeballs removed. Fluorescence microscopy revealed green fluorescence on the corneal surface and in the corneal epithelium of tissue sections from all three transplant groups, confirming that the epithelial cells transplanted into the rabbit corneas were tissue-engineered corneal epithelial cells expanded in vitro according to the present invention. However, fluorescence was also found to be weaker in all three transplant groups compared to pre-transplantation levels. This is believed to be primarily due to the proliferation of corneal stem cells and the continuous bisection of the fluorescent agent caused by cell nuclear division.

[0072] (6) The animals were killed 2 weeks after transplantation, and the rabbit cornea specimens were frozen sectioned and stained with H&E. Figure 6 As shown, the DAM transplant group had 2-3 layers of corneal epithelial cells in the central cornea and 4-5 layers of corneal epithelial cells in the peripheral limbus. The UAM transplant group had 3-4 layers of corneal epithelial cells in the central cornea and 5-6 layers of corneal epithelial cells in the peripheral limbus. In the UAM-MSC transplant group, the central corneal epithelial cells reached 5-6 layers, while the peripheral limbus had 6-7 layers of corneal epithelial cells.

[0073] (7) In order to further observe the phenotype of corneal epithelial cells after transplantation, the present invention performed immunofluorescence staining and Real-time PCR detection on the corneal specimens of rabbits sacrificed 2 weeks after transplantation to detect the expression of markers related to cell differentiation (K12, K3) and progenitor cells (K14, PAX6, ABCG2). Figure 7 As shown in the immunofluorescence results in Figure A, the corneal epithelial progenitor cell markers K14 and PAX6 were expressed throughout the epithelial sheet in the UAM-MSCs group, with stronger expression than in the other two groups. K12, a marker for terminal corneal epithelial cell differentiation, was expressed at low levels in the central cornea and essentially absent at the limbus in the UAM-MSCs group, but was highly expressed in both the UAM and DAM groups. Figure 7 Real-time PCR results in Figure B further demonstrate that the corneal epithelial progenitor cell markers ABCG2 and PAX6 were more strongly expressed in the epithelial sheets of the UAM-MSCs group compared to the other two groups, while the expression of K12, a marker for terminal differentiation of corneal epithelial cells, was significantly lower than in the other two groups. This suggests that the stem cell properties of the rabbit corneal epithelium transplanted with UAM-MSCs were better preserved.

[0074] (8) To further observe the expression of corneal epithelial cell connections in the three groups of tissue-engineered corneal epithelial sheet transplants, the present invention performed transmission electron microscopy and Real-time PCR on the corneal specimens of rabbits sacrificed 2 weeks after transplantation to detect the RNA expression level of Connexin 43, a marker related to cell connection markers, as shown in Figure 3. Figure 8As shown: Transmission electron microscopy results showed that the connections between cells and between basal cells and basement membrane in the UAM-MSCs group were tighter, and the expression level of Connexin 43RNA was higher than that in the other two groups, indicating that the connections between epithelial cells in the UAM-MSCs group were tighter.

[0075] (9) TIMPs and thrombospondin are factors related to anti-angiogenesis. Previous studies have found that these two proteins are mainly expressed in the basal cells of normal corneal epithelium. These factors are closely related to the neovascularization of the peripheral cornea after transplantation. The present invention collected RNA from three groups of rabbit corneal epithelium after tissue-engineered corneal epithelial sheet transplantation and found through real-time PCR detection that, Figure 9 As shown in Figure 2, the expression levels of TIMP-1 and thrombospondin-1 in rabbit corneal epithelial cells in the UAM-MSCs group were higher than those in the other two groups. Figure 7 The results are consistent with the high expression levels of K14, PAX6 and ABCG2 in the UAM-MSCs group, indicating that when there are more limbal stem cells and the cell activity is better, the secretion of anti-angiogenic factors in the cells will also increase, reducing the risk of corneal neovascularization after transplantation.

[0076] Summarize:

[0077] In this example, collagenase was first used to remove the basement membrane, spongy layer, and fibroblast layer beneath the epithelial AM (AM) to obtain the UAM, which consists solely of a highly transparent, acellular, uniformly dense layer. Mesenchymal stem cells were then seeded onto the stromal surface of the UAM to form UAM-MSCs. This preparation is highly advantageous in corneal epithelial reconstruction surgery because it remains transparent even after serving as a tissue carrier, resulting in significantly better vision for the recipient during this surgical procedure than with a full-thickness AM. Furthermore, the mesenchymal stem cells on the stromal surface secrete various factors that better mimic the limbal microenvironment than the UAM. Therefore, UAM-MSCs are an ideal carrier for constructing tissue-engineered corneal epithelial cells.

[0078] Studies have shown that during in vitro expansion of the limbal epithelium (AM) by amniotic membrane (UM), the amniotic basement membrane is initially degraded and then replaced by newly synthesized basement membrane within 4 weeks by the expanded epithelial cells. Following collagenase digestion, basement membrane components on the UAM, such as type IV collagen, type VII collagen, laminin 5, and perlecan, are largely removed. Mesenchymal stem cells can also successfully differentiate into corneal stromal and corneal epithelial cells. MSCs possess significant potential for tissue regeneration: they secrete signaling molecules, such as neurotrophic factors, growth factors, and cytokines, which diffuse within the local tissue medium and interact with nearby cells. MSCs also provide cytokines that promote extracellular matrix deposition, supporting the remodeling of the cellular microenvironment and playing a crucial role in the repair and reconstruction of tissue-engineered living corneas. Therefore, prior to the synthesis of new basement membrane, the various factors secreted by stromal MSCs can support and promote epithelial cell proliferation, maintain the integrity and function of the tissue-engineered epithelium, and prevent the outcome of corneal epithelial reconstruction surgery from being compromised by transient loss of basement membrane support.

[0079] In terms of transparency, UAM-MSCs are slightly less transparent than UAM due to the inoculation of mesenchymal stem cells on the matrix surface, but significantly better than the DAM group. AM fibroblasts and spongy layers are rich in hyaluronic acid, which tends to absorb liquid after transplantation, resulting in increased thickness and decreased transparency (e.g. Figure 1 The tissue-engineered corneal epithelium cultured on UAM-MSCs was thicker than that on DAM and UAM (as shown). Figure 2 (As shown). Although the epithelial thickness was slightly thicker than normal, there were other signs that the engineered epithelial layer more closely resembled normal in vivo conditions. The expanded cells had normal epithelial cell polarity, with flat cells in the surface layer and columnar cells in the basal layer. One reason for the better simulation of in vivo conditions may be that UAM-MSCs provide better epithelial support than UAM and DAM. This difference may exist because: 1) the thinner UAM has easier access to nutrients; 2) the hyaluronic acid in the thicker stroma of DAM may absorb growth factors, potentially inhibiting cell proliferation and metabolism; and 3) the mesenchymal stem cells on the matrix surface of UAM-MSCs have anti-apoptotic, immunomodulatory, anti-inflammatory, chemokine secretion, and damage repair-promoting functions, which can better repair or regenerate damaged corneal limbus / corneal epithelium and improve the ocular surface microenvironment.

[0080] The preservation of the corneal epithelial cell phenotype transplanted onto UAM-MSCs tissue was verified by detecting the expression of K12, K3, K14, PAX6, and ABCG2. The presence of these markers indicates that the engineered epithelium on UAM-MSCs undergoes normal differentiation, which mimics the response of limbal epithelial cells in vivo (e.g., Figure 3Basal cells are negative for K3 and K12, but highly express K14. This cytokeratin expression pattern indicates an undifferentiated progenitor cell phenotype. Cells above the basal layer are positive for K3 and L12, but low in K14, suggesting a differentiated corneal cell phenotype. High expression of PAX6 and ABCG2 in UAM-MSC-based tissue-engineered epithelium supports the concept that UAM-MSCs contribute to the maintenance of the limbal progenitor cell state.

[0081] The tissue-engineered epithelium constructed by DAM, UAM and UAM-MSCs was transplanted into the rabbit corneal limbal stem cell deficiency model. Two weeks after surgery, the ocular surface epithelial cells formed a complete continuous layer. The results of UAM-MSCs transplantation showed high transparency and better corneal epithelial repair (such as Figure 4 Transmission electron microscopy results showed that the connection between cells and between basal cells and basement membrane was tighter (as shown in Figure 8 The poorer results obtained with DAM and UAM surgeries are consistent with poorly organized epithelial structures, indicating defective tissue function. Cell tracking results showed that these cells were identical to those transplanted two weeks earlier (e.g. Figure 5 MSCs can prevent the formation of peroxynitrite, a toxic reaction product between nitric oxide and superoxide, reduce cell apoptosis, reduce the level of matrix metalloproteinases, and induce the production of proinflammatory cytokines. This leads to the inhibition of corneal inflammation and neovascularization, and significantly promotes corneal healing. This is consistent with the research results of the present invention. By detecting the RNA expression levels of anti-angiogenic factors TIMP-1 and Thrombospondin-1 in the cornea after tissue engineering corneal epithelial sheet transplantation for the treatment of LSCD model, the UAM-MSCs group was significantly increased (as shown). Figure 9 (as shown). In addition, previous studies have shown that neovascularization gradually increases after AM tissue transplantation. Compared with UAM, the hyaluronic acid-softened DAM matrix may affect the attachment of the graft to the host corneal stroma, because the transplanted corneal stroma is dense and relatively rigid. It is also obvious that vascular endothelial cells are more likely to migrate to the loose interface between the AM matrix and the corneal stroma. Therefore, in this study, the exposed corneas that received UAM and UAM-MSCs corneal epithelial cell transplants are likely to recover their transparency for a longer period of time after two weeks. Due to the high expression of anti-angiogenic factors, the rabbit corneas that received UAM-MSCs corneal epithelial sheet transplants will be able to maintain transparency for a longer period of time compared with the UAM group.

[0082] In summary, the present invention has designed a new tissue-engineered corneal epithelial carrier that, through a controlled digestion protocol, is used to thin the epithelial-stripped amniotic membrane to increase its transparency. Meanwhile, inoculating MSCs on its stromal surface can better repair or regenerate damaged limbus / corneal epithelium and improve the ocular surface microenvironment. The resulting ultrathin amniotic membrane combined with mesenchymal stem cell carrier significantly improved the results of rabbit limbal tissue transplantation surgery. The cornea implanted with tissue-engineered corneal epithelium on the UAM-MSCs carrier was transparent, and this significant improvement, combined with the effect of the stromal surface mesenchymal stem cells, demonstrates that UAM-MSCs are an ideal carrier for constructing tissue-engineered corneal epithelial cells.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing tissue-engineered corneal epithelial cells, characterized in that: include: Obtain limbal tissue blocks; The tissue blocks were digested in SHEM medium containing Dispase II to isolate and obtain epithelial cells from the tissue blocks; Epithelial cells are attached to ultra-thin amniotic membrane-umbilical cord-derived mesenchymal stem cells and cultured in SHEM medium. The SHEM medium is changed every two days. After a period of culture, the epithelial cells are cultured for a period of time using the air-liquid interface culture method to promote the growth of epithelial cells in multiple layers, thereby obtaining tissue-engineered corneal epithelial cells.

2. The method for constructing tissue-engineered corneal epithelial cells according to claim 1, characterized in that: The limbal tissue was cut into 2 mm × 2 mm tissue blocks, and then the tissue blocks were incubated in SHEM medium containing 2 mg / ml Dispase II at 4°C for 14 h to separate and obtain epithelial cells from the tissue blocks.

3. The method for constructing tissue-engineered corneal epithelial cells according to claim 1, characterized in that: A method for preparing ultrathin amniotic membrane-umbilical cord-derived mesenchymal stem cells, comprising: Umbilical cord-derived mesenchymal stem cells are inoculated on the reverse side of the ultra-thin amniotic membrane to form ultra-thin amniotic membrane-umbilical cord-derived mesenchymal stem cells.

4. The method for constructing tissue-engineered corneal epithelial cells according to claim 3, characterized in that: A method for preparing umbilical cord-derived mesenchymal stem cells, comprising: The umbilical cord was cleaned under sterile conditions, the arteries and veins were stripped, the Warburg jelly was separated, and the tissue blocks were cut into 1×1 mm pieces. After digestion with type IV collagenase overnight, the cells were cultured in DMEM medium to obtain umbilical cord-derived mesenchymal stem cells.

5. The method for constructing tissue-engineered corneal epithelial cells according to claim 3, characterized in that: The method for preparing an ultra-thin amniotic membrane comprises: The amniotic membrane was separated from the placenta and rinsed with HBSS buffer until there was no blood stain; The amniotic membrane is spread flat on the amniotic ring, and after digestion and removal of the amniotic epithelium, the de-epithelialized amniotic membrane is obtained; then a sterilized cylindrical nanosponge is immersed in 1.5 mg / mL type IV collagenase and placed under the amniotic ring. The circular surface of the nanosponge is tightly attached to the matrix surface of the de-epithelialized amniotic membrane. After digestion, the ultra-thin amniotic membrane is obtained.

6. The method for constructing tissue-engineered corneal epithelial cells according to claim 5, characterized in that: The amniotic epithelium was removed by digestion with 0.02% EDTA.

7. The method for constructing tissue-engineered corneal epithelial cells according to claim 5, characterized in that: The round surface of the nanosponge was closely attached to the matrix surface of the de-epithelialized amniotic membrane, and after digestion at 37°C for 75-90 minutes, the ultra-thin amniotic membrane was obtained.

8. The method for constructing tissue-engineered corneal epithelial cells according to claim 1, characterized in that: The cells were cultured in SHEM medium, which was changed every two days. After 8-10 days of culture, the epithelial cells were cultured using an air-liquid interface method for 3 days to promote the stratified growth of the epithelial cells and obtain tissue-engineered corneal epithelial cells.

Images