干细胞 z-bo 2020-11-23 328

　　Abstract: Human corneal stromal stem cells (CSSC) inhibit corneal stromal scar formation in a mouse wound healing model, and promote the regeneration of natural transparent tissue. This study explored compressed collagen gel (CGG) as a carrier to provide CSSC corneal treatment. CSSC separated from the limbal stroma of a human donor was embedded in soluble rat tendon collagen, gelled at 37°C, and partially dehydrated to a thickness of 100 μm by passive absorption. The CCG disc is dimensionally stable, easy to handle, and can be firmly adhered to the deepithelized mouse cornea with fibrin adhesive. The survival rate of CCSG in the culture medium is more than 80%, and it is maintained in the culture medium for more than 1 week. In 20% fetal calf serum, 10% DMSO is cryopreserved in liquid nitrogen. CCG containing 500 CSSCs can effectively prevent visible scar formation and inhibit fibrotic col3a1 mRNA expression. CSC is more effective in preventing scar formation in CSG. Collagen embedded cells can still inhibit corneal scar formation after routine cryopreservation. This study demonstrates the use of a common biological material that can promote the storage and processing of stem cells. This method can provide ready-made delivery of stem cells as a treatment for corneal scars.

　　Introduction: Corneal blindness, cataracts, glaucoma and age-related macular degeneration are one of the main causes of blindness worldwide. This is especially true in developing countries, where it is estimated that 35%-50% of blindness is caused by corneal scars. The standard treatment for corneal scars is corneal transplantation, which replaces the scarred corneal tissue with allogeneic donor tissue. The immune privileged position of the cornea has made transplants a significant success, making the cornea the most frequent transplant of all organs. However, this treatment relies on the availability of donated corneal tissue, which is severely limited in many parts of the world. It is estimated that only one of 70 patients with corneal scars has access to donor tissue. Therefore, in many parts of the world, donor tissue is not available and corneal blindness is not treated. Some strategies have been proposed to overcome the shortage of donors, including artificial cornea and various biological materials to replace scar tissue, but so far there is no long-term effective solution. Another way to solve this problem is to use stem cell therapy to prevent or repair corneal scar tissue deposition. Our research team recently confirmed that the application of human corneal stromal stem cells (CSSC) can prevent the deposition of scar tissue during stromal wound healing in mice and promote the regeneration of transparent stromal tissue. CSSC is a mesenchymal stem cell isolated from the corneal stroma, which is different from limbal epithelial cells that maintain corneal epithelium homeostasis. The addition of CSSC to the fibrin gel immediately after injury resulted in increased transparency, reduced fibrotic matrix components, and remodeled matrix layer tissue. Subsequent studies revealed that the tumor necrosis factor alpha stimulating gene 6 protein (TSG-6) secreted by CSSC plays an important role in preventing neutrophils from infiltrating the damaged cornea. CSSC can be obtained from minimally invasive biopsies that have been performed clinically, which suggests that they may provide effective autologous treatment for corneal scars. At the same time, clinical trials using CSSC to treat existing corneal scars are underway. Collagen gel scaffolds have been successfully applied in tissue engineering due to their biocompatibility, low antigenicity, and high biodegradability. However, a potential defect of using collagen gel in tissue engineering is its mechanical strength and deformation. Sex. Various methods for improving the mechanical stability of collagen gels have been tested. The chemical cross-linking of collagen can increase the strength of the material, and cross-linked collagen has been used in implants to replace corneal tissue. However, crosslinking agents, such as glutaraldehyde, are cytotoxic to embedded cells. In order to increase the hardness of collagen gel without affecting its biocompatibility, Brown et al. Others have developed a technique to produce plastic compressed collagen gel (CCG) by dehydrating the gel through compression and passive absorption. These plastic CCGs greatly improve the strength and hardness, and can produce embedded cells without causing cytotoxicity. Corneal stromal cells, including CSSC, have been successfully embedded in CCG and act as a feeder layer for corneal epithelial cells. Therefore, the technology is obviously compatible with CSSC's survivability. In this study, we examined CCG as a vehicle to transport CSSC to the injured cornea. CCG improves the processing of CSSC, increases the effectiveness of treatment, and allows CSSC to be stored in a treatment-ready state.

　　Preparation of CCG: Compressed collagen hydrogel is using RAFT reagent according to the manufacturer's protocol. Briefly, the acid-soluble rat tail collagen was neutralized and diluted to a concentration of 1.6 mg/ml in a solution containing 1× basal medium and CSSC cells. Transfer the collagen solution (usually 1 ml) to the culture wells of a 24-well petri dish containing a 12 mm round glass cover slip. The collagen was gelled in a carbon dioxide incubator at 37°C for 1 hour, and then dehydrated with a fiber absorbent in a laminar flow hood at room temperature for 15 minutes. Transfer the collagen gel to a 60 mm Petri dish containing sterile phosphate buffered saline (PBS). Prepare 2mm college gels, and store the cell-free CCG gels in sterile PBS. The CCG containing cells was cultured in a carbon dioxide incubator at 37°C for 2 weeks. .Before sectioning, use Dylight 633 to stain part of the cell-free CCG gel. Rinse the gel with 0.1 M NaHCO3, and stain it with 0.25 mg/ml Dylight 633 NHS ester reactive dye in 0.1 M NaHCO3 (pH 8.5) for 2 hours at room temperature. Wash CCG in sterile PBS and store at 4°C until use.

　　CSSC separation: obtained from the Organ Recovery and Education Center in Pittsburgh, Pennsylvania, a human cornea-scleral limbus approved by an accredited donor under the age of 60 for research purposes. Use the tissue within 5 days after harvesting. CSSC was obtained from the separated marginal tissues by collagenase digestion. CSSC was inoculated into 2% (VOL/VOL) mixed human serum stem cell growth medium. The medium is changed every 3 days. When 80% of the cells are fused into the culture flask, trypsin is used to quickly and quickly digest the passaged cells, and cryopreserve during passage. To freeze the gel, CGG filled with CSSC (2000 cells per 2 mm gel) was transferred to DMEM medium containing 20% fetal bovine serum and 10% DMSO. The solution is slowly cooled in the NMR machine. Place the freeze-thaw gel in a freezer at 80°C overnight and transfer to liquid nitrogen for long-term storage. For repeated use, the freeze-thaw gel is quickly thawed in a 37°C water bath. 4-16 hours before use, the gel is balanced in the serum free stem cell growth medium at 37°C.

　　Cell staining: CSSC cells were collected after trypsinization and centrifugation, suspended in serum-free DMEM/F12, 106 cells per ml, at 37°C, stained with Vybrant DiO at 50ug/ml. After incubating for 20 minutes, the cells were centrifuged, the supernatant was removed, and fresh medium was added. Live and dead CSSC were incubated in AM medium containing 50μg/ml calcinin at 37°C for 20 minutes, and then incubated in 5μg/ml iodide for 5 minutes.

　　Mouse corneal injury model: Female C57/BL6 mice, 7-8 weeks old, raised in an ABSL2 facility approved by AALAC and provided with unlimited standard diet. 6 groups of mice were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). In the visible scar analysis, at least 6 eyes are required for statistical significance analysis, and 2 weeks provides a suitable time point for gene expression and fibrosis analysis. Before debridement with local anesthesia, add one drop of procaine hydrochloride (0.5%) to each eye. Pass Algerbrush II through the center of the mouse cornea 2 mm to debride the corneal epithelium. After removing the epithelium, apply Algerbrush II again, this time applying more pressure to remove the basement membrane and 10-15 μm anterior matrix tissue. The mice were given ketoprofen (3 mg/kg) for analgesia immediately after the operation. Both eyes received the same damage and treatment.

　　Application of fibrin gel and CSSC: CSSC and human fibrinogen at a specific concentration are mixed 1:1 in PBS, 70 mg/ml in PBS, and stored on ice. After the cornea is injured, add 0.5μl of thrombin (100 U/ml) to the wound, and then immediately add 1μl of fibrinogen (with or without CSSC). Fibrin gels for 1-2 minutes, and a second round of thrombin and fibrinogen is applied. Gentamicin eye drops (0.3%) were used to treat the wound. The corneal epithelium heals within 24-36 hours. Check the eyes for signs of rejection and infection every day for 1 week, and then once a week thereafter.

　　Placement of CCG on the cornea: initially on the excised mouse eyes, and then on the eyes after live corneal injury, rinse the eyes with PBS, and add 1 microliter of thrombin to the wound. As mentioned above, gently place the 2 mm CCG disc on the cornea, and then place 1 μl of fibrinogen. This process causes the compressed collagen to adhere firmly to the surface of the eye.

　　CCG corneal unilateral imaging: use Olympus FV1000 confocal microscope to image the labeled cells in CCG, use Olympus SZX dissecting microscope and epifluorescence optics to image the gel of mouse eye. The excised mouse eyes were fixed overnight in 3.2% paraformaldehyde PBS at 4°C, and frozen section or paraffin histological examination was performed.

　　Scar assessment: Two weeks after corneal debridement, all eyes were collected, and the entire sphere was imaged with an indirect lighting dissecting microscope. Determine the scar area based on these images. Corneal staining: The paraffin sections (8μm) of the cornea injured for 2 weeks were deparaffinized and stained with H&E to observe the tissue morphology. In order to prove type III collagen, part of the arylation was removed by microwave treatment in 10 mM sodium citrate (pH 6), which contained 0.5% wt/vol Tween 20.

　　Results: Embedding CSSC in compressed collagen: To evaluate the potential use of CCG as a CSSC delivery vehicle, we first tested the ability to produce a gel containing living cells, which is so thin that it can create a light path on the mouse cornea . . The thickness of the mouse's central cornea is only 120 microns. Therefore, we strive to produce a gel with the smallest thickness to limit eye irritation and removal of eyelid gel during blink reflex. As a result, the thickness of CCG varies with the amount of collagen added, but at least reaches about 100 μm, with about 3 mg collagen per hole. Reducing the collagen content below 3 mg can reduce the concentration of collagen in the compressed gel, but not the thickness. The addition of CSSC to the collagen solution during the gelation process showed that the survival rate of the cells after dehydration remained above 90%, similar to the cells added to the gel. CSSC is distributed in aggregates of 20-50μm in CCG.

　　The adhesion of CCG to mouse cornea: To test the adhesion of CGG to the cornea, the corneal epithelium was isolated with 70% EtOH for 10 seconds, and then the corneal epithelium was scraped off with a foam tip applicator. The gel labeled with dye 633 can be firmly attached to the exposed corneal stroma with fibrinogen and thrombin. With Algerbrush debridement, it was found that CCG can adhere to the cornea very well. The marked CSSC is embedded in the CCG and can be seen on the eyes after the CCG is adhered. Interestingly, after CCG adhered to the body for 2 days, CCG no longer appeared on the eye surface, and no labeled cells were visible.

　　CSSC implantation to prevent corneal scar formation: After debridement of epithelial cells and corneal basement membrane, CSSC is transplanted into the matrix with fibrin gel to prevent corneal fibrosis in mice. In order to observe whether CSSC maintains the ability to inhibit scar formation when CCG is implanted, the CCG with or without CSSC implanted is adhered to the surface of the mouse cornea immediately after matrix injury. Most eyes treated with acellular CCG (CCG only) showed visible stromal scars 14 days after injury. When CCGS contains CSSC, scars rarely appear. Although the ratio between experiments is different, statistically, the presence of CSSC in CCG can always significantly inhibit corneal scar formation. Corneal scars contain extracellular matrix and unusual connective tissue components. This fibrotic matrix is believed to be a source of light scattering and contributes to the interruption of vision caused by corneal scarring. The two molecular markers of corneal scars are collagen type III and smooth muscle actin, which are the products of mouse col3a1 and acta2 genes. As we have demonstrated before, both of these genes were significantly up-regulated in the cornea 14 days after injury. When CSSC appeared in the CCG covering the injured eye, the activation was significantly reduced.

　　CSSC induced matrix morphological repair in CCG: To evaluate the regeneration of corneal stroma with or without CSSC CCG treatment, tissue sections were analyzed by H&E staining 14 days after injury. The results showed that CCG treatment only resulted in tissue edema, cell proliferation, and uneven epithelial coverage in the wound healing area. The cornea exposed to the CCG loaded with CSSC showed a histology indistinguishable from the normal cornea, showing regeneration of native corneal tissue in the area removed by Algerbrush debridement. The corneal edema area healed without CSSC cells and contained collagen III, which is a well-known sign of corneal scar formation, while the tissues treated with CCG containing stem cells did not stain collagen III.

　　CSSC dose response in CCG: In previous studies, 50,000 CSC cells were used in fibrin gel to inhibit corneal wound scar formation. This concentration represents the maximum number of cells in the fibrin gel remaining on the surface of the mouse cornea during the healing process. In order to evaluate the effective dose of CSSC needed to inhibit corneal scarring, CSSC cells were embedded in CCG at different concentrations (50-2500 cells). Corneal scar analysis showed that 50 cells had no inhibitory effect on scars, while 125, 500 and 2500 cells had significant inhibitory effects on scars. Comparing 500 CSSCs in the CCG with the same number of fibrinogen cells, it was observed that the effectiveness of each CSSC cell embedded in the CCG was significantly increased.

　　The cryopreservation of CSSC in CCG: In order to evaluate the effect of CSSC after cryopreservation, we used a method commonly used for culturing cells, freezing CCG containing cells in liquid nitrogen. After storage for 2 weeks, the gel was frozen and thawed, cultured in serum-free medium overnight, and labeled with calcein AM activity dye. Under the microscope, these cells are alive and have condensed into aggregates of 50-100μm. The use of frozen/thawed CCG in a mouse wound healing model showed that the preserved cells were highly effective in inhibiting corneal scarring.

　　Conclusion: Implanting CSSC into CCG is a quick and simple method, which can prevent the formation of transparent corneal tissue scar and induce its regeneration. This new method may bring therapeutic potential to a large number of patients suffering from corneal scars.