윤부줄기세포가 결핍된 토끼 모델에서. 효소에 민감한 세포판을 기반으로 한 인간 각막 윤부 상피 세포의 이식. 연구 목적: 각막 상피 및 시력 회복을 위한 배양된 인간 윤부 각막 상피 세포 시트 이식.
각막 윤부 상피 세포의 배양은 여러 가지 방법으로 수행되었습니다. 이에 저자들은 효소 민감도를 기반으로 한 새로운 배양 기반을 개발하고, 토끼 윤부줄기세포 결핍 모델을 통해 이 방법의 유효성을 확인하고자 했다. 4주 후 따오기의 각막 상태를 평가하여 토끼 윤부줄기세포 결핍 모델을 분류하였습니다.
카르복시메틸셀룰로오스-도파민(CMC-DOPA)이 코팅된 트랜스웰을 사용하여 기증자의 각막에서 얻은 인간 윤부상피세포를 배양하였고, 배양액은 3, 5, 7일 후에 매일 교체하였다. 5명의 피험자를 조직학적 검사와 면역화학적 염색을 통해 검사한 결과, 4명의 피험자에서 인간 윤부 상피 세포 시트가 4주 동안 생존했습니다. 결론: 토끼 윤부 줄기 세포 결핍 모델에서 효소 민감성 세포 시트 기반 배양된 인간 윤부 상피 세포.
이를 토대로 배양된 윤부상피세포시트의 이식이 향후 윤부줄기세포 결핍증 치료에 중요한 역할을 할 것으로 사료된다.
Introduction
Materials & Methods
- Preparation of rabbit LSCD model and classification of LSCD grading
- Method of carboxymethyl cellulose-dopamine (CMC-DOPA) coating
- Preparation of cultivated human limbal epithelial sheets
- Evaluation of cultivated human limbal epithelial sheet status by histology and immunohistochemistry
- Method of cell sheet transplantation
- Evaluation of transplantation status
Limbal epithelium was isolated by treatment with 0.2% Dispase II (Roche) in a 35 mm dish) at 4°C overnight. Subsequently, the epithelium was treated with 0.25% trypsin/0.01% EDTA (Gibco, USA) for 1 hour at 37°C to generate isolated cells, followed by the dissociation by pipetting to obtain the mixture of single cells and cell aggregates. to acquire. Cell culture inserts were coated with 1 mg/ml CMC-DA and human limbal epithelial cells were placed on the 6 well at 3-5x10 5 cells per well.
After the epithelial cells reached confluence, human limbal epithelial cells were cultured for two weeks with underlying medium replacement daily. To obtain cellular sheets, medium containing 100U/ml cellulose (Cellulase from Trichoderma sp. Sigmaaldrich) was treated on the underside of the insert for 1 hour at 37℃. After detaching cell sheets from the insert, they were aspirated with a pipette and washed with PBS and transferred to a fresh cell culture dish.
Paraffin sections or formalin-fixed cryosections were stained with hematoxylin and eosin (HE) for histological examination. After observing the rabbit LSCD models, the scar tissue consisting of conjunctiva and neovascularization was removed. The prepared limbal epithelial cell sheet was placed on the cornea with scar tissue removed, and a 4-point peripheral corneal suture was performed.
After cell sheet transplantation, the rabbits were treated with eye ointment (Neomycin Sulfate 3.5 mg/g, Dexamethasone 1 mg/g, Polymyxin B Sulfate 6000 IU/g daily), and triamcinolone acetonide (0.1 ml injected subconjunctivally every 1 week). A week later, the sutures of cornea and eyelid were removed and the contact lens was also removed. In group 1, two weeks after transplantation, the rabbits were sacrificed, and in group 2, four weeks after transplantation, the rabbits were sacrificed.
In group 2, clinical outcome was observed in the first, second and fourth week after surgery. Clinical outcome included grade of corneal opacity and corneal neovascularization, and status of fluorescence stained corneal epithelium. According to the ocular surface standard grading table [42], two indices of corneal opacity and neovascularization grading were compared before transplantation and after second or fourth week.
Results
Clinical outcome
Histology and immunohistochemistry
In immunohistochemistry (anti-human mitochondria; Alexa Fluor 647), after 4 weeks human corneal epithelium was observed in the total cornea. In histology at 4 weeks, the rabbit cornea was covered with human corneal epithelium and goblet cells were not observed. In immunohistochemistry (anti-human mitochondria; Alexa Fluor 647) after 4 weeks, human corneal epithelium was observed throughout the cornea, but the surface was irregular.
In general, corneal opacity was similar to pre-operative condition, and neovascularization was slightly better than pre-.
Discussion
However, in all cases without change in groups 1 and 2, the degree of corneal opacity and neovascularization was the same, but microscopic findings showed a decrease in the extent and severity of corneal opacity and neovascularization. The result of histology and immunohistochemistry in this study showed that only in 1 case, the human limbal epithelial cell did not survive, but in the remaining 4 cases, the human limbal epithelial sheet survived for 4 weeks. This confirms that enzyme-based cell sheets survived relatively well in rabbit LSCD eyes immunosuppressed for 4 weeks.
We used the immunosuppressive treatment for only 1 week to improve the survival rate of rabbits. Due to immunosuppressive treatment for 1 week, corneal opacity and neovascularization after 2 weeks appear to be worse than after 1 week. Second, the rabbit LSCD model in this study was a severe case of grade 3 or more in corneal opacity and neovascularization, except for 1 case in group 2 (shown in Table 1).
Third, we did not use amniotic membrane transplantation (AMT) because we designed the experiment to confirm the sole effect of human limbal epithelial transplantation. AMT is an excellent option for the treatment of ocular surface disease in particular chemical burns [50]. In the future, it will be necessary to experiment with the combination of AMT and human limbal epithelial transplantation.
In conclusion, transplantation of cultured limbal epithelial cell sheet will play a significant role for LSCD treatment in the future. Tissue-sparing surgery through cultured limbal epithelial cell skin is beneficial in preserving a healthy eye and reducing or eliminating the need for systemic immunosuppression, especially in the case of homologous transplantation. It is important to note that even with this potential treatment, presurgical planning will still play a catalytic role in the success of such procedures.
Clinical results of xeno-free allogeneic cultured limbal epithelial transplantation for bilateral limbal stem cell deficiency. Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface in patients with total limbal stem cell deficiency. Development of a surface-modified contact lens for transfer of cultured limbal epithelial cells to the cornea for ocular surface diseases.
Transplantation of tissue-engineered epithelial cell sheets after excimer laser photoablation reduces postoperative corneal haze. Reconstruction of functional ocular surface by acellular porcine corneal matrix scaffold and limbal stem cells derived from human embryonic stem cells. Concise review: transplantation of cultured oral mucosal epithelial cells for the treatment of limbal stem cell deficiency-current status and future perspectives.
Successful use of ex vivo expanded human autologous oral mucosal epithelium for the treatment of complete bilateral limbal stem cell deficiency. Regeneration of corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell-free media. Cultivation and characterization of limbal epithelial stem cells on nutrient-coated contact lenses: towards the treatment of limbal stem cell deficiency.
In vivo morphology of vogt limbal palisades correlates with progressive stem cell deficiency in aniridia-associated keratopathy. Delayed loss of corneal epithelial stem cells in a chemical injury model associated with limbal stem cell deficiency in rabbits. Fixed amniotic membrane cell delivery reconstructs the corneal epithelium in limbal stem cell-deficient rabbits.
Transplantation of autologous serum-derived cultured corneal epithelial equivalents for the treatment of severe ocular surface disease. A contact lens-based technique for expansion and transplantation of autologous epithelial progenitor cells for ocular surface reconstruction. Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results.
Abstract
Conclusions: Cultured human limbal epithelial graft based on enzyme-sensitive cell sheet in a rabbit LSCD model was effective in reducing corneal opacity for 4 weeks. As a result, transplantation of cultured limbal epithelial cells will play a significant role for LSCD treatment in the future.
