Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics
2.2. Isolation and Cultivation of hRPEs
2.3. Ultrathin Nanofibrous Carrier for RPE Transplantation
2.4. Trans-Epithelial Electrical Resistance (TEER)
2.5. Implantation Injector
2.6. Animals Used for the Experimental Study
2.7. Immunosuppression of Minipigs
2.8. Implantation of hRPEs on a Nanofibrous Carrier into Minipigs
2.9. Surgical Technique
2.10. In Vivo Optical Coherence Tomography
2.11. Euthanasia and Tissue Processing
2.12. Immunohistochemical Analysis
2.13. Laser Scanning Confocal Microscopy
3. Results
3.1. Production of a Monolayer of hRPEs on a Nanofibrous Carrier
3.2. Trans-Epithelial Electrical Resistance (TEER)
3.3. Implantation of the hRPEs
3.4. Immunohistochemical Analysis of the Implanted hRPEs
4. Discussion
4.1. RPE Preparation for Transplantation and Post-Implantation Characterization
4.2. RPE Surgical Transplantation
4.3. Injection of RPE Cell Monolayer
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Pig Number | Time of Euthanasia (Weeks) | Right Eye | Left Eye | Time of OCT Scans and Fundus Photography (Weeks) |
---|---|---|---|---|
4 (T149) | 1 | Treated eye | Control eye | 1 |
1 (T141) | 2 | Treated eye * | Treated eye | 1, 2 |
3 (T146) | 6 | Treated eye | Control eye | 1, 2, 6 |
6 (T192) | 6 | Treated eye | Control eye | 1, 2, 6 |
5 (T145A) | 8 | Treated eye | Control eye | 1, 2, 6, 8 |
Protein | Source | Manufacturer | Working Dilution |
---|---|---|---|
HNAA | mAb mouse | Novus Biologicals, UK, Abingdon, (235-1) NBP2-34342-0.1 mg | 1:300 |
STEM121 | mAb mouse | Takara Bio Inc., Kusatsu, Japan, cat. No. Y40410 | 1:100 |
Bestrophin | mAb mouse | Santa Cruz Inc., Germany, Heidelberg, cat. No. sc-32792 | 1:50 |
CRALBP | mAb mouse | Novus Biologicals UK, Abingdon, (B2) cat. No. NB100-74392 | 1:100 |
Vimentin | mAb mouse | Cell Signaling, (D21H3), USA, cat. No. 5741 | 1:100 |
GFAP | pAb rabbit | Merck Millipore, Germany, Darmstadt, rabbit polyclonal antibody, cat. No. AB5804, lot No. 2464502 | 1:1000 |
IbaI, Microglial Marker | mAb mouse | Abcam, Cambridge, UK, mouse monoclonal antibody, cat. No. ab15690, clone 1022-5 | 1:250 |
PKC alpha | pAb rabbit | Sigma-Aldrich, Germany, Taufkirchen, rabbit polyclonal, cat. No. P4334 | 1:10,000 |
Lectin PNA Conjugate Alexa Fluor 488 | Molecular probes, cat. No. L-21409 | 1:300 |
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Lytvynchuk, L.; Ebbert, A.; Studenovska, H.; Nagymihály, R.; Josifovska, N.; Rais, D.; Popelka, Š.; Tichotová, L.; Nemesh, Y.; Čížková, J.; et al. Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs. Biomedicines 2022, 10, 669. https://doi.org/10.3390/biomedicines10030669
Lytvynchuk L, Ebbert A, Studenovska H, Nagymihály R, Josifovska N, Rais D, Popelka Š, Tichotová L, Nemesh Y, Čížková J, et al. Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs. Biomedicines. 2022; 10(3):669. https://doi.org/10.3390/biomedicines10030669
Chicago/Turabian StyleLytvynchuk, Lyubomyr, Annabelle Ebbert, Hana Studenovska, Richárd Nagymihály, Natasha Josifovska, David Rais, Štěpán Popelka, Lucie Tichotová, Yaroslav Nemesh, Jana Čížková, and et al. 2022. "Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs" Biomedicines 10, no. 3: 669. https://doi.org/10.3390/biomedicines10030669