A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy
Donor corneas with low endothelial cell densities (ECD) are deemed unsuitable for corneal endothelial transplantation. This study evaluated a two-step incubation and dissociation harvesting approach to isolate single corneal endothelial cells (CECs) from donor corneas for corneal endothelial cell-in...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-06-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/9/6/1428 |
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| author | Hon Shing Ong Gary Peh Dawn Jin Hui Neo Heng-Pei Ang Khadijah Adnan Chan Lwin Nyein Fernando Morales-Wong Maninder Bhogal Viridiana Kocaba Jodhbir S. Mehta |
| author_facet | Hon Shing Ong Gary Peh Dawn Jin Hui Neo Heng-Pei Ang Khadijah Adnan Chan Lwin Nyein Fernando Morales-Wong Maninder Bhogal Viridiana Kocaba Jodhbir S. Mehta |
| author_sort | Hon Shing Ong |
| collection | DOAJ |
| description | Donor corneas with low endothelial cell densities (ECD) are deemed unsuitable for corneal endothelial transplantation. This study evaluated a two-step incubation and dissociation harvesting approach to isolate single corneal endothelial cells (CECs) from donor corneas for corneal endothelial cell-injection (CE-CI) therapy. To isolate CECs directly from donor corneas, optimization studies were performed where donor Descemet’s membrane/corneal endothelium (DM/CE) were peeled and incubated in either M4-F99 or M5-Endo media before enzymatic digestion. Morphometric analyses were performed on the isolated single cells. The functional capacities of these cells, isolated using the optimized simple non-cultured endothelial cells (SNEC) harvesting technique, for CE-CI therapy were investigated using a rabbit bullous keratopathy model. The two control groups were the positive controls, where rabbits received cultured CECs, and the negative controls, where rabbits received no CECs. Whilst it took longer for CECs to dislodge as single cells following donor DM/CE incubation in M5-Endo medium, CECs harvested were morphologically more homogenous and smaller compared to CECs obtained from DM/CE incubated in M4-F99 medium (<i>p</i> < 0.05). M5-Endo medium was hence selected as the DM/CE incubation medium prior to enzymatic digestion to harvest CECs for the in vivo cell-injection studies. Following SNEC injection, mean central corneal thickness (CCT) of rabbits increased to 802.9 ± 147.8 μm on day 1, gradually thinned, and remained clear with a CCT of 385.5 ± 38.6 μm at week 3. Recovery of corneas was comparable to rabbits receiving cultured CE-CI (<i>p</i> = 0.40, <i>p</i> = 0.17, and <i>p</i> = 0.08 at weeks 1, 2, and 3, respectively). Corneas that did not receive any cells remained significantly thicker compared to both SNEC injection and cultured CE-CI groups (<i>p</i> < 0.05). This study concluded that direct harvesting of single CECs from donor corneas for SNEC injection allows the utilization of donor corneas unsuitable for conventional endothelial transplantation. |
| first_indexed | 2024-03-10T19:16:43Z |
| format | Article |
| id | doaj.art-23499d319308455181355fa05f04743f |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-10T19:16:43Z |
| publishDate | 2020-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-23499d319308455181355fa05f04743f2023-11-20T03:16:52ZengMDPI AGCells2073-44092020-06-0196142810.3390/cells9061428A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection TherapyHon Shing Ong0Gary Peh1Dawn Jin Hui Neo2Heng-Pei Ang3Khadijah Adnan4Chan Lwin Nyein5Fernando Morales-Wong6Maninder Bhogal7Viridiana Kocaba8Jodhbir S. Mehta9Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, SingaporeTissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, SingaporeTissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, SingaporeTissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, SingaporeTissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, SingaporeTissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, SingaporeTissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, SingaporeTissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, SingaporeTissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, SingaporeTissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, SingaporeDonor corneas with low endothelial cell densities (ECD) are deemed unsuitable for corneal endothelial transplantation. This study evaluated a two-step incubation and dissociation harvesting approach to isolate single corneal endothelial cells (CECs) from donor corneas for corneal endothelial cell-injection (CE-CI) therapy. To isolate CECs directly from donor corneas, optimization studies were performed where donor Descemet’s membrane/corneal endothelium (DM/CE) were peeled and incubated in either M4-F99 or M5-Endo media before enzymatic digestion. Morphometric analyses were performed on the isolated single cells. The functional capacities of these cells, isolated using the optimized simple non-cultured endothelial cells (SNEC) harvesting technique, for CE-CI therapy were investigated using a rabbit bullous keratopathy model. The two control groups were the positive controls, where rabbits received cultured CECs, and the negative controls, where rabbits received no CECs. Whilst it took longer for CECs to dislodge as single cells following donor DM/CE incubation in M5-Endo medium, CECs harvested were morphologically more homogenous and smaller compared to CECs obtained from DM/CE incubated in M4-F99 medium (<i>p</i> < 0.05). M5-Endo medium was hence selected as the DM/CE incubation medium prior to enzymatic digestion to harvest CECs for the in vivo cell-injection studies. Following SNEC injection, mean central corneal thickness (CCT) of rabbits increased to 802.9 ± 147.8 μm on day 1, gradually thinned, and remained clear with a CCT of 385.5 ± 38.6 μm at week 3. Recovery of corneas was comparable to rabbits receiving cultured CE-CI (<i>p</i> = 0.40, <i>p</i> = 0.17, and <i>p</i> = 0.08 at weeks 1, 2, and 3, respectively). Corneas that did not receive any cells remained significantly thicker compared to both SNEC injection and cultured CE-CI groups (<i>p</i> < 0.05). This study concluded that direct harvesting of single CECs from donor corneas for SNEC injection allows the utilization of donor corneas unsuitable for conventional endothelial transplantation.https://www.mdpi.com/2073-4409/9/6/1428ophthalmologycorneacell injectioncell therapycorneal endotheliumregenerative medicine |
| spellingShingle | Hon Shing Ong Gary Peh Dawn Jin Hui Neo Heng-Pei Ang Khadijah Adnan Chan Lwin Nyein Fernando Morales-Wong Maninder Bhogal Viridiana Kocaba Jodhbir S. Mehta A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy Cells ophthalmology cornea cell injection cell therapy corneal endothelium regenerative medicine |
| title | A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy |
| title_full | A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy |
| title_fullStr | A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy |
| title_full_unstemmed | A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy |
| title_short | A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy |
| title_sort | novel approach of harvesting viable single cells from donor corneal endothelium for cell injection therapy |
| topic | ophthalmology cornea cell injection cell therapy corneal endothelium regenerative medicine |
| url | https://www.mdpi.com/2073-4409/9/6/1428 |
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