Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.
Omdannelsen af lys til elektriske impulser forekommer i den ydre retina og opnås i høj grad af stang og kegle fotoreceptorer og retinale pigmentepitel (RPE) celler. RPE giver kritisk støtte til fotoreceptorer og død eller dysfunktion af RPE-celler er karakteristisk for aldersrelateret makuladegeneration (AMD), den førende årsag til permanent synstab i mennesker 55 og ældre. Mens ingen helbredelse for AMD er blevet identificeret, kan implantation af sunde RPE i syge øjne vise sig at være en effektiv behandling, og store antal af RPE-celler kan let genereres ud fra pluripotente stamceller. Flere interessante spørgsmål vedrørende sikkerhed og effekt af RPE-celle levering kan stadig blive undersøgt i dyremodeller, og godt anerkendte protokoller, der anvendes til at injicere RPE er blevet udviklet. Den her beskrevne teknik er blevet anvendt af flere grupper i forskellige undersøgelser og involverer først at skabe et hul i øjet med en skarp nål. Derefter en sprøjte med en blunt nål fyldt med celler indsættes gennem hullet og føres gennem glaslegemet, indtil det rører ved RPE. Ved hjælp af denne injektion metode, der er forholdsvis enkel og kræver minimal udstyr, vi opnå en ensartet og effektiv integration af stamceller celleafledte RPE celler mellem værten RPE der forhindrer betydelig mængde fotoreceptordegenerering i dyremodeller. Mens ikke en del af selve protokollen, vi også beskrive, hvordan at bestemme omfanget af traumer som følge af injektion, og hvordan at kontrollere, at cellerne blev injiceret i subretinarummet ved hjælp af in vivo imaging modaliteter. Endelig er anvendelsen af denne protokol ikke begrænset til RPE celler; den kan anvendes til at injicere enhver forbindelse eller celle i det subretinale rum.
The sensory retina is organized in functional tiers of neurons, glia, and endothelial cells. Photoreceptors at the back of the retina are activated by light; through phototransduction they convert photons into electrical signals that are refined by interneurons and transmitted to the visual cortex in the brain. Phototransduction cannot occur without the coordinated efforts of Mueller glia and retinal pigment epithelium (RPE) cells. RPE are organized in a monolayer directly behind the photoreceptors and perform multiple and diverse functions integral to photoreceptor function and homeostasis. In fact, RPE and photoreceptors are so co-dependent that they are considered to be one functional unit. Death or dysfunction of RPE results in devastating secondary effects on photoreceptors and is associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly1,2.
While no cure has been discovered for AMD, several clinical studies have shown that RPE cell replacement may be a promising therapeutic option3-13. With the advent of stem cell technology, it is now possible to generate large numbers of RPE cells in vitro from embryonic and induced pluripotent stem cells (hES and hiPS) that strongly resemble their somatic counterparts functionally and anatomically14-26. Stem cell-derived RPE have also been shown to function in vivo by multiple independent groups, including our own, to significantly slow retinal degeneration in rat and mouse lines with spontaneous retinal degeneration16,18,21,22,25,28,29. This combination of clinical and preclinical supporting evidence is so compelling that several clinical trials to prevent retinal degeneration using stem cell-derived RPE cells are now ongoing30,31.
RPE can be readily derived from hES and/or hiPS and implanted in the subretinal space of rodents using various derivation and injection techniques32,33. (See Westenskow et al. for a methods paper in multimedia format demonstrating the directed differentiation protocol we employ)34. There are critical remaining questions regarding the safety, survival, and functional capacity of exogenously delivered RPE cells upon implantation, therefore the ability to perform subretinal injections in rodents is a critical skill16,18,21,29,36,37. The delivery of RPE is not trivial, and the field is divided on the most effective injection technique. The protocol we describe here is a simple and effective way to deliver of bolus of RPE cells subretinally, and was used in the first clinical trial for stem cell-derived RPE transplantation31. (The reader may also refer to another JoVE article by Eberle et al. for an alternative depiction of subretinal injections in rodents.38)
The technique outlined in this manuscript cannot be visualized and trauma is unavoidable (as with any subretinal injection technique). It is performed by making a hole just under the limbus vessels and inserting a blunt needle along a transscleral route to inject a bolus of cells under the diametrically opposed retina. The person doing the injection will feel resistance as the blunt needle touches the retina. The cells may be directly visualized after the injection, however, and the degree of the induced retinal detachment can be determined by labeling the RPE cells with a transient fluorescent marker and detecting them with a confocal scanning ophthalmoscope (cSLO). An optical coherence tomography (OCT) system can also be used to monitor the trauma and easily identify the injection site.
I denne artikel beskriver vi en forholdsvis enkel fremgangsmåde til udførelse af subretinal injektioner af RPE-celler i suspension i rotter og mus. Protokollen er let at lære og mere erfaring med teknikken vil oversætte færre traumer (figur 3; dette repræsenterer en af de bedre injektioner), især hvis en mikromanipulator anvendes (figur 1A). Enhver traumer kan overvåges in vivo med en cSLO og oktober (figur 2), hvis de er tilgængelige. Hvis der ønskes…
The authors have nothing to disclose.
We wish to thank Alison Dorsey for helping to develop the subretinal injection technique. We also acknowledge the National Eye Institute (NEI grants EY11254 and EY021416), California Institute for Regenerative Medicine (CIRM grant TR1-01219), and the Lowy Medical Research Institute (LMRI) for very generous funding for this project.
Name of Material/ Equipment (A-Z) | Company | Catalog Number | Comments/Description |
2-Mercaptoethanol (55 mM) | Gibco | 21985-023 | 50 mL x 1 |
Cell Scapers | VWR | 89260-222 | Case x 1 |
CellTracker Green CMFDA | Molecular Probes | C34552 | 50 ug x 20 |
DPBS, no calcium, no magnesium | Gibco | 14190-144 | 500 mL x 1 |
Fast Green | Sigma-Aldrich | F7258 | 25 g x 1 |
Genteal Geldrops Moderate to Severe Lubricant Eye Drops | Walmart | 4060941 | 25 mL x 1 |
Hamilton Model 62 RN SYR | Hamilton | 87942 | Syringe x 1 |
Hamilton Needle 33 gauge, 0.5", point 3 (304 stainless steel) | Hamilton | 7803-05 | Needles x 6 |
Knockout DMEM | Gibco | 10829-018 | 500 mL x 1 |
KnockOut Serum Replacement | Gibco | 10828-028 | 500 mL x 1 |
L-Glutamine 200 mM | Gibco | 25030-081 | 100 mL x 1 |
Magnetic Stand | Leica Biosystems | 39430216 | Stand x 1 |
MEM Non-Essential Amino Acids Solution 100X | Gibco | 11140-050 | 100 mL x 1 |
Micromanipulator | Leica Biosystems | 3943001 | Manipulator x 1 |
Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140-122 | 100 mL x 1 |
Slip Tip Syringes without Needles BD (3 mL) | VWR | BD309656 | Pack x 1 |
Specialty-Use Needles BD (30 gauge, 1") | VWR | BD305128 | Box x 1 |
TrypLE Express Enzyme (1X), no phenol red | Gibco | 12604013 | 100 mL x 1 |