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 av lys til elektriske impulser inntreffer i den ytre retina, og utføres i stor grad av stang- og kjeglefotoreseptorene og retinalt pigmentepitel (RPE) celler. RPE gi kritisk støtte for fotoreseptorene og død eller dysfunksjon av RPE celler er karakteristisk for aldersrelatert makuladegenerasjon (AMD), den ledende årsak til permanent synstap hos personer 55 år og eldre. Mens ingen kur for AMD har blitt identifisert, kan implantasjon av sunn RPE i syke øyne vise seg å være en effektiv behandling, og et stort antall RPE-celler kan lett genereres fra pluripotente stamceller. Flere interessante spørsmål angående sikkerhet og effekt av RPE celle levering kan fortsatt bli undersøkt i dyremodeller, og godt anerkjente protokoller som brukes til å injisere RPE har blitt utviklet. Den teknikk som er beskrevet her har blitt brukt av flere grupper i forskjellige studier og innebærer først å lage et hull i øyet med en skarp nål. Deretter en sprøyte med en blunt p lastet med cellene er satt inn gjennom hullet og føres gjennom glasslegemet inntil den så vidt berører RPE. Ved hjelp av denne metoden injeksjon, noe som er relativt enkel og krever minimalt utstyr, oppnår vi konsekvent og effektiv integrering av stamcelle-avledet RPE-celler i mellom verts RPE som hindrer betydelig mengde av fotoreseptoren degenerasjon i dyremodeller. Selv ikke en del av selve protokollen beskriver vi også hvordan å fastslå omfanget av traumer forårsaket av injeksjon, og hvordan du kontrollerer at cellene ble injisert i subretinal plass ved hjelp av in vivo bildediagnostikk. Til slutt er anvendelse av denne protokollen ikke begrenset til RPE-celler; Det kan anvendes for å injisere en hvilken som helst forbindelse eller celle inn i subretinal plass.
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 artikkelen beskriver vi en relativt enkel fremgangsmåte for å utføre subretinal injeksjoner av RPE-celler i suspensjon i rotter og mus. Protokollen er lett å lære og mer erfaring med teknikken vil oversette i færre traumer (figur 3, og dette representerer en av de bedre injeksjoner), spesielt hvis en micromanipulator brukes (figur 1A). Noen traumer kan overvåkes in vivo med en cSLO og OCT-systemet (figur 2) hvis tilgjengelig. Hvis høyere oppløs…
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 |