Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

Summary

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.

Abstract

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Introduction

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 elderly^1,2.

While no cure has been discovered for AMD, several clinical studies have shown that RPE cell replacement may be a promising therapeutic option^3-13. 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 anatomically^14-26. 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 degeneration^{16,18,21,22,25,28,29}. 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 ongoing^30,31.

RPE can be readily derived from hES and/or hiPS and implanted in the subretinal space of rodents using various derivation and injection techniques^32,33. (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 skill^{16,18,21,29,36,37}. 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.

Protocol

NOTE: All animals were treated in accordance with the ethical guidelines established by the Scripps Research Institute.

1. Preparation of Materials for the Injection (~20 min)

1. Pre-warm cell dissociation solution (preferably one that is inactivated through dilution, not with serum), sterile PBS, and culture media (Table 1).
2. Sterilize the syringe with a blunt needle by disassembling it and boiling the parts in water for 15 min.

2. Preparation of the RPE Cells for Injection (~30 min to 1 hr)

1. Detach the RPE cells using pre-warmed cell dissociation solution for 5-8 min at 37 °C.
2. Scrape the cells gently to release any that are still attached.
3. Dilute the cells with a large volume of culture media (fill up a 15 ml tube) to inactivate the dissociation solution and count them.
4. Centrifuge at 800 x g for 5 min to pellet the cells.
5. Resuspend the cells at 200,000 cells/µl (to deliver 100,000 cells in a 0.5 µl volume) in sterile pre-warmed PBS and transfer them into a 1.5 ml microcentrifuge tube.
6. Optionally, add a live cell transient fluorescent marker and incubate at 37 °C for 30-45 min.
7. Load the syringe with a blunt needle with 0.5 µl of cells. Inject the cells as soon as possible.

3. Sub-retinal Injection (~5 min per Injection)

NOTE: If possible, learn the technique with adult albino rats since the limbus vessels are much easier to visualize. Inject Fast Green solution when learning (before trying to inject cells) to more easily facilitate visualization of the injection site.

1. Anesthetize the rodent. Use intraperitoneal injections of 100 mg/ml ketamine and 10 mg/ml xylazine (20 µl/10 g body weight) over isofluorane inhalation since it is difficult to maneuver the rodent and inject into the eye with the snout in the inhaler.
   1. Ensure that the animal is deeply anesthetized by pinching one of its paws. If it flinches, wait several more minutes and try again before beginning the subretinal injection.
2. Position the rodent onto its side so that the eye that will be injected is facing the ceiling.
3. Under a dissecting microscope gently stretch the skin so the eye pops slightly up out of the socket (temporary proptosis) and becomes more accessible by holding its head with two fingers just above the ear and by its jaw and gently stretch the skin parallel to the eyelids so that the eye pops slightly up out of the socket (See Figure 1C). Do not grasp the rodent too close to the throat.
4. With a 30 G sharp disposable pre-sterilized needle, make a hole immediately below the limbus (if the vessels are hit, bleeding will be significant and it be difficult to find the hole later) and at an angle to avoid touching the lens with the needle (Figure 1D). Avoid touching the lens with the sharp (or blunt) needle or immediate cataract formation will occur.
   NOTE: Injections work better with two people. In this way one person can pass the syringe with the blunt needle to the person performing the injection after they have created the first hole with the sharp disposable needle to maintain focus on the where the hole is.
5. Retract the disposable sharp needle from the eye while maintaining the grip on the head. Remember exactly where the hole is.
6. After either mounting the pre-loaded syringe with a blunt needle on a micromanipulator or holding it by hand, insert the tip of the syringe with the blunt needle through the hole, taking care again not to touch the lens and gently push it through the eye very gently until feeling resistance (Figure 1D).
7. Keeping all movements to a minimum, carefully inject the RPE cells slowly into the subretinal space.
   NOTE: RPE/retina detachment will be induced; this is unavoidable. However, a cleaner injection minimizes the detachment and greatly improves the chances of reattachment (Figure 1E). Any exaggerated movements may move the needle back into the retina, and sideways movements can damage the retina. The use of an injection pump is optional but allows for a very precise delivery.
8. Retract the syringe slowly. Apply eye moisturizing drops to keep the eye hydrated.
9. Continue to monitor the animal until it regains sternal recumbency. Do not leave animal unattended or return to a cage with other alert animals until it regains sternal recumbency.

Results

We can deliver a suspension of RPE cells into the subretinal space of rodents quickly and consistently using the technique described in this manuscript. While not required, traumas can be greatly minimized using the setup shown with a micromanipulator in Figure 1A&B. Hold the rodent as shown in Figure 1C for temporary proptosis. The steps are the same if performed with the micromanipulator or by hand; these are depicted in the cartoon in Figure 1D. When performed cle...

Discussion

In this article we describe a relatively simple method for performing subretinal injections of RPE cells in suspension in rats and mice. The protocol is easy to learn and more experience with the technique will translate in fewer traumas (Figure 3; this represents one of the better injections), especially if a micromanipulator is used (Figure 1A). Any trauma can be monitored in vivo with a cSLO and OCT system (Figure 2) if available. If higher resolution and les...

Materials

Name	Company	Catalog Number	Comments
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 µg 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 G, 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 G, 1")	VWR	BD305128	Box x 1
TrypLE Express Enzyme (1X), no phenol red	Gibco	12604013	100 ml x 1