US20170362567A1

US20170362567A1 - Methods for storage and retrieval of functional mature retinal cells

Info

Publication number: US20170362567A1
Application number: US15/692,843
Authority: US
Inventors: Peter R. MACLEISH; Xiaoming Chen
Original assignee: Morehouse School of Medicine Inc
Current assignee: Morehouse School of Medicine Inc
Priority date: 2010-12-14
Filing date: 2017-08-31
Publication date: 2017-12-21
Also published as: US9777255B2; US20120148536A1

Abstract

A method for the isolation, storage and retrieval of mature retinal cells is disclosed. The Method is applicable to adult mammalian cone cells, and more particularly human cone cells, and to healthy as well as pathological or otherwise altered cone cells. A kit for the isolation, storage and retrieval of mature retinal cells is also described.

Description

This application is a Continuation of U.S. application Ser. No. 12/967,696, filed Dec. 14, 2010. The entirety of the aforementioned applications is incorporated herein by reference.

FIELD

The present application relates generally to the field of cell isolation and storage and, in particular, to the isolation, storage and retrieval of functional mature retinal cells.

BACKGROUND

Nearly 10 million Americans are blind or suffer visual impairment due to glaucoma, retinitis pigmentosa, age-related macular degeneration and diabetic retinopathies. These diseases are all due to the loss of one or more retinal cell type and according to the most recent statistics represent 36% of the existing cases of legal blindness in the United States. Every year an additional 230,000 patients are diagnosed with these diseases. Current treatments can slow disease progression, but cannot replace lost retinal cells.
The isolation of retinal neurons from the adult retina provided an opportunity to perform electrophysiological experiments that is virtually impossible in the intact retina. Transplantation of retinal cells, especially adult retinal stem cells, have been used in the treatment for diseases involving the loss of retinal neurons, such as glaucoma, retinitis pigmentosa, and age macular degeneration. However, successful use of adult retinal cells for research and transplantation has been impeded by the difficulty in propagating and maintaining these cells. Following isolation, retinal cells remain viable for only a couple of days thereby limiting the use of the majority of cells that were obtained as a result of the retinal dissociation. Therefore, there still exists a need for new methods for of isolating, storing and retrieving functional mature retinal cells.

SUMMARY

One aspect of the present invention relates a method for the isolation and storage of mature retinal cells. The method includes incubating freshly isolated retinal tissue with a retinal tissue digestion solution containing a protease, removing the retinal tissue digestion solution, triturating enzyme-treated retinal tissue in a retinal cell suspension medium to form a retinal cell suspension, adding fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) to the retinal cell suspension, and storing the retinal cell suspension in liquid nitrogen.
In an embodiment, the retinal cells are selected from the group consisting of cone cells, rod bipolar cells, rod cells, ganglion cells, glial cell and mixtures thereof.
In another embodiment, the retinal tissue is retina or retinal pigmented epithelium.
In another embodiment, the method further comprises dissecting the isolated retinal tissue into small fragments.
In another embodiment, the protease is selected from the group consisting of trypsin, chymotrypsin, pepsin, papain, elastase A and mixtures thereof.
In one embodiment, the protease is papain.
In another embodiment, the tissue digestion solution further contains a collagenase.
In another embodiment, the tissue digestion solution further contains an enzyme activator.
In one embodiment, the enzyme activator is cysteine.
In another embodiment, the tissue digestion solution comprises NaCl, NaHCO₃, sodium pyruvate, KCl, NaH₂PO₄and CaCl₂.
In one embodiment, the tissue digestion solution comprises 114 mM NaCl, 25 mM NaHCO₃, 1 mM soduim pyruvate, 3 mM KCl, 0.5 mM NaH₂PO₄, 0.5 mM CaCl₂, and 0.02 mM Phenol red.
In another embodiment, the tissue digestion solution has a pH of 7.2-7.3.
In another embodiment, the freshly isolated retinal tissue is incubated with a retinal tissue digestion solution containing a protease at a temperature between about 18° C. and 42° C. for about 15-90 min.
In another embodiment, the freshly isolated retinal tissue is incubated with a retinal tissue digestion solution containing a protease at room temperature for about 30-40 min.
In another embodiment, the method further comprises thawing stored retinal cells; and mixing thawed cells with a retinal cell culture medium.
In a related embodiment, the retinal cell culture medium comprises a growth factor selected from the group consisting of human epidermal growth factor, human fibroblast growth factor-2 and platelet-derived growth factor.
Another aspect of the present invention relates to a method for treating retinal dysfunction in a subject. The method includes introducing into an eye of the subject an effective amount of retinal cells stored and retrieved using the method of the present invention.
In one embodiment, the retinal dysfunction is selected from the group consisting of photoreceptor degeneration, retinal detachment, retinal trauma, photic lesions caused by laser or sunlight; a macular hole, macular edema; night blindness and color blindness, ischemic retinopathy, and inflammatory conditions.
In another embodiment, the retinal cells are introduced into said eye with a substance that stimulate differentiation of neuroretina-derived stem cells into photoreceptor cells.
Another aspect of the present invention relates to a kit for isolating, storing and retrieving retinal cells. The kit includes a retinal cell digestion solution; a retinal cell suspension medium; a retinal cell culture medium, and instructions on how to isolate, store and retrieve retinal cells.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a flow chart showing a method for isolating, storing and retrieving retina cells.

FIG. 2 is a picture of a retinal cone cell recovered from frozen cell suspension.

FIG. 3 is a picture of a freshly isolated retinal cone cell.

FIG. 4 is a diagram showing the average I_Depvalue measured in freshly isolated retinal cone cells and retinal cone cell recovered from frozen cell suspension.

FIG. 5 is a diagram showing the average I_hvalue measured in freshly isolated retinal cone cells and retinal cone cell recovered from frozen cell suspension.

FIG. 6 is a diagram showing the average I_ClCavalue measured in freshly isolated retinal cone cells and retinal cone cell recovered from frozen cell suspension.

DETAILED DESCRIPTION

The practice of the embodiments described in further detail below will employ, unless other wise indicated, conventional methods cell biology, molecular biology, biochemistry, immunology and ophthalmology within the skill of the art. Such techniques are explained fully in the literature. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Method for Harvesting, Storing and Retrieving Retinal Cells

One aspect of the present invention relates to a method for the isolation, storage and retrieval of mature retinal cells. The method 100 (FIG. 1) comprises the steps of incubating (110) a freshly isolated retinal tissue with a retinal tissue digestion solution containing a protease, removing (120) the retinal tissue digestion solution, triturating (130) the digested retinal tissue in a retinal cell suspension solution to form a retinal cell suspension, adding (140) fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) to the retinal cell suspension, storing (150) the cell suspension in liquid nitrogen, and thawing (160) stored cells and mixing thawed cells with a retinal cell culture medium.

Retinal Tissue

The term “retinal tissue” includes the retina or retinal pigmented epithelium. The retinal tissue can be a mammalian retinal tissue (primate or non-primate retinal tissue), e.g. a human retinal tissue, or an animal but non-human retinal tissue such as pig, rat, mouse retinal tissue. The retinal tissue can be a healthy tissue or a pathologic tissue, e.g. a retinal tissue undergoes or has undergone photoreceptor degeneration (cone and/or rod dysfunction). Isolation and storage of diseased retinal cells allows for screening for compounds capable of showing a protective and/or anti-disease effect on retinal cells, as well as genomics and proteomics applications, e.g,. identifying genes and proteins that are specific or characteristic of the diseased retinal cells compared to healthy retinal cells (and vice versa).
The pathologic retinal tissue can originate from an individual afflicted with an inherited or acquired disease involving photoreceptor degeneration, such as retinitis piginentosa and age macular degeneration, or other maculopathies. The freshly isolated retinal tissue may optionally be dissected into small fragments (e.g., 1-2 mm by 1-2 mm, 2-3 mm by 2-3 mm or 3-5 mm by 3-5 mm), preferably under conditions non deleterious to retinal cell biology, i.e. under conditions that do not substantially impair the growth potential of the retinal cells, for example, in cold CO₂-independent medium.

Retinal Cell Digestion Solution

The retinal cell digestion solution contains one or more a proteolytie enzymes. Examples of the proteolytic enzymes include, but are not limited to, trypsin, chymotrypsin, pepsin, papain, elastase A and mixtures thereof. skilled person can adjust the dissociation conditions, such as temperature, enzyme concentration, and dissociation duration, as a function of the enzyme(s) used and of the retinal tissue to which it is applied. Illustrative conditions are mentioned in the Example below. The skilled person may also use an enzyme activator, such as cysteine, if desired. In certain embodiments, the retinal cell digestion solution further contains a collogenase.
The retinal cell digestion solution also contains a buffer and/or at least one salt or a combination of salts. In some embodiments, the pH of the retinal cell digestion solution ranges from about 5 to about 8, from about 6 to 8, or from about 7 to about 7.5. A variety of pH buffers may be used to achieve the desired pH. Suitable buffers include, but are not limited to, Tris, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Tris propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, HEPPSO, POPSO, TEA, HEPPS, Tricine, Gly-Gly, Bicine, and a phosphate buffer (e.g., sodium phosphate or sodium-potassium phosphate, among others). The retinal cell digestion solution may comprise from about 10 mM to about 100 mM buffer, about 25 mM to about 75 mM buffer, or from about 40 mM to about 60 mM buffer, among others. The type and amount of the buffer used in the retinal cell digestion solution can vary from application to application. In some embodiments, the retinal cell digestion solution has a pH of about 7.4, which can be achieved using about 50 mM Tris buffer. In another embodiment, the retinal cell digestion solution has a pH of about 7.2-7.3.
In one embodiment, the retinal cell digestion solution contains NaCl, NaHCO₃, sodium pyruvate, KCl, NaH₂PO₄and CaCl₂. In another embodiment, the retinal cell digestion solution contains: 114 mM NaCl, 25 mM NaHCO₃, 1 mM soduim pyruvate, 3 mM KCl, 0.5 mM NaH₂PO₄, 0.5 mM CaCl₂, and 0.02 mM Phenol red. In another embodiment, the retinal cell digestion solution is prepared by adding 3 mg cysteine HCl, 20 mg glucose, 10-14 units/ml papain into 7 ml salt solution, bubbling solution with 5% CO2 and 95% O₂for 10 min to reach pH 7.2-7.3.

Digestion Conditions

The freshly isolated tissue is treated with the retinal cell digestion solution to enzymatically digest the extracellular matrix of the retinal tissue or retinal tissue fragments, under conditions to dissociate the retinal cells from each other without substantially altering their cellular integrity, i.e. without lysing the retinal cells.
In certain embodiments, the freshly isolated retinal tissue is incubated at a temperature between 18° C. and 42° C. for a period of 15-90 minutes. In one embodiment, the freshly isolated retinal tissue is incubated in the retinal tissue digestion solution at room temperature for 30-40 minutes. After digestion, the retinal tissue digestion solution may be removed by aspiration. The digested retinal tissue is washed once or twice with the retinal cell storage medium and is triturated in 10-100 volumes of the retinal cell storage medium (i.e., if the original retinal tissue has a volume of 0.1 cm³, the digested retinal tissue will be suspended in 1-10 ml of the retinal cell storage medium) to form a retinal cell suspension. The cell population obtained by the present invention typically comprises cone cells, rod bipolar cells, rod cells, ganglion cells and filial cells.

Retinal Cell Suspension Medium and Retinal Cell Storage Medium

The retinal cell suspension medium is selected as being suitable for retinal cell storage and survival. Suitable medium include, but are not limited to, DMEM/F12 medium (available e.g. from Invitrogen), RPM11640 (available e.g. from Invitrogen), Ames (available e.g. from Sigma), NBA™ (Neurobasal Medium, available from Invitrogen) and X-VIVO 15 serum-free medium.
The retinal cell storage medium is retinal cell suspension medium with 10% serum and 10% DMSO.
In one embodiment, the retinal cell suspension medium comprises 139 mM NaCl, 2 mM HEPES, 1 mM sodium pyruvate, 0.5 mM MgCl₂, 0.5 mM MgSO₄, 0.5 mM NaH₂PO₄, 1.8 mM CaCl₂, 3 mM KCl, 1 mM NaHCO₃, 0.1 mM Choline Cl, 0.02 mM Phenol red, 15 mM glucose, with 1 N NaOH added to adjust to pH 7.2-7.3. The retinal cell storage medium is prepared by adding 10% fetal bovine serum(FBS) and 10% DMSO to the above-described retinal cell suspension medium.

Storage Conditions

The retinal cell suspension is stored in the presence of fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO). In certain embodiments, the retinal cells are suspended in the retinal cell suspension medium. FBS and DMSO are then added to the cell suspension to a final concentration of 5-20% (v/v) each, preferably 10% (v/v) each (e.g., 10 ml FBS and 10 ml DMSO for 80 ml cell suspension). In other embodiments, the retinal cells are suspended directly in the retinal cell storage medium.
The cell suspension is then aliquoted and stored in liquid nitrogen. In one embodiment, the aliquots are stored at −80° C. overnight and then transferred to liquid nitrogen. In another embodiment, the aliquots are snap-frozen in dry ice, stored at −80° C. overnight, and then transferred to liquid nitrogen. In yet another embodiment, the aliquots are snap-frozen in dry ice and then transferred to liquid nitrogen.

Retrieval Conditions

To preserve viability, the frozen retinal cells are thawed quickly in a water bath at 37° C. The thawed cells are washed with a warm retinal cell culture medium to remove DMSO and cultured in an incubator with 5% CO₂at 37° C. In one embodiment, the thawed cells are mixed gently with 10-20 fold volume of the retinal cell culture medium and centrifuged at 100 xg for 3 min. The supernatant is discarded. The pellet is resuspended in the retinal cell culture medium.

Retinal Cell Culture Medium

In one embodiment, the retinal cell storage medium contains a serum-free culture medium; at least one growth factor at a final concentration of about 0.1 ng/mL to about 40 ng/mL, preferably 10 ng/mL to about 30 ng/mL, at least one neural supplement at a final concentration of about 0.1% v/v to about 10% v/v, preferably about 0.5% v/v to about 3% v/v of the retinal cell storage medium, and optionally a heat-inactivated serum comprising from about 0.1% v/v to about 20% v/v, preferably from about 5% v/v to about 15% v/v of the retinal cell storage medium.
As used herein, the term “% v/v” refers percentage by volume. For example, 1% v/v is equivalent to 1 part volume per every 100 part volume of the retinal cell culture medium.
In one embodiment, the retina cell culture medium contains NaCl, HEPES, sodium pyruvate, MgCl₂, MgSO₄, NaH₂PO₄, CaCl₂, KCl, NaHCO₃, and Choline Cl, In another embodiment, the retina cell culture medium contains 139 mM NaCl, 2 mM HEPES, 1 mM sodium pyruvate, 0.5 mM MgCl₂, 0.5 mM MgSO₄, 0.5 mM NaH₂PO₄, 1.8 mM CaCl₂, 3 mM KCl, 1 mM NaHCO₃, 0.1 mM Choline Cl, 0.02 mM Phenol red, and 15 mM glucose, adjusted to pH 7.2-7.3 with 1 N NaOH. In another embodiment, the retina cell culture medium further contains 0.01% BSA for short term culture.
The retinal cell culture medium may further contain a growth factor, a neural supplement, glutamine and/or serum.
In some embodiments, the growth factor is recombinant human epidermal growth factor (hrEGF), recombinant human fibroblast growth factor-2 (hrFGF-2), or platelet-derived growth factor (PDGF). Recombinant human, fibroblast growth factor-2 (hrFGF-2) is also known as basic fibroblast growth factor (bFGF). In some embodiments, the first growth factor is recombinant human basic fibroblast growth factor-2 (hrbFGF-2).
In some embodiments, the neural supplement is N-2 supplement (Gibco, Invitrogen) or B-27 supplement (Gibco, Invitrogen). N-2 supplement contains 500 μg/ml insulin, 10 mg/ml h-transferrin, 0.63 μg/ml progesterone, 1.611 mg/ml putrascine, and 0.52 μg/ml selenite.
In some other embodiments, the neural supplement comprises insulin at about 100 μg/ml to about 1000 μg/ml of the culture medium; h-transferrin at about 0.1 mg/ml to about 100 mg/ml of the culture medium; progesterone at about 0.1 μg/ml to about 10 μg/ml of the culture medium; putrascine at about 0.1 mg/ml to about 10 mg/ml of the culture medium; and selenite at about 0.01 μg/ml to about 10 μg/ml of the culture medium.
In some other embodiments, the neural supplement comprises insulin at about 300 μg/ml to about 700 μg/ml of the culture medium; h-transferrin at about 5 mg/ml to about 15 mg/ml of the culture medium; progesterone at about 0.3 μg/ml to about 0.9 μg/ml of the culture medium; putrascine at about 1 mg/ml to about 2 mg/ml of the culture medium; and selenite at about 0.2 μg/ml to about 1 μg/ml of the culture medium.
In certain embodiments, the retinal cell culture medium is a preconditioned culture medium that is adapted to the in vitro culture of cone photoreceptor cells. Briefly, the culture medium is pre-conditioned by culturing Muller glial cells in a culture medium that is suitable for cone survival, and collecting the resulting culture medium. In one embodiment, the pre-conditioned culture medium is prepared by culturing Muller glial cells in NBATM culture medium, optionally supplemented by B27 and glutamine, or DMEM/F12 medium, or Ames medium. The conditioned medium is typically collected at day 1 or 2 of the Muller glial cell culture on pre-conditioned medium.

Method for Treating Retinal Dysfunction

Another aspect of the present invention provides a method for treating retinal dysfunction in a mammal. The stored retinal cells can be used to treat a mammalian recipient suffering from a lack or diminution of photoreceptor cell function. Examples of retinal dysfunction that can be treated by the stored retinal cells include, but are not limited to: photoreceptor degeneration (as occurs in, e.g., hereditary or acquired retinitis pigmentosa, cone dystrophies, cone-rod and/or rod-cone dystrophies, and macular degeneration, including age-related and early onset macular degeneration); retinal detachment and retinal trauma; photic lesions caused by laser or sunlight; a macular hole; a macular edema; night blindness and color blindness; ischemic retinopathy as caused by diabetes or vascular occlusion; retinopathy due to prematurity/premature birth; infectious conditions, such as, e.g., CMV (cytomegalovirus) retinitis, herpes type 1 retinitis, Ebstein-Barr virus retinitis, toxoplasmosis, rubella and pox virus; inflammatory conditions, such as the uveitidies, multifocal choroiditis and uveitis, birdshot chorioretinopathy, collagen vascular diseases affecting the posterior segment of the eye, including Wegener's granulomatosis, uveitis associated with systemic lupus erythematosus, uveitis associated with polyarteritis nodosa, peripheral or intermediate uveitis, chronic central serous chorioretinopathy, and myopic choroidal neovascular membranes and scars. Inflammatory disorders also include Behcet syndrome, intermediate uveitis (pars planitis), masquerade syndromes, peripheral uveitis, ocular syphilis, ocular tuberculosis, viral-related chorioretinitis (ARN) syndrome, HIV-related uveitis, progressive outer retinal necrosis syndrome, sympathetic ophthalmia, white dot syndromes, presumed ocular histoplasmosis syndrome, acute macular neuroretinopathy, diffuse unilateral subacute neuroretinitis, ophthalmomyiasis, serpiginous choroidopathy, panuveitis, birdshot retinochoroidopathy, and uveitis associated with disorders such as juvenile rheumatoid arthritis, Kawasaki syndrome, multiple sclerosis, sarcoidosis, toxocariasis, toxoplasmosis, Vogt-Koyanagi-Harada (VKH), and HLA-B27 seropositive spondylopathy syndromes.
Other disorders include tumors, such as retinoblastoma and ocular melanoma. Additionally, stored retinal cells can be used for replacement of inner retinal neurons, which are affected in ocular neuropathies including glaucoma, traumatic optic neuropathy, degenerative optic neuropathy, ischemic optic neuropathy, optic neuropathy from multiple sclerosis, and radiation optic neuropathy and retinopathy.
The methods can also be used to treat optic nerve diseases such as optic atrophy, ischemic optic neuropathy, diabetes induced optic atrophy, optic nerve hypoplasia, morning glory syndrome, Graves ophthalmopathy, optic neuritis, cytomegalovirus neuritis, arteritic optic neuropathy, compressive neuropathy, diabetic neuropathy, giant cell arteritis, infiltrative neuropathy, nutriotional, ischemic neuropathy, retrobulbar optic neuritis, retrobulbar ischemic neuropathy, toxic neuropathy, traumatic neuropathy; optic nerve diseases resulting from causes such as syphilis, Lyme disease, toxoplasmosis, cat scratch disease, systemic lupus erythematosus, paraneoplastic syndrome, multiple sclerosis, and autoimmune disease; degenerative optic diseases such as age-related macular degeneration, early onset macular degeneration, Usher Syndrome, retinitis pigmentosa, cone-road dystrophy, and choroideremia; and congenital optical diseases such as Leber's congential amaurosis, congential stationary night blindness, and optic nerve hypoplasia. One of skill in the art will recognize that there is overlap between the various classifications of the disorders and conditions listed herein.
In some embodiments, the retinal dysfunction is a result of photoreceptor degeneration, retinal detachment, retinal trauma, a photic lesion, a macular hole, a macular edema, night blindness, color blindness, ischemic retinopathy, retinopathy due to premature birth, infection, inflammatory condition, or an ocular neuropathy. In some embodiments, the retinal dysfunction is a result of a tumor, a degenerative optic disease, or a congenital optical disease.
In some embodiments, the retinal dysfunction is a result of an ocular neuropathy. In some embodiments, the optic neuropathy is glaucoma, traumatic optic neuropathy, degenerative optic neuropathy, ischemic optic neuropathy, optic neuropathy from multiple sclerosis, or radiation optic neuropathy, or retinopathy.
In some embodiments, the retinal dysfunction is the result of multifocal choroiditis, birdshot chorioretinopathy, collagen vascular diseases affecting the posterior segment of the eye, Wegener's granulomatosis, peripheral uveitis, intermediate uveitis, chronic central serous chorioretinopathy, myopic choroidal neovascular membranes, myopic choroidal neovascular membranes scars, Behcet syndrome, a masquerade syndrome, ocular syphilis, ocular tuberculosis, viral-related chorioretinitis (ARN) syndrome, HIV-related uveitis, progressive outer retinal necrosis syndrome, sympathetic ophthalmia, a white dot syndrome, presumed ocular histoplasmosis syndrome, acute macular neuroretinopathy, diffuse unilateral subacute neuroretinitis, ophthalmomyiasis, serpiginous choroidopathy, panuveitis, birdshot retinochoroidopathy, uveitis associated with juvenile rheumatoid arthritis, uveitis associated with Kawasaki syndrome, uveitis associated with multiple sclerosis, uveitis associated with sarcoidosis, uveitis associated with toxocariasis, uveitis associated with toxoplasmosis, uveitis associated with systemic lupus erythematosus, uveitis associated with polyarteritis nodosa, uveitis associated with Vogt-Koyanagi-Harada, or uveitis associated with a HLA-B27 seropositive spondylopathy syndrome.
In some embodiments, the retinal dysfunction is the result of optic atrophy, ischemic optic neuropathy, diabetes induced optic atrophy, optic nerve hypoplasia, morning glory syndrome, Graves ophthalmopathy, optic neuritis, cytomegalovirus neuritis, arteritic optic neuropathy, compressive neuropathy, diabetic neuropathy, giant cell arteritis, infiltrative neuropathy, nutriotional, ischemic neuropathy, retrobulbar optic neuritis, retrobulbar ischemic neuropathy, toxic neuropathy, or traumatic neuropathy.
In some embodiments, the retinal dysfunction is the result of an optic nerve disease associated with syphilis, Lyme disease, toxoplasmosis, cat scratch disease, systemic lupus erythematosus, paraneoplastic syndrome, multiple sclerosis, or autoimmune disease.
In some embodiments, the retinal dysfunction is the result of age-related macular degeneration, early onset macular degeneration, Usher Syndrome, retinitis pigmentosa, choroideremia, cone dystrophy, cone-rod dystrophy, rod-cone dystrophy, Leber's congential amaurosis, congential stationary night blindness, Sticklers Syndrome, colobomas, vitreoretinal dysplasia, achromatopsia, or optic nerve hypoplasia.
In some embodiments, the inflammatory condition is multifocal choroiditis, birdshot chorioretinopathy, collagen vascular diseases affecting the posterior segment of the eye, Wegener's granulomatosis, peripheral uveitis, intermediate uveitis, chronic central serous chorioretinopathy, myopic choroidal neovascular membranes, myopic choroidal neovascular membranes scars, Behcet syndrome, a masquerade syndrome, ocular syphilis, ocular tuberculosis, viral-related chorioretinitis (ARN) syndrome, HIV-related uveitis, progressive outer retinal necrosis syndrome, sympathetic ophthalmia, a white dot syndrome, presumed ocular histoplasmosis syndrome, acute macular neuroretinopathy, diffuse unilateral subacute neuroretinitis, ophthalmomyiasis, serpiginous choroidopathy, panuveitis, or birdshot retinochoroidopathy.
In some embodiments, the inflammatory condition is uveitis associated with a disorder selected from the group consisting of juvenile rheumatoid arthritis, Kawasaki syndrome, multiple sclerosis, sarcoidosis, toxocariasis, toxoplasmosis, systemic lupus erythematosus, polyarteritis nodosa, Vogt-Koyanagi-Harada (VKH), or a HLA-B27 seropositive spondylopathy syndrome.
In some embodiments, the infection is cytomegalovirus retinitis, herpes type I retinitis, Ebstein-Barr virus retinitis, toxoplasmosis, rubella, or pox virus.
In some embodiments, the optic nerve disease is optic atrophy, ischemic optic neuropathy, diabetes induced optic atrophy, optic nerve hypoplasia, morning glory syndrome, Graves ophthalmopathy, optic neuritis, cytomegalovirus neuritis, arteritic optic neuropathy, compressive neuropathy, diabetic neuropathy, giant cell arteritis, infiltrative neuropathy, nutriotional, ischemic neuropathy, retrobulbar optic neuritis, retrobulbar ischemic neuropathy, toxic neuropathy, or traumatic neuropathy.
In some embodiments, the optic nerve disease results from a cause selected from the group consisting of syphilis, Lyme disease, toxoplasmosis, cat scratch disease, systemic lupus erythematosus, paraneoplastic syndrome, multiple sclerosis, and autoimmune disease.
In some embodiments, the degenerative optic disease is the result of age-related macular degeneration, early onset macular degeneration, Usher Syndrome, retinitis pigmentosa, cone-road dystrophy, or choroideremia.
In some embodiments, the congential optic disease is Leber's congential amaurosis, congential stationary night blindness, or optic nerve hypoplasia.
In some embodiments, the tumor is retinoblastoma or ocular melanoma.
In some embodiments, the dystrophic eye is the result of glaucoma.
In using the stored retinal cells to treat retinal dysfunction, one can, in conjunction with introducing the retinal cells into a recipient's eye, administer a substance that stimulates differentiation of the neuroretina-derived stem cells in the stored retinal cells into photoreceptors cells or other retinal cell types (e.g., bipolar cells, ganglion cells, horizontal cells, amacrine cells, Mueller cells). When stored retinal cells are introduced to treat a neural dysfunction of the eye, one can also utilize a substance (or combination of substances) that stimulates differentiation of the neuroretina-derived stem cells into neurons, astrocytes, or oligodendrocytes.

Kits

Also encompassed by the present invention are kits for isolating, storing and retrieving retinal cells. The kit comprises a protease, a retinal tissue digestion solution, and a retinal cell storage medium. In certain embodiments, the kit may further include a retinal cell culture medium. In certain other embodiment, the kit may further include a sterile container and a harvesting solution for harvesting retinal tissue. The harvesting solution allows the survival of the retinal tissue until later dissociation of cells from the tissue.
The harvesting solution may be any solution suitable for preserving ocular tissue. The harvesting solution may be a commercially available solution, or may be separately prepared from a serum-free tissue culture medium. Any serum-free tissue culture medium suitable for preserving ocular tissue may be used in the kits of the invention.
In some embodiments, the harvesting solution is a solution suitable for preserving corneal tissue. In some embodiments, the harvesting solution is Optisol.
In some embodiments, the harvesting solution is X-VIVO serum-free medium.
In some embodiments, the harvesting comprises an antibiotic component. As used herein, the term “antibiotic component” refers to a single antibiotic or a combination of two or more antibiotics. In some embodiments, the antibiotic combination comprises penicillin, streptomycin, or gentamicin, or a combination thereof. In some embodiments, the antibiotic component comprises penicillin and streptomycin. In some embodiments, the antibiotic component comprises pencillin and gentamicin. In some embodiments, the harvesting solution comprises X-VIVO serum-free medium and an antibiotic component comprising penicillin, streptomycin, or gentamicin or a combination thereof.
In some embodiments, the harvesting solution comprises three times the normal dosage of antibiotics. In some embodiments, the harvesting solution comprises 300 IU/ml of a combination of penicillin and streptomycin, such as that available from Gibco, Invitrogen. In some embodiments, the harvesting solution comprises 300 IU/ml of penicillin and 150 μg/ml of gentamicin. As used herein, the abbreviation “IU/ml” refers to international units of the antibiotic per ml of harvesting solution.
In some embodiments, the kit allows the survival of the tissue for up to about 7 days. In some embodiments, the kit allows the survival of the tissue for up to about 4 days. In some embodiments, the kit allows the survival of the tissue for up to about 2 days. As used herein, the term “survival” indicates that the condition of the tissue is such that retinal cells can still be isolated from the tissue.
Any sterile container can be used with the kits of the invention, including sterile vials and ampoules. In some embodiments, the sterile container comprises two or more collection vessels. In some embodiments, the two or more collection vessels are separate sterile compartments within one container, allowing the preservation of different samples of extra-ocular tissue in each collection vessel with its own supply of harvesting solution.
Certain features of the invention which are, for clarity, described herein in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.
The present invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and Tables, are incorporated herein by reference.

EXAMPLE 1

Isolation. Storage and Retrieval of Monkey Retina Cft,t,s

Isolation

Fresh monkey eyeballs were obtained from Yerkes National Primate Research Center, Atlanta,Ga. A piece of retina tissue (5 mm×5 mm) was removed from the fovea area, transferred to a 15 ml conical tube with 7 ml of retinal tissue digestion solution containing: 114 mM NaCl, 25 mM NaHCO₃, 1 mM sodium pyruvate, 3 mM KCl, 0.5 mM NaH₂PO₄, 0.5 mM CaCl₂, 0.02 mM phenol red. In 7 ml solution, add 3 mg cysteine HCl, 20 mg glucose, 10-14 units/ml of papain. Solution is then bubbled with 5% CO2 and 95% 02 until the pH is 7.2-7.3. Tissue is incubated with enzyme solution at room temperature for 30-40 minutes. After incubation, the retinal tissue digestion solution was carefully removed by aspiration. The digested retina tissue was rinsed twice with a retinal cell suspension medium containing: 139 mM NaCl, 2 mM HEPES, 1 mM sodium pyruvate, 0.5 mM MgCl₂, 0.5 mM MgSO₄, 0.5 mM NaH₂PO₄, 1.8 mM CaCl₂, 3 mM KCl, 1 mM NaHCO₃, 0.1 mM Choline Cl, 0.02 mM Phenol red, 15 mM glucose, with 1 N NaOH added to adjust to pH 7.2-7.3, and triturated gently in 6-14 ml of retinal cell storage medium using a 5 ml glass serological pipet to form a cell suspension containing mostly single cells.

Storage

FBS and DMSO were added to the retinal cell suspension to a final concentration of 10% (v/v) each. The cell suspension was mixed gently with FBS and DMSO, and 1 ml was aliquoted into cyroprotective vials at a cell densityof 10⁴-10⁶. The cyroprotective vials were bathed in isopropyl alcohol and placed in the −80° C. freezer, in order to cool the vials at a rate of approximately 1° C. per minute. After cooling in isopropyl alcohol, the vials were placed in a −80° C. freezer overnight. The frozen vials were then placed in a liquid nitrogen freezer in designated boxes.

Retrieval

A vial of frozen retinal cells was thawed quickly in a 37° C. water bath,. The thawed cells (1 ml) were mixed gently with 10 ml warm (e.g., 37° C.) retinal cell culture medium: 139 mM NaCl, 2 mM HEPES, 1 mM sodium pyruvate, 0.5 mM MgCl₂, 0.5 mM MgSO₄, 0.5 mM NaH₂PO₄, 1.8mM CaCl₂, 3 mM KCl, 1 mM NaHCO₃, 0.1 mM Choline Cl, 0,02 mM Phenol red, 15 mM glucose, add 1 N NaOH to adjust pH 7.2-7.3 and spun at 100 xg for 3 min. The supernatant was removed and the cell pellet was gently resuspended in 1-2 ml retinal cell culture medium. A few drops of the suspended retinal cells were plated in a coated cell culture dish that was coated with an antibody, 9B5, that labeled living retinal cells. The cells were allowed to settle down for a few minute, then covered with more retinal cell culture medium, and incubated in the incubator at 37° C. with 5% CO₂.

EXAMPLE 2

Patch Clamp Analysis of Freshly Isolated and Frozen/Thawed Retinal Cone Cells

Freshly isolated monkey retinal cone cells and retinal cone cells recovered from frozen retinal cells (stored for at least 6 months and at −170° C.) were subjected to patch clamp analysis. As shown in FIGS. 2 and 3, the retinal cone cell recovered from the frozen cell suspension (FIG. 2) is morphologically indistinguishable from the freshly isolated retinal cone cell (FIG. 3).
Tables 1 and 2 show individual I_Depmeasurements in fresh retinal cone cells (Table 1) and recovered frozen retinal cone cells (Table 2). As shown in FIG. 4 and Table 3, there is no statistically significant difference (at p=0.05 level) in the I_Depmeasurements between the freshly isolated retinal cone cells and the cone cells recovered from the frozen stock.
Tables 4 and 5 show individual I_hmeasurements in fresh retinal cone cells (Table 4) and recovered frozen retinal cone cells (Table 5). As shown in FIG. 5 and Table 6, there is no statistically significant difference (at p=0.05 level) in the I_hmeasurements between the freshly isolated retinal cone cells and the cone cells recovered from the frozen stock.
Tables 7 and 8 show individual I_Clcameasurements in fresh retinal cone cells (Table 7) and recovered frozen retinal cone cells (Table 8). As shown in FIG. 6 and Table 9, there is a statistically significant difference (at p=0.05 level) in the Icica measurements between the freshly isolated retinal cone cells and the cone cells recovered from the frozen stock.
Whole-cell recording: Membrane currents were recorded in the whole-cell mode of the patch-clamp technique using an Axopatch 200 B amplifier (Axon Instruments). Dishes were mounted on a warmed (30° C.) stage of a Zeiss Axiovert 200 M microscope equipped with phase-contrast optics. Patch-clamp pipettes were pulled on a Flaming/Brown P-97 (Sutter Instruments Co. Novato, Calif.) electrode puller and had a tip diameter of about 1 μm and a resistance of approximately 6-10 MQ. Membrane rupture was achieved by gentle suction applied to the inside of the pipette following seal formation. For most experiments, the composition of the pipette solution was as follows: 139 mM KCl, 10 mM NaCl, 10 mM HEPES, 0.05 mM EGTA, 5 mM MgCl₂, and 1 mM ATP. The pH was adjusted to 7.0-7.2. Data acquisition and analysis were carried out using pCLAMP software version 6.0.3 or 9.2 (Axon Instruments, Inc., Union City, Calif.).
I_Depis the total voltage current recorded when the membrane is depolarized from a holding potential of −70 mV. A number of different currents contribute to I_Depand the relative contributions differ depending on the magnitude of the voltage during the step. The individual currents are: a voltage-gated calcium current, calcium-activated chloride current, voltage-gated potassium currents. The presence of this net current indicates that the cell is alive and that the cell membrane contains active ionic conductances.
I_his a characteristic conductance found on a variety of cells including photoreceptors. It is activated by hyperpolarization and has relatively slow onset kinetics. The conductance activates at voltages negative to the holding potential of −70 mV. The presence of I_hindicated that the cell has the expected properties of healthy photoreceptors.
I_Clcais a current that is activated when levels of calcium rise in the cell and activate a conductance that is sensitive to high levels of intracellular calcium. In our experiments shown here, calcium entry is achieved by opening voltage-gated calcium channels by depolarization for a brief amount of time. When the membrane potential is returned to the holding level of −70 mV, calcium entry is shut off and the high levels of calcium inside the cell are reduced by restorative mechanisms in the cell. The inward current seen after the voltage step is referred to as a tail current and its magnitude is controlled by the concentration of free calcium inside the cell. The size of the tail current and the time course of restoration are properties associated with healthy cells.
The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following embodiments. The embodiments are intended to cover the components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

TABLE 1

Fresh Monkey Cone I_h

				Measured			Frozen/
Number	Date	Trace number	Cell #	voltage(mV)	Current I_h(pA)	Fresh	Thawed

1	Jul. 10, 2009	000	1	−120	−317.4	x
2	Aug. 8, 2008	005	1	−120	−372	x
3	Aug. 8, 2008	029	3	−120	−306.1	x
4	Aug. 8, 2008	039	4	−120	−363.5	x
5	Aug. 8, 2008	074	9	−120	−118	x
6	Aug. 8, 2008	078	10	−120	−322.3	x
7	Aug. 8, 2008	091	12	−120	−277.1	x
8	Apr. 14, 2009	001	1	−120	−365	x
9	Apr. 14, 2009	007	2	−120	−458	x
10	Apr. 15, 2009	002	1	−120	−419.6	x
11	Apr. 15, 2009	010	2	−120	−532	x
12	Apr. 15, 2009	024	5	−120	−535	x
13	Apr. 15, 2009	034	7	−120	−610.7	x
14	Apr. 16, 2009	013	4	−120	−375	x
15	Apr. 16, 2009	019	5	−120	−248.1	x
16	Apr. 16, 2009	042	9	−120	−326.2	x

TABLE 2

Frozen/Thawed Monkey Cone I_h

1	Jun. 2, 2009	000	1	−120	−117.8	x
2	Jun. 2, 2009	007	2	−120	−589	x
3	Jun. 2, 2009	013	4	−120	−735	x
4	Jun. 3, 2009	001	1	−120	−324	x
5	Jun. 3, 2009	019	3	−120	−171.8	x
6	Jun. 3, 2009	046	4	−120	−122.7	x
7	Jun. 3, 2009	073	5	−120	−302.7	x
8	Jun. 9, 2009	001	1	−120	−149.2	x
9	Jun. 9, 2009	008	3	−120	−402.2	x
10	Jun. 9, 2009	023	4	−120	−265.8	x
11	Jun. 10, 2009	003	3	−120	−236.2	x
12	Jun. 10, 2009	022	4	−120	−311.9	x
13	Jun. 10, 2009	062	8	−120	−204.8	x
14	Jun. 23, 2009	002	2	−120	−499	x
15	Jun. 24, 2009	055	13	−120	−430.6	x
16	Jun. 24, 2009	066	14	−120	−447.4	x
17	Jun. 24, 2009	080	16	−120	−276.8	x
18	Jun. 24, 2009	094	17	−120	−351.9	x
19	Jun. 24, 2009	113	19	−120	−296.6	x
20	Jun. 25, 2009	006	3	−120	−294.8	x
21	Jun. 25, 2009	018	6	−120	−296.3	x
22	Jun. 25, 2009	025	8	−120	−218.2	x
23	Jun. 25, 2009	031	9	−120	−150	x
24	Jun. 25, 2009	038	10	−120	−283.2	x
25	Jun. 25, 2009	045	12	−120	−286.3	x
26	Jun. 25, 2009	051	14	−120	−237.1	x
27	Jun. 25, 2009	057	15	−120	−265.2	x
28	Jun. 25, 2009	067	17	−120	−339.4	x
29	Jun. 25, 2009	074	18	−120	−259.4	x
30	Jun. 25, 2009	104	25	−120	−298.5	x
31	Jun. 25, 2009	110	26	−120	−295.4	x
32	Jun. 25, 2009	115	27	−120	−170.9	x
33	Jun. 25, 2009	121	29	−120	−213.6	x

TABLE 3

At the 0.05 level,
the population means are not significantly different.
Means Comparison using Tukey Test

		Simultaneous
	Difference	Confidence Intervals	Significant

		between		Upper	at 0.05
Dataset	Mean	Means	Lower Limit	Limit	Level

Data8_A	967.31667
Data8_B	967.89167	−0.575	−160.37786	159.22786	No

Power Analysis

	Total
Alpha	Sample Size	Power

0.05	54	0.05001 (actual)

TABLE 4

Fresh Monkey Cone I_Dep

				Meas-
				ured
Num-		Trace	Cell	voltage	Current		Frozen/
ber	Date	number	#	(mV)	I_Dep(pA)	Fresh	Thawed

1	Jul. 10, 2009	003	1	50	833	x
2	Aug. 8, 2008	002	1	50	945.1	x
3	Aug. 8, 2008	024	3	50	1111.3	x
4	Aug. 8, 2008	038	4	50	671.7	x
5	Aug. 8, 2008	069	8	50	574.6	x
6	Aug. 8, 2008	073	9	50	548.4	x
7	Aug. 8, 2008	077	10	50	805.8	x
8	Aug. 8, 2008	099	12	50	809.3	x
9	Aug. 8, 2008	096	13	50	1029.1	x
10	Apr. 14, 2009	002	1	50	1658.9	x
11	Apr. 14, 2009	008	2	50	1327.8	x
12	Apr. 15, 2009	003	1	50	1004	x
13	Apr. 15, 2009	011	2	50	542.3	x
14	Apr. 15, 2009	025	5	50	1345.8	x
15	Apr. 15, 2009	035	7	50	1021.7	x
16	Apr. 16, 2009	014	4	50	957.6	x
17	Apr. 16, 2009	020	5	50	1064.1	x
18	Apr. 16, 2009	044			1161.2	x

TABLE 5

Frozen/Thawed Monkey Cone I_Dep

				Measured	Current		Frozen/
Number	Date	Trace number	Cell #	voltage	I_Dep(pA)	Fresh	Thawed

1	Jun. 2, 2009	002	1	50	377.8	x
2	Jun. 2, 2009	008	2	50	1233.2	x
3	Jun. 2, 2009	012	4	50	1496	x
4	Jun. 3, 2009	003	1	50	808.7	x
5	Jun. 3, 2009	018	3	50	1182.3	x
6	Jun. 3, 2009	045	4	50	937.2	x
7	Jun. 3, 2009	074	5	50	947.9	x
8	Jun. 9, 2009	000	1	50	1091.3	x
9	Jun. 9, 2009	009	3	50	853	x
10	Jun. 9, 2009	022	4	50	850.8	x
11	Jun. 10, 2009	004	3	50	1134.3	x
12	Jun. 10, 2009	023	4	50	760.8	x
13	Jun. 10, 2009	056	8	50	1022.3	x
14	Jun. 19, 2009	009	3	50	1448.7	x
15	Jun. 19, 2009	014	4	50	1640.6	x
16	Jun. 19, 2009	022	6	50	1287.5	x
17	Jun. 23, 2009	001	2	50	1046.1	x
18	Jun. 24, 2009	053	13	50	1056.2	x
19	Jun. 24, 2009	065	14	50	1084.6	x
20	Jun. 24, 2009	081	16	50	527.6	x
21	Jun. 24, 2009	091	17	50	1048.3	x
22	Jun. 24, 2009	112	19	50	754.7	x
23	Jun. 25, 2009	005	3	50	939.3	x
24	Jun. 25, 2009	016	6	50	1063.5	x
25	Jun. 25, 2009	023	8	50	995.8	x
26	Jun. 25, 2009	030	9	50	1004.6	x
27	Jun. 25, 2009	036	10	50	1142	x
28	Jun. 25, 2009	043	12	50	859.7	x
29	Jun. 25, 2009	049	14	50	783.7	x
30	Jun. 25, 2009	056	15	50	580.1	x
31	Jun. 25, 2009	066	17	50	802.6	x
32	Jun. 25, 2009	072	18	50	1059.3	x
33	Jun. 25, 2009	102	25	50	799	x
34	Jun. 25, 2009	108	26	50	606	x
35	Jun. 25, 2009	113	27	50	939.3	x
36	Jun. 25, 2009	119	29	50	679.3	x

TABLE 6

At the 0.05 level,
the population means are not significantly different.
Means Comparison using Tukey Test

		Simultaneous
	Difference	Confidence Intervals	Significant

		between		Upper	at 0.05
Dataset	Mean	Means	Lower Limit	Limit	Level

Data5_A	−371.625
Data5_B	−298.29394	−73.33106	−151.543	4.88088	No

Power Analysis

	Total
Alpha	Sample Size	Power

0.05	49	0.45535 (actual)

TABLE 7

Fresh Monkey Cone I_Cl(Ca)

				Measured	Current I_ClCa		Frozen/
Number	Date	Trace number	Cell #	voltage(mV)	(pA)	Fresh	Thawed

1	Jul. 10, 2009	001	1	−70	−650	x
2	Aug. 8, 2008	000	1	−70	−1036.6	x
3	Aug. 8, 2008	023	3	−70	−690	x
4	Aug. 8, 2008	040	4	−70	−471	x
5	Aug. 8, 2008	067	8	−70	−425	x
6	Aug. 8, 2008	075	10	−70	−444.3	x
7	Aug. 8, 2008	088	12	−70	−313.9	x
8	Aug. 8, 2008	096	13	−70	−786.9	x
9	Apr. 14, 2009	000	1	−70	−1588.7	x
10	Apr. 14, 2009	006	2	−70	−1146.5	x
11	Apr. 15, 2009	000	1	−70	−412.3	x
12	Apr. 15, 2009	009	2	−70	−813.6	x
13	Apr. 15, 2009	023	5	−70	−931.4	x
14	Apr. 15, 2009	033	7	−70	−491.6	x
15	Apr. 16, 2009	012	4	−70	−656.4	x
16	Apr. 16, 2009	018	5	−70	−835	x
17	Apr. 16, 2009	041	9	−70	−564.9	x

TABLE 8

				Measured	Current I_ClCa		Frozen/
Number	Date	Trace number	Cell #	voltage(mV)	(pA)	Fresh	Thawed

1	Jun. 2, 2009	001	1	−1192	x
2	Jun. 2, 2009	005	2	−2883.2	x
2	Jun. 2, 2009	014	4	−2671.5	x
4	Jun. 3, 2009	002	1	−606.7	x
5	Jun. 3, 2009	020	3	−639.6	x
6	Jun. 3, 2009	047	4	−494.4	x
7	Jun. 3, 2009	075	5	−1001	x
8	Jun. 9, 2009	002	1	−1418.5	x
9	Jun. 9, 2009	010	3	−612.8	x
10	Jun. 9, 2009	021	4	−378.4	x
11	Jun. 10, 2009	005	3	−1359.9	x
12	Jun. 10, 2009	024	4	−620.7	x
13	Jun. 10, 2009	063	8	−412	x
14	Jun. 19, 2009	007	3	−1511.2	x
15	Jun. 19, 2009	015	4	−1946.4	x
16	Jun. 19, 2009	021	6	−1185.9	x
17	Jun. 23, 2009	003	2	−1099.1	x
18	Jun. 24, 2009	054	13	−2035.8	x
19	Jun. 24, 2009	068	14	−2345.3	x
20	Jun. 24, 2009	079	16	−620.7	x
21	Jun. 24, 2009	090	17	−1835.3	x
22	Jun. 24, 2009	114	19	−1717.2	x
23	Jun. 25, 2009	003	3	−1870.1	x
24	Jun. 25, 2009	015	6	−1443.5	x
25	Jun. 25, 2009	021	8	−2568.1	x
26	Jun. 25, 2009	028	9	−743.2	x
27	Jun. 25, 2009	035	10	−1517.6	x
28	Jun. 25, 2009	042	12	−1361.7	x
29	Jun. 25, 2009	048	14	−281	x
30	Jun. 25, 2009	061	15	−390	x
31	Jun. 25, 2009	068	17	−803.8	x
32	Jun. 25, 2009	071	18	−1661.4	x
33	Jun. 25, 2009	105	25	−892.8	x
34	Jun. 25, 2009	107	26	−949.6	x
35	Jun. 25, 2009	112	27	−865.5	x
36	Jun. 25, 2009	118	29	−991.8	x

TABLE 9

At the 0.05 level,
the population means are significantly different.
Means Comparison using Tukey Test

		Simultaneous
	Difference	Confidence Intervals	Significant

		between	Lower	Upper	at 0.05
Dataset	Mean	Means	Limit	Limit	Level

Data2_A	−721.06471
Data2_B	−1247.99167	526.92696	171.80147	882.05245	Yes

Power Analysis

	Total
Alpha	Sample Size	Power

0.05	53	0.83206 (actual)

Claims

1-16. (canceled)

17. A method for treating retinal dysfunction in a subject, comprising:

introducing into an eye of said subject an effective amount of retinal cone cells stored and retrieved using a method comprising the steps of:

incubating freshly isolated retinal tissue with a retinal tissue digestion solution comprising a protease and CaCl₂;

removing said retinal tissue digestion solution to yield an enzyme-treated retinal tissue;

triturating said enzyme-treated retinal tissue in a retinal cell suspension medium to form a retinal cell suspension comprising retinal cone cells;

adding fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) to said retinal cell suspension;

freezing and storing said retinal cell suspension comprising FBS and DMSO in liquid nitrogen;

thawing said retinal cell suspension;

washing and culturing said thawed retinal cells with a retinal cell culture medium comprising Choline Cl; and

isolating mature retinal cone cells recovered from said frozen retinal cell suspension.

18. The method of claim 17, wherein said retinal dysfunction is selected from the group consisting of photoreceptor degeneration, retinal detachment, retinal trauma, photic lesions caused by laser or sunlight; a macular hole, macular edema; night blindness and color blindness, ischemic retinopathy, and inflammatory conditions.

19. The method of claim 17, wherein said retinal cells are introduced into said eye with a substance that stimulate differentiation of neuroretina-derived stem cells into photoreceptor cells.

20. (canceled)

21. The method of claim 17, wherein said isolated mature retinal cone cells are capable of maintaining I_Depand I_hlevels that are not statistically different from I_Depand I_hlevels of freshly isolated retinal cone cells not subjected to freezing and thawing as measured by patch clamp analysis after being frozen for at least 6 months at −170° C.

22. The method of claim 17, wherein said protease is selected from the group consisting of trypsin, chymotrypsin, pepsin, papain, elastase A and mixtures thereof.

23. The method of claim 22, wherein said protease is papain.

24. The method of claim 17, wherein said tissue digestion solution further contains a collagenase.

25. The method of claim 17, wherein said tissue digestion solution further contains an enzyme activator.

26. The method of claim 25, wherein said enzyme activator is cysteine.

27. The method of claim 17, wherein said tissue digestion solution comprises NaCl, NaHCO₃, sodium pyruvate, KCl and NaH₂PO₄.

28. The method of claim 17, wherein said tissue digestion solution comprises 114 mM NaCl, 25 mM NaHCO₃, 1 mM soduim pyruvate, 3 mM KCl, 0.5 mM NaH₂PO₄, 0.5 mM CaCl₂, and 0.02 mM Phenol red.

29. The method of claim 17, wherein said tissue digestion solution has a pH of 7.2-7.3.

30. The method of claim 17, wherein said freshly isolated retinal tissue is incubated with a retinal tissue digestion solution containing a protease at a temperature between about 18° C. and 42° C. for about 15-90 min.

31. The method of claim 17, wherein said freshly isolated retinal tissue is incubated with a retinal tissue digestion solution containing a protease at room temperature for about 30-40 min.

32. The method of claim 17, wherein said retinal cell culture medium comprises a growth factor selected from the group consisting of human epidermal growth factor, human fibroblast growth factor-2 and platelet-derived growth factor.

33. The method of claim 17, wherein said mature retinal cone cells have an I_Clcalevel that is statistically lower than the I_Clcalevel of freshly isolated retinal cone cells as measured by patch clamp analysis, after being frozen for at least 6 months at −170° C.

34. The method of claim 17, wherein the retinal cell culture medium comprises preconditioned culture medium prepared by culturing Muller glial cells in a culture medium suitable for cone survival.

35. The method of claim 34, wherein the retinal cell culture medium further comprises insulin, h-transferrin, putrascine, selenite and glutamine.

36. The method of claim 17, wherein said retinal cell culture medium comprises platelet-derived growth factor.

37. The method of claim 17, further comprising the step of dissecting freshly isolated retinal tissue into small fragments in cold CO₂-independent medium, prior to incubation with the retinal tissue digest solution comprising the protease and CaCl₂.