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:
[0001]    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 
       [0002]    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 
       [0003]    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. 
         [0004]    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 
       [0005]    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. 
         [0006]    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. 
         [0007]    In another embodiment, the retinal tissue is retina or retinal pigmented epithelium. 
         [0008]    In another embodiment, the method further comprises dissecting the isolated retinal tissue into small fragments. 
         [0009]    In another embodiment, the protease is selected from the group consisting of trypsin, chymotrypsin, pepsin, papain, elastase A and mixtures thereof. 
         [0010]    In one embodiment, the protease is papain. 
         [0011]    In another embodiment, the tissue digestion solution further contains a collagenase. 
         [0012]    In another embodiment, the tissue digestion solution further contains an enzyme activator. 
         [0013]    In one embodiment, the enzyme activator is cysteine. 
         [0014]    In another embodiment, the tissue digestion solution comprises NaCl, NaHCO 3 , sodium pyruvate, KCl, NaH 2 PO 4  and CaCl 2 . 
         [0015]    In one embodiment, the tissue digestion solution comprises 114 mM NaCl, 25 mM NaHCO 3 , 1 mM soduim pyruvate, 3 mM KCl, 0.5 mM NaH 2 PO 4 , 0.5 mM CaCl 2 , and 0.02 mM Phenol red. 
         [0016]    In another embodiment, the tissue digestion solution has a pH of 7.2-7.3. 
         [0017]    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. 
         [0018]    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. 
         [0019]    In another embodiment, the method further comprises thawing stored retinal cells; and mixing thawed cells with a retinal cell culture medium. 
         [0020]    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. 
         [0021]    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. 
         [0022]    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. 
         [0023]    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. 
         [0024]    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 
         [0025]      FIG. 1  is a flow chart showing a method for isolating, storing and retrieving retina cells. 
           [0026]      FIG. 2  is a picture of a retinal cone cell recovered from frozen cell suspension. 
           [0027]      FIG. 3  is a picture of a freshly isolated retinal cone cell. 
           [0028]      FIG. 4  is a diagram showing the average I Dep  value measured in freshly isolated retinal cone cells and retinal cone cell recovered from frozen cell suspension. 
           [0029]      FIG. 5  is a diagram showing the average I h  value measured in freshly isolated retinal cone cells and retinal cone cell recovered from frozen cell suspension. 
           [0030]      FIG. 6  is a diagram showing the average I ClCa  value measured in freshly isolated retinal cone cells and retinal cone cell recovered from frozen cell suspension. 
       
    
    
     DETAILED DESCRIPTION 
       [0031]    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 
       [0032]    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 
       [0033]    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). 
         [0034]    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 CO 2 -independent medium. 
       Retinal Cell Digestion Solution 
       [0035]    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. 
         [0036]    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. 
         [0037]    In one embodiment, the retinal cell digestion solution contains NaCl, NaHCO 3 , sodium pyruvate, KCl, NaH 2 PO 4  and CaCl 2 . In another embodiment, the retinal cell digestion solution contains: 114 mM NaCl, 25 mM NaHCO 3 , 1 mM soduim pyruvate, 3 mM KCl, 0.5 mM NaH 2 PO 4 , 0.5 mM CaCl 2 , 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% O 2  for 10 min to reach pH 7.2-7.3. 
       Digestion Conditions 
       [0038]    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. 
         [0039]    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 cm 3 , 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 
       [0040]    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. 
         [0041]    The retinal cell storage medium is retinal cell suspension medium with 10% serum and 10% DMSO. 
         [0042]    In one embodiment, the retinal cell suspension medium comprises 139 mM NaCl, 2 mM HEPES, 1 mM sodium pyruvate, 0.5 mM MgCl 2 , 0.5 mM MgSO 4 , 0.5 mM NaH 2 PO 4 , 1.8 mM CaCl 2 , 3 mM KCl, 1 mM NaHCO 3 , 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 
       [0043]    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. 
         [0044]    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 
       [0045]    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% CO 2  at 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 
       [0046]    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. 
         [0047]    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. 
         [0048]    In one embodiment, the retina cell culture medium contains NaCl, HEPES, sodium pyruvate, MgCl 2 , MgSO 4 , NaH 2 PO 4 , CaCl 2 , KCl, NaHCO 3 , 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 MgCl 2 , 0.5 mM MgSO 4 , 0.5 mM NaH 2 PO 4 , 1.8 mM CaCl 2 , 3 mM KCl, 1 mM NaHCO 3 , 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. 
         [0049]    The retinal cell culture medium may further contain a growth factor, a neural supplement, glutamine and/or serum. 
         [0050]    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). 
         [0051]    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. 
         [0052]    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. 
         [0053]    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. 
         [0054]    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 
       [0055]    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&#39;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. 
         [0056]    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. 
         [0057]    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&#39;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. 
         [0058]    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. 
         [0059]    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. 
         [0060]    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&#39;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. 
         [0061]    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. 
         [0062]    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. 
         [0063]    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&#39;s congential amaurosis, congential stationary night blindness, Sticklers Syndrome, colobomas, vitreoretinal dysplasia, achromatopsia, or optic nerve hypoplasia. 
         [0064]    In some embodiments, the inflammatory condition is multifocal choroiditis, birdshot chorioretinopathy, collagen vascular diseases affecting the posterior segment of the eye, Wegener&#39;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. 
         [0065]    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. 
         [0066]    In some embodiments, the infection is cytomegalovirus retinitis, herpes type I retinitis, Ebstein-Barr virus retinitis, toxoplasmosis, rubella, or pox virus. 
         [0067]    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. 
         [0068]    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. 
         [0069]    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. 
         [0070]    In some embodiments, the congential optic disease is Leber&#39;s congential amaurosis, congential stationary night blindness, or optic nerve hypoplasia. 
         [0071]    In some embodiments, the tumor is retinoblastoma or ocular melanoma. 
         [0072]    In some embodiments, the dystrophic eye is the result of glaucoma. 
         [0073]    In using the stored retinal cells to treat retinal dysfunction, one can, in conjunction with introducing the retinal cells into a recipient&#39;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 
       [0074]    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. 
         [0075]    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. 
         [0076]    In some embodiments, the harvesting solution is a solution suitable for preserving corneal tissue. In some embodiments, the harvesting solution is Optisol. 
         [0077]    In some embodiments, the harvesting solution is X-VIVO serum-free medium. 
         [0078]    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. 
         [0079]    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. 
         [0080]    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. 
         [0081]    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. 
         [0082]    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. 
         [0083]    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 
       [0084]    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 NaHCO 3 , 1 mM sodium pyruvate, 3 mM KCl, 0.5 mM NaH 2 PO 4 , 0.5 mM CaCl 2 , 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 MgCl 2 , 0.5 mM MgSO 4 , 0.5 mM NaH 2 PO 4 , 1.8 mM CaCl 2 , 3 mM KCl, 1 mM NaHCO 3 , 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 
       [0085]    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 10 4 -10 6 . 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 
       [0086]    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 MgCl 2 , 0.5 mM MgSO 4 , 0.5 mM NaH 2 PO 4 , 1.8mM CaCl 2 , 3 mM KCl, 1 mM NaHCO 3 , 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% CO 2 . 
       EXAMPLE 2 
     Patch Clamp Analysis of Freshly Isolated and Frozen/Thawed Retinal Cone Cells 
       [0087]    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 ). 
         [0088]    Tables 1 and 2 show individual I Dep  measurements 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 Dep  measurements between the freshly isolated retinal cone cells and the cone cells recovered from the frozen stock. 
         [0089]    Tables 4 and 5 show individual I h  measurements 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 h  measurements between the freshly isolated retinal cone cells and the cone cells recovered from the frozen stock. 
         [0090]    Tables 7 and 8 show individual I Clca  measurements 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. 
         [0091]    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 MgCl 2 , 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.). 
         [0092]    I Dep  is 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 Dep  and 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. 
         [0093]    I h  is 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 h  indicated that the cell has the expected properties of healthy photoreceptors. 
         [0094]    I Clca  is 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. 
         [0095]    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. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 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 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Frozen/Thawed Monkey Cone I h   
               
             
          
           
               
                   
                   
                   
                   
                 Measured 
                   
                   
                 Frozen/ 
               
               
                 Number 
                 Date 
                 Trace number 
                 Cell #  
                 voltage(mV) 
                 Current I h (pA) 
                 Fresh 
                 Thawed 
               
               
                   
               
             
          
           
               
                 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 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
             
               
               
               
             
           
               
                 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) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 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 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 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 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
             
               
               
               
             
           
               
                 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) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 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 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 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 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
             
               
               
               
             
           
               
                 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)