Patent Description:
Retinal pigment epithelial (RPE) cells exist in the outermost layer of the retina as one layer of epithelial cell tissue accompanied by pigment, and play an extremely important role in maintaining the function of the eye ball retina which is responsible for vision. Representative functions thereof include neogenesis of outer segment of retinal photoreceptor cell due to phagocytotic function, recycling of visual substances that are photosensitive proteins specifically present in the outer segment of photoreceptor cell, protection effect on photoreceptor cells and choroid membrane, which are neighboring tissues of RPE, by secretion of various cytokines, and the like. It is known, therefore, that when RPE cells become dysfunctional due to aging, genetic abnormality and the like, resulting in degeneration and cell death associated therewith, serious retinal denaturation such as macular degeneration such as age-related macular degeneration (AMD), Stargardt disease and the like or retinitis pigmentosa (RP) and the like is developed. In particular, AMD is an ophthalmic disease that causes impairment of central vision and sight loss in elderly people, and poses an important social problem in developed countries including Japan, which will become unprecedented aging societies hereafter. Presently, the treatment method for AMD is generally an intraocular administration of antibody medicine, which is a symptomatic treatment, and an effective treatment method has still not been established. Thus, the development of an alternative curative treatment method is desired. Furthermore, an effective treatment method for Stargardt disease and RP has not been established at all to date.

In recent years, a cell transplantation therapy including supplementing or substituting RPE cells induced to differentiate from pluripotent stem cells is attracting attention as a new treatment method of AMD and RP. Thus, utilization of RPE cells as a graft material for cell therapy is expected. For example, Ocata Therapeutics, Inc. (formerly Advanced Cell Technology (ACT), Inc. ) is undertaking clinical research on age-related macular degeneration (AMD) and Stargardt disease using RPE cells derived from human embryonic stem cells (ES cells) (non-patent document <NUM>). In Japan, surgery to transplant an RPE cell sheet derived from human induced pluripotent stem cells (iPS cells) to exudative AMD patient was conducted in <NUM>. The surgery attracted much attention as the world's first iPS cell transplantation therapy, and it is reported that the treatment progress is still good now.

Currently, transplantation of RPE cells includes (<NUM>) a method of transplanting a prepared RPE cell sheet or an RPE cell sheet with scaffold, which is prepared by seeding RPE cells on a scaffold material, to a part where the retinal pigment epithelium is denatured or defective from an incision formed in the retina, or (<NUM>) a method of injecting an RPE cell suspension into a similar part. Cultivation of cells for transplantation needs to be performed at a GMP level. In the former case, therefore, the RPE cell sheet after production is transported from a cell processing center (CPC) to a facility (hospital) where transplant operation is performed. On the other hand, in the latter case, in a clinical trial conducted by Ocata Therapeutics, for example, cryopreserved RPE cells produced in CPC are transported to a hospital and, in the hospital, the cells are thawed, suspended in a transplantation medium, immediately brought into an operating room, and transplanted. The latter is considered highly convenient since the cells can be transported from CPC to the hospital in a frozen state.

Nevertheless, the possibility that the freeze-thaw treatment causes damage on the graft cells cannot be denied. Conventionally, terminally differentiated cells and tissues used for topical transplantation were generally provided by transplantation between living bodies or production free of freezing step. When cryopreserved cells were transplanted unavoidably, the freeze-thaw step was feared to cause damage on the structure of the cells and tissues and deterioration of functions thereof. Suppression of decline in the function of cryopreserved cells is a big task in realizing localized cell transplantation therapy. An influence of transplantation of RPE cells immediately after thawing on the therapeutic effect has not been studied so far.

Poloxamer <NUM> (polyoxyethylene(<NUM>)polyoxypropylene (<NUM>)glycol) is known to exhibit cell protection effects by repairing physically or chemically damaged cell membranes. Several patents and patent applications have heretofore been made relating to the use of poloxamer <NUM> as a cell membrane sealing agent in the treatment of pathological conditions such as muscular dystrophy, heart failure, neurodegenerative diseases, electrical damage and the like and medical applications such as transplantation and the like (non-patent document <NUM>). For example, Austen et al. discloses a method of sealing cell membrane of adipose tissues damaged during liposuction with poloxamer <NUM> (patent document <NUM>). They further disclose a method of improving the survival rate of cryopreserved cells by thawing the cryopreserved cells in the presence of poloxamer <NUM> (patent document <NUM>).

However, there has been no report to date on the use of poloxamer <NUM> during implantation of RPE cells or thawing of cryopreserved RPE cells.

Any references in the description to methods of treatment refer to the products of the present invention for use in a method for treatment. An object of the present invention is to provide the pharmaceutical composition according to claims <NUM> and <NUM> which can be used for the treatment of retinal degenerative diseases such as macular degeneration, retinitis pigmentosa (RP) and the like, and to provide a method according to claim <NUM>.

The present inventors have conducted intensive studies in an attempt to achieve the aforementioned objects and found that the post-thawing survival rate and the post-thawing recovery rate of cryopreserved RPE cells are improved by adding poloxamer <NUM> to a transplantation medium (suspending agent) to be added after thawing.

In clinical trials on macular degeneration by using ES cell-derived RPE cells, cryopreserved RPE cells were transplanted immediately after thawing (the above-mentioned non-patent document <NUM>). From the above-mentioned results, the present inventors have had an idea that transplantation of RPE cells immediately after thawing may decrease the cell viability and deteriorate the cell condition, which in turn may impair the transplantation effects (e.g., photoreceptor cell protection effect). The photoreceptor cell protection effect was compared between transplantation of RPE cells immediately after thawing and that after culturing for a certain period after thawing to RCS (Royal College of Surgeons) rats, which are retinal degeneration models. As a result, it was clarified that the latter showed a higher photoreceptor cell protection effect.

The present inventors have conducted intensive studies in an attempt to prevent reduction of a photoreceptor cell protection effect in the RPE cells immediately after thawing. As a result, RPE cells showed, even immediately after thawing, a high photoreceptor cell protection effect equivalent or superior to that after incubation for a certain period, when poloxamer <NUM> was added to the transplantation medium. The results show that, by the use of poloxamer, a high transplantation effect can be obtained even when RPE cells in a cryopreserved state are transported into a hospital and transplanted immediately after thawing, without once culturing the RPE cells in CPC after thawing from cryopreservation.

Furthermore, the present inventors have tested whether the addition of poloxamer <NUM> to the transplantation medium can contribute to not only the survival rate and transplantation effect of RPE cells after thawing but also reduction of the loss of RPE cells during various operations from thawing to transplantation (improvement of cell recovery rate). As a result, it was clarified that all of the loss of cells after thawing, loss of cells by a centrifugation operation, and loss of cells when passing a transplantation device can be reduced by using a poloxamer <NUM>-containing medium as a suspending agent from the steps of dilution and washing operation immediately after thawing.

Based on these findings, the present inventors have found that use of a suspending agent containing poloxamer <NUM> as a medium for transplantation is useful for simplification and speeding up of transplantation protocol, improvement and equalizing of transplantation effect, and further, cost reduction in the RPE cell transplantation treatment, which resulted in the completion of the present invention.

That is, the present invention provides the embodiments as defined in the claims.

According to the present invention, the post-thawing survival rate and the post-thawing recovery rate of cryopreserved RPE cells can be improved using a poloxamer. The effect lasts for a long time even at an ordinary temperature and enables preservation and transportation at an ordinary temperature after preparation of a cell suspension for transplantation.

Using a poloxamer, RPE cells cryopreserved and transplanted immediately after thawing show a photoreceptor cell protection effect equal to or higher than that of RPE cells transplanted after culturing for a given period after thawing. Thus, RPE cells in a frozen state can be transported from CPC to a hospital and transplanted immediately after thawing in the hospital. This enables simplification of transplantation protocol and shortening of the time before performing transplantation.

Furthermore, using a poloxamer, loss of RPE cells in each step from thawing to transplantation can be reduced and the number of cells can be equalized. Thus, the number of RPE cells to be prepared for ensuring a sufficient number of cells for transplantation can be reduced. In addition, variability of the survival rate and the number of transplanted cells can be decreased in every transplantation. Thus, the treatment effect can be equalized and safety in terms of prevention of administration of damaged cells can be improved.

The present invention provides a pharmaceutical composition for transplantation as defined in claims <NUM>, <NUM> and <NUM>.

In the present specification, "retinal pigment epithelial (RPE) cell" refers to an epithelial cell constituting the retinal pigment epithelium, and a progenitor cell thereof. Whether a cell is a retinal pigment epithelial cell can be confirmed by, for example, expression of cell marker (RPE65, CRALBP, MERTK, BEST1 etc.), cell morphology (intracellular melanin pigment deposition, polygonal, flat epithelium-like cell morphology, polygonal actin bundle formation, etc.) and the like. Progenitor cell of retinal pigment epithelial cell means a cell in which induction of differentiation into retinal cell is programmed. Whether a cell is the progenitor cell can be confirmed by expression of cell marker (Mitf (pigment epithelial cell, pigment epithelial progenitor cell), Pax6 (pigment epithelial progenitor cell), Rx (retinal progenitor cell), OTX2 (retinal progenitor cell), RPE65 (pigment epithelial cell), BEST1 (pigment epithelial cell)) and the like. Functional evaluation of retinal pigment epithelial cell can be confirmed, for example, using the secretory activity of cytokines (VEGF, PEDF, etc.), phagocytotic activity and the like as indexes. Those skilled in the art can perform such functional evaluation and confirmation operation by setting conditions as appropriate.

RPE cell can be obtained from any animal having RPE cells or can also be obtained from pluripotent stem cell, etc. by inducing differentiation according to a method known per se. Examples of the RPE cell include one obtained by, for example, inducing differentiation from pluripotent stem cell. The pluripotent stem cell is not particularly limited as long as it has pluripotency permitting differentiation into any cell present in living organisms, and also has proliferative capacity. For example, embryonic stem cell (ES cell), induced pluripotent stem cell (iPS cell), pluripotent cell derived from cultured fibroblast or myelogenic stem cell (Muse cell), and the like are included. Preferable pluripotent stem cells are ES cell and iPS cell. The derivation of the pluripotent stem cell is not particularly limited and, for example, any animal, preferably mammal, more preferably human, mouse, rat and the like, in which establishment of any of the following pluripotent stem cells has been reported, can be mentioned.

An iPS cell is a somatic cell-derived artificial stem cell having properties almost equivalent to those of ES cells, such as pluripotency and proliferation potency by self-renewal, and can be produced by introducing a particular reprogramming factor in the form of a DNA or protein into a somatic cell (<NPL>; <NPL>; <NPL>; <NPL>); <CIT>).

The term "somatic cell" used in the present specification refers to any animal cell (preferably, cells of mammals inclusive of human) except germ line cells and totipotent cells such as ovum, oocyte, ES cell and the like. The somatic cell nonlimitatively encompasses any of somatic cells of the fetus (pup), somatic cells of the newborn (pup), and matured healthy or diseased somatic cells and encompasses any of primary culture cells, subcultured cells and established lines of cells. To be specific, examples of the somatic cell include (<NUM>) tissue stem cell (somatic stem cell) such as neural stem cell, hematopoietic stem cell, mesenchymal stem cell, pulp stem cell and the like, (<NUM>) tissue progenitor cell, (<NUM>) differentiated cell such as lymphocyte, epithelial cell, endothelial cell, muscle cell, fibroblast (skin cell etc.), hair cell, hepatocyte, gastric mucosa cell, enterocyte, splenocyte, pancreatic cell (pancreatic exocrine cell etc.), brain cell, lung cell, kidney cell, fat cell and the like, and the like.

The reprogramming factor may be composed of a gene that is specifically expressed in ES cells, a gene product or non-coding RNA thereof or a gene that plays an important role in maintaining undifferentiation state of ES cell, a gene product or non-coding RNA thereof, or a low molecular weight compound. Examples of the gene contained in the reprogramming factor include Oct3/<NUM>, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-<NUM>, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1 and the like, and these reprogramming factors may be used alone or in combination. Examples of the combinations of the reprogramming factors include those recited in <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <NPL>, <NPL>, <NPL>, <NPL>, <NPL>, <NPL>, <NPL>, <NPL>, <NPL>, <NPL>, <NPL>, <NPL>, <NPL>, <NPL>, <NPL>, and <NPL>.

Differentiation of RPE cell from iPS cell can be induced by a known method (e.g., <NPL>; <NPL>). Alternatively, the methods described in <CIT>, <CIT>, <CIT>, <CIT> and the like can also be used.

As the RPE cell in the present specification, RPE cell induced to differentiate from human ES cell or human iPS cell can be specifically mentioned.

RPE cell to which the suspending agent used in the present invention is applied or RPE cell contained in the pharmaceutical composition of the present invention is preferably an RPE cell within <NUM> hr or <NUM> after thawing, or immediately after thawing from a cryopreserved state. The RPE cell prepared as mentioned above is cryopreserved according to a method known per se and thawed immediately before suspending in the suspending agent used in the present invention. Examples of the cryopreservation method of RPE cell include, but are not limited to, a method including recovering RPE cells in a centrifugation tube or the like, pelletizing the cells by centrifugation, suspending the cells in a cryopreservation solution containing a cryoprotective agent, placing the suspension in a cryopreservation tube, freezing same by a freezer at -<NUM> and preserving same in a gaseous phase or liquid phase in a nitrogen tank, and the like. As the cryoprotective agent, for example, DMSO, glycerol, antifreeze protein, antifreeze glycoprotein and the like can be used as appropriate.

As a method for thawing the cryopreserved RPE cells, a method well known in the pertinent technical field can be used (e.g., <NPL>). Preferably, the cells are rapidly thawed in a hot-water bath at about <NUM>. When a highly cytotoxic cryoprotective agent such as DMSO is used, it is desirable to dilute DMSO immediately after thawing with a suitable diluent to a concentration free of an adverse influence on the cell. It is desirable to remove toxic cryoprotective agents and the like by removing the supernatant by centrifugation. As the diluent, serum-containing or serum-free medium, saline or PBS can be used, and a pharmaceutically acceptable medium is also preferably used as the ocular irrigating/washing solution in the suspending agent used in the present invention.

In the present DESCRIPTION, the "poloxamer" is a triblock copolymer represented by the following structural formula:
<CHM>.

Examples of the poloxamer include poloxamer <NUM>, poloxamer <NUM>, poloxamer <NUM> and poloxamer <NUM>, and preferred are poloxamer <NUM> and poloxamer <NUM>, and more preferred is poloxamer <NUM>.

Poloxamer <NUM> (in the formula, a=<NUM>, b=<NUM>) is used as a pharmaceutical additive for applications such as stable (stabilizing) agent, surfactant, lubricant, soluble (solubilizing) agent, base, binder, suspension (suspending) agent, coating agent, wetting agent, emulsifier, thickening agent, excipient, dispersing agent, disintegrant, solubilizing agent and the like and can be used safely.

Poloxamer <NUM> can be produced by a method known per se. Poloxamer <NUM> is commercially available under the trade names of, for example, Pluronic (registered trade mark) F68(BASF), Pluronic F68 <NUM>% (<NUM>×) (Thermo Fisher Scientific (former Life Technologies)), PRONON (registered trade mark) #188P (NOF CORPORATION) and the like.

The concentration of the poloxamer, for example, poloxamer <NUM>, contained in the suspending agent used in the present invention, or the medium in the pharmaceutical composition of the present invention is not particularly limited as long as it is sufficient for, for example, protecting RPE cells, particularly, RPE cells thawed from cryopreservation, suppressing cell damage or cell death (RPE cell protection effect), improving photoreceptor cell protection effect (prevention of decrease) of RPE cell, and/or preventing a loss in the number of RPE cells in various steps from thawing to transplantation. It is preferably <NUM>%(w/v) - <NUM>%(w/v), more preferably <NUM>%(w/v) - <NUM>%(w/v) (e.g., <NUM>%(w/v) - less than <NUM>%(w/v), <NUM>%(w/v) - <NUM>%(w/v), <NUM>%(w/v), <NUM>%(w/v), <NUM>%(w/v) or <NUM>%(w/v) etc.). In consideration of the concentration of poloxamer <NUM> necessary for preventing cell death during "thawing" of cryopreserved adipose tissue, which is <NUM> to <NUM>%(w/v) or not less than that (the above-mentioned patent document <NUM>), it is a surprising finding that a superior RPE cell protection effect is achieved by adding a poloxamer at a low concentration of less than <NUM>%(w/v) "after thawing".

The "medium pharmaceutically acceptable as ocular irrigating/washing solution" contained in the suspending agent used in the present invention and the pharmaceutical composition of the present invention is not particularly limited as long as it is a suspension in which RPE cells are suspended in a medium and direct injection thereof into the disease site, namely, degenerative or defective site of the subretinal pigmented epithelium in macular degeneration or retinitis pigmentosa is pharmaceutically acceptable. For example, buffering agent, isotonicity agent, viscosity base, chelating agent, pH adjuster, antioxidant and the like can be appropriately selected and contained at a range free from an influence on the survival rate of the RPE cell.

Examples of the buffering agent include phosphoric acid buffering agent, boric acid buffering agent, citrate buffering agent, tartaric acid buffering agent, acetate buffering agent, amino acid and the like.

Examples of the tonicity agent include saccharides such as sorbitol, glucose, mannitol and the like, polyhydric alcohols such as glycerol, propylene glycol and the like, salts such as sodium chloride and the like, boric acid and the like.

Examples of the viscous base include water-soluble polymers such as polyvinylpyrrolidone, polyethylene glycol, poly(vinyl alcohol) and the like, celluloses such as hydroxyethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and the like, and the like.

Examples of the chelating agent include sodium edetate, citric acid and the like.

Examples of the pH adjuster include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, boric acid or a salt thereof (borax), hydrochloric acid, citric acid or a salt thereof (sodium citrate, citric acid dihydrogen sodium etc.), phosphoric acid or a salt thereof (disodium hydrogen phosphate, potassium dihydrogen phosphate etc.), acetic acid or a salt thereof (sodium acetate, ammonium acetate etc.), tartaric acid or a salt thereof (sodium tartrate etc.) and the like.

Examples of the antioxidant include glutathione, sodium hydrogen sulfite, dry sodium sulfite, sodium pyrrosulfite, tocopherol and the like.

The pH of the agent of the present invention is generally adjusted to about <NUM> - about <NUM>, preferably about <NUM> - about <NUM>. Preferably, a sterilization treatment such as sterilization by filtration using a membrane filter and the like, and the like can be performed.

In one preferable embodiment of the present invention, as the "medium pharmaceutically acceptable as ocular irrigating/washing solution", modified Hank's Balanced Salt Solution (HBSS) or oxyglutathione-containing ocular irrigating/washing solution can be used. Examples of the modified HBSS include phenol red-free HBSS(-) (<NUM>/L KCl, <NUM>/L NaCl, <NUM>/L NaHCO<NUM>, <NUM>/L KH<NUM>PO<NUM>, <NUM>/L anhydrous Na<NUM>HPO<NUM>, <NUM>/L D-glucose; hereinafter to be also simply referred to as "HBSS(-)"), phenol red-free HBSS(+) (HBSS(-) added with <NUM>/L anhydrous CaCl<NUM>, <NUM>/L MgCl<NUM> • <NUM><NUM>O, <NUM>/L MgSO<NUM> • <NUM><NUM>O; hereinafter to be also simply referred to as "HBSS(+)") and the like. Examples of the modified ocular irrigating/washing solution containing oxyglutathione include BSS plus (registered trade mark) intraocular irrigating solution <NUM>% (Nihon Alcon) (hereinafter to be also simply referred to as "BSS") and the like.

The suspending agent used in the present invention can be prepared by adding an appropriate amount of a poloxamer (e.g., poloxamer <NUM>) to the medium pharmaceutically acceptable as ocular irrigating/washing solution of the above-mentioned (C).

The pharmaceutical composition of the present invention can be prepared by suspending the above-mentioned RPE cells in the suspending agent used in the present invention.

As mentioned above, in one preferable embodiment of the present invention, the RPE cells suspended in the suspending agent used in the present invention and prepared as the pharmaceutical composition of the present invention are cells immediately after thawing from a cryopreserved state. The cryopreserved RPE cells immediately after thawing by the above-mentioned method are desirably diluted with a suitable diluent and washed by centrifugation. As the diluent here, as mentioned above, saline, PBS, and the medium (e.g., HBSS(+), BSS etc.) pharmaceutically acceptable as the ocular irrigating/washing solution of the above-mentioned (C) can be used. The diluent may or may not contain a poloxamer (e.g., poloxamer <NUM>). To suppress damage on or cell death of RPE cells in the washing step by centrifugation, and prevent a loss in the number of cells during operations, it is preferable to add poloxamer <NUM> to the diluent. That is, in one embodiment, the dilution and washing steps are performed in a medium pharmaceutically acceptable as an ocular irrigating/washing solution containing a poloxamer (e.g., poloxamer <NUM>), whereby an RPE cell protection effect, or a suppressive effect on the damage on or cell death of RPE cell is acknowledged, and the recovery rate of the RPE cells is improved. Therefore, a method of protecting RPE cells, a method of suppressing damage on or cell death of RPE cells, and a method of improving the recovery rate of RPE cells, by suspending the RPE cells in a medium pharmaceutically acceptable as an ocular irrigating/washing solution and containing a poloxamer (e.g., poloxamer <NUM>), are also encompassed in the present invention. The addition concentration of the poloxamer is similarly preferably exemplified by the concentration range of the poloxamer in the suspension of the present invention. The dilution and washing steps may be performed from room temperature to about <NUM>, or under cooling at about <NUM>. The washing operation may be performed only once, or can be repeated two to several times.

By resuspending the diluted and washed RPE cells in the suspending agent used in the present invention, the pharmaceutical composition of the present invention, namely, an RPE cell-containing composition, can be obtained. The RPE cell in the pharmaceutical composition of the present invention is an RPE cell within <NUM> hr, further preferably <NUM>, after thawing from a cryopreserved state. The density of the RPE cell in the pharmaceutical composition of the present invention is not particularly limited as long as a therapeutically effective amount of RPE cell is contained in a suspension (e.g., <NUM> - <NUM>µL, preferably <NUM> - <NUM>µL) to be injected into a diseased site, namely, a defective site of retinal pigment epithelium in macular degeneration and retinitis pigmentosa. For example, the RPE cells can be suspended to a cell density of <NUM> - <NUM>,<NUM> cells/µL, preferably <NUM>,<NUM> - <NUM>,<NUM> cells/µL. In a clinical trial using ES cell-derived RPE cells on macular degeneration, live RPE cells were suspended in BSS finally to <NUM> cells/µL and the cell suspension (<NUM>µL, total number of live RPE cells, <NUM>,<NUM> cells) was injected into a macular area (the above-mentioned non-patent document <NUM>). The pharmaceutical composition of the present invention containing a poloxamer (e.g., poloxamer <NUM>) can protect RPE cells, remarkably suppress damage on and cell death of RPE cells, and remarkably prevent cell loss during a centrifugation step for the dilution and the washing, resuspending thereof, and passage of a transplantation device (syringe etc.) during transplantation thereof more than when RPE cells were suspended in a medium (e.g., BSS) alone. Therefore, the number of starting RPE cells required to make an equal amount of live RPE cells as in the conventional method arrive at the transplant site can be decreased, and the cost and time required for preparation of RPE cells necessary for transplantation can be reduced.

The pharmaceutical composition of the present invention obtained by suspending cryopreserved RPE cells immediately after thawing in the suspending agent used in the present invention does not show a decrease in the survival rate of RPE cells even when stood at <NUM> for at least <NUM> hr or at ordinary temperature for at least <NUM> hr. This means advantages are present that RPE cells cryopreserved in a CPC can be thawed, prepared as a preparation for transplantation, and transported under cooling or at ordinary temperature to a hospital where transplantation is performed, as a result of which complications in transportation can be eliminated and transplantation can be performed immediately after arrival at the hospital. That is, in one embodiment, cryopreserved RPE cells immediately after thawing are suspended in the suspending agent used in the present invention, whereby the RPE cells can be transplanted to a subject patient within <NUM> hr after thawing.

Therefore, the composition according to the invention can be used in a method for transplanting RPE cell to a patient, including the following steps:.

On the other hand, even when the pharmaceutical composition of the present invention is used for transplantation immediately after suspending cryopreserved RPE cells immediately after thawing in the suspending agent used in the present invention, it shows a photoreceptor cell protection effect equal to or higher than that of transplantation after culturing the thawed cells for a certain period of time. That is, in one embodiment, cryopreserved RPE cells can be transplanted without recovery culturing after thawing. That is, the above-mentioned composition for use in a method for transplanting to a patient, which is characterized by the absence of a step of culturing the RPE cells after thawing, is also within the scope of the present invention.

As used herein, the "photoreceptor cell protection effect" by RPE cell refers to an effect of maintaining survival of the photoreceptor cell and protecting and normalizing retinal functions by transplantation of RPE cells.

In a clinical trial using ES cell-derived RPE cells on macular degeneration, RPE cells immediately after thawing were suspended in poloxamer-free BSS and used as they were for transplantation without involving a culturing step (the above-mentioned non-patent document <NUM>), whereby a given treatment effect was obtained. The present invention has simultaneously provided a new problem that such conventional method has a high possibility of causing a remarkable decrease in the photoreceptor cell protection effect of RPE cell, and a means for solving the problem by adding a poloxamer (e.g., poloxamer <NUM>) to a suspending agent. This new problem prompts reconsideration of the easy transplantation protocol performed conventionally in which, for the purpose of improving the treatment effect (photoreceptor cell protection effect) of RPE cell, RPE cells in a cryopreserved state are transported from CPC to a hospital, thawed and transplanted immediately thereafter in the hospital. The present disclosure has demonstrated that even when RPE cells immediately after thawing are used for transplantation, a treatment effect equivalent to that achieved by transplantation of RPE cells after culturing for a given period can be obtained by the addition of poloxamer <NUM>, whereby it has been shown that a conventional simple transplantation protocol can be performed without impairing the treatment effect. That is, the pharmaceutical composition of the present invention can be used to realize a convenient and effective transplantation treatment with RPE cells.

The pharmaceutical composition of the present invention can be transplanted by subretinally injecting with a suitable transplantation device containing a syringe and a needle (e.g., MedOne0 (registered trade mark) Poly Tip (registered trade mark) Cannula <NUM>/<NUM> etc.) into, for example, mammals (e.g., human, mouse, rat, etc., preferably human) with a retinal disease such as macular degeneration (e.g., atrophic and exudative age-related macular degeneration, Stargardt disease), Retinitis pigmentosa and the like. The pharmaceutical composition of the present invention containing a poloxamer (e.g., poloxamer <NUM>) can improve flowability in a transplantation device and remarkably reduce the number of RPE cells remaining in the device upon injection. Therefore, the transplantation dose can be accurately determined, and evaluation judgment of a dose-dependent treatment effect can be performed accurately in clinical research, clinical trial and clinical use after approval, thus greatly contributing to the practicalization of products such as regenerative medicine and the like.

While the present invention is explained in more detail in the following by referring to Examples.

The protection effect of poloxamer <NUM>, which is known to have a cell protection effect, on RPE cell was verified by measuring the cell survival rate of RPE cells over time when the RPE cells are suspended in HBSS (+) containing poloxamer <NUM> at various concentrations and preserved in the state of cell suspension.

Cryopreserved (STEM-CELL BANKER (registered trade mark) GMP grade) RPE cells (1120C7 iPS cell-derived RPE cells; iPS cell source: Kyoto University) were thawed at <NUM>±<NUM>, live cell number and cell survival rate were measured by a Trypan Blue staining method, and the cells were dispensed to a cell number of <NUM> × <NUM><NUM> cells/tube. The RPE cells were centrifuged (<NUM>, <NUM>, room temperature) and the supernatant was removed. HBSS (+) (<NUM>µL) containing poloxamer <NUM> at a concentration of <NUM>%(w/v), <NUM>%(w/v), <NUM>%(w/v), or <NUM>%(w/v) was added and the resuspended RPE cell suspension was stood at <NUM>, and the cell survival rate was measured over time. The poloxamer <NUM>-containing HBSS(+) was prepared by diluting Pluronic F-<NUM>, <NUM>% (100X) (Thermo Fisher Scientific (former Life Technologies)) with HBSS(+) (in all Examples hereafter, poloxamer <NUM>-containing medium was prepared by a similar method). Sampling was performed at each measurement time point of immediately after suspension preparation as <NUM> hr, <NUM> hr later, <NUM> hr later, and cell survival rate was measured by the Trypan Blue staining method.

The results are shown in <FIG> shows a plot of variation of cell survival rates as a measured value, and <FIG> shows variation of the cell survival rate after standing at <NUM> as a relative value in which the cell survival rate immediately after (<NUM> hr later) suspension preparation is <NUM>%. When compared to suspending in plain HBSS (+), a decrease in the cell survival rate of RPE cells suspended in <NUM>%(w/v), <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+) was suppressed up to <NUM> standing <NUM> hr (<FIG>). On the other hand, when compared to suspending in plain HBSS (+), the slope of decrease in the cell survival rate of RPE cells suspended in <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+) is steep, and the decrease in the cell survival rate was not be suppressed (<FIG>). These results reveal that poloxamer <NUM> at addition concentrations of <NUM>%(w/v) and <NUM>%(w/v) suppresses a decrease in the cell survival rate. Furthermore, the addition concentration <NUM>%(w/v) of poloxamer <NUM> could not suppress a decrease in the cell survival rate. It was suggested that cytotoxicity appears at this concentration due to the cellular membrane solubilization action of the surfactant.

Considering that the RPE cell suspension preparation operation is performed at room temperature in clinical practice, the following experiment was performed with the aim to evaluate the preservation stability of RPE cell suspension suspended in plain HBSS or <NUM>%(w/v) poloxamer <NUM>-containing BSS at room temperature and <NUM>.

Cryopreserved RPE cells (1120C7 iPS cell-derived RPE cells; iPS cell source: Kyoto University) were thawed at <NUM>±<NUM>, diluted with BSS, washed, live cell number and cell survival rate were measured by Trypan Blue staining method, and the cells were dispensed to a cell number of <NUM> × <NUM><NUM> cells/tube. The RPE cells were centrifuged (<NUM>, <NUM>, room temperature) and the supernatant was removed. Plain HBSS or <NUM>%(w/v) poloxamer <NUM>-containing BSS (<NUM>) was added and the resuspended RPE cell suspension was stood at room temperature or <NUM>, and the cell survival rate was measured <NUM> hr later by a nucleocounter (registered trade mark) NC-<NUM> (produced by: ChemoMetec).

The results are shown in <FIG>. When compared to suspending in plain HBSS, the cell survival rate of RPE cells suspended in poloxamer <NUM>-containing BSS was maintained high at room temperature and <NUM> after standing for <NUM> hr. In addition, it was shown that the preservation stability of RPE cell suspension suspended in poloxamer <NUM>-containing BSS did not change until <NUM> hr later when stood at room temperature and <NUM>. Therefore, it was clarified that the protection effect of poloxamer <NUM> on RPE cell is found even at room temperature. Use of poloxamer <NUM>-containing BSS as a medium for transplantation is also effective for preparing, storing or transporting RPE cell suspension under room temperature conditions where temperature control is easy in a special environment such as hospital CPC etc. and a remote location.

Furthermore, the following experiment was performed to verify in detail the effective concentration for the protection effect of poloxamer <NUM> on RPE cells.

Cryopreserved RPE cells (Ff-I01 iPS cell derived from RPE cell; iPS cell source: Kyoto University) were thawed at <NUM> ± <NUM>, diluted with HBSS (+) and washed. The live cell number and cell survival rate were measured using a nucleocounter (registered trade mark) NC-<NUM> (produced by: ChemoMetec), and the cells were dispensed to <NUM> × <NUM><NUM> cells/tube, centrifuged (<NUM>, <NUM>, room temperature), and the supernatant was removed. HBSS (+) (<NUM>) containing poloxamer <NUM> at a concentration of <NUM>%(w/v), <NUM>%(w/v), <NUM>%(w/v), <NUM>%(w/v), <NUM>%(w/v), or <NUM>%(w/v) was added and the resuspended RPE cell suspension was stood at room temperature, and the cell survival rate was measured over time. Sampling was performed at each measurement time point of immediately after suspension preparation, <NUM> hr later, <NUM> hr later, <NUM> hr later, and cell survival rate was measured using a nucleocounter (registered trade mark) NC-<NUM> (produced by: ChemoMetec).

When compared to suspending in plain HBSS (+), the cell survival rate of RPE cells suspended in poloxamer <NUM>-containing BSS (+) was maintained high at room temperature after standing for <NUM> hr at all tested poloxamer <NUM> concentrations (<FIG>). A particularly high cell survival rate was shown when the concentration of poloxamer <NUM> was <NUM>%(w/v) - <NUM>%(w/v). These results reveal that a concentration of poloxamer <NUM> preferable for RPE cell protection effect as a medium for transplantation is <NUM>%(w/v) - <NUM>%(w/v). Consequently, under the environment during general preparation in the hospital where special conditions are not necessary, it is possible to stably maintain the cell survival rate of RPE cells for a time period presumed to be sufficient for transfer and transportation after preparation from the site of preparation, and ophthalmic surgery for transplantation, and safely perform transplantation without impairing the quality as an RPE cell preparation. In addition, use of this medium for transplantation is also effective for preparing RPE cell suspension, and storing or transporting same at ordinary temperature in a special environment such as hospital CPC etc. and a remote location.

As the effectiveness of a cell suspension using human iPS cell-derived RPE cells, the difference in the effect due to the presence or absence of a culture step after thawing and the presence or absence of addition of poloxamer <NUM> to a medium for transplantation was evaluated by the retina photoreceptor cell protection effect after subretinal transplantation to RCS rat as a retina denaturation rat model.

the maximum number of photoreceptor cells remaining in the external granular layer for each slide in each region was scored and mean of the maximum number of each area was calculated.

The results are shown in <FIG>. When BSS was used as a medium for transplantation, the number of remaining photoreceptor cell layers (ONL score) was significantly high in the cultured RPE cell suspension (test substance-<NUM>) administration group as compared to the BSS administration group. However, when compared to the BSS administration group, the thawed RPE cell suspension (test substance-1A) administration group tended to show high values but a significant difference was not found. The results reveal that the transplanted RPE cells have a photoreceptor cell protection effect but the effect is higher in the cultured RPE cells than in the thawed RPE cells. On the other hand, when poloxamer <NUM>-containing BSS was used as a medium for transplantation, the number of remaining photoreceptor cell layers (ONL score) was significantly and remarkably higher in the thawed RPE cell suspension (test substance-1B) and cultured RPE cell suspension (test substance-<NUM>) administration groups compared to the poloxamer <NUM>-containing BSS administration group. More surprisingly, the thawed RPE cell suspension (test substance-1B) administration group tended to show higher values than the cultured RPE cell suspension (test substance-<NUM>) administration group.

The results reveal that use of poloxamer <NUM>-containing BSS as a medium for transplantation results in equivalent or rather reversed photoreceptor cell protection effect of the thawed RPE cells and the cultured RPE cells. Using poloxamer <NUM>-containing BSS as a medium for transplantation, it is possible to omit a step of culturing freeze-thawed cells for <NUM> days, subsequent series of steps of detaching and recovering cultured cells, preparing the cells as a cell suspension and transporting the suspension, and all tests for ensuring stable quality and performance during this period, when in use for every transplantation. Considering environmental and technical variables such as clinical setting and in-hospital preparation and the like, it was difficult to thaw frozen cells and culture the cells for a certain period in the hospital. However, using the present method, it has become possible to provide cells that retain or exhibit photoreceptor cell protection function equivalent to or higher than that of cultured cells even when a cell pharmaceutical product formulated by freezing in a production center (CPC) is thawed and immediately used in a medical front.

Influence of plain HBSS (+) or <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+) as a medium for transplantation to be used for thawing cryopreserved cells on the cell recovery rate after thawing was verified.

RPE cells (QHJI01 iPS cell-derived RPE cell; iPS cell source: Kyoto University) cryopreserved after preparation were thawed at <NUM> ± <NUM> and suspended in plain HBSS (+) or <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+). Then, the suspension was centrifuged (<NUM>, <NUM>, room temperature) and the supernatant was removed. The cells were resuspended in the same plain HBSS (+) or poloxamer <NUM>-containing HBSS (+), and the live cell number and the dead cell number were measured using a nucleocounter (registered trade mark) NC-<NUM> (produced by: ChemoMetec) or a Trypan Blue staining method. The live cells in the cells cryopreserved at <NUM> × <NUM><NUM> cells/vial, the recovery rate of dead cells when <NUM> × <NUM><NUM> cells was <NUM>% and the ratio of lost cells were calculated.

The results are shown in <FIG>. When live cell number and dead cell number were measured using a nucleocounter (registered trade mark) NC-<NUM> (produced by: ChemoMetec), the post-thawing recovery rate of those resuspended in plain HBSS (+) was <NUM>% for live cells, <NUM>% for dead cells, with the lost cells of <NUM>%. On the other hand, the post-thawing recovery rate of those resuspended in <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+) was <NUM>% for live cells, <NUM>% for dead cells, with the lost cells of <NUM>%. When live cell number and dead cell number were measured by a Trypan Blue staining method, the post-thawing recovery rate of those resuspended in plain HBSS (+) was <NUM>% for live cells, <NUM>% for dead cells, with the lost cells of <NUM>%. On the other hand, the recovery rate of those resuspended in <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+) was <NUM>% for live cells, <NUM>% for dead cells, with the lost cells of <NUM>%.

From these results, it was demonstrated that the recovery rate of live cells decreased and the ratio of lost cells increased since the cryopreserved RPE cells received great damage when thawed. However, it was found that the ratio of lost cells can be greatly decreased by suspending RPE cells in <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+) immediately after thawing. Reduction of cell loss by the use of <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+) leads to an industrial merit of possible strict control of the number of cells in a cryopreserved preparation, as well as securing the necessary number of cells per patient and reduction of the burden cost.

As a transplantation medium, a cell suspension in which RPE cells are suspended in plain HBSS (+) or <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+) was prepared. The cell suspension was centrifuged, the supernatant was removed, the number of live cells and the number of dead cells were counted, and the recovery rate of RPE cells was calculated, based on which the influence of poloxamer <NUM> addition on the cell recovery rate was verified.

After thawing, RPE cells (QHJI01 iPS cell derived from RPE cells; iPS cell source: Kyoto University) washed once with a transplantation medium were suspended in the same plain HBSS (+) or <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+) as in the previous step. The number of live cells was counted using a nucleocounter (registered trade mark) NC-<NUM> (produced by: ChemoMetec) or a Trypan Blue staining method, and the RPE cell suspension was dispensed to a cell number of <NUM> × <NUM><NUM> cells/tube. Then, as the cell number before centrifugation, the number of live cells and the number of dead cells were counted using a nucleocounter (registered trade mark) NC-<NUM> (produced by: ChemoMetec) or a Trypan Blue staining method. After centrifugation (<NUM>, <NUM>, room temperature), the supernatant was removed. The RPE cells were resuspended in plain HBSS (+) or poloxamer <NUM>-containing HBSS (+). Then, as a cell number after centrifugation, the live cell number and the dead cell number were measured using a nucleocounter (registered trade mark) NC-<NUM> (produced by: ChemoMetec) or a Trypan Blue staining method. With the number of live cells before centrifugation as <NUM>%, the recovery rate of the cells after centrifugation was calculated.

The results are shown in <FIG>. When live cell number and dead cell number were measured using a nucleocounter (registered trade mark) NC-<NUM> (produced by: ChemoMetec), the recovery rate of those resuspended in plain HBSS (+) was <NUM>% for live cells, <NUM>% for dead cells, with the lost cells of <NUM>%. On the other hand, the recovery rate of those resuspended in <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+) was <NUM>% for live cells, <NUM>% for dead cells, with the lost cells of <NUM>%. When live cell number and dead cell number were measured by a Trypan Blue staining method, the recovery rate of those resuspended in plain HBSS (+) was <NUM>% for live cells, <NUM>% for dead cells, with the lost cells of <NUM>%. On the other hand, the recovery rate of those resuspended in <NUM>%(w/v) poloxamer <NUM>-containing HBSS (+) was <NUM>% for live cells, <NUM>% for dead cells, with the lost cells of <NUM>%.

These results have demonstrated that the addition of poloxamer <NUM> to a transplantation medium greatly decreases the number of dead cells or the ratio of lost cells due to the centrifugation operation in the cell suspension preparation step, and improves the recovery rate of the live cells. Consequently, it is possible to reduce influences of work environment of the cell suspension preparation, for example, use instrument, and cell injury due to variations in the manipulation, etc., which are dependent on workers, of thawing and washing cells, and counting cells in the suspension, not only ensure the number of effective cells but accurately grasp the number, which in turn enables stabilization of the preparation operation of the cell suspension to be transplanted in the clinical site.

As a transplantation medium, a cell suspension in which RPE cells are suspended in plain BSS or <NUM>%(w/v) poloxamer <NUM>-containing BSS was prepared. The passage efficiency of the cell suspension through a transplantation device was evaluated by measuring the number of live cells after passing through the transplantation device.

RPE cell (Ff-I01 iPS cell-derived RPE cells; iPS cell source: Kyoto University) suspension obtained by suspending in plain HBSS or <NUM>% (w/v) poloxamer <NUM>-containing BSS was prepared. As cells before passing through a transplantation device, the number of live cells was measured using a nucleocounter (registered trade mark) NC-<NUM> (produced by: ChemoMetec). The cell suspensions after passing through a transplantation device were collected, and the number of live cells was measured using a nucleocounter (registered trade mark) NC-<NUM> (produced by: ChemoMetec). With the number of live cells before passing through a transplantation device as the initial value (<NUM>%), the ratio of the number of live cells after passing through the transplantation device was calculated and the efficiency of passage through the transplantation device was evaluated.

The results are shown in Table <NUM>. The number of live cells resuspended in plain HBSS was <NUM> × <NUM><NUM> cells/mL/tube (initial value) before passing through the transplantation device and <NUM> × <NUM><NUM> cells/mL/tube (passage efficiency <NUM>%) after passing through the transplantation device. On the other hand, the number of live cells resuspended in <NUM>%(w/v) poloxamer <NUM>-containing BSS was <NUM> × <NUM><NUM> cells/mL/tube (initial value) before passing through the transplantation device and <NUM> × <NUM><NUM> cells/mL/tube (passage efficiency <NUM>%) after passing through the transplantation device.

These results have demonstrated that the use of <NUM>%(w/v) poloxamer <NUM>-containing BSS as a transplantation medium improves flowability of RPE cell suspension in the transplantation device, decreases the number of cells trapped in the transplantation device, and improves passage efficiency through the transplantation device. Heretofore, there was a deviation of about <NUM>% between the number of cells to be transplanted and the number of cells after passing through the transplantation device. However, it was clarified that the addition of poloxamer <NUM> greatly decreases the deviation. Consequently, the concern over a decrease in the cell survival rate of transplanted cells due to the passage through a transplantation device has been alleviated. In addition, accurate determination of the transplantation dose becomes possible, evaluation judgment of a dose-dependent treatment effect can be performed accurately in clinical study and clinical trial, and clinical use after approval, and great contribution can be made to the practicalization of products such as regenerative medicine and the like.

The pharmaceutical composition of the present invention containing a poloxamer (e.g., poloxamer <NUM>) affords an improvement effect on the post-thawing survival rate of cryopreserved RPE cells, an improvement effect on the photoreceptor cell protection effect when RPE cells are transplanted immediately after thawing, and a preventive effect on cell loss in various steps from thawing to transplantation. Therefore, the pharmaceutical composition of the present invention containing RPE cells suspended in the suspending agent used in the present invention is convenient for handling, also superior in a treatment effect, and extremely useful in a transplantation therapy of retinal diseases including macular degeneration.

Claim 1:
A pharmaceutical composition for use in a method of transplantation, wherein the composition is prepared by a preparation method comprising the following steps:
(<NUM>) a step of thawing cryopreserved retinal pigment epithelial (RPE) cells, and
(<NUM>) a step of suspending the thawed RPE cells in a suspending agent comprising a poloxamer, preferably poloxamer <NUM>; and
wherein the method of transplantation comprises a step of administering the suspension containing the RPE cells obtained in step (<NUM>) to an eye tissue of the patient within <NUM> hr after thawing.