Corneal endothelial cell preparation containing no culture-derived component, and method for producing same

Abstract

The present disclosure provides: a method for washing corneal endothelial cells; and a method for producing a corneal endothelial cell preparation. In one aspect, the present disclosure provides a method for producing a corneal endothelial cell preparation, the method comprising a step for washing a corneal endothelial cell or a corneal endothelium-like cell with a solution containing about 0.01% by weight to about 10% by weight of a protein and a step for mixing the corneal endothelial cell or the corneal endothelium-like cell with a composition for preparation production use to produce a corneal endothelial cell preparation, in which the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin and combinations thereof.

Description

Corneal endothelial cell preparation free of culture-derived components and its manufacturing method

[0001] The present disclosure relates to a method for washing corneal endothelial cells and a method for producing a corneal endothelial cell preparation, and a corneal endothelial cell preparation produced by such a method.

[0002] Damage to corneal endothelial cells leads to corneal opacity and severe visual impairment due to bullous keratopathy. Corneal transplantation is the only treatment for bullous keratopathy, but issues such as donor shortages and transplant rejection pose a pressing need for the development of new regenerative medicine treatments. The present inventors established a treatment method for regenerating corneal endothelium by growing corneal endothelial cells isolated from donor corneas in the presence of a ROCK inhibitor and injecting these cells into multiple patients. However, to safely administer the cells to patients, it is necessary to recover the cells from the culture vessel and remove fetal bovine serum and other additives added during culture. However, conventional methods have resulted in only approximately 50% recovery of cells due to cell damage during washing or adhesion to the vessel.

[0003] The present inventors have conducted extensive research into methods for adding stabilizing components during cell washing and have discovered a method that allows cells to be washed efficiently without reducing the cell recovery rate by adding an appropriate amount of a clinically usable protein component, such as human serum albumin.

[0004] The present disclosure provides, for example, the following: (Item 1) A method for producing a corneal endothelial cell preparation, the method comprising: washing corneal endothelial cells or corneal endothelial-like cells with a solution containing about 0.01% to about 10% by weight of protein; and mixing the corneal endothelial cells or corneal endothelial-like cells with a formulation composition to form a corneal endothelial cell preparation, wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof. (Item 2) The method according to the above items, wherein the washing step comprises suspending the corneal endothelial cells or corneal endothelial-like cells in the solution to provide a suspension of the corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells. (Item 3) The method according to any one of the above items, wherein impurities in the corneal endothelial cell preparation are removed by the washing. (Item 4) The method according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells in the washing step is 20% or less. (Item 5) The method according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells in the washing step is 15% or less. (Item 6) The method according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells in the washing step is 10% or less. (Item 7) The method according to any one of the above items, wherein the protein is human serum albumin. (Item 8) The method according to any one of the above items, wherein the solution comprises about 0.5% to about 10% by weight of human serum albumin. (Item 9) The method according to any one of the above items, wherein the solution comprises about 1% to about 5% by weight of human serum albumin. (Item 10) The method according to any one of the above items, wherein the solution comprises about 1% to about 3% by weight of human serum albumin. (Item 11) The method of any one of the above items, wherein the solution comprises about 2% by weight of human serum albumin. (Item 12) The method of any one of the above items, wherein the solution is a culture medium, a buffer solution, or an intraocular irrigation solution, or comprises one or more components of a culture medium, a buffer solution, or an intraocular irrigation solution.(Item 13) The method of any one of the above items, wherein the culture medium or intraocular irrigation solution is Opti-MEM (registered trademark), DMEM, MEM, RPMI 1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, oxyglutathione solution, or Opeguard. (Item 14) The method of any one of the above items, wherein the components of the culture medium include calcium, magnesium, potassium, sodium, phosphate, bicarbonate, vitamins, essential amino acids, or a combination thereof. (Item 15) The method of any one of the above items, wherein the components of the culture medium further include transferrin, insulin, hypoxanthine, thymidine, sodium bicarbonate, sodium pyruvate, L-glutamic acid, or a combination thereof. (Item 16) The method of any one of the above items, wherein the formulation composition is a cell preservation solution, a culture medium, a buffer solution, or an intraocular irrigation solution, or comprises one or more components of a cell preservation solution, a culture medium, a buffer solution, or an intraocular irrigation solution. (Item 17) The method of any one of the above items, wherein the cell preservation solution is CryoStor® CS2, CryoStor® CS5, Bambanker®, or Cellbanker®. (Item 18) The method of any one of the above items, wherein a component of the cell preservation solution comprises DMSO, glycerin, polyethylene glycol, or a combination thereof. (Item 19) A method for washing corneal endothelial cells or corneal endothelial-like cells, the method comprising the steps of: suspending corneal endothelial cells or corneal endothelial-like cells in a solution containing about 0.01% to about 10% by weight of protein to provide a suspension of corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells, wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof. (Item 19A) The method according to any one of the above items, wherein impurities to the corneal endothelial cell preparation are removed by the washing.(Item 19B) The method according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 20% or less. (Item 19C) The method according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 15% or less. (Item 19D) The method according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 10% or less. (Item 19E) The method according to any one of the above items, wherein the protein is human serum albumin. (Item 19F) The method according to any one of the above items, wherein the solution comprises about 0.5% to about 10% by weight of human serum albumin. (Item 19G) The method according to any one of the above items, wherein the solution comprises about 1% to about 5% by weight of human serum albumin. (Item 19H) The method of any one of the above items, wherein the solution comprises about 1% to about 3% by weight of human serum albumin. (Item 19I) The method of any one of the above items, wherein the solution comprises about 2% by weight of human serum albumin. (Item 19J) The method of any one of the above items, wherein the solution is a culture medium, a buffer solution, or an intraocular irrigation solution, or comprises one or more components of a culture medium, a buffer solution, or an intraocular irrigation solution. (Item 19K) The method of any one of the above items, wherein the culture medium or intraocular irrigation solution is Opti-MEM (registered trademark), DMEM, MEM, RPMI 1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, oxyglutathione solution, or Opeguard. (Item 19L) The method according to any one of the preceding items, wherein the components of the culture medium include calcium, magnesium, potassium, sodium, phosphate, bicarbonate, vitamins, essential amino acids, or a combination thereof. (Item 20) A cell washing solution for corneal endothelial cells or corneal endothelial-like cells, comprising about 0.01% to about 10% by weight of protein, wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof.(Item 21) The cell washing solution according to any one of the above items, wherein the washing of the corneal endothelial cells is carried out by suspending the corneal endothelial cells or corneal endothelial-like cells in the cell washing solution to provide a suspension of the corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells. (Item 22) The cell washing solution according to any one of the above items, wherein impurities in the corneal endothelial cell preparation are removed by the washing. (Item 23) The cell washing solution according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 20% or less. (Item 24) The cell washing solution according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 15% or less. (Item 25) The cell washing solution according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 10% or less. (Item 26) The cell washing solution of any one of the preceding items, wherein the protein is human serum albumin. (Item 27) The cell washing solution of any one of the preceding items, comprising about 0.5% to about 10% by weight of human serum albumin. (Item 28) The cell washing solution of any one of the preceding items, comprising about 1% to about 5% by weight of human serum albumin. (Item 29) The cell washing solution of any one of the preceding items, comprising about 1% to about 3% by weight of human serum albumin. (Item 30) The cell washing solution of any one of the preceding items, comprising about 2% by weight of human serum albumin. (Item 31) The cell washing solution of any one of the preceding items, wherein the solution is a culture medium or buffer solution or comprises one or more components of a culture medium or buffer solution. (Item 32) The cell washing solution according to any one of the preceding items, wherein the culture medium is Opti-MEM (registered trademark), DMEM, MEM, RPMI 1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, or oxyglutathione solution.(Item 33) The cell washing solution according to any one of the preceding items, wherein the culture medium component comprises calcium, magnesium, potassium, sodium, phosphate, bicarbonate, a vitamin, an essential amino acid, or a combination thereof. (Item 34) The cell washing solution according to any one of the preceding items, wherein the culture medium component further comprises transferrin, insulin, hypoxanthine, thymidine, sodium bicarbonate, sodium pyruvate, L-glutamic acid, or a combination thereof. (Item 35) A composition for producing a suspended corneal endothelial cell preparation, comprising about 0.01% to about 10% by weight of protein, wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof. (Item 35A) The composition according to any one of the above items, wherein the composition is contacted with cultured corneal endothelial cells or corneal endothelial-like cells, the corneal endothelial cells are washed, and the washing of the corneal endothelial cells is carried out by suspending the corneal endothelial cells or corneal endothelial-like cells in the composition to provide a suspension of the corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells. (Item 35B) The composition according to any one of the above items, wherein impurities in the corneal endothelial cell preparation are removed by the washing. (Item 35C) The composition according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 20% or less. (Item 35D) The composition according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 15% or less. (Item 35E) The composition according to any one of the preceding items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 10% or less. (Item 35F) The composition according to any one of the preceding items, wherein the protein is human serum albumin. (Item 35G) The composition according to any one of the preceding items, comprising about 0.5% to about 10% by weight of human serum albumin. (Item 35H) The composition according to any one of the preceding items, comprising about 1% to about 5% by weight of human serum albumin.(Item 35I) The composition of any one of the above items, comprising about 1% to about 3% by weight of human serum albumin. (Item 35J) The composition of any one of the above items, comprising about 2% by weight of human serum albumin. (Item 35K) The composition of any one of the above items, wherein the composition is a culture medium, a buffer solution, or an intraocular irrigation solution, or comprises one or more components of a culture medium, a buffer solution, or an intraocular irrigation solution. (Item 35L) The composition of any one of the above items, wherein the culture medium or intraocular irrigation solution is Opti-MEM (registered trademark), DMEM, MEM, RPMI 1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, or oxyglutathione solution. (Item 35M) The composition of any one of the preceding items, wherein the culture medium components include calcium, magnesium, potassium, sodium, phosphate, bicarbonate, vitamins, essential amino acids, or a combination thereof. (Item 35N) The composition of any one of the preceding items, wherein the culture medium components further include transferrin, insulin, hypoxanthine, thymidine, sodium bicarbonate, sodium pyruvate, L-glutamic acid, or a combination thereof. (Item A1) Use of a protein in cell washing of corneal endothelial cells or corneal endothelial-like cells, wherein the protein is used in a cell washing solution present at about 0.01% to about 10% by weight, and the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof. (Item A2) The use according to any one of the above items, wherein the washing of the corneal endothelial cells is carried out by suspending the corneal endothelial cells or corneal endothelial-like cells in the cell washing solution to provide a suspension of the corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells. (Item A3) The use according to any one of the above items, wherein impurities in the corneal endothelial cell preparation are removed by the washing.(Item A4) The use according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 20% or less. (Item A5) The use according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 15% or less. (Item A6) The use according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 10% or less. (Item A7) The use according to any one of the above items, wherein the protein is human serum albumin. (Item A8) The use according to any one of the above items, wherein the cell washing solution comprises about 0.5% to about 10% by weight of human serum albumin. (Item A9) The use according to any one of the above items, wherein the cell washing solution comprises about 1% to about 5% by weight of human serum albumin. (Item A10) The use according to any one of the above items, wherein the cell washing solution comprises about 1% to about 3% by weight of human serum albumin. (Item A11) The use according to any one of the above items, wherein the cell washing solution comprises about 2% by weight of human serum albumin. (Item A12) The use according to any one of the above items, wherein the solution is a culture medium or buffer solution, or comprises one or more components of a culture medium or buffer solution. (Item A13) The use according to any one of the above items, wherein the culture medium is Opti-MEM (registered trademark), DMEM, MEM, RPMI 1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, or oxyglutathione solution. (Item A14) The use according to any one of the above items, wherein the components of the culture medium include calcium, magnesium, potassium, sodium, phosphate, bicarbonate, vitamins, essential amino acids, or a combination thereof. (Item A15) The use according to any one of the above items, wherein the components of the culture medium further include transferrin, insulin, hypoxanthine, thymidine, sodium bicarbonate, sodium pyruvate, L-glutamic acid, or a combination thereof.(Item A16) Use of a protein in the manufacture of a suspended corneal endothelial cell preparation, wherein the protein is used in a composition present at about 0.01% to about 10% by weight, and the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof. (Item A17) The use according to any one of the above items, wherein the composition is contacted with cultured corneal endothelial cells or corneal endothelial-like cells and the corneal endothelial cells are washed, and the washing of the corneal endothelial cells is carried out by suspending the corneal endothelial cells or corneal endothelial-like cells in the composition to provide a suspension of the corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells. (Item A18) The use according to any one of the above items, wherein impurities in the corneal endothelial cell preparation are removed by the washing. (Item A19) The use according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 20% or less. (Item A20) The use according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 15% or less. (Item A21) The use according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 10% or less. (Item A22) The use according to any one of the above items, wherein the protein is human serum albumin. (Item A23) The use according to any one of the above items, wherein the composition comprises about 0.5% to about 10% by weight of human serum albumin. (Item A24) The use according to any one of the above items, wherein the composition comprises about 1% to about 5% by weight of human serum albumin. (Item A25) The use according to any one of the above items, wherein the composition comprises about 1% to about 3% by weight of human serum albumin. (Item A26) The use according to any one of the above items, wherein the composition comprises about 2% by weight of human serum albumin. (Item A27) The use according to any one of the above items, wherein the composition is a culture medium, a buffer solution, or an intraocular irrigation solution, or comprises one or more components of a culture medium, a buffer solution, or an intraocular irrigation solution.(Item A28) The use according to any one of the above items, wherein the culture medium or intraocular irrigation solution is Opti-MEM (registered trademark), DMEM, MEM, RPMI 1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, or oxyglutathione solution. (Item A29) The use according to any one of the above items, wherein the components of the culture medium include calcium, magnesium, potassium, sodium, phosphate, bicarbonate, vitamins, essential amino acids, or a combination thereof. (Item A30) The use according to any one of the above items, wherein the components of the culture medium further include transferrin, insulin, hypoxanthine, thymidine, sodium bicarbonate, sodium pyruvate, L-glutamic acid, or a combination thereof. (Item B1) A protein for cell washing of corneal endothelial cells or corneal endothelial-like cells, the protein being used in a cell washing solution present at about 0.01% to about 10% by weight, the protein being selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof. (Item B2) The protein according to any one of the above items, wherein the washing of the corneal endothelial cells is carried out by suspending the corneal endothelial cells or corneal endothelial-like cells in the cell washing solution to provide a suspension of the corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells. (Item B3) The protein according to any one of the above items, wherein impurities in the corneal endothelial cell preparation are removed by the washing. (Item B4) The protein according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 20% or less. (Item B5) The protein according to any one of the above items, wherein the rate of loss of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 15% or less. (Item B6) The protein according to any one of the above items, wherein the rate of loss of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 10% or less.(Item B7) The protein according to any one of the preceding items, wherein the protein is human serum albumin. (Item B8) The protein according to any one of the preceding items, wherein the cell washing solution comprises about 0.5% to about 10% by weight of human serum albumin. (Item B9) The protein according to any one of the preceding items, wherein the cell washing solution comprises about 1% to about 5% by weight of human serum albumin. (Item B10) The protein according to any one of the preceding items, wherein the cell washing solution comprises about 1% to about 3% by weight of human serum albumin. (Item B11) The protein according to any one of the preceding items, wherein the cell washing solution comprises about 2% by weight of human serum albumin. (Item B12) The protein according to any one of the preceding items, wherein the solution is a culture medium or buffer solution or comprises one or more components of a culture medium or buffer solution. (Item B13) The protein according to any one of the above items, wherein the culture medium is Opti-MEM (registered trademark), DMEM, MEM, RPMI 1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, or oxyglutathione solution. (Item B14) The protein according to any one of the above items, wherein the components of the culture medium include calcium, magnesium, potassium, sodium, phosphate, bicarbonate, vitamins, essential amino acids, or a combination thereof. (Item B15) The protein according to any one of the above items, wherein the components of the culture medium further include transferrin, insulin, hypoxanthine, thymidine, sodium bicarbonate, sodium pyruvate, L-glutamic acid, or a combination thereof. (Item B16) A protein for use in the production of a preparation of corneal endothelial cells in a suspension state, wherein the protein is used in a composition present at about 0.01% by weight to about 10% by weight, and the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof.(Item B17) The protein according to any one of the above items, wherein the composition is contacted with cultured corneal endothelial cells or corneal endothelial-like cells, the corneal endothelial cells are washed, and the washing of the corneal endothelial cells is carried out by suspending the corneal endothelial cells or corneal endothelial-like cells in the composition to provide a suspension of the corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells. (Item B18) The protein according to any one of the above items, wherein impurities in the corneal endothelial cell preparation are removed by the washing. (Item B19) The protein according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 20% or less. (Item B20) The protein according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 15% or less. (Item B21) The protein according to any one of the above items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 10% or less. (Item B22) The protein according to any one of the above items, wherein the protein is human serum albumin. (Item B23) The protein according to any one of the above items, wherein the composition comprises about 0.5% to about 10% by weight of human serum albumin. (Item B24) The protein according to any one of the above items, wherein the composition comprises about 1% to about 5% by weight of human serum albumin. (Item B25) The protein according to any one of the above items, wherein the composition comprises about 1% to about 3% by weight of human serum albumin. (Item B26) The protein according to any one of the above items, wherein the composition comprises about 2% by weight of human serum albumin. (Item B27) The protein according to any one of the above items, wherein the composition is a culture medium, a buffer solution, or an intraocular irrigation solution, or comprises one or more components of a culture medium, a buffer solution, or an intraocular irrigation solution.(Item B28) The protein according to any one of the above items, wherein the culture medium or intraocular irrigation solution is Opti-MEM (registered trademark), DMEM, MEM, RPMI 1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, or oxyglutathione solution. (Item B29) The protein according to any one of the above items, wherein the components of the culture medium include calcium, magnesium, potassium, sodium, phosphate, bicarbonate, vitamins, essential amino acids, or a combination thereof. (Item B30) The protein according to any one of the above items, wherein the components of the culture medium further include transferrin, insulin, hypoxanthine, thymidine, sodium bicarbonate, sodium pyruvate, L-glutamic acid, or a combination thereof. (Item C1) A corneal endothelial cell preparation, the corneal endothelial cell preparation being produced by a step of washing corneal endothelial cells or corneal endothelial-like cells with a solution containing about 0.01% by weight to about 10% by weight of protein, and a step of mixing the corneal endothelial cells or corneal endothelial-like cells with a formulation composition to form a corneal endothelial cell preparation, the protein being selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof. (Item C2) A corneal endothelial cell preparation according to the above items, wherein the washing step comprises suspending the corneal endothelial cells or corneal endothelial-like cells in the solution to provide a suspension of the corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells. (Item C3) A corneal endothelial cell preparation according to any one of the preceding items, wherein impurities in the corneal endothelial cell preparation are removed by the washing. (Item C4) A corneal endothelial cell preparation according to any one of the preceding items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells in the washing step is 20% or less. (Item C5) A corneal endothelial cell preparation according to any one of the preceding items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells in the washing step is 15% or less.(Item C6) A corneal endothelial cell preparation according to any one of the preceding items, wherein the loss rate of the corneal endothelial cells or corneal endothelial-like cells in the washing step is 10% or less. (Item C7) A corneal endothelial cell preparation according to any one of the preceding items, wherein the protein is human serum albumin. (Item C8) A corneal endothelial cell preparation according to any one of the preceding items, wherein the solution contains about 0.5% to about 10% by weight of human serum albumin. (Item C9) A corneal endothelial cell preparation according to any one of the preceding items, wherein the solution contains about 1% to about 5% by weight of human serum albumin. (Item C10) A corneal endothelial cell preparation according to any one of the preceding items, wherein the solution contains about 1% to about 3% by weight of human serum albumin. (Item C11) A corneal endothelial cell preparation according to any one of the preceding items, wherein the solution contains about 2% by weight of human serum albumin. (Item C12) The corneal endothelial cell preparation according to any one of the preceding items, wherein the solution is a culture medium, a buffer solution, or an intraocular irrigation solution, or comprises one or more components of a culture medium, a buffer solution, or an intraocular irrigation solution. (Item C13) The corneal endothelial cell preparation according to any one of the preceding items, wherein the culture medium or intraocular irrigation solution is Opti-MEM (registered trademark), DMEM, MEM, RPMI 1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, oxyglutathione solution, or Opeguard. (Item C14) The corneal endothelial cell preparation according to any one of the preceding items, wherein a component of the culture medium comprises calcium, magnesium, potassium, sodium, phosphate, bicarbonate, a vitamin, an essential amino acid, or a combination thereof. (Item C15) A corneal endothelial cell preparation according to any one of the preceding items, wherein the components of the culture medium further comprise transferrin, insulin, hypoxanthine, thymidine, sodium bicarbonate, sodium pyruvate, L-glutamic acid, or a combination thereof. (Item C16) A corneal endothelial cell preparation according to any one of the preceding items, wherein the formulation composition is a cell preservation solution, a culture medium, a buffer solution, or an intraocular irrigation solution, or comprises one or more components of a cell preservation solution, a culture medium, a buffer solution, or an intraocular irrigation solution.(Item C17) The corneal endothelial cell preparation according to any one of the preceding items, wherein the cell preservation solution is CryoStor (registered trademark) CS2, CryoStor (registered trademark) CS5, Bambanker (registered trademark), or Cellbanker (registered trademark). (Item C18) The corneal endothelial cell preparation according to any one of the preceding items, wherein a component of the cell preservation solution includes DMSO, glycerin, polyethylene glycol, or a combination thereof.

[0005] It is contemplated that the present invention may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0006] The present disclosure provides a cell preparation from which xenogeneic components such as fetal bovine serum are removed by washing with a protein that can be administered to the human body during the preparation of a cell preparation for regenerative medicine. The present invention makes it possible to reduce cell loss during cell washing by adding a protein that can be administered to the human body to the cell washing solution.

[0007] FIG. 1 shows an overview of Examples 1 to 4. FIG. 2 shows a graph of the loss rate due to the addition of casein to a cell washing solution using an immortalized corneal endothelial cell line (iHCEC). Error bars indicate the mean ± SD. Statistical significance is based on Dunnett's t-test (vs. 0%) (n=3, **p<0.01, *p<0.05). FIG. 3 shows a graph of the loss rate due to the addition of lactoferrin to a cell washing solution using an immortalized corneal endothelial cell line (iHCEC). Error bars indicate the mean ± SD. Statistical significance is based on Dunnett's t-test (vs. 0%) (n=3, **p<0.01, *p<0.05). FIG. 4 shows a graph of the loss rate due to the addition of ovalbumin to a cell washing solution using an immortalized corneal endothelial cell line (iHCEC). Error bars indicate the mean ± SD. Statistical significance is based on Dunnett's t-test (vs. 0%) (n=3, **p<0.01, *p<0.05). FIG. 5 shows a graph of the loss rate due to the addition of human serum albumin to a cell washing solution, using an immortalized corneal endothelial cell line (iHCEC). Error bars indicate the mean ± SD. Statistical significance is based on the Dunnett's t-test (vs. 0%) (n=3, **p<0.01, *p<0.05). FIG. 6 shows an overview of Example 5. FIG. 7 shows a graph of the loss rate due to the addition of human serum albumin to a cell washing solution, using human corneal endothelial cells after four passages. Error bars indicate the mean ± SD. Statistical significance is based on the Dunnett's t-test (vs. 0%) (n=3, **p<0.01). FIG. 8 shows representative photographs of the results of Example 7.

[0008] The present invention will be described below. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that the terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the event of conflict, the present specification (including definitions) will prevail. In this specification, "about" means ±10% of the following value.

[0009] (Definition) In this specification, the term "corneal endothelial cells" is used in the usual sense used in the relevant field. The cornea is one of the layered tissues that make up the eye, is transparent, and is located closest to the outside world. In humans, the cornea is said to be made up of five layers, starting from the outside (body surface), and is composed of, from the outside, the corneal epithelium, Bowman's membrane, lamina propria, Descemet's membrane (corneal endothelial basement membrane), and corneal endothelium. Unless otherwise specified, parts other than the epithelium and endothelium are sometimes collectively referred to as the "corneal stroma," and will be referred to as such in this specification. In this specification, "HCECs" (human corneal endothelial cells) is an abbreviation for human corneal endothelial cells.

[0010] As used herein, the term "corneal endothelial-like cells" refers to cells differentiated from stem cells, for example, cells differentiated from iPS cells, and which have substantially the same functions as corneal endothelial cells. Methods for differentiating stem cells, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc., into corneal endothelial-like cells are well known in the art (McCabe et al., PLoS One. 2015 Dec 21; 10(12): e0145266; Ali et al., Invest Ophthalmol Vis Sci. 2018 May 1; 59(6): 2437-2444). Briefly, in a typical example, iPS cells are seeded onto 35 mm Matrigel-coated plates (Corning) at a 1:12 dilution using cell dissociation buffer (Life Technologies) on day 0 (80% confluent plates are split into 12 plates). iPS cells are grown in culture medium (mTeSR1; STEMCELL Technologies Inc.) for 4 days. On day 4, mTeSR1 medium was supplemented with 80% DMEM-F12 (Life Technologies), 20% KSR (Life Technologies), 1% non-essential amino acids (Life Technologies), 1 mM L-glutamine (STEMCELL Technologies, Inc.), 0.1 mM β-mercaptoethanol (MilliporeSigma), and 8 ng / mL βFGF (MilliporeSigma) in a basal medium containing 500 ng / mL human recombinant Noggin (R&D The medium is replaced with Smad inhibitor medium containing 10 μM SB431542 (MilliporeSigma) and 10 μM SB431542 (Scientific Systems, Minneapolis, MN, USA).On day 6, Smad inhibitor medium was prepared by adding 80% DMEM-F12 (Life Technologies), 20% KSR (Life Technologies), 1% non-essential amino acids (Life Technologies), 1 mM L-glutamine (STEMCELL Technologies, Inc.), 0.1 mM β-mercaptoethanol (MilliporeSigma), and 8 ng / mL βFGF (MilliporeSigma) to the basal medium, supplemented with 0.1× B27 supplement (Life Technologies), 10 ng / mL recombinant human platelet-derived growth factor-BB (PDGF-BB; PeproTech, Rocky Mountain). The corneal medium is replaced with corneal medium containing 10 ng / mL recombinant human Dickkopf-related protein-2 (DKK-2; R&D Systems) and 10 ng / mL recombinant human Dickkopf-related protein-2 (DKK-2; R&D Systems). On day 7, the differentiating CECs are transferred to a new Matrigel-coated plate (35 mm) and grown in corneal medium for an additional 13 days. The differentiated CECs are harvested on day 20. The above example is a typical example, and those skilled in the art may also use other methods well known in the art (Fukuta et al., PLoS One. 2014 Dec 2;9(12):e112291; Hayashi et al., Nature. 2016 Mar 17;531(7594):376-80). Furthermore, those skilled in the art can prepare corneal endothelial-like cells by appropriately adjusting the conditions of methods well known in the art.

[0011] "Corneal endothelial cells" and "corneal endothelial-like cells" may contain a magnetic material (e.g., iron). For example, when corneal endothelial cells containing a magnetic substance are injected into the anterior chamber, they can be attracted to the inside of the cornea (e.g., Descemet's membrane) by magnetic force, promoting adhesion (Patel et al., Invest Ophthalmol Vis Sci. 2009 May; 50(5): 2123-31; Mimura et al., Exp Eye Res. 2003 Jun; 76(6): 745-51; and Mimura et al., Exp Eye Res. 2005 Feb; 80(2): 149-57). A "magnetic material" refers to a substance that can be magnetized by a magnetic field, and examples include iron, cobalt, nickel, and ferrite.

[0012] As used herein, the term "corneal endothelial cell preparation" refers to a preparation containing corneal endothelial cells or corneal endothelial-like cells, which is in a form suitable for use or in a form prepared just before use. "Prepared just before use" refers to preparing a preparation suitable for use by adding a drug or by diluting with a solvent just before administration.

[0013] As used herein, "washing" refers to the removal of impurities from a corneal endothelial cell preparation. "Impurities from a corneal endothelial cell preparation" refers to xenogeneic components added during cell culture (e.g., fetal bovine serum), components undesirable for administration to humans, for example, antibiotics (gentamicin, etc.) that may be added to the culture medium, enzyme inhibitors (e.g., p38 MAPK inhibitors such as SB203580), and waste products and cytokines (e.g., IL-6, IL-8, etc.) released by cells into the medium.

[0014] As used herein, the term "loss rate" refers to the percentage of cells lost after washing with the cell washing solution of the present disclosure. The percentage is calculated as the percentage of the number of cells after washing, with the number of cells before washing set at 100%.

[0015] (Preferred Embodiments) A description of preferred embodiments will be given below, but it should be understood that these embodiments are merely examples of the present invention and that the scope of the present invention is not limited to such preferred embodiments. It should also be understood that those skilled in the art can easily make modifications, changes, etc. within the scope of the present invention by referring to the following preferred examples. Those skilled in the art can combine any of these embodiments as appropriate.

[0016] (Method for Producing Corneal Endothelial Cell Preparation and Method for Washing Corneal Endothelial Cells) In one aspect, the present disclosure provides a method for producing a corneal endothelial cell preparation, the method comprising the steps of washing corneal endothelial cells or corneal endothelial-like cells with a solution containing a protein that can be administered to humans, and mixing the corneal endothelial cells or corneal endothelial-like cells with a formulation composition to form a corneal endothelial cell preparation. In some embodiments, the protein that can be administered to humans may be selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof. In some embodiments, the protein that can be administered to humans may be contained in the solution at about 0.01% by weight to about 10% by weight.

[0017] In one aspect, the present disclosure provides a method for producing a corneal endothelial cell preparation, the method comprising the steps of washing corneal endothelial cells or corneal endothelial-like cells with a solution containing about 0.01% by weight to about 10% by weight of protein, and mixing the corneal endothelial cells or corneal endothelial-like cells with a formulation composition to form a corneal endothelial cell preparation, wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof.

[0018] In one aspect, the present disclosure provides a method for washing corneal endothelial cells or corneal endothelial-like cells, the method comprising the steps of suspending corneal endothelial cells or corneal endothelial-like cells in a solution containing a protein that is administrable to humans to provide a suspension of corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to collect the corneal endothelial cells or corneal endothelial-like cells. In some embodiments, the protein that is administrable to humans may be selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof. In some embodiments, the protein that is administrable to humans may be included in the solution at about 0.01% to about 10% by weight.

[0019] In one aspect, the present disclosure provides a method for washing corneal endothelial cells or corneal endothelial-like cells, the method comprising the steps of suspending corneal endothelial cells or corneal endothelial-like cells in a solution containing about 0.01% to about 10% by weight of a protein to provide a suspension of corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells, wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof.

[0020] In some embodiments, washing may be performed once or multiple times, preferably twice, with a maximum of five washes being preferred to reduce washing losses.

[0021] In some embodiments, the cell concentration when suspending the cells in the cell washing solution is at least about 0.5 x 10 ４ cells / mL, at least about 1.0 x 10 ４ cells / mL, at least about 0.5 x 10 ５ cells / mL, at least about 1.0 x 10 ６ cells / mL, at least about 0.5 x 10 ７ cells / mL, at least about 1.0 x 10 ７ The preferred cell concentration is about 1.0 x 10 cells / mL. ６ pieces / mL ~ approx. 1.0 x 10 ７ / mL, and the maximum is about 0.5 × 10 ８ It may be cells / mL.

[0022] The cells of the present disclosure can be used without re-culturing after washing because there is little cell loss during washing and impurities are significantly reduced. This is particularly advantageous for clinical use. In some embodiments, the cells of the present disclosure can be suspended in a suspension appropriate for the intended use after washing without re-culturing. In some embodiments, the cells of the present disclosure are cells for clinical use (e.g., cell infusion therapy), and can be suspended and administered after washing without re-culturing.

[0023] In some embodiments, the washing step may include suspending the corneal endothelial cells or corneal endothelial-like cells in the solution to provide a suspension of the corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells. The washing step may remove impurities from the corneal endothelial cell preparation. Impurities from the corneal endothelial cell preparation include, but are not limited to, any xenogeneic component (e.g., fetal bovine serum), any antibiotic (gentamicin, etc.), any enzyme inhibitor (e.g., p38 MAPK inhibitor such as SB203580, etc.), any waste product released by the cells into the medium, or any cytokines (e.g., IL-6, IL-8, etc.). In some embodiments, washing may remove at least one of fetal bovine serum, gentamicin, p38 MAPK inhibitor, IL-6, and IL-8.

[0024] The method of the present disclosure can reduce the rate of loss of corneal endothelial cells or corneal endothelial-like cells during cleaning. In some embodiments, the rate of loss of corneal endothelial cells or corneal endothelial-like cells during the cleaning step can be 20% or less, preferably 15% or less, and more preferably 10% or less.

[0025] In some embodiments, the protein in the wash solution can be human serum albumin. In certain embodiments, the amount of human serum albumin in the wash solution can be about 0.5% to about 10% by weight, preferably about 1% to about 5% by weight, more preferably about 1% to about 3% by weight, and most preferably about 2% by weight.

[0026] In some embodiments, the protein in the wash solution may be casein, hi certain embodiments, the amount of casein in the wash solution may be from about 0.01% to about 1% by weight, preferably from about 0.03% to about 0.3% by weight, and more preferably about 0.1% by weight.

[0027] In some embodiments, the protein in the washing solution can be lactoferrin. In certain embodiments, the amount of lactoferrin in the washing solution can be about 0.5% to about 5% by weight, preferably about 1% to about 4% by weight, more preferably about 2% to about 3% by weight, and most preferably about 3% by weight.

[0028] In some embodiments, the protein in the cleaning solution can be ovalbumin. In certain embodiments, the amount of ovalbumin in the cleaning solution can be about 0.01% to about 1% by weight, preferably about 0.03% to about 0.5% by weight, more preferably about 0.03% to about 0.3% by weight, even more preferably about 0.03% to about 0.1% by weight, and most preferably about 0.03% by weight.

[0029] In some embodiments, the solution used in the methods of the present disclosure may be a culture medium or buffer, or may contain one or more components of a culture medium or buffer. Examples of culture media, buffers, or intraocular irrigation solutions include, but are not limited to, Opti-MEM®, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), RPMI 1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, and oxyglutathione solution. In certain embodiments, the components of the culture medium may include calcium, magnesium, potassium, sodium, phosphate, bicarbonate, vitamins, essential amino acids, or combinations thereof. In further embodiments, the culture medium components may include transferrin, insulin, hypoxanthine, thymidine, sodium bicarbonate, sodium pyruvate, L-glutamic acid, or combinations thereof in addition to the culture medium or culture medium components described above.

[0030] Examples of buffer solutions include, but are not limited to, acetate buffer, phosphate buffer, citrate buffer, HEPES buffer, Tris buffer, ammonium chloride buffer, and phosphate buffered saline. In some embodiments, the pH may be adjusted to about 6.0 to about 8.0, preferably about 6.2 to about 7.7. In a preferred embodiment, the buffer solution may be a phosphate buffer.

[0031] Intraocular irrigating solutions include, but are not limited to, oxyglutathione or Opeguard.

[0032] In some embodiments, the formulation composition may be a cell preservation solution, culture medium, buffer solution, or intraocular irrigation solution, or may contain one or more components of a cell preservation solution, culture medium, buffer solution, or intraocular irrigation solution. Cell preservation solutions include, but are not limited to, CryoStor® CS2, CryoStor® CS5, Bambanker®, and Cellbanker®. In certain embodiments, dimethyl sulfoxide (DMSO), glycerin, polyethylene glycol, or a combination thereof may be included. In some embodiments, DMSO, glycerin, polyethylene glycol, or a combination thereof may be added to the culture medium to form a cell preservation solution.

[0033] In certain embodiments, the amount of DMSO contained in the cell preservation solution may be about 1 to about 10% by weight, preferably about 1 to about 5% by weight, more preferably about 1 to about 3% by weight, and most preferably about 2% by weight.

[0034] In certain embodiments, the amount of glycerin contained in the cell preservation solution may be about 1 to about 20% by weight, preferably about 5 to about 15% by weight, more preferably about 7 to about 13% by weight, and most preferably about 10% by weight.

[0035] In certain embodiments, the amount of polyethylene glycol contained in the cell preservation solution may be about 1 to about 20% by weight, preferably about 5 to about 15% by weight, more preferably about 7 to about 13% by weight, and most preferably about 10% by weight.

[0036] (Corneal endothelial cell washing solution and composition for manufacturing a formulation) In one aspect, the present disclosure provides a cell washing solution for corneal endothelial cells or corneal endothelial-like cells comprising about 0.01% by weight to about 10% by weight of protein, wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof.

[0037] In another aspect, the present disclosure provides a composition for producing a corneal endothelial cell preparation in a suspended state, comprising about 0.01% to about 10% by weight of a protein, wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof.

[0038] In some embodiments, washing of the corneal endothelial cells can be carried out by suspending the corneal endothelial cells or corneal endothelial-like cells in a cell washing solution to provide a suspension of corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells. This washing can remove impurities from the corneal endothelial cell preparation.

[0039] The loss rate of corneal endothelial cells or corneal endothelial-like cells, the amount of protein, the culture medium, and the buffer solution are as described above.

[0040] The present invention has been described above by showing preferred embodiments for ease of understanding. The present invention will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

[0041] The present disclosure will be described in more detail below with reference to examples. It will be understood that the various reagents used in these examples may include those specifically shown, as well as those available from Sigma-Aldrich, BASF Japan Ltd., and the like.

[0042] (Example 1: Reduction of cell loss rate by adding casein to cell washing solution) (Preparation example: production of immortalized corneal endothelial cell line (iHCEC)) In this example, an immortalized corneal endothelial cell line (iHCEC) was produced from research donor corneas derived from healthy donors.

[0043] (Culture Method) Corneal endothelial cells were mechanically detached together with the basement membrane from research corneas purchased from the Seattle Eye Bank, and the basement membrane was dissolved using collagenase to collect the corneal endothelial cells, which were then subjected to primary culture. The culture medium was Opti-MEM I Reduced-Serum Medium, Liquid (INVITROGEN, Catalog No.: 31985-070), supplemented with 8% FBS (BIOWEST, Catalog No.: S1820-500), 200 mg / ml CaCl ２ ・2H ２The medium used was a basal medium conditioned for 3T3 feeder cells supplemented with 0 (SIGMA catalog number: C7902-500G), 0.08% chondroitin sulfate (SIGMA catalog number: C9819-5G), 20 μg / ml ascorbic acid (SIGMA catalog number: A4544-25G), 50 μg / ml gentamicin (INVITROGEN catalog number: 15710-064), and 5 ng / ml EGF (INVITROGEN catalog number: PHG0311). Alternatively, the cells were cultured in a basal medium supplemented with SB431542 (1 μmol / L) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5(4-pyridyl)imidazole < 4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine) (1 μmol / L) (also referred to herein as "SB203580 + SB431542 + 3T3 conditioned medium"). The SV40 large T antigen and hTERT genes of the cultured corneal endothelial cells were amplified by PCR and transfected into a lentiviral vector (pLenti6.3_V5-TOPO; Life Technologies Inc.). Subsequently, 293T cells (RCB2202; Riken Bioresource Center, Ibaraki, Japan) were infected with the lentiviral vector together with three helper plasmids (pLP1, pLP2, pLP / VSVG; Life Technologies Inc.) using a transfection reagent (Fugene HD; Promega Corp., Madison, WI). After 48 hours of infection, the virus-containing culture supernatant was collected and added to the culture medium of corneal endothelial cells derived from a healthy donor, cultured using 5 μg / ml polybrene. The cells were immortalized by introducing SV40 large T antigen and hTERT genes to obtain iHCECs. The iHCECs were maintained and cultured in DMEM + 10% FBS.

[0044] (Materials) - Immortalized human corneal endothelial cells (iHCEC) - 150mm dish (Corning 430599) - Dulbecco's Modified Eagle Medium: DMEM (Nacalai tesque 08456-36)・0.5g / L-Trypsin-0.53mmol / L-EDTA Solution, with Phenol Red (Nacalai tesque 32778-34) ・Casein, milk-derived (Fuji Film Wako Pure Chemical Industries, Ltd. 030-01505)・1 x Phosphatase-Buffered Saline (-): 1 x PBS (-) (Nissui Pharma 05913) ・50 mL centrifuge tubes (Corning 430829) ・0.5% trypan blue staining solution (Nakarai Tesque 29853-34) (Method) 1. Cell washing solutions with various casein concentrations were prepared by adding casein to DMEM. 2. Immortalized human corneal endothelial cells were used. The medium was removed from the culture dish during cultivation, and 1x PBS (-) pre-warmed to 37°C was added and washed. This process was repeated twice. This washing step was performed to remove the culture medium while the cells remained adhered to the bottom of the culture vessel. This is because remaining protein components such as fetal bovine serum in the culture medium weaken the cell detachment effect of trypsin in the next step. Because the cells remained firmly adhered to the culture base while in culture, no cell loss occurred. This is also true in the following examples. 3. After removing the 1x PBS(-), 0.05% Trypsin-EDTA was added and the cells were incubated at 37°C (5% CO ２ ) for 3 minutes. 4. After 3 minutes, the cells were suspended in 10 mL of cell washing solution and collected in a 50 mL centrifuge tube. The cell concentration in the suspension varied depending on the experiment, but was generally 0.5 × 10 ７ ~1 x 10 ７cells / mL. 5. The number of viable cells was counted by trypan blue staining. 6. Centrifuged at 300G for 5 minutes. 7. The supernatant was removed and the cells were suspended in 10 mL of cell washing solution. 8. Centrifuged at 300G for 5 minutes. 9. Steps 7 and 8 were repeated again. 10. The supernatant was removed and the cells were suspended in DMEM, and the number of viable cells was counted again by trypan blue staining. 11. The number of viable cells counted in step 5 was set to 100%, and the cell loss rate was calculated from the number of viable cells counted in step 10.

[0045] An outline of Example 1 is shown in FIG.

[0046] (Results) Figure 2 shows the loss rate when the fetal bovine serum washing process was performed using cell washing solutions containing different concentrations of casein. In the conventional washing method using the 0% casein group, approximately 20% of the cells were lost during washing, and only 78.8% of the cells were recovered. Compared to the 0% casein group, the cell recovery rate after washing was significantly improved in the 0.03%, 0.1%, and 0.3% casein groups. In particular, the addition of 0.1% casein allowed almost all cells to be recovered, at 97.9%.

[0047] (Example 2: Reduction of cell loss rate by adding lactoferrin to cell washing solution) (Materials) Immortalized human corneal endothelial cells (iHCEC) 150 mm dish (Corning 430599) Dulbecco's Modified Eagle Medium: DMEM (Nacalai tesque 08456-36) 0.5 g / L-Trypsin-0.53 mmol / L-EDTA Solution, with Phenol Red (Nacalai tesque 32778-34) Lactoferrin, derived from cow's milk (Fujifilm Wako Pure Chemical Industries, Ltd. 127-04122) 1× Phosphatase-Buffered Saline (-): 1x PBS (-) (Nissui Pharma 05913) ・Centrifuge tube 50 mL (Corning 430829) ・0.5% trypan blue staining solution (Nakarai tesque 29853-34) (Method) 1. Cell washing solutions with various lactoferrin concentrations were prepared by adding lactoferrin to DMEM. 2. Immortalized human corneal endothelial cells were used. The medium was removed from the culture dish during cultivation, and 1x PBS (-) that had been pre-warmed to 37°C was added and washed. This procedure was repeated twice. 3. After removing the 1x PBS (-), 0.05% trypsin-EDTA was added, and the cells were incubated at 37°C (5% CO ２ ) for 3 minutes. 4. After 3 minutes, the cells were suspended in 10 mL of cell washing solution and collected in a 50 mL centrifuge tube. The cell concentration in the suspension varied depending on the experiment, but was generally 0.5 × 10 ７ ~1 x 10 ７ cells / mL. 5. The number of viable cells was counted by trypan blue staining. 6. Centrifuged at 300G for 5 minutes. 7. The supernatant was removed and the cells were suspended in 10 mL of cell washing solution. 8. Centrifuged at 300G for 5 minutes. 9. Steps 7 and 8 were repeated again. 10. The supernatant was removed and the cells were suspended in DMEM, and the number of viable cells was counted again by trypan blue staining. 11. The number of viable cells counted in step 5 was set to 100%, and the loss rate was calculated from the number of viable cells counted in step 10.

[0048] An outline of Example 2 is shown in FIG.

[0049] (Results) Figure 3 shows the loss rate when the fetal bovine serum washing process was performed using cell washing solutions containing various concentrations of lactoferrin. Compared to the 0% group (no lactoferrin added), the recovery rate was improved in the 1%, 2%, 3%, and 4% groups.

[0050] (Example 3: Reduction of cell loss rate by adding ovalbumin to cell washing solution) (Materials) Immortalized human corneal endothelial cells (iHCEC) 150 mm dish (Corning 430599) Dulbecco's Modified Eagle Medium: DMEM (Nacalai tesque 08456-36) 0.5 g / L-Trypsin-0.53 mmol / L-EDTA Solution, with Phenol Red (Nacalai tesque 32778-34) Albumin, egg-derived (Fujifilm Wako Pure Chemical Industries, Ltd. 018-09882)・1x Phosphatase-Buffered Saline (-): 1x PBS (-) (Nissui Pharma 05913) ・50 mL Centrifuge Tube (Corning 430829) ・0.5% Trypan Blue Staining Solution (Nakarai Tesque 29853-34) (Method) 1. Ovalbumin was added to 1x PBS (-) to prepare a 1.5% solution. 2. Cell washing solutions with various ovalbumin concentrations were prepared by adding the solution prepared in 1 to DMEM. 3. Immortalized human corneal endothelial cells were used. The medium was removed from the culture dish during cultivation, and 1x PBS (-) pre-warmed to 37°C was added and washed. This process was repeated twice. 4. After removing the 1x PBS(-), 0.05% Trypsin-EDTA was added and the cells were incubated at 37°C (5% CO ２ ) for 3 minutes. 5. After 3 minutes, the cells were suspended in 10 mL of cell washing solution and collected in a 50 mL centrifuge tube. The cell concentration in the suspension varied depending on the experiment, but was generally 0.5 × 10 ７ ~1 x 10 ７cells / mL. 6. The number of viable cells was counted by trypan blue staining. 7. Centrifuged at 300G for 5 minutes. 8. The supernatant was removed and the cells were suspended in 10 mL of cell washing solution. 9. Centrifuged at 300G for 5 minutes. 10. Steps 8-9 were repeated again. 11. The supernatant was removed and the cells were suspended in DMEM, and the number of viable cells was counted again by trypan blue staining. 12. The number of viable cells counted in step 6 was set to 100%, and the loss rate was calculated from the number of viable cells counted in step 11.

[0051] An outline of Example 3 is shown in FIG.

[0052] (Results) Figure 4 shows the loss rate when the fetal bovine serum washing step was performed using cell washing solutions containing different concentrations of ovalbumin. Compared to the 0% group (no ovalbumin added), an improvement in recovery rate was confirmed in the 0.01%, 0.03%, 0.1%, and 0.3% ovalbumin added groups.

[0053] (Example 4: Reduction of cell loss rate by adding human serum albumin to cell washing solution) (Materials) Immortalized human corneal endothelial cells (iHCEC) 150 mm dish (Corning 430599) OptiMEM ＴＭ -I (Invitrogen 21585-070) ・Tryple ＴＭ Select Enzyme (10x) (Thermo Fisher Scientific A12177-01) ・Red Cross Albumin 25% Intravenous Injection 12.5g / 50mL (Japan Blood Products Organization) ・1x Phosphatase-Buffered Saline (-): 1x PBS (-) (Nissui Pharma 05913) ・Centrifuge tube 50mL (Corning 430829) ・0.5% Trypan Blue Staining Solution (Nakarai Tesque 29853-34) (Method) 1. Cell washing solutions with various human serum albumin concentrations were prepared by adding human serum albumin to OptiMEM. 2. Immortalized human corneal endothelial cells were used. The medium was removed from the culture dish during culturing, and OptiMEM pre-warmed to 37°C was added and washed. This procedure was repeated twice. 3. After removing OptiMEM, ＴＭSelect Enzyme (10x) was added, and the mixture was incubated at 37°C (5% CO ２ 4. After 20 minutes, the cells were suspended in 10 mL of cell washing solution and collected in a 50 mL centrifuge tube. The cell concentration in the suspension varied depending on the experiment, but was generally 0.5 × 10 ７ ~1 x 10 ７ cells / mL. 5. The number of viable cells was counted by trypan blue staining. 6. Centrifuged at 300G for 5 minutes. 7. The supernatant was removed and the cells were suspended in 10 mL of cell washing solution. 8. Centrifuged at 300G for 5 minutes. 9. Steps 7 and 8 were repeated again. 10. The supernatant was removed and the cells were suspended in DMEM, and the number of viable cells was counted again by trypan blue staining. 11. The number of viable cells counted in step 5 was set to 100%, and the loss rate was calculated from the number of viable cells counted in step 10.

[0054] An outline of Example 4 is shown in FIG.

[0055] (Results) Figure 5 shows the graph of cell recovery rate when the fetal bovine serum washing step was performed using cell washing solutions containing various concentrations of human serum albumin. Compared to the 0% group (no human serum albumin added), improved recovery rates were observed in the 0.4%, 0.5%, 1%, 2%, and 5% added groups.

[0056] (Example 5: Reduction in the loss rate of non-immortalized corneal endothelial cells for clinical use by adding human serum albumin to a cell washing solution) (Method for culturing human corneal endothelial cells) Corneal endothelial cells were collected from human corneal tissue and subjected to primary culture and subculture. First, 0.4 w / v% trypan blue solution for cell staining (Fujifilm Wako Pure Chemical Industries, Ltd., 207-17081) was added to the center of the donor cornea to stain it. After staining, the cells were cultured in OptiMEM supplemented with gentamicin (Invitrogen, 15710-064) to a final concentration of 50 μg / mL. ＴＭThe trypan blue staining solution was washed away using ointment-I (Invitrogen, 31985-088). The cornea was observed using a microscope and monitor installed in a clean bench, and the Descemet's membrane stained with trypan blue was peeled off from the cornea with forceps. The peeled Descemet's membrane was transferred to a 12-well plate containing collagenase (Amano Enzyme Co., Ltd., 639-44651) and incubated at 37°C (5% CO ２ ) for 16 hours. After incubation, the Descemet's membrane was transferred to a 15 mL centrifuge tube and centrifuged at 300 G for 3 minutes. After centrifugation, the supernatant was removed, and OptiMEM supplemented with gentamicin to a final concentration of 50 μg / mL was added, followed by centrifugation at 300 G for 3 minutes. This procedure was repeated twice. After centrifugation, the supernatant was removed, and the pellet was resuspended in medium supplemented with Y-27632 (WAKO, 259-00613) to a final concentration of 10 μM. The resuspended cell suspension was seeded onto a 6-well plate coated with laminin E8 fragment (iMatrix-511; Nippi Corporation, 892012). After 24 hours, the medium was replaced with one without Y-27632, and thereafter the medium was replaced every two days and the cells were cultured for 10 days. OptiMEM was used as the medium. ＴＭ The medium used was medium I supplemented with 8% fetal bovine serum (FBS) (Thermo Fisher Scientific, GVJ0081), 50 μg / mL gentamicin, 200 mg / L calcium chloride (Otsuka Pharmaceutical, 873215), 0.08% sodium chondroitin sulfate C (WAKO, 032-14613), 5 ng / mL epidermal growth factor (EGF; Invitrogen, PHG0311), 20 μg / mL ascorbic acid (nipro 21F01), and 10 μM SB203580 (Cayman Chemical, 13067). (Materials) Human corneal endothelial cells after passage 4 T25 flask (Corning 430168) OptiMEM ＴＭ -I (Invitrogen 21585-070) ・Tryple ＴＭSelect Enzyme (10x) (Thermo Fisher Scientific A12177-01) ・Red Cross Albumin 25% Intravenous Injection 12.5g / 50mL (Japan Blood Products Organization) ・Stemful Centrifuge Tube 15mL (Sumitomo Bakelite MS-90150) ・0.5% Trypan Blue Staining Solution (Nakarai Tesque 29853-34) (Method) 1. Cell washing solutions with various human serum albumin concentrations were prepared by adding human serum albumin to OptiMEM. 2. Human corneal endothelial cells after passage 4 were used. The medium was removed from a T25 flask during culture, and OptiMEM that had been pre-warmed to 37°C was added. ＴＭ 3. After removing OptiMEM, Tryple ＴＭ Select Enzyme (10x) was added, and the mixture was incubated at 37°C (5% CO ２ 4. After 20 minutes, the cells were suspended in 10 mL of cell washing solution and collected in a 15 mL stem-full. The cell concentration in the suspension was approximately 9 x 10 ６ 5. The number of viable cells was counted by trypan blue staining. 6. The cells were centrifuged at 300 G for 5 minutes. 7. The supernatant was removed, and the cells were suspended in 10 mL of cell washing solution. The cell concentration in the suspension varied depending on the experiment, but was generally 0.5 x 10 ７ ~1 x 10 ７ The cell concentration was 100 cells / mL. 8. Centrifuged at 300 G for 5 minutes. 9. Steps 7 and 8 were repeated again. 10. The supernatant was removed, the cells were suspended in OptiMEM, and the viable cells were counted again using trypan blue staining. 11. The viable cell count in step 5 was set as 100%, and the loss rate was calculated from the viable cell count in step 10.

[0057] An outline of Example 5 is shown in FIG.

[0058] (Results) Figure 7 shows the loss rate when performing the fetal bovine serum washing process using cell washing solutions containing varying concentrations of human serum albumin. In the conventional washing method using the 0% group without added human serum albumin, approximately 20% of the cells were lost during washing, and only 82.5% of the cells were recovered. Compared to the 0% group without added human serum albumin, improved recovery rates were observed in the 2% and 5% groups. In particular, adding 2% human serum albumin to the cell washing solution allowed almost all cells to be recovered without loss.

[0059] Example 6-1: Production Example 1 of Corneal Endothelial Cell Preparation In this example, a production example of a corneal endothelial cell preparation (cryopreserved preparation) is shown.

[0060] (Materials) 2% human serum albumin / CTS-OPTI-MEM 25% human serum albumin 3.6 mL CTS-OPTI-MEM 41.4 mL Total 45 mL Two 45 mL / 50 mL tubes Sterile vial Y27632 (Method) - Collect the culture supernatant from the corneal endothelial cell culture. - Suspend the corneal endothelial cells in 2% human serum albumin / CTS-OPTI-MEM (30 mL). - Centrifuge at 300 x g for 5 minutes. - Suspend in 2% human serum albumin / CTS-OPTI-MEM (20 mL). - Centrifuge at 300 x g for 5 minutes. - Suspend in 2% human serum albumin / CTS-OPTI-MEM (20 mL). - Centrifuge at 300 x g for 5 minutes. Y27632 was dissolved in CryoStor CS2 to a concentration of 100 μM and suspended in 2.6 × 10 ６ Dilute the cell suspension to a concentration of 10 ...

[0061] Example 6-2: Production Example 2 of Corneal Endothelial Cell Preparation In this example, a production example of a corneal endothelial cell preparation (cryopreserved preparation) is shown.

[0062] (Material) TrypLE ＴＭSelect Enzyme (10x) (Thermo Fisher Scientific; A1217701) 2%HSA+CTS ＴＭ Opti-MEM ＴＭ I Medium (37℃) CryoStor CS2 (Charles River; 202102) Corning for adherent cell culture 75cm ２ Flask: CryoStor CS2 (Charles River; 202102) Y27632

[0063] (Method) Corning 75cm for adherent cell culture ２ In the flask, TrypLE ＴＭ Add 5 mL of Select Enzyme (10x) (warmed to 37°C) and incubate at 37°C for 20 minutes in an incubator. ２ The entire cell suspension was collected from the flask using an autopipettor and placed in a 50 mL centrifuge tube (liquid volume: 15 mL). ２ The flask was filled with 2% HSA + CTS ＴＭ Opti-MEM ＴＭ Add 10 mL of I Medium (37°C) and suspend the corneal endothelial cells. Repeat this process twice (35 mL volume). Centrifuge the 50 mL centrifuge tube (300 x g, 5 min, room temperature). Add 2% HSA + CTS to the pellet using a micropipette. ＴＭ Opti-MEM ＴＭ Add 1000 μL of I Medium and suspend the cells by pipetting. Then, without changing the tip, rinse with 1000 μL of 2% HSA + CTS. ＴＭ Opti-MEM ＴＭ Add 18 mL of I Medium (total 20 mL). Centrifuge the 50 mL centrifuge tube (300 x g, 5 min, room temperature). 2% HSA + CTS ＴＭ Opti-MEM ＴＭ Add 20 mL of I Medium. Centrifuge the 50 mL centrifuge tube (300 x g, 5 min, room temperature). Add 4 mL of CryoStor CS2 depending on the cell number (350-450 x 10 ４cells / mL). - Suspend Y27632 in CryoStor CS2 to a concentration of 450 μM. - Dispense 450 μL of the above cell suspension into formulation vials, and use a programmed freezer to cool each vial to -80°C at a rate of -0.1°C / min → -2.0°C / min to freeze the cells. - Store in a -80°C freezer.

[0064] (Example 7: Example of use of corneal endothelial cell preparation) In this example, an example of use of a corneal endothelial cell preparation is shown. (Materials) - Japanese white rabbits - Frozen corneal endothelial cell preparation prepared in Example 6-2 (Method) - Lensectomy was performed on the rabbits 7 to 14 days before cell transfer. - On the day of cell transfer, the rabbit corneal endothelium was removed over an area of ​​8 mm in diameter using a scraper. - After the rabbit anterior chamber fluid was removed by suction using a syringe equipped with a 30-gauge needle, a corneal endothelial cell preparation (8.0 x 10 cells) that had been rapidly thawed in advance in a water bath at 37°C was added. ５ A 27-gauge nanoneedle syringe was used to aspirate a solution of 0.3 mL of HCECs and 0.3 mL of CS2 (100 μM Y-27632) and 300 μL of the corneal endothelial preparation was transferred into the anterior chamber. After cell transfer, the rabbits were fixed with their faces (eye surfaces) facing downward for three hours to allow the corneal endothelial cells to settle on the corneal endothelium. Five days after transfer, the rabbits were euthanized and subjected to immunohistological examination.

[0065] (Results) Representative results are shown in Figure 8. As indicated by the barrier function marker ZO-1 and the human corneal endothelial cell marker CD166, it was confirmed that normal corneal endothelial cells had engrafted onto the corneal endothelium. Furthermore, corneal transparency had returned to normal after 5 days.

[0066] As described above, the present disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present disclosure should be interpreted only by the claims. It is understood that the patents, patent applications, and literature cited in this specification should be incorporated by reference into this specification in the same manner as if the contents themselves were specifically set forth herein. This application claims the benefit of priority from Japanese Patent Application No. 2022-60148, filed on March 31, 2022, the contents of which are incorporated herein by reference.

[0067] In the process of preparing cultured corneal endothelial cells for use in regenerative medicine, it is necessary to wash out the xenogeneic component, fetal bovine serum. By adding a protein that can be administered to the human body to the washing solution, it is possible to reduce the loss of cells recovered during cell washing. Cell preparations washed using a protein that can be administered to the human body can be used for cell transplantation, and are therefore applicable in fields such as pharmaceuticals.

Claims

1. A method for producing a corneal endothelial cell preparation, the method comprising: Washing the corneal endothelial cells or corneal endothelial-like cells with a solution containing about 0.01% to about 10% by weight of protein; and a step of mixing the corneal endothelial cells or corneal endothelial-like cells with a composition for formulation to prepare a corneal endothelial cell formulation. wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof.

2. 2. The method of claim 1, wherein the washing step comprises suspending the corneal endothelial cells or corneal endothelial-like cells in the solution to provide a suspension of the corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells.

3. The method according to claim 1 or 2, wherein impurities in the corneal endothelial cell preparation are removed by the washing.

4. The method according to claim 1 or 2, wherein the rate of loss of the corneal endothelial cells or corneal endothelial-like cells in the washing step is 20% or less.

5. 3. The method according to claim 1, wherein the rate of loss of the corneal endothelial cells or corneal endothelial-like cells in the washing step is 15% or less.

6. The method according to claim 1 or 2, wherein the rate of loss of the corneal endothelial cells or corneal endothelial-like cells in the washing step is 10% or less.

7. 3. The method of claim 1 or 2, wherein the protein is human serum albumin.

8. 8. The method of claim 7, wherein the solution comprises about 0.5% to about 10% by weight of human serum albumin.

9. 8. The method of claim 7, wherein the solution comprises about 1% to about 5% by weight of human serum albumin.

10. 8. The method of claim 7, wherein the solution comprises about 1% to about 3% by weight of human serum albumin.

11. 8. The method of claim 7, wherein the solution comprises about 2% by weight human serum albumin.

12. 3. The method of claim 1 or 2, wherein the solution is a culture medium, a buffer solution, or an intraocular irrigation solution, or comprises one or more components of a culture medium, a buffer solution, or an intraocular irrigation solution.

13. The method according to claim 12, wherein the culture medium or intraocular irrigation solution is Opti-MEM (registered trademark), DMEM, MEM, RPMI 1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, oxyglutathione solution, or Opeguard.

14. 13. The method of claim 12, wherein the components of the culture medium include calcium, magnesium, potassium, sodium, phosphate, bicarbonate, vitamins, essential amino acids, or combinations thereof.

15. 15. The method of claim 14, wherein the culture medium components further comprise transferrin, insulin, hypoxanthine, thymidine, sodium bicarbonate, sodium pyruvate, L-glutamic acid, or a combination thereof.

16. 3. The method of claim 1 or 2, wherein the formulation composition is a cell preservation solution, a culture medium, a buffer solution, or an intraocular irrigation solution, or comprises one or more components of a cell preservation solution, a culture medium, a buffer solution, or an intraocular irrigation solution.

17. 17. The method of claim 16, wherein the cell preservation solution is CryoStor® CS2, CryoStor® CS5, Bambanker®, or Cellbanker®.

18. 17. The method of claim 16, wherein the components of the cell preservation solution include DMSO, glycerin, polyethylene glycol, or a combination thereof.

19. A method for washing corneal endothelial cells or corneal endothelial-like cells, the method comprising: a step of suspending corneal endothelial cells or corneal endothelial-like cells in a solution containing about 0.01% to about 10% by weight of protein to provide a suspension of corneal endothelial cells or corneal endothelial-like cells; a step of centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells. wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof.

20. A cell washing solution for corneal endothelial cells or corneal endothelial-like cells, comprising about 0.01% by weight to about 10% by weight of protein, wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof.

21. The cell washing solution according to claim 20, wherein the washing of the corneal endothelial cells is carried out by suspending the corneal endothelial cells or corneal endothelial-like cells in the cell washing solution to provide a suspension of the corneal endothelial cells or corneal endothelial-like cells, and centrifuging the suspension to recover the corneal endothelial cells or corneal endothelial-like cells.

22. The cell washing solution according to claim 20 or 21, wherein impurities in the corneal endothelial cell preparation are removed by the washing.

23. The cell washing solution according to claim 20 or 21, wherein the rate of loss of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 20% or less.

24. The cell washing solution according to claim 20 or 21, wherein the rate of loss of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 15% or less.

25. The cell washing solution according to claim 20 or 21, wherein the rate of loss of the corneal endothelial cells or corneal endothelial-like cells due to the washing is 10% or less.

26. 22. The cell washing solution of claim 20 or 21, wherein the protein is human serum albumin.

27. 27. The cell wash solution of claim 26, comprising about 0.5% to about 10% by weight human serum albumin.

28. 27. The cell wash solution of claim 26, comprising about 1% to about 5% by weight of human serum albumin.

29. 27. The cell wash solution of claim 26, comprising about 1% to about 3% by weight of human serum albumin.

30. 27. The cell wash solution of claim 26, comprising about 2% by weight human serum albumin.

31. 22. The cell washing solution of claim 20 or 21, wherein the solution is a culture medium, a buffer solution, or an intraocular irrigation solution, or comprises one or more components of a culture medium, a buffer solution, or an intraocular irrigation solution.

32. The cell washing solution according to claim 31, wherein the culture medium or intraocular irrigation solution is Opti-MEM (registered trademark), DMEM, MEM, RPMI1640, Ham's F-12, PBS, HEPES, Hanks' Balanced Salt Solution (HBSS), physiological saline, or oxyglutathione solution.

33. 32. The cell wash solution of claim 31, wherein the culture medium components include calcium, magnesium, potassium, sodium, phosphate, bicarbonate, vitamins, essential amino acids, or combinations thereof.

34. 32. The cell wash solution of claim 31, wherein the culture medium components further comprise transferrin, insulin, hypoxanthine, thymidine, sodium bicarbonate, sodium pyruvate, L-glutamic acid, or a combination thereof.

35. A composition for producing a preparation of corneal endothelial cells in a suspended state, comprising about 0.01% by weight to about 10% by weight of a protein, wherein the protein is selected from the group consisting of human serum albumin, casein, lactoferrin, ovalbumin, and combinations thereof.