Cell lines and their use for the production plasminogen activators

Claim 2

A process for producing tissue plasminogen activator, pro-tissue plasminogen activator, or mixtures thereof, comprising culturing, in a culture medium deprived of serum and exogenous macromolecular growth factors, cells of a human cell line, derived from the tissue plasminogen activator-producing human melanoma cell line Bowes, which cells are capable of reproducibly and indefinitely propagating in culture in a medium deprived of serum and exogenous macromolecular growth factors and which product tissue plasminogen activator and/or pro-tissue plasminogen activator, harvesting fluid from said culture, said isolating the activator or mixture from said harvested fluid.

Claim 3

A process for producing pro-tissue plasminogen activator essentially free of tissue plasminogen activator, comprising culturing, in a culture medium deprived of serum and exogenous macromolecular growth factors, cells of a human cell line, derived from the tissue plasminogen activator-producting human melanoma cell line Bowes, which cells are capable of reproducibly and indefinitely propagating in culture in a medium deprived of serum and exogenous macromolecular growth factors and which produce tissue plasminogen activator and/or pro-tissue plasminogen activator, harvesting fluid from said culture, subjecting said harvested fluid, in the presence of protease inhibitor, to protein purification procedures, and effecting a final purification of said purified harvested fluid in the presence of an inhibitor which selectively inhibits tissue plasminogen activator.

Claim 4

A process for producing pro-tissue plasminogen activator essentially free of tissue plasminogen activator, comprising culturing, in a culture medium deprived of serum and exogenous macromolecular growth factors, cells of a human cell line, derived from the tissue plasminogen activator-producting human melanoma cell line Bowes, which cells are capable of reproducibly and indefinitely propagating in culture in a medium deprived of serum and exogenous macromolecular growth factors and which produce tissue plasminogen activator and/or pro-tissue plasminogen activator, harvesting fluid from said culture, subjecting said harvested fluid, in the presence of protease inhibitor, to protein purification procedures, and effecting a final purification of said purified harvested fluid in the presence of an inhibitor which selectively inhibits tissue plasminogen activator.

Claim 5

A process for producing tissue plasminogen activator essentially free of pro-tissue plasminogen activator comprising culturing, in a culture medium deprived of serum and exogenous macromolecular growth factors, cells of a human cell line, derived from the tissue plasminogen activator-producing human melanoma cell line Bowes, which cells are capable of reproducibly and indefinitely propagating in culture in a medium deprived of serum and exogenous macromolecular growth factors and which produce tissue plasminogen activator and/or pro-tissue plasminogen activator, harvesting fluid from said culture, treating said harvested fluid with an enzyme effective of convert pro-tissue plasminogen activator to tissue plasminogen activator, and thereafter isolating tissue plasminogen activator.

Claim 6

A process for producing tissue plasminogen activator essentially free of pro-tissue plasminogen activator comprising culturing, in a culture medium deprived of serum and exogenous macromolecular growth factors, cells of a human cell line, derived from the tissue plasminogen activator-producing human melanoma cell line Bowes, which cells are capable of reproducibly and indefinitely propagating in culture in a medium deprived of serum and exogenous macromolecular growth factors and which produce tissue plasminogen activator and/or pro-tissue plasminogen activator, harvesting fluid from said culture, treating said harvested fluid with an enzyme effective of convert pro-tissue plasminogen activator to tissue plasminogen activator, and thereafter isolating tissue plasminogen activator.

Claim 7

A human cell line derived from the tissue plasminogen activator-producing human melanoma cell line Bowes, capable of reproducibly and indefinitely propagating in culture in a medium deprived of serum and exogenous macromolecular growth factor, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator.

Claim 8

A human cell line derived from the tissue plasminogen activator-producing human melanoma cell line Bowes, capable of reproducibly and indefinitely propagating in culture in a medium deprived of serum and exogenous macromolecular growth factor, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator.

Claim 9

A process for the production of a human cell line capable of reproducibly and indefinitely propagating in culture in the absence of serum and in the absence of exogenous macromolecular growth factors, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator, comprising the steps of: (a) establishing a culture of the tissue plasminogen activator-producing human melanoma cell line Bowes in a culture medium containing serum; (b) removing said culture medium containng serum from said culture and replacing it with a culture medium deprived of serum and deprived of exogenous macromolecular growth factors; (c) feeing the adherent and any non-adherent cells with culture medium deprived of serum and deprived of exogenous macromolecular growth factors; and (d) culturing the cells until they propagate reproducibly and indefinitely in culture medium deprived of serum and deprived of exogenous macromolecular growth factors, thereby establishing a human cell line which is capable of reproducibly and indefinitely propagating in medium deprived of serum and deprived of exogenous macromolecular growth factors and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator.

Claim 10

A process for the production of a human cell line capable of reproducibly and indefinitely propagating in culture in the absence of serum and in the absence of exogenous macromolecular growth factors, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator, comprising the steps of: (a) establishing a culture of the tissue plasminogen activator-producing human melanoma cell line Bowes in a culture medium containing serum; (b) removing said culture medium containng serum from said culture and replacing it with a culture medium deprived of serum and deprived of exogenous macromolecular growth factors; (c) feeing the adherent and any non-adherent cells with culture medium deprived of serum and deprived of exogenous macromolecular growth factors; and (d) culturing the cells until they propagate reproducibly and indefinitely in culture medium deprived of serum and deprived of exogenous macromolecular growth factors, thereby establishing a human cell line which is capable of reproducibly and indefinitely propagating in medium deprived of serum and deprived of exogenous macromolecular growth factors and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator.

Claim 11

A process according to claim 5 comprising the further step of subjecting the established human cell line of step (e) to a treatment selected from the group consisting of treatment with a mutagen, exposure to X-rays, and exposure to ultraviolet radiation, to produce an establishemutant cell line thereof capable of reproducibly and indefinitely propagating in medium deprived of serum and deprived of any exogenous macromolecular growth factor, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator.

Claim 12

A process for the production of a human cell line capable of reproducibly and indefintely propagating in culture in the absence of serum and in the absence of exogenous macromolecular growth factors, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator, comprising the steps of: (a) establishing a culture of the tissue plasminogen activator-producing human melanoma cell line Bowes in a culture medium containing serum; (b) removing said culture medium containing serum from said culture and replacing it with a culture medium deprived of serum and deprived of exogenous macromolecular growth factors; (c) periodically removing from said culture essentially the entire medium and any non-adherent cells, separating the non-adherent cells from said removed medium, and adding back to the remaining adherent cells in culture said non-adherent cells and fresh medium deprived of serum and exogenous macromolecular growth factors; and (d) repeating the removal, separating and addition back procedure of step (c) until the adherent cells in culture propagate reproducibly and indefintely in fresh culture medium deprived of serum and deprived of exogenous macromolecular growth factors, thereby establishing a human cell line capable of reproducibly and indefinitely propagating in medium deprived of serum and exogenous macromolecular growth factors, and which produces tissue plasminogen activator and/or pro-tissue plasminoqen activator.

Claim 13

A process for the production of a human cell line capable of reproducibly and indefintely propagating in culture in the absence of serum and in the absence of exogenous macromolecular growth factors, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator, comprising the steps of: (a) establishing a culture of the tissue plasminogen activator-producing human melanoma cell line Bowes in a culture medium containing serum; (b) removing said culture medium containing serum from said culture and replacing it with a culture medium deprived of serum and deprived of exogenous macromolecular growth factors; (c) periodically removing from said culture essentially the entire medium and any non-adherent cells, separating the non-adherent cells from said removed medium, and adding back to the remaining adherent cells in culture said non-adherent cells and fresh medium deprived of serum and exogenous macromolecular growth factors; and (d) repeating the removal, separating and addition back procedure of step (c) until the adherent cells in culture propagate reproducibly and indefintely in fresh culture medium deprived of serum and deprived of exogenous macromolecular growth factors, thereby establishing a human cell line capable of reproducibly and indefinitely propagating in medium deprived of serum and exogenous macromolecular growth factors, and which produces tissue plasminogen activator and/or pro-tissue plasminoqen activator.

Claim 14

A process for the production of a human cell line capable of reproducibly and indefintely propagating in culture in the absence of serum and in the absence of exogenous macromolecular growth factors, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator, comprising the steps of: (a) establishing a culture of the tissue plasminogen activator-producing human melanoma cell line Bowes in a culture medium containing serum; (b) removing said culture medium containing serum from said culture and replacing it with a culture medium deprived of serum and deprived of exogenous macromolecular growth factors; (c) therafter periodically removing at least a portion of the medium from said culture and replacing it with fresh medium deprived of serum and exogenous macromolecular growth factors; (d) repeating the removal and replacement of step (c) until a confluency of adherent cells in culture of at least about 30% is obtained; and (e) dislodging the adherent cells and and culturing them in the presence of medium comprised of a mixture of a portion of the medium removed from the culture prior to said dislodging and fresh medium deprived of serum and deprived of exogenous macromolecular growth factors to produce an established cell line capable of reproducibly and indefinitely propagating in medium deprived of serum and exogenous macromolecualr growth factors and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator.

Claim 15

A process for the production of a human cell line capable of reproducibly and indefintely propagating in culture in the absence of serum and in the absence of exogenous macromolecular growth factors, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator, comprising the steps of: (a) establishing a culture of the tissue plasminogen activator-producing human melanoma cell line Bowes in a culture medium containing serum; (b) removing said culture medium containing serum from said culture and replacing it with a culture medium deprived of serum and deprived of exogenous macromolecular growth factors; (c) therafter periodically removing at least a portion of the medium from said culture and replacing it with fresh medium deprived of serum and exogenous macromolecular growth factors; (d) repeating the removal and replacement of step (c) until a confluency of adherent cells in culture of at least about 30% is obtained; and (e) dislodging the adherent cells and and culturing them in the presence of medium comprised of a mixture of a portion of the medium removed from the culture prior to said dislodging and fresh medium deprived of serum and deprived of exogenous macromolecular growth factors to produce an established cell line capable of reproducibly and indefinitely propagating in medium deprived of serum and exogenous macromolecualr growth factors and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator.

Claim 16

The process according to claim 8 wherein said detachment of adherent cells is performed after a confluency of at least about 60% is obtained.

Claim 17

A process for producing a human cell line capable of reproducibly and indefinitely propagating in culture in the absence of serum and in the absence of oxogenous macromolecular growth factors, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator, comprising the steps of: (a) establishing a culture of the tissue plasminogen activator-producing human melanoma cell line Bowes in a culture medium containing serum; (b) removing said culture medium containing serum from said culture and replacing it with a culture medium deprived of serum and exogenous macromolecular growth factors; (c) periodically adding to the culture a medium deprived of serum and exogenous macromolecular growth factors and comprised of a medium harvested from a companion cell culture of Bowes cells after replacement therein of medium containing serum with medium deprived of serum and exogenous macromolecular growth factor; and (d) continuing said periodic addition of step (c) until there is obtained an established cell line capable of reproducibly and indefinitely propagating in culture in a medium deprived of serum and exogenous macromolecular growth actors, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator.

Claim 18

A process for producing a human cell line capable of reproducibly and indefinitely propagating in culture in the absence of serum and in the absence of oxogenous macromolecular growth factors, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator, comprising the steps of: (a) establishing a culture of the tissue plasminogen activator-producing human melanoma cell line Bowes in a culture medium containing serum; (b) removing said culture medium containing serum from said culture and replacing it with a culture medium deprived of serum and exogenous macromolecular growth factors; (c) periodically adding to the culture a medium deprived of serum and exogenous macromolecular growth factors and comprised of a medium harvested from a companion cell culture of Bowes cells after replacement therein of medium containing serum with medium deprived of serum and exogenous macromolecular growth factor; and (d) continuing said periodic addition of step (c) until there is obtained an established cell line capable of reproducibly and indefinitely propagating in culture in a medium deprived of serum and exogenous macromolecular growth actors, and which produces tissue plasminogen activator and/or pro-tissue plasminogen activator.