Process for the production of an insulin-producing cell line of pancreatic beta cells

Claim 2

A process for producing an insulin-producing pancreatic cell line by conditionally transforming pancreatic cells with a temperature sensitive mutant strain of Rous sarcoma virus, said process comprising the steps of (a) selectively isolating insulin-producing beta cells cells from a pancreas and culturing said insulin-producing cells to produce a homogeneous population thereof; (b) growing a culture of a mutant strain of Rous sarcoma virus in a manner to produce a supernatant portion which contains the virion of said mutant strain of said virus, said mutant strain of virus containing in the transforming gene thereof a temperature sensitive lesion which causes said transforming gene to be expressed at a permissive environmental temperature and to be inactivated at a non-permissive environmental temperature; (c) harvesting the virion-containing supernatant portion of said mutant virus culture including said temperature sensitive transforming gene and infecting said insulin-producing pancreatic cells with said virion of said mutant virus so that the expression of said temperature sensitive transforming gene causes the conditional transformation of said insulin-producing pancreatic cells at said permissive temperature; (d) culturing the resulting infected insulin-producing pancreatic cells at the permissive temperature for the expression of the temperature sensitive transforming gene of said mutant virus strain in said insulin-producing pancreatic cells for a time sufficient to cause the conditional transformation of said insulin-producing pancreatic cells by said temperature sensitive transforming gene and to produce a desirably large population of said insulin-producing pancreatic cells; and (e) shifting the temperature of the culture produced in step (d) to the nonpermissive temperature for the inactivation of the temperature sensitive transforming gene of said mutant virus strain in said insulin-producing pancreatic cells for a time sufficient to cause said conditionally transformed insulin-producing pancreatic cells to revert to a non-transformed state and to produce insulin.

Claim 3

The process described in claim 1, wherein said insulin-producing pancreatic cells are obtained from a human pancreas.

Claim 4

The process described in claim 1, wherein said insulin-producing pancreatic cells are obtained from a bovine pancreas.

Claim 5

The process described in claim 1, wherein said insulin-producing pancreatic cells are obtained from a porcine pancreas.

Claim 6

The process described in claim 1, wherein said mutant strain of Rous sarcoma virus is selected from the group consisting of the viral strains ts-sarc-SR-RSV-D and ts-sarc-Bryan-RSV.

Claim 7

The process described in claim 5, wherein said mutant strain of Rous sarcoma virus comprises the strain ts-sarc-SR-RSV-D mutant FU-19.

Claim 8

The process described in claim 5, wherein said mutant strain of Rous sarcoma virus comprises the strain ts-sarc-Bryan-RSV.

Claim 9

The process described in claim 7, further including the step of fusing the membrane of said mutant viral strain ts-sarc-Bryan-RSV and the membrane of said insulin-producing cells with inactivated Sendai virus.

Claim 10

The process described in claim 7, further including the step of adding a nontransforming helper RNA tumor virus of subgroup C or D during step (b).

Claim 11

The process described in claim 9, wherein said nontransforming helper RNA tumor virus is Rous associated virus (RAV)-50.

Claim 12

The process described in claim 1, further including the steps of: (a) enzymatically dispersing said conditionally transformed insulin-producing pancreatic cells; (b) diluting said dispersed cells with nutrient medium and plating a single cell in each of the wells of a standard tissue culture plate; (c) incubating said plated cells at the permissive temperature for the expression of the temperature sensitive transforming gene of said mutant virus strain in said insulin-producing pancreatic cells for at least about 24 hours; and (d) selecting those of said insulin-producing pancreatic cells showing no evidence of morphological transformation and culturing said morphologically normal pancreatic cells to obtain an insulin-producing cell population.

Claim 13

A process for producing an insulin-producing cell line of pancreatic cells by conditionally transforming pancreatic cells with a temperature sensitive strain of Rous sarcoma virus, said process comprising the steps of (a) selectively isolating insulin-producing beta cells from a human pancreas to produce a suspension of single cells rich in insulin producing beta cells, pelleting said cells by low speed centrifugation, suspending the pellet thus formed in nutrient medium and plating said cells; (b) growing a culture of a mutant strain of Rous sarcoma virus including a temperature sensitive lesion in the transforming gene thereof which causes the transforming gene to be expressed at a permissive environmental temperature and to be inactivated at a nonpermissive environmental temperature by infecting chick embryo fibroblasts with said mutant strain and incubating the infected fibroblasts in a nutrient medium in a manner to produce a supernatant portion which contains the virion of said mutant strain of Rous sarcoma virus for a time sufficient to confluently transform said culture; (c) harvesting said supernatant portion of said culture and adjusting the pH of said supernatant to a pH in the range of about 7.2 to 7.8; (d) infecting the pancreatic cells obtained in step (a) with the virion-containing supernatant obtained in step (c); (e) culturing said infected pancreatic cells at the permissive temperature for the expression of the temperature sensitive transforming gene of said mutant virus strain in insulin-producing pancreatic beta cells for a time sufficient to cause said insulin-producing pancreatic cells to be conditionally transformed by the expression of said temperature sensitive transforming gene; (f) culturing in a culture medium the conditionally transformed insulin-producing pancreatic cells at the permissive temperature for a time sufficient to produce a desirably large population of said insulin-producing pancreatic cells; and (g) replacing the culture medium containing the conditionally transformed insulin-producing pancreatic cells with a culture medium containing glucose, incubating said cells in the glucose medium at the nonpermissive temperature for the inactivation of the temperature sensitive transforming gene of said mutant virus strain in said insulin-producing pancreatic cells for a time sufficient to cause said conditionally transformed insulin-producing pancreatic cells to revert to a nontransformed state and to produce insulin, and harvesting the insulin-produced by said cells in said nontransformed state from said glucose medium.

Claim 14

A process for producing an insulin-producing cell line of pancreatic cells by conditionally transforming pancreatic cells with a temperature sensitive strain of Rous sarcoma virus, said process comprising the steps of (a) selectively isolating insulin-producing beta cells from a human pancreas to produce a suspension of single cells rich in insulin producing beta cells, pelleting said cells by low speed centrifugation, suspending the pellet thus formed in nutrient medium and plating said cells; (b) growing a culture of a mutant strain of Rous sarcoma virus including a temperature sensitive lesion in the transforming gene thereof which causes the transforming gene to be expressed at a permissive environmental temperature and to be inactivated at a nonpermissive environmental temperature by infecting chick embryo fibroblasts with said mutant strain and incubating the infected fibroblasts in a nutrient medium in a manner to produce a supernatant portion which contains the virion of said mutant strain of Rous sarcoma virus for a time sufficient to confluently transform said culture; (c) harvesting said supernatant portion of said culture and adjusting the pH of said supernatant to a pH in the range of about 7.2 to 7.8; (d) infecting the pancreatic cells obtained in step (a) with the virion-containing supernatant obtained in step (c); (e) culturing said infected pancreatic cells at the permissive temperature for the expression of the temperature sensitive transforming gene of said mutant virus strain in insulin-producing pancreatic beta cells for a time sufficient to cause said insulin-producing pancreatic cells to be conditionally transformed by the expression of said temperature sensitive transforming gene; (f) culturing in a culture medium the conditionally transformed insulin-producing pancreatic cells at the permissive temperature for a time sufficient to produce a desirably large population of said insulin-producing pancreatic cells; and (g) replacing the culture medium containing the conditionally transformed insulin-producing pancreatic cells with a culture medium containing glucose, incubating said cells in the glucose medium at the nonpermissive temperature for the inactivation of the temperature sensitive transforming gene of said mutant virus strain in said insulin-producing pancreatic cells for a time sufficient to cause said conditionally transformed insulin-producing pancreatic cells to revert to a nontransformed state and to produce insulin, and harvesting the insulin-produced by said cells in said nontransformed state from said glucose medium.

Claim 15

The process described in claim 12, wherein said mutant strain of Rous sarcoma virus is selected from the group consisting of the viral strains ts-sarc-SR-RSV-D and ts-sarc-Bryan-RSV.

Claim 16

The process described in claim 13, wherein said mutant strain of Rous sarcoma virus comprises the strain ts-sarc-SR-RSV-D mutant FU-19.

Claim 17

The process described in claim 13, wherein said mutant strain of Rous sarcoma virus comprises the strain ts-sarc-Bryan-RSV.

Claim 18

The process described in claim 12, further including the steps of: (a) enzymatically dispersing said conditionally transformed insulin-producing pancreatic cells; (b) diluting said dispersed cells with nutrient medium and plating a single cell in each of the wells of a standard tissue culture plate; (c) incubating said plated cells at the permissive temperature for the expression of the temperature sensitive transforming gene of said mutant virus strain in said insulin-producing pancreatic cells for at least about 24 hours; and (d) selecting those of said insulin-producing pancreatic cells showing no evidence of morphological transformation and culturing said morphologically normal pancreatic cells to obtain an insulin-producing cell population.