Mammalian cell population and medicaments for cell therapies in humans and improved cell cultivation methods
Compositions of matter, including cell populations and medicaments, are provided that are derived from human gingival fibroblasts and have cell phenotypes that occur in proportions not found in natural gum tissue, but rather, are preferentially are selected to express proteins favoring angiogenesis and anti-inflammatory effects, while reducing cell populations that promote tumorigenicity and/or formation of metalloproteinases that inhibit tissue regeneration. Methods of generating such compositions are provided that increase proliferation many-fold compared to previously known methods, and methods of using such compositions in a wide range of human cell therapies are provided.
1 . A pharmaceutical dose for treating a human ailment comprising:
an injectable volume of about 1.0 ml to 2.5 ml containing:
a medium comprising 10% dimethyl sulfoxide, 88% DMEM, and 1% non-essential amino acids; and
between 2 and 40 million viable cultured human gingival fibroblast cells having phenotypes such that at least about 90% of the cell population expresses CD90 and CD63 and about 10% or less of the cell population expresses CD146,
wherein the viable cultured human gingival fibroblast cells are cultured by a process comprising the steps of:
obtaining a population of viable gingival fibroblast cells representative of naturally occurring gingival fibroblast cells in human gum tissue;
culturing the viable gingival fibroblast cells through four passages in a culture medium that includes at least 20% fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF), without isolating gingival fibroblast cell subpopulations, wherein during the first two passages, the population of viable gingival fibroblast cells monotonically increases; and
culturing the viable gingival fibroblast cells to a fifth passage, wherein during the fifth passage, omitting the 20% FBS and bFGF from the culture medium and adding interleukin 1β in a concentration of 0.1 ng/ml to 10 ng/ml,
wherein by completion of the fifth passage, the gingival fibroblast cells correspond to cell phenotypes cultivated so that at least about 90% of the cell population expresses CD90 and CD63 and about 10% or less of the cell population expresses CD146.
2 . The pharmaceutical dose of claim 1 , wherein a duration of the first of the four passages of culturing the viable gingival fibroblast cells is about 6 days.
3 . The pharmaceutical dose of claim 2 , wherein each of the second, the third, and the fourth passage of the four passages of culturing the viable gingival fibroblast cells has a duration of about 6 days.
4 . The pharmaceutical dose of claim 1 , wherein during at least the fifth passage of culturing the viable gingival fibroblast cells, non-essential amino acids are added to the culture medium.
5 . The pharmaceutical dose of claim 1 , wherein culturing the viable gingival fibroblast cells during each of the five passages expands the cells more than six-fold.
6 . The pharmaceutical dose of claim 1 , wherein the pharmaceutical dose contains between 10 and 20 million viable cultured human gingival fibroblast cells.
7 . The pharmaceutical dose of claim 1 , wherein about 10% or less of the cell population expresses at least one mRNA selected from the group consisting of MCAM, VCAM1, CD19, ITGAM, CD3D, CD4, FZD9, NGFR, NANOG, POU5F1, SOX2, KLF4, MYC, TNF, IL1A, IL1B, IL17A, IL23A, OSM, IF127, IFI44L, RSAD2, IFIT1, IFNA1 and IFNG mRNAs.
8 . The pharmaceutical dose of claim 1 , wherein about 50% or more of the cell population expresses at least one mRNA selected from the group consisting of TIMP1, CD9, CD81, THY, ITGB1, FST, and COL1A2 mRNAs.
9 . The pharmaceutical dose of claim 1 , wherein the viable human gingival fibroblast cells are allogeneic.
10 . A pharmaceutical dose for treating a human ailment comprising:
an injectable volume of about 1.0 ml to 2.5 ml containing:
a medium comprising 10% dimethyl sulfoxide, 88% DMEM, and 1% non-essential amino acids; and
at least 2 million viable cultured human gingival fibroblast cells having phenotypes such that at least about 90% of the cell population expresses CD90 and CD63 and about 10% or less of the cell population expresses CD146,
wherein the viable cultured human gingival fibroblast cells are cultured by a process comprising the steps of:
obtaining a population of viable gingival fibroblast cells representative of naturally occurring gingival fibroblast cells in human gum tissue;
culturing the population of viable gingival fibroblast cells through four passages in a culture medium that includes at least 20% fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF), without isolating gingival fibroblast cell subpopulations, wherein during the first two passages, the population of viable gingival fibroblast cells monotonically increases; and
culturing the population of viable gingival fibroblast cells to a fifth passage, wherein during the fifth passage, omitting the 20% FBS and bFGF from the culture medium and adding interleukin 1β in a concentration of 0.1 ng/ml to 10 ng/ml and nonessential amino acids,
wherein by completion of the fifth passage, the gingival fibroblast cells correspond to cell phenotypes cultivated so that at least about 90% of the cell population expresses CD90 and CD63 and about 10% or less of the cell population expresses CD146.
11 . The pharmaceutical dose of claim 10 , wherein a duration of the first of the four passages of culturing the viable gingival fibroblast cells is about 6 days.
12 . The pharmaceutical dose of claim 10 , wherein each of the second, the third, and the fourth passage of the four passages of culturing the viable gingival fibroblast cells has a duration of about 6 days.
13 . The pharmaceutical dose of claim 10 , wherein culturing the viable gingival fibroblast cells during each of the five passages expands the cells more than six-fold.
14 . The pharmaceutical dose of claim 10 , wherein the pharmaceutical dose contains up to 40 million cells.
15 . The pharmaceutical dose of claim 10 , wherein about 10% or less of the cell population expresses at least one mRNA selected from the group consisting of MCAM, VCAM1, CD19, ITGAM, CD3D, CD4, FZD9, NGFR, NANOG, POU5F1, SOX2, KLF4, MYC, TNF, IL1A, IL1B, IL17A, IL23A, OSM, IF127, IFI44L, RSAD2, JFIT1, IFNA1 and IFNG mRNAs.
16 . The pharmaceutical dose of claim 10 , wherein about 50% or more of the cell population expresses at least one mRNA selected from the group consisting of TIMP1, CD9, CD81, THY, ITGB1, FST, and COL1A2 mRNAs.
17 . The pharmaceutical dose of claim 10 , wherein the viable human gingival fibroblast cells are allogeneic.
18 . The pharmaceutical dose of claim 10 , wherein the pharmaceutical dose is configured for injection with a syringe.