Mammalian cell population and medicaments for cell therapies in equines and improved cell cultivation methods
Compositions of matter, including cell populations and medicaments, are provided that are derived from equine 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 equine cell therapies are provided.
1. A pharmaceutical or cosmetic composition produced by a process comprising the steps of:
obtaining equine gingival fibroblast cells representative of a naturally occurring population of equine gingival fibroblast cells in equine gum tissue;
culturing the equine gingival fibroblast cells through four passages in a culture medium comprising 20% Fetal Bovine Serum (FBS) and Basic Fibroblast Growth Factor (bFGF); and
during a fifth passage of the process, omitting the 20% FBS from the culture medium and adding exogenous Interleukin 1 Beta (IL-1β) in a concentration from 0.1 ng/ml to 10 ng/ml, wherein by completion of the fifth passage the equine gingival fibroblast cells correspond to cell phenotypes cultivated so that at least about 90% of the cells of the cultivated population express CD90 and CD63 and about 20% or less of the cells of the cultivated population express CD146; and
collecting the culture medium having a protein expression produced by the fifth passage for use as the pharmaceutical or cosmetic composition.
2. The composition of claim 1 , wherein about 10% or less of the cells of the cultivated population express at least one mRNA selected from the group consisting of CD146, VCAM1, CD19, ITGAM, CD3D, CD4, FZD9, NGFR, NANOG, POU5F1, SOX2, KLF4, MYC, TNF, IL1A, IL1B, IL17A, IL23A, OSM, IFI27, IF144L, RSAD2, IFIT1, IFNA1 and IFNG mRNAs.
3. The composition of claim 1 , wherein at least about 50% of the cells of the cultivated population express at least one mRNA selected from the group consisting of TIMP1, CD9, CD81, THY, ITGB1, FST, and COL1A2 mRNAs.
4. The composition of claim 1 , wherein the gingival fibroblasts are allogeneic.
5. The composition of claim 1 , wherein the gingival fibroblasts are autologous.
6. The composition of claim 1 , further including at least one pharmaceutically acceptable carrier or excipient.
7. A pharmaceutical or cosmetic composition made by a process comprising the steps of:
obtaining equine gingival fibroblast cells representative of a naturally occurring population of equine gingival fibroblast cells in equine gum tissue;
culturing the equine gingival fibroblast cells through four passages in a culture medium including 20% Fetal Bovine Serum (FBS) and Basic Fibroblast Growth Factor (bFGF); and
during a fifth passage of the process, omitting the 20% FBS and adding exogenous Interleukin 1 Beta (IL-1β) in a concentration from 0.5 ng/ml to 2 ng/ml to the culture medium, wherein by completion of the fifth passage the equine gingival fibroblast cells correspond to cell phenotypes cultivated so that at least about 90% of the cells of the cultivated population express CD90 and CD63 and about 10% or less of the cells of the cultivated population express CD146; and
collecting the culture medium having a protein expression produced by the fifth passage for use as the pharmaceutical or cosmetic composition.
8. The composition of claim 7 , wherein about 10% or less of the cells of the cultivated population express at least one mRNA selected from the group consisting of VCAM1, CD19, ITGAM, CD3D, CD4, FZD9, NGFR, NANOG, POU5F1, SOX2, KLF4, MYC, TNF, IL1A, IL1B, IL17A, IL23A, OSM, IFI27, IFI44L, RSAD2, IFIT1, IFNA1 and IFNG mRNAs.
9. The composition of claim 7 , wherein at least about 50% of the cells of the cultivated population express at least one mRNA selected from the group consisting of TIMP1, CD9, CD81, THY, ITGB1, FST, and COL1A2 mRNAs.
10. The composition of claim 7 , wherein the gingival fibroblasts are allogeneic.
11. The composition of claim 7 , wherein the gingival fibroblasts are autologous.
12. The composition of claim 7 , wherein the composition comprises at least one pharmaceutically acceptable carrier or excipient.
13. A pharmaceutical or cosmetic composition made by a process comprising the steps of:
obtaining equine gingival fibroblast cells representative of a naturally occurring population of equine gingival fibroblast cells in equine gum tissue;
culturing the equine gingival fibroblast cells through four passages in a culture medium comprising 20% Fetal Bovine Serum (FBS) and Basic Fibroblast Growth Factor (bFGF); and
during a fifth passage of the process, omitting the 20% FBS from the culture medium and adding exogenous Interleukin 1 Beta (IL-1β) in a concentration from 0.1 ng/ml to 10 ng/ml, wherein by completion of the fifth passage the equine gingival fibroblast cells correspond to cell phenotypes cultivated so that at least about 90% of the cells of the cultivated population express CD90 and CD63 and about 20% or less of the cells of the cultivated population express CD146; and
collecting the culture medium having a protein expression produced by the fifth passage and adding at least one pharmaceutically acceptable carrier or excipient.
14. The composition of claim 13 , wherein about 10% or less of the cells of the cultivated population express at least one mRNA selected from the group consisting of CD146, VCAM1, CD19, ITGAM, CD3D, CD4, FZD9, NGFR, NANOG, POU5F1, SOX2, KLF4, MYC, TNF, IL1A, IL1B, IL17A, IL23A, OSM, IFI27, IFI44L, RSAD2, IFIT1, IFNA1 and IFNG mRNAs.
15. The composition of claim 13 , wherein at least about 50% of the cells of the cultivated population express at least one mRNA selected from the group consisting of TIMP1, CD9, CD81, THY, ITGB1, FST, and COL1A2 mRNAs.
16. The composition of claim 13 , wherein the gingival fibroblasts are allogeneic or autologous.
17. The composition of claim 13 , wherein the composition further comprises the cultured cell population of equine gingival fibroblast cells in which the cells are cultivated so that at least about 90% of the cells of the population express CD90 and CD63 and about 20% or less of the cells of the population express CD146.