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US-12624341-B2 - Mammalian cell population and medicaments for cell therapies in humans and improved cell cultivation methods

US12624341B2US 12624341 B2US12624341 B2US 12624341B2US-12624341-B2

Abstract

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.

Inventors

  • Antoine Lafont
  • Mathieu CASTELA

Assignees

  • SCARCELL THERAPEUTICS

Dates

Publication Date
20260512
Application Date
20250127
Priority Date
20230315

Claims (18)

  1. 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. 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. 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. 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. 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. 6 . The pharmaceutical dose of claim 1 , wherein the pharmaceutical dose contains between 10 and 20 million viable cultured human gingival fibroblast cells.
  7. 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. 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. 9 . The pharmaceutical dose of claim 1 , wherein the viable human gingival fibroblast cells are allogeneic.
  10. 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. 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. 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. 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. 14 . The pharmaceutical dose of claim 10 , wherein the pharmaceutical dose contains up to 40 million cells.
  15. 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. 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. 17 . The pharmaceutical dose of claim 10 , wherein the viable human gingival fibroblast cells are allogeneic.
  18. 18 . The pharmaceutical dose of claim 10 , wherein the pharmaceutical dose is configured for injection with a syringe.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional application of U.S. patent application Ser. No. 18/301,839, filed Apr. 17, 2023, now U.S. Pat. No. 12,258,579, and claims priority to U.S. Provisional Patent Application Ser. No. 63/490,489, filed Mar. 15, 2023, and EP patent application Ser. No. 23/305,350.3, filed Mar. 15, 2023, the entire contents of each of which are incorporated herein by reference. FIELD OF THE INVENTION The present invention relates generally to compositions of matter and medicaments derived from gingival fibroblasts to use in cell therapies and improved methods for cultivating such compositions to create specialized phenotype populations that advantageously vary from naturally occurring tissue. In particular, the inventive methods are designed to enhance cell proliferation to facilitate generation of the inventive cell compositions in sufficient quantities to realize commercial scale production. Also included are methods of using the novel composition to treat a variety of mammalian afflictions, including those affecting epi- and endo-thelial surfaces (e.g., skin-aging, pressure ulcers, alopecia, immune and vascular diseases) and of osteopathic origin (e.g., osteoarthritis, cartilage defects, etc.). BACKGROUND For at least the last two decades, uses of gingival fibroblasts have been investigated for a wide range of cell therapy treatments due to the multipotent nature of gingival fibroblasts and the ability of such cells to differentiate into a broad spectrum of cells than other types of mesenchymal stem cells. In addition, while the latter are harvested from bone marrow, which poses some risks to the donor, gingival fibroblasts are readily harvested from gum tissue, e.g., during wisdom tooth extraction and other dental procedures. Use of gingival fibroblast (GF) derived compositions, both cultured GF cells and GF conditioned media, have been described for treatment of a number of mammalian ailments, as set forth in the following U.S. patents and publications: U.S. Pat. No. 8,609,085 (atherosclerosis); U.S. Pat. No. 8,303,948 (skin wounds); U.S. Patent Application Publication No. 2011/0097421 (skin aging); U.S. Pat. No. 10,624,838 (alopecia); U.S. Pat. No. 11,229,670 (allergic reactions, atopic dermatitis and asthma); U.S. Patent Application Publication No. 2018/0028571 (cancer); U.S. Patent Application Publication No. 2016/0256496 (human orthopedic pathologies, including osteoarthritis and cartilage defects); U.S. Patent Application Publication No. 2020/0268805 (equine musculoskeletal diseases, including tendinitis, arthropathy and arthrosis) and International Publication No. WO2023/007244A1 (pressure ulcers). All of following patents and applications are licensed or assigned to the assignee of the present application, the entireties of which are incorporated herein by reference. Methods of cultivating gingival fibroblasts are described in a number of previously known articles, including for example, Hou et al., “Autologous Transplantation of Gingival Fibroblast-Like Cells and a Hydroxylapatite Complex Graft in the Treatment of Periodontal Osseous Defects: Cell Cultivation and Long-Term Report of Cases,” Cell Transplantation, 12:787-797 (2003); Ferré et al., “Formation of Cartilage and Synovial Tissue by Human Gingival Stem Cells,” Stem Cells & Dev., 23 (23): 2895-2907 (2014); Linard et al., “Therapeutic Potential of Gingival Fibroblasts for Cutaneous Radiation Syndrome: Comparison to Bone Marrow-Mesenchymal Stem Cell Grafts,” Stem Cells & Dev., 24 (10): 1182-1193 (2015) and Ahangar et al., “Human gingival fibroblast secretome accelerates wound healing through anti-inflammatory and pro-angiogenic mechanisms,” npj Regenerative Medicine 5 (24) 1-10 (2020). All of the foregoing articles describe previously known methods of cultivating cells, including use of at least a culture medium, e.g., Eagle's minimally essential medium (EMEM) or Dulbecco's Modified Eagle's Medium (DMEM), an antibiotic (e.g., penicillin, streptomycin or gentamicin), and a mammalian blood derived serum, such as 10% Fetal Bovine Serum (FBS). In general, previously known methods of cultivating gingival fibroblasts involved explanting gum tissue, followed either by mincing or enzymatic digestion. Cells then are incubated in a medium as described above in a humidified incubator with 5% carbon dioxide atmosphere for a period of about two to three weeks until confluence is attained, and then trypsinized to create single cell suspensions. In some cultivation methods, the culture medium may be refreshed every 72 hours and after the initial cultivation period, gingival fibroblast colonies may again be trypsinized to create single cell suspensions that are seeded in fresh culture for additional two week periods, often referred to as “passages.” After a final passage and trypsinization step, the cultivated cells may be washed and collected for use in a desired application. A number of drawbacks exist fo