Methods for culturing minimally-passaged fibroblasts and uses thereof
Inventors
Maslowski, John • Thomas, Myrna F. • Lindner, Marie A.
Assignees
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Publication Number
US-8728819-B2
Publication Date
2014-05-20
Expiration Date
Abstract
The present invention relates to novel methods of growing at least 100 million minimally-passaged fibroblasts from a small biopsy specimen. The invention includes methods wherein a small biopsy specimen is seeded directly into a large tissue culture flask, and passaged no more than three times.
Core Innovation
The invention relates to a cell-free topical composition derived from conditioned medium produced by culturing fibroblasts. Fibroblasts are derived from a single full-thickness biopsy specimen that is not more than 5 mm thick and not more than 12 cm2, and the biopsy specimen is digested with a dissociative or digestive enzyme to form a fibroblast cell suspension.
The fibroblast cell suspension is seeded into a first tissue culture flask containing a medium, and a fibroblast cell population is cultured through at most three passages into sequentially larger tissue culture flasks in the medium, thereby generating at least 100 million minimally-passaged fibroblasts and a conditioned medium. The minimally-passaged fibroblasts are harvested to obtain the conditioned medium, and the conditioned medium is formulated for topical administration to obtain the cell-free topical composition.
The document states that the conditioned medium is used as a cell-free topical treatment for tissue repair and regeneration of skin, bone, and other connective-tissue defects. It further states that the minimally-passaged fibroblasts can be shipped fresh or cryopreserved, and that culture conditions incorporate antibiotics and antifungal components.
Claims Coverage
The independent claim covers a cell-free topical composition produced from conditioned medium generated by culturing minimally-passaged fibroblasts derived from a single small full-thickness biopsy, with at most three passages and topical formulation. The inventive features are: biopsy digestion, controlled sequential culturing with limited passaging to reach specified minimally-passaged fibroblast quantity, harvesting conditioned medium, and formulation into a cell-free topical composition.
Cell-free topical composition from conditioned medium
A cell-free topical composition comprising conditioned medium produced by digesting a tissue biopsy specimen, seeding and culturing fibroblast cells through at most three passages, harvesting minimally-passaged fibroblasts to obtain the conditioned medium, and formulating the conditioned medium for topical administration.
Single full-thickness biopsy digestion and suspension formation
Digesting a tissue biopsy specimen with a dissociative or digestive enzyme to form a fibroblast cell suspension, wherein the biopsy specimen is a single full-thickness biopsy not more than 5 mm thick and not more than 12 cm2.
At most three passages into sequentially larger tissue culture flasks
Seeding the fibroblast cell suspension into a first tissue culture flask containing a medium; and culturing a fibroblast cell population in the first tissue culture flask through at most three passages into sequentially larger tissue culture flasks in the medium, thereby generating at least 100 million minimally-passaged fibroblasts and a conditioned medium.
Harvesting conditioned medium and topical formulation
Harvesting said at least 100 million minimally-passaged fibroblasts to obtain a cell-free conditioned medium; and formulating the conditioned medium for topical administration to obtain the cell-free topical composition.
Overall, the claim coverage centers on producing a cell-free topical composition from conditioned medium derived from minimally-passaged fibroblasts generated by digesting a single small full-thickness biopsy and culturing fibroblasts through at most three passages into sequentially larger tissue culture flasks, followed by harvesting conditioned medium and topical formulation.
Stated Advantages
Improved viability and effectiveness compared with multi-passaging.
Documented Applications
Tissue repair and regeneration of skin, bone, and other connective-tissue defects.
Topical cell-free treatment using conditioned medium.
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