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-10900020-B2
Publication Date
2021-01-26
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 method of preparing a composition comprising a cell-free fibroblast conditioned medium. The method obtains a small tissue biopsy specimen from a human, digests the biopsy specimen with a dissociative or digestive enzyme to form a fibroblast cell suspension, and uses a limited passaging workflow through sequentially larger tissue culture flasks. The conditioned medium is harvested during culturing and then formulated into the composition.
The biopsy specimen is characterized as no more than 5 mm in thickness and not more than 12 cm2 in area. The fibroblast cell suspension is seeded into a first tissue culture flask and cultured through at most three passages into sequentially larger tissue culture flasks. At harvest, the fibroblast cell suspension comprises at least 3×10^8 cells, and the harvested fibroblast conditioned medium is used as the basis for the final composition.
Dependent refinements further specify the sequentially larger tissue culture vessel formats and digestion enzymes. The sequentially larger tissue culture flask can be a T-500 flask or a multi-layer culture stack, and the dissociative/digestive enzyme can be collagenase, including Liberase, or trypsin. Further use-related refinements specify administration of the composition to the human, including topical administration, for treating skin and other connective tissue defects.
Claims Coverage
The disclosed independent claim covers preparing a composition from harvested fibroblast conditioned medium produced from a human biopsy, with at most three passages into sequentially larger culture flasks and harvesting conditioned medium when at least 3×10^8 cells are present, followed by formulation into the composition. Dependent claims then narrow the culture vessel format, specify digestive enzymes, and specify topical administration to the human.
Small human biopsy limited by thickness and area
Obtaining a small tissue biopsy specimen from a human, wherein the biopsy specimen is no more than 5 mm in thickness and not more than 12 cm2 in area.
Enzyme digestion to form a fibroblast cell suspension
Digesting the biopsy specimen with a dissociative or digestive enzyme to form a fibroblast cell suspension.
Sequentially larger tissue culture flasks with at most three passages
Seeding the fibroblast cell suspension into a first tissue culture flask and culturing the fibroblast cell suspension in the first tissue culture flask through at most three passages into a sequentially larger tissue culture flask.
Harvesting cell-free fibroblast conditioned medium at high cell level
Harvesting the fibroblast conditioned medium from the cell suspension during the culturing step, wherein the fibroblast cell suspension comprises at least 3×10^8 cells at harvest.
Formulating the conditioned medium into the composition
Formulating the conditioned medium into the composition comprising a cell-free fibroblast conditioned medium.
Sequentially larger vessel as T-500 or multi-layer culture stack
The sequentially larger tissue culture flask is a T-500 flask or a multi-layer culture stack.
Digestive enzyme as collagenase (including Liberase)
Using an enzyme that is collagenase, wherein the collagenase used is Liberase.
Digestive enzyme as trypsin
Using the enzyme trypsin.
Topical administration to the human
Administering the composition to the human, wherein the composition is administered topically.
Overall, the claim set is directed to producing a cell-free fibroblast conditioned medium composition from a small human biopsy using enzyme digestion, limiting culturing to at most three passages into sequentially larger vessels, and harvesting conditioned medium when at least 3×10^8 cells are present. Dependent claims further specify vessel formats, digestion enzymes, and administration as topical to the human.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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