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-9902937-B2
Publication Date
2018-02-27
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 topical composition produced from fibroblast conditioned medium. The topical composition is produced by digesting a dermal biopsy specimen obtained from a human and comprising fibroblasts with a dissociative or digestive enzyme to form a fibroblast cell suspension, then seeding the fibroblast cell suspension into a first tissue culture flask.
The fibroblast cell suspension is cultured through at most three passages into a sequentially larger tissue culture flask, and the fibroblast conditioned medium is harvested during the culturing step. The harvested conditioned medium is then formulated into the topical composition, and the fibroblast cell suspension comprises 3×108 or more cells at harvest.
In further embodiments, the dissociative or digestive enzyme includes collagenase, collagenase subtype liberase, or trypsin, and the sequentially larger tissue culture vessel may include a T-500 flask or a multi-layer culture stack. The document also describes shipping fresh or cryopreserved cells and the use of cryopreservation for harvested cells, while maintaining use of autologous minimally-passaged fibroblasts associated with improved viability/effectiveness.
Claims Coverage
The partial claims provided include one independent claim, which defines a topical composition based on fibroblast conditioned medium produced from an enzyme-digested human dermal biopsy. This independent claim includes four inventive features tied to biopsy size, enzymatic dissociation, limited passaging, and harvest cell number.
Topical fibroblast conditioned medium composition
A topical composition comprising fibroblast conditioned medium, wherein the topical composition is produced by digesting a dermal biopsy specimen obtained from a human and comprising fibroblasts with a dissociative or digestive enzyme to form a fibroblast cell suspension, seeding the fibroblast cell suspension into a first tissue culture flask, culturing the fibroblast cell suspension through at most three passages into a sequentially larger tissue culture flask, harvesting the fibroblast conditioned medium during the culturing step when the fibroblast cell suspension comprises 3×108 or more cells at harvest, and formulating the conditioned medium into the topical composition.
Enzyme digestion of a sized dermal biopsy specimen
Digesting a dermal biopsy specimen obtained from a human and comprising fibroblasts with a dissociative or digestive enzyme, wherein the dermal biopsy specimen is from 1 to 5 mm in thickness and between 4 and 12 cm2 in area to form a fibroblast cell suspension.
Limited culturing through at most three passages
Culturing the fibroblast cell suspension in the first tissue culture flask through at most three passages into a sequentially larger tissue culture flask.
Harvest at a defined cell number for conditioned-medium collection
Harvesting the fibroblast conditioned medium from the fibroblast cell suspension during the culturing step, wherein the fibroblast cell suspension comprises 3×108 or more cells at harvest.
Overall, the claim coverage is centered on producing a topical composition from fibroblast conditioned medium generated by enzyme-digesting a defined-size human dermal biopsy, culturing through no more than three passages in sequentially larger tissue culture flasks, harvesting conditioned medium when the cell suspension comprises 3×108 or more cells, and formulating the conditioned medium into the topical composition.
Stated Advantages
Improved viability/effectiveness
Documented Applications
Treating skin/bone/connective tissue defects via injection or topical delivery
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