Methods for culturing minimally-passaged fibroblasts and uses thereof
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.
1. A topical composition comprising fibroblast conditioned medium, wherein the topical composition is produced by the method comprising the steps of:
digesting a dermal biopsy specimen obtained from a human and comprising fibroblasts with a dissociative or digestive enzyme to form a fibroblast cell suspension, wherein the dermal biopsy specimen is from 1 to 5 mm in thickness, and between 4 and 12 cm 2 in area;
seeding the fibroblast cell suspension into a first tissue culture flask;
culturing the fibroblast cell suspension in the first tissue culture flask through at most three passages into a sequentially larger tissue culture flask;
harvesting the fibroblast conditioned medium from the fibroblast cell suspension during the culturing step, wherein the fibroblast cell suspension comprises 3×10 8 or more cells at harvest; and
formulating the conditioned medium into the topical composition.
2. The composition of claim 1 , wherein the fibroblast cell suspension is passaged twice.
3. The composition of claim 1 , wherein the fibroblast cell suspension is passaged once.
4. The composition of claim 1 , wherein the enzyme is a collagenase.
5. The composition of claim 4 , wherein the collagenase is liberase.
6. The composition of claim 1 , wherein the enzyme is trypsin.
7. The composition of claim 1 , wherein the first tissue culture flask is a T-125 flask, T-150 flask, T-175 flask, or T-225 flask.
8. The composition of claim 1 , wherein the sequentially larger tissue culture flask is a T-500 flask or multi-layer culture stack.