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 cell-free topical composition comprising derived from at most passage three fibroblasts conditioned medium produced by a method comprising the steps of:
a) 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 cm 2 ;
b) seeding the fibroblast cell suspension into a first tissue culture flask containing a medium;
c) 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;
d) harvesting said at least 100 million minimally-passaged fibroblasts to obtain a cell-free conditioned medium; and
e) formulating the conditioned medium for topical administration to obtain the cell-free topical composition.
2. The composition of claim 1 , wherein the enzyme is a collagenase.
3. The composition of claim 2 , wherein the collagenase is liberase.
4. The composition of claim 1 , wherein the enzyme is trypsin.
5. The composition of claim 1 , wherein at least 200 million minimally-passaged fibroblasts are generated.
6. The composition of claim 1 , wherein at least one billion minimally-passaged fibroblasts are generated.
7. The composition of claim 1 , wherein the first tissue culture flask is a T-175 or a T-225 flask.
8. The composition of claim 7 , wherein the sequentially larger tissue culture flasks are a T-500 flask or a 10-layer cell culture stack.
9. The composition of claim 1 , wherein the first tissue culture flask is a T-175 or a T-225 flask, the sequentially larger tissue culture flasks are a T-500 flask or multilayer cell culture stacks cell culture stack, and wherein the multilayer stacks are 10-layer stacks.
10. The composition of claim 1 , wherein the at least 100 million minimally-passaged fibroblasts are generated by culturing the fibroblast cell population in the first tissue culture flask through no more than three passages into sequentially larger tissue culture flasks.