Figure 3.

Images of P0-P1 primary human keratinocytes in culture

(A) Image of P0 keratinocytes in hemacytometer, viewed through phase-contrast microscope. The 16-square area for counting is indicated by white arrows. Note that cell morphology is heterogeneous and only refractory (bright) cells of roughly uniform size should be counted. Dark looking cells and debris (black arrows) should not be counted.

(B) Human P0 keratinocytes at day 1, ∼24 h after inoculation into collagen-coated flask. Note that the cell population appears heterogeneous at this stage, with many cells appearing bright and raised from culture surface. Cells have irregular shapes, and some 2-cell colonies or dividing cells may be observed (white arrows).

(C) Human P0 keratinocytes at day 2 of culture. The cells exhibit diverse morphologies: rounded-up cells, which may be in the process of cell division (white arrow), can be seen, in addition to more flattened cells. This appearance is normal at P0 in KGM-LC. In KGM-LC, P0 keratinocytes may appear large and flat, and form loosely-associated colonies. Occasional elongated cells, most likely melanocytes, may be observed (black arrow).

(D) By culture day 6, P0 keratinocyte morphology appears more uniform, with numerous flattened, widely separated cells. A melanocyte is identified by its dendritic morphology (black arrow).

(E) P0 keratinocytes at day 7, immediately prior to melanocyte removal. Melanocytes are indicated by black arrows.

(F) Nearly confluent P0 keratinocytes at the time of harvesting for passage.

(G) P1 keratinocytes shown 48 h after inoculation.

(H) P1 keratinocytes 6 days after inoculation, at time of harvest (∼80%–85% confluent). Note tighter colonies and more uniform morphology after passage and culture in “regular” calcium (0.2 mM) KGM. Original image magnification: 10× (B–D); 4× (A, E–H).