Figure 1.
Keratinocyte behaviour in monolayer cultures.
(A–D) Live cell images of p3 NHEKs grown in standard tissue culture flasks. Cells were grown in either KGM-Gold, serum-free media (A,C) or in complete FAD with feeder-support (B,D). (C,D) Trace of individual cells over a period of 24 hours. Axis scale in µM. (E,F) Equal numbers of p4 NHEKs were seeded onto cover-slips in a 24 well plate and grown in either KGM-Gold, serum-free media (E) or in complete FAD with feeder-support (F). Once confluent, the cells were fixed and then stained with phalloidin (red) and DAPI (blue). Scale bars: 50 microns.
Figure 2.
Keratinocyte media sufficient to support feeder-free, serum-free epidermal growth fails to support stratification in skin equivalents.
(A, B, D–F) Hematoxylin and eosin-stained sections of p4 NHEKs grown on DEDs in the following culture media: (A) complete FAD (high Ca++/10%FCS), (B) KGM-Gold (low Ca++), (D) low-Ca++ FAD with 10% serum (low Ca++/10%FCS), (E) KGM-gold plus added Ca++ (high Ca++), (F) KGM-Gold supplemented with 10% fetal bovine serum (low Ca++/10%FCS) and (G) KGM supplemented with 10% fetal bovine serum and added Ca++ (high Ca++/10%FCS). Insets show higher magnification images. Layers of the stratified epidermis indicated: B, basal; S, spinous; G, granular; C, cornified. (C) Graph indicates the average depth of the epithelium layer in the different types of media. Statistical significance calculated relative to control, complete FAD (high Ca++/10%FCS) cultured samples. Black arrow in B marks keratinocytes on DED surface. Blue arrows in A and G marks granular cells. ** p value<0.005; *** p value<0.0005 (Student's t-test); scale bars: 50 microns.
Figure 3.
Expression of keratinocyte differentiation markers.
(A–L) Eight micron sections of NHEKs grown on DEDs stained with antibodies to Keratin 14 (A–D, green), Keratin 10 (E–H, green) and Involucrin (I–L, green). All sections were counterstained with DAPI (blue). DEDs were grown in complete FAD (A,E,I), KGM-Gold plus added Ca++ (high Ca++; B,F,J), KGM-Gold supplemented with 10% fetal bovine serum (low Ca++/10%FCS; C,G,K) or KGM supplemented with 10% fetal bovine serum and added Ca++ (high Ca++/10%FCS; D,H,L). Layers of the stratified epidermis indicated: B, basal; S, spinous; G, granular; C, cornified. Scale bars: 50 microns.
Figure 4.
EpiLife media supplemented with calcium and 10% serum supports keratinocyte stratification.
(A–C) Hematoxylin and eosin-stained sections of p4 NHEKs grown on DEDs in the following culture media: (A) EpiLife plus added Ca++ (high Ca++), (B) EpiLife supplemented with 10% fetal bovine serum (low Ca++/10%FCS) or (C) EpiLife supplemented with 10% fetal bovine serum and added Ca++ (high Ca++/10%FCS). Scale bars: 50 microns.
Figure 5.
Heat-treating serum increases epidermal thickness and organisation.
Eight micron sections of p4 NHEKs grown on DEDs in complete FAD with 10% serum heated to 56°C (A, D, F; FAD+HI serum) or KGM supplemented with 10% serum heated to 56°C and added Ca++ (B, E, G; KGM+HI serum). (A, B, D–H) Sections were stained with hematoxylin and eosin or with antibodies to Keratin 14 (D, E, green) or Keratin 10 (F, G, green). Sections were counterstained with DAPI (blue). (C) Graph indicates the average depth of the epithelium layer in the two types of media. Statistical significance calculated relative to control, complete FAD with 10% serum (high Ca++/10%FCS) cultured samples (Figure 2). ** p value<0.005; *** p value<0.0005 (Student's t-test); scale bars: 50 microns.
Figure 6.
Heat-denaturation removes key serum factors needed to support epidermal stratification.
(A–C) Eight micron sections of p4 NHEKs grown on DEDs in complete FAD (A), KGM-Gold (B) or EpiLife (C) supplemented with 10% serum boiled at 95°C and 1.4 mM Ca++. Scale bars: 50 microns.