For dermal reconstruction, 1 ml of a cell-free buffered collagen solution consisting of bovine collagen, type I (Organogenesis, Canton, MA, Cat. No. 200/50) at a final concentration of 0.78-1.01 mg/ml in Minimal Essential Medium with Earle's Salts (Cambrex, Cat. No. 12-684F), 200 mM L-glutamine (Invitrogen, Cat. No. 25030-081), FCS, and 7.5% sodium bicarbonate (Cambrex, Cat. No. 17-613E) was added to tissue reconstruct trays (Organogenesis, Canton, MA) (Fig. 1). This precoated acellular layer was then overlaid with 3 ml of fibroblast-containing collagen (2.4 x 104 /ml). After 4-7 days of incubation at 37°C, the fibroblasts had contracted the collagen gel, which formed a concave central area for subsequent seeding of epidermal cells.
Epidermis
For epidermal reconstruction, the mature dermal reconstruct was rinsed and equilibrated with 14 ml Ca2+, Mg2+ -free HBSS containing 1% dialyzed FCS (12 ml outside and 2 ml on top of the polycarbonate insert). After 1 hour, HBSS + 1% dFCS was removed, and replaced with Melanocyte Reconstruct Media (MRM) (1.1) (12 ml and 1.7 ml outside and inside of the insert, respectively). Neonatal foreskin melanocytes and keratinocytes in a ratio of 1:5 (500,000 cells total/reconstruct) were dispensed on top in a total volume of 200 μl MRM. Seeded melanocytes and keratinocytes were allowed to attach and proliferate. After 4 days of submerged cultures, with a feeding after 2 days, skin reconstructs were lifted to the air-liquid interface and medium was switched to maintenance medium with omitted EGF and increased CaCl2 concentration.
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Components |
Melanocyte Reconstruct Media (MRM) |
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Medium I (Days 1-2) |
Medium II (Days 3-4) |
Medium III (Day 5+) |
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Keratinocyte Serum-Free Media (Invitrogen Cat. No 17005042 includes BPE and EGF) |
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Bovine Pituitary Extract (BPE) |
60 ug/ul |
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Dialyzed Fetal Bovine Serum (Hyclone Cat. No. SH30079.03) |
2% |
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bFGF (recombinant human bFGF- obtained from E. coli) |
1.1 ng/ml |
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ET-3 (Peninsula Laboratories #6911) |
100 nM |
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SCF (R&D #2555C10) 10 ng/ml |
10 ng/ml |
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Epidermal Growth Factor (EGF) |
1 ng/ml |
0.2 ng/ml |
None |
CaCl2 |
None |
None |
2.4 mM |
Sterilized by using 0.22 μm filter |
|||
Notes: All
concentrations and percentages are written
as final concentrations in the media. All
concentrations of ingredients stay constant
for Medias I, II, III except for EGF and
CaCl2 which vary as indicated above.
After 10-14 days of air exposure, skin reconstructs were harvested. Skin reconstructs were fixed for 4-6 hours with 10% buffered formalin, and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E).
For incorporation of melanomas, cells were seeded together with keratinocytes onto dermal reconstructs at a 1:5 ratio of melanoma cells:keratinocytes. Culture conditions were the same as for reconstructs containing keratinocytes and normal melanocytes, with modified media.
|
Days 1 and 2 |
Days 3 and 4 |
Days 5+ |
Epidermalization I |
Epidermalization II |
Cerrif. III medium |
|
DME |
725 |
725 |
474 |
F12 |
240 |
240 |
474 |
L-glutamine |
20 |
20 |
20 |
Hydrocortisone |
2 |
2 |
2 |
ITT |
2 |
2 |
2 |
EOP |
2 |
2 |
2 |
Adenine |
2 |
2 |
2 |
Selenium |
2 |
2 |
2 |
CaCl2 |
2 |
2 |
2 |
Progesterone |
2 |
2 |
- |
Serum |
1 (NBCS) |
1 (NBCS) |
20 (NBCS) |
| All volumes are in ml. | |||
DMEM
Order from JRH Biosciences (Cat. #56430-10L)
Stock concentration: 1X
Final concentration: 75%
Preparation: Dissolve 43 g of DME in 4.75 l ddH2O, add 18.5 g NaHCO3 and 0.5 g MgSO4, adjust pH to 7.4 with approximately 3 ml of HCl, then filter-sterilize and incubate for 24 hours at 37°C (done by Cell Center)Store at 4°C (up to 3 weeks)
Use 725 ml/1 l EPI II medium
F-12 (HAM's) with glutamine
Order from Gibco (Cat. #11765-054)
Stock concentration: 1X
Final concentration: 25%
Store at 4°C, light-protected (up to 3 weeks)
Use 240 ml/1 l EPI II medium
L-Glutamine
Order from Cellgro (Cat. #MT25-005-CI)
Stock concentration: 200 mM (50x)
Final concentration: 4 mM
Preparation: Aliquot into 10 ml aliquots
Store at -20°C
Add 20 ml/1 l EPI II medium
Hydrocortisone
Order from Sigma (Cat. #H-0888)
1 g = $15.30 FW 362.5
Stock concentration: 500X (7.4 x 10-4 M)
(269 g/ml)
Final concentration: 1.48 x 10-6 M (0.5
g/ml)
Preparation: Dissolve 0.0269 g in 2.5 ml
absolute ethanol, then add 97.5 ml DMEM,
filter-sterilize (0.2 m filter) Aliquot 20 ml into 1 ml aliquots and 80
ml into 15 ml aliquots
Store at -20°C
Use 2 ml/1 l EPI II medium
Insulin, Transferrin, Ethanolamine,
Selenium (ITES)
Order from BioWhittaker (Cat. #17839Z)
5 ml = $41.40
Stock concentration: 500X (Insulin: 5 mg/ml,
Transferrin: 5 mg/ml, Ethanolamine: 5 mM,
Selenium: 5 g/ml)
Final concentration: 1X
Aliquot into 1 ml aliquots
O-Phosphorylethanolamine
Order from Sigma (Cat. #P0503)
FW 141.1
1 g = $6.80
Stock concentration: 0.05 M (7.05 mg/ml)
Final concentration: 10-4 M (0.1 mM) (14.1
g/ml)
Preparation: Dissolve 705 mg in 100 ml DMEM
and filter-sterilize
Aliquot 20 ml into 1 ml aliquots and 80
ml into 10 ml aliquots
Store at -20°C
Add 2 ml/1 l EPI II medium
Adenine
Hydrochloride, min. 99%
FW 171.6
Order from Sigma (Cat. #A9795)
5 g = $24.65
Stock concentration: 90 mM (15.5 mg/ml)
Final concentration: 0.18 mM (31 g/ml)
Preparation: Dissolve 1.55 g in 100 ml warm
(37°C) ddH2O, filter-sterilize
Aliquot into 2 ml aliquots
Store at -20°C
Add 2 ml/1 l EPI II medium
CaCl2
CaCl2,2H2O: FW 147
Order from Sigma (Cat. #C-7902)
500 g = $26.95
Stock concentration: 500X
Final concentration: 2.4 mM 0.5 mM
Preparation: Dissolve 3.55 g in 20 ml of
ddH2O and filter-sterilize
Aliquot into 2 ml aliquots
Store at -20°C
Add 2 ml/1 I EPI II medium
Progesterone
FW 314.5
Order from Sigma (Cat. #P-8783)
1 g = $8.80
Stock concentration: 2 nM (500X) (635.9
ng/ml)
Final concentration: 4 pM (1.27 ng/ml)
Preparation: Dissolve 1 mg in 1 ml absolute
ethanol and add 14.7 ml ddH2O, then dilute
1 ml in 100 ml DMEM, filter-sterilize and
aliquot 20 ml into 1 ml aliquots and 80
ml into 10 ml aliquots
Store at =20°C
Triiodothyronine
FW 637
Order from Sigma (Cat. #T-5516)
1 mg = $30.00
Stock concentration: 10 nM (6.5 g/l)
Final concentration: 20 pM (13 ng/l)
Preparation: Dissolve 1 mg in 1 ml 1 N NaOH
and add 19 ml DMEM (= 50 g/ml), then dilute
4 l in 31 ml DMEM, filter-sterilize and
aliquot into 2 ml aliquots
Store at -20°C
Add 2 ml/1 l EPI II medium
Newborn Calf Serum, Non-Chelated
Order from Hyclone (Cat. #SH 3011802)
Chelex 100
Order from Sigma (Cat. #C-7901)
Preparation: Mix 10 g of Chelex with 100 ml of NBCS and stir for 3 hours at 4°C, filter-sterilize supernatant (top layer)
Aliquot into
Store at -20°C
Human melanoma cell lines, originated from RGP (WM35, Sbcl2) and VGP primary (WM793, WM115), and metastatic melanomas (451Lu, 1205 Lu, WM852) were tested. All experiments were performed in duplicate. Paraffin sections of reconstructs were stained immunohistochemically with anti-type IV collage, anti-laminin, anti-HMB 45 and anti-S100 monoclonal antibodies (DAKO Carpriteria, CA) using standard methods. Apoptosis in paraffin sections was evaluated using an in situ apoptosis detection kit (ApopTag, Oncor, Gaithersburg, MD).
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