Application Note for KerCT Immortalized Keratinocyte Cells
(CRL-4048™)
Abstract
Primary and immortal keratinocyte cultures are important cell models for the study of normal and
pathological biology of the cutaneous epithelia. Primary keratinocytes can form organotypic skin
equivalents that mimic the architectural features and behavior of normal skin in an advanced threedimensional (3D) culture model. However, primary keratinocytes have finite lifespan in culture,
which greatly restricts their use as in vitro cellular model. In this study, we confirmed that an
immortal keratinocyte cell line, Ker-CT (ATCC® CRL-4048™), is able to fully differentiate into
organotypic skin equivalents in a 3D culture model. The immortality of Ker-CT cell line makes it an
invaluable model for the research of keratinocyte biology.
Introduction
Human keratinocytes are instrumental for the study of skin biology and the pathogenesis of skinrelated disease, however, the limited lifespan of primary keratinocyte cultures greatly limit their
potentials for advanced mechanism studies, as genetic manipulations in keratinocytes entail
extended lifespan of the cultured cells. The Ker-CT (ATCC® CRL-4048™) cell line was developed
by sequential introduction of retroviruses expressing human telomerase (hTERT) and mouse cyclindependent kinase 4 (CDK4) genes into human neonatal foreskin keratinocytes (Ramirez et al.,
2003). The Ker-CT cells express basal epithelial stem cell markers such as Tumor Protein P63
(TP63) and Keratin 5 (KRT5). These cells are positive for telomerase expression, fail to senesce
and continue proliferation even after more than 250 population doublings, and up-regulate the
expression of differentiation markers in response to increased calcium concentration in conventional
2D culture (Ramirez et al., 2003).
To confirm that the Ker-CT cells can differentiate in 3D organotypic culture model even after an
extended culture period as reported previously (Ramirez et al., 2003; Vaughan et al., 2009), we
seeded Ker-CT cells at both early and late passages on top of collagen gels embedded with primary
dermal fibroblasts isolated from neonatal foreskin (ATCC® PCS-201-010™), following a published
protocol (Kalabis et al., 2012). Histology staining indicated that the Ker-CT cells, even at late
passage, are able to fully differentiate into stratified skin equivalent structures similar to primary
neonatal foreskin epidermal keratinocytes (ATCC® PCS-200-010™). These data render the
immortal Ker-CT cells an excellent and reproducible in vitro cell model for the examination of
mechanisms regulating keratinocyte biology in the processes of wound healing, dermal remodeling,
cancer development and drug toxicology evaluation in a tissue-physiology-relevant context.
American Type Culture Collection
P.O. Box 1549
Manassas, VA 20108 USA
http://www.atcc.org
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Application Note for KerCT Immortalized Keratinocyte Cells
(CRL-4048™)
Materials and Methods
1. Place a 3.0µm transwell inserts (BD 354573) in deep 6-well plates (BD 355467).
2. Add the following on ice for one 6-well plate to a tube and mix well after each addition:
EMEM
FBS
L-Glutamine
Sodium
Bicarbonate
Rat Tail Collagen
3.
4.
5.
6.
Stock
Concentration
Volume
(6-wells)
Final
Concentration
Company
Catalogue
Number
10X
690µL
0.94X
Lonza
12-684F
100%
700µL
14.3%
ATCC
30-2020
200mM
60µL
1.6mM
ATCC
30-2214
7.5%
300µL
0.31%
Lonza
17-613E
1.2mg/ml
5.6mL
0.91mg/ml
BD
354236
Add 1mL to the apical chamber (on top of the insert).
Incubate at 37oC for 30-60 minutes until it solidifies. A color change from yellow to pink should be observed.
Trypsinize fibroblast cells, count, and resuspend them at 6x105 cells/mL.
Add the following on ice ,for one 6-well plate to a tube, and mix well after each addition:
10x EMEM
FBS
L-Glutamine
Sodium
Bicarbonate
Rat Tail Collagen
Fibroblast cells
Stock
Concentration
Volume
(6-wells)
Final
Concentration
Company
Catalogue
Number
10X
1.8mL
0.84X
Lonza
12-684F
100%
2.0mL
9.28%
ATCC
30-2020
200mM
160µL
1.48mM
ATCC
30-2214
7.5%
800µL
0.28%
Lonza
17-613E
1.2mg/mL
15.2mL
0.84mg/ml
BD
354236
6 x 105
cells/mL
1.6mL
4.4 x 104
cells/mL
--------
--------
7. Add 3mL to the apical chamber.
8. Incubate at 37oC for 60-90 minutes until it solidifies.
________________
American Type Culture Collection
P.O. Box 1549
Manassas, VA 20108 USA
http://www.atcc.org
___
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800-683-6597 or 703-365-2700
Fax: 703-365-2750
E-mail: [email protected]
Or contact your local distributor.
Application Note for KerCT Immortalized Keratinocyte Cells
(CRL-4048™)
9. Add 10mL DMEM (ATCC 30-2002) supplemented with 10% FBS to the basal chamber (below insert) and 2mL
into the apical chamber.
10. Incubate at 37oC incubator for 24 hours.
11. Loosen the plug from the wall of the insert using a glass Pasteur pipette. Move the pipette along the inside
edge of the insert three times without piercing the insert.
12. Add 2mL of DMEM supplemented with 10% FBS into the apical chamber.
13. Incubate at 37oC for 4-6 days. Allowing the plug to contract forming a crater-like top. No media change is
necessary.
14. Mix DMEM and Hams F12 (Invitrogen 11765-054) at a 3:1 ratio to make presaturation media.
15. Aspirate media from apical and basal chamber of inserts.
16. Add 10mL of presaturation media to basal chamber and 2mL to apical chamber.
17. Incubate at 37oC incubator for 60 minutes.
18. Trypsinize keratinocytes as normal.
19. Resuspend cells at 4x106 cells/mL in complete growth media.
20. Aspirate media from apical and basal chamber of inserts.
21. Place a cloning ring (corning 3166-6) into the center of the insert plug, a surface area of 0.126cm2.
22. Add 50µL of keratinocytes into each cloning ring, 2x10 5 total cells or 1.6x106 cells/cm2.
23. Incubate at 37oC incubator for two hours.
24. Make EPM1 and EPM2 as follows:
Reagent
Stock
concentration
EPM1
(6-wells)
Final
concentration
EPM2
(6-wells)
Final
concentration
Company
Catalogue
Number
DMEM
1x
109mL
0.73x
0.48X
ATCC
30-2002
Ham’s F12
1x
36mL
0.24x
0.48X
200mM
3mL
4mM
Invitroge
n
ATCC
11765-054
L-Glutamine
71.25m
L
71.25m
L
3mL
Hydrocortisone
74.2µM
300µL
0.15µM
300µL
0.15µM
H0888
ITES
500x
300µL
1X
300µL
1X
SigmaAldrich
Lonza
OPhosphorylethanolamine
Adenine
5mM
300µL
0.01mM
300µL
0.01mM
90mM
300µL
0.18mM
300µL
0.18mM
Progesterone
2µM
300µL
0.004µM
--------
--------
Triiodothyronine
10nM
300µL
0.02nM
300µL
0.02nM
FBS
100%
150µL
0.1%
3mL
2.0%
________________
American Type Culture Collection
P.O. Box 1549
Manassas, VA 20108 USA
http://www.atcc.org
4mM
SigmaAldrich
SigmaAldrich
SigmaAldrich
SigmaAldrich
ATCC
30-2214
17839Z
P0503
A9795
P8783
T5516
30-2020
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800-683-6597 or 703-365-2700
Fax: 703-365-2750
E-mail: [email protected]
Or contact your local distributor.
Application Note for KerCT Immortalized Keratinocyte Cells
(CRL-4048™)
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
Remove cloning ring
Add 10mL EPM1 to basal and 2mL EPM1 to apical chamber.
Incubate at 37oC for two days.
Change media with 10mL EPM1 to basal and 2mL EPM1 to apical chamber.
Incubate at 37oC for two days.
Change media with 8.5mL EPM2 to basal chamber and remove media in the apical chamber1.
Change media every two to three days with 8.5mL EPM2 to the basal chamber for 14-21 days total.
Move inserts to a new 6-well plate (non-deep well).
Fix the cells by adding 2mL 4% formaldehyde to basal and 2mL 4% formaldehyde to apical chamber.
Incubate for one hour at 4oC.
Wash two times with 1x PBS leaving in PBS while processing the remaining samples.
Cut out the membrane from the insert using a scalpel.
Place onto a flat plastic surface.
Cut in half to ensure that the keratinocytes can be sectioned.
Place sections into tubes completely filled with PBS stored at 4oC or 70% ethanol stored at room temperature.
Process samples for desired staining.
Results and Discussion
To evaluate the differentiation capability of Ker-CT cells in a 3D organotypic skin equivalent model
after consecutive sub-cultivations, we tested the Ker-CT cells at early passage (passage 6) and late
passage (passage 15) after recovery from cryopreservation, following the recommended culture
protocol listed in the product sheet. Calculation of cell population growth indicated that Ker-CT cells
at passage 15 were more than 25 population doublings over the cells at passage 6 (data not
shown). Neonatal foreskin dermal keratinocytes (ATCC® PCS-200-010™) at passage 2 after
recovery from cryopreservation were used as positive control for skin equivalent differentiation,
since primary keratinocyte culture lost their differentiation capability after passage 5 (data not
shown).
As shown by hematoxylin and eosin stain (H&E stain) in Figure 1, 11 days after exposing to airliquid interface culture, primary keratinocytes developed into characteristic stratified epidermis
architecture composed of proliferating basal and differentiated suprabasal keratinocytes, and an
evident stratum croneum at the very top (Figure 1). Ker-CT cells also developed into similar
stratified epidermis architecture, although the stratum croneum was thinner than primary
keratinocytes. No apparent difference was seen between the early and late passages of Ker-CT
cells (Figure 1). Furthermore, after 21 days of air-liquid interface culture, Ker-CT cells at both early
and late passages differentiated into characteristic epidermis architectures that are indistinguishable
from primary keratinocytes (Figure 2).
These data indicated that the immortal Ker-CT cells retain intact differentiation capability after
extended in vitro culture period beyond the capacity of normal primary keratinocyte culture. The
1
This is day one for the airlift.
________________
American Type Culture Collection
P.O. Box 1549
Manassas, VA 20108 USA
http://www.atcc.org
___
© 2017 ATCC. All rights reserved.
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800-683-6597 or 703-365-2700
Fax: 703-365-2750
E-mail: [email protected]
Or contact your local distributor.
Application Note for KerCT Immortalized Keratinocyte Cells
(CRL-4048™)
Ker-CT cells may have significant potential value for a wide variety of investigations of human
epithelial biology in a tissue-physiology-relevant context.
To verify the fully differentiation of functionality of the cells, frozen cross sections were stained with
Filaggrin, which stains for well differentiated keratinocytes and KRT14, which stains all epidermal
cells. Filaggrin in both primary keratinocytes and Ker-CT shows increasing positivity as the cells
are closer to the surface nearing terminal differentiation. KRT14 shows positivity in both primary
keratinocytes and Ker-CT throughout the epithelial cells.
*For Figures, please see the CRL-4048 webpage for attachments
Figure 1: H&E stain of cross sections revealed a stratified epidermis architecture formed by
culturing A) primary foreskin keratinocytes at passage 2, B) Ker-CT at passage 6, and C) Ker-CT at
passage 15, on top of a collage gel embedded with primary foreskin dermal fibroblasts, after 11
days post air-liquid interface culture.
Figure 2: H&E stain of cross sections revealed a stratified epidermis architecture formed by
culturing A) primary foreskin keratinocytes at passage 2, B) Ker-CT at passage 6, and C) Ker-CT at
passage 15, on top of a collage gel embedded with primary foreskin dermal fibroblasts, after 21
days post air-liquid interface culture.
Figure3: Filaggrin(red), KRT14(green), and DAPI(blue) stained cross sections at 11 days post
airlift for Primary Keratinocytes.
Figure4: Filaggrin(red), KRT14(green), and DAPI(blue) stained cross sections at 11 days post
airlift for Ker-CT at P+5.
References
Kalabis, J., Wong, G.S., Vega, M.E., Natsuizaka, M., Robertson, E.S., Herlyn, M., Nakagawa, H.,
and Rustgi, A.K. (2012). Isolation and characterization of mouse and human esophageal epithelial
cells in 3D organotypic culture. Nat. Protoc. 7, 235–246.
Ramirez, R.D., Herbert, B.-S., Vaughan, M.B., Zou, Y., Gandia, K., Morales, C.P., Wright, W.E., and
Shay, J.W. (2003). Bypass of telomere-dependent replicative senescence (M1) upon
overexpression of Cdk4 in normal human epithelial cells. Oncogene 22, 433–444.
Vaughan, M.B., Ramirez, R.D., Andrews, C.M., Wright, W.E., and Shay, J.W. (2009). H-Ras
Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin
Equivalents. PLoS ONE 4, e7908.
________________
American Type Culture Collection
P.O. Box 1549
Manassas, VA 20108 USA
http://www.atcc.org
___
© 2017 ATCC. All rights reserved.
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800-683-6597 or 703-365-2700
Fax: 703-365-2750
E-mail: [email protected]
Or contact your local distributor.