-

2. Keratinocytes (Basal cells)
Highly specialized epithelial cells found in basal layer
Perform specific functions, separation of organism
from its environment
Serve as chemical immune role and activate Langerhans
cells in response to injury
Keratinocyte stem cells divide to produce transient
amplifying cells which differentiate to produce
compounds important for skin integrity
Figure 1: Human epidermal keratinocytes (Volker 2017)

3. Lonza KGM-Gold bullet kit
Contents:
● Bovine pituitary extract
● hEGF
● Insulin (recombinant human)
● Hydrocortisone
● GA-1000 (gentamicin, amphotericin B)
Application (research):
● Skin grafting
● Cancer
● Gene therapy
● Cosmetics & personal
care
● Drug testing
● Dermal disorders
● Serum free media
● Low risk of contamination
● Formulated for high and low density NHEK cultures
● Minimal presence of enlarged and vacuolated cells enabling
more than 15 population doublings
● Supports clonal growth and high density keratinocyte
proliferation
● Supports superior growth and morphology of keratinocytes
Figure 2: KGM Gold bullet kit

4. Figure 3: Morphology of keratinocytes at high density in Clonetics KGM-Gold
Medium vs. Clonetics KGM-2 Medium (100X) (Willstaedt & Damian 2018)

5. Rheinwald & Green Complete FAD medium
Principle used by Rheinwald & Green:
Co-culturing human keratinocytes with irradiated 3T3 J2 fibroblast of mouse that acts as a nurse
cell in the feeder layer
Feeder layer contains non-dividing,
metabolically active fibroblast
It supports the keratinocyte
proliferation & tissue organization
Double paracrine pathway:
Keratinocytes will release IL-1 &
induce fibroblast to increase expression
for keratinocyte growth factor Figure 4: Shows an organotypic culture - 3D skin culture system
with human keratinocytes cultured on top of feeder layer

6. Basic components of basal media:
The basal media is a combination of DMEM + Ham’s F12 in ratio of 3:1
*DMEM = Dulbecco’s modified Eagle medium
1. Bovine serum (10%)
2. Buffering system (HEPES)- maintains physiological pH in culture medium
3. Mitomycin C - inhibits proliferation of fibroblast in feeder layer
4. EDTA - sequester Ca2+ ions and loosen up the adhesion molecule; eg. cadherin
5. Trypsin - proteolytic enzyme that degrades the adhesion molecules and detaches cells
6. PBS - phosphate buffer solution for washing the cells
7. Antibiotics: Penicillin & Streptomycin - prevent & eliminate bacterial
Additional Growth Factors - stimulates the growth & morphology of keratinocytes:
Hydrocortisone, Cholera enterotoxin, Insulin, Calcium, Adenine, Glutamine, Pyruvate,
Epidermal Growth Factor(EGF)

7. Lonza KGM Gold Bullet Kit Rheinwald and Green
Complete FAD Medium
Advantages ● Consistent performance
● Lower risk of contamination
● Cost-effective
● High availability
● High reliability
● Serum may act as buffering agent
that helps to maintain pH
● High albumin content in serum
protect the cells from adverse
conditions
Disadvantages ● Slow cell proliferation
● Cells are undifferentiated
● pH needs to be maintained regularly
● Inconsistent performance
● Susceptible to contamination
● Time consuming
● Low availability
● Low reliability

8. Lonza KGM Gold Bullet Kit
(Serum-free)
Rheinwald and Green Complete FAD
Medium (with serum)
❏ Single cells/loosely-connected groups
❏ High cell motility
❏ No differentiation of cells occurs.
❏ Unable to form stratified epidermis. As a
result, monolayer epidermis (2D) is formed.
❏ Tight clusters
❏ Low cell motility
❏ Differentiation occurs.
❏ Able to form stratified epidermis, thus
forming multi-layered epidermis (3D).

9. Current Developments
(Willstaedt & Damian, 2010)
(Richards, et al., 2008)

10. Challenges
1) Keratinocytes are only able to grow as a 2D culture and failed to show any stratified
epidermis due to extracellular calcium supplementation failed to improve epidermis
development. Cell potential is not loss due to the ability to form stratified epidermis
when re-introduced serum-containing media. (Lamb & Ambler, 2013)
1) Risk of infection with virus from bovine serum in the serum-containing media. If the
reconstituted skin model were to used for infection diagnosis, the infected skin model
may interfere with the diagnosis. (SJ & RL, 1999)

11. References
Eckert R L and Rorke E R, 1989, ‘Molecular biology of keratinocyte differentiation’, NCBI,
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1567608/
‘Keratinocytes-structure, function, immunity and differentiation’, n.d, Keratinocyte Transfection Kit,
http://www.keratinocyte-transfection.com/
Volker J H, 2017, ‘Cells and layers of the epidermis’, Earth’s Lab,
https://www.earthslab.com/physiology/cells-layers-epidermis/
Willstaedt T and Damian Z, 2010, ‘Clonetics KGM-Gold keratinocyte growth medium: A new robust and
versatile serum-free medium formulation for high and low density normal human epidermal keratinocyte
(NHEK) cultures’, Labonline,
https://www.labonline.com.au/content/consumables/article/clonetics-kgm-gold-keratinocyte-growth-
medium-a-new-robust-and-versatile-serum-free-medium-formulation-for-high-and-low-density-normal-
human-epidermal-keratinocyte-nhek-cultures-45357728

12. Choi, M. & Lee,C. (2015), ‘Immortalization of Primary Keratinocyte and It’s Application to Skin Research’,
23(5),pp.391-399.
https://www.e-sciencecentral.org/articles/?scid=SC000012059
Stark, H., Fusenig, N. & Szabowski, N.( 2000), ‘ Keratinocyte Growth Regulation in Defined Organotypic
Cultures Through IL-1-Induced Keratinocyte Growth Factor Expression in Resting Fibroblasts’,
114(6),pp.1075-1084.
https://www.sciencedirect.com/science/article/pii/S0022202X15408851
Watt,F.M., Broad, S. & Prowse, D.M. (2006), ‘Cultivation and Retroviral Infection of Human Epidermal
Keratinocytes’, in Cell Biology, Elsevier Science, pp.133-138
http://www.wattlab.org/storeddocs/Cultivation_and_Retroviral.pdf

13. Lamb, R. & Ambler, C. A., 2013. Keratinocytes Propagated in Serum-Free, Feeder-Free Culture
Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model.
Richards, S., Leaveesley, D., Topping, G. & Upton, Z., 2008. Development of Defined Media for the
Serum-Free Expansion of Primary Keratinocytes and Human Embryonic Stem Cells. Tissue Engineering
Part C: Methods, 3(14), pp. 221-232.
SJ, W. & RL, L., 1999. Benefits and risks due to animal serum used in cell culture production..
Developments in biological standardization, Issue 99, pp. 3-8.
Willstaedt, T. & Damian, Z., 2010. Clonetics KGM-Gold Keratinocyte Growth Medium: A new robust and
versatile serum-free medium formulation for high and low density normal human epidermal keratinocyte
(NHEK) cultures. [Online]
Available at: https://www.labonline.com.au/content/consumables/article/clonetics-kgm-gold-keratinocyte-
growth-medium-a-new-robust-and-versatile-serum-free-medium-formulation-for-high-and-low-density-
normal-human-epidermal-keratinocyte-nhek-cultures-45357728
[Accessed 21 04 2018].