Compare the use of Lonza KGM Gold Bullet kit and Rheinwald and Green complete FAD medium in primary human epidermal keratinocytes culture and its applicability cells cultured by these medium in the construction of reconstituted skin equivalent model

1. Comparing Lonza KGM-Gold™ Keratinocyte
Growth Medium & Rheinwald and Green
Complete FAD Medium
A presentation by:
Bryan Yap Jin Hang
Calvin Chan Yoong Liang
Chai Jun Yang
Julian Kee Zheng Yuan
Leong Weng Hhin

2. What We Will Be Covering:
• The Basics
• Applications
• Comparison
• Current Development
• Challenges
KGM-Gold™ Keratinocyte Growth Medium

3. The Basics

4. Composition
KGM-Gold™ Keratinocyte Growth Medium Rheinwald and Green Complete FAD
Medium
• BPE (Bovine Pitutary Extract)
• hEGF
• Insulin
• Hydrocortisone
• GA-1000 (Gentamicin, amphotericin-B)
• Epinephrine
• Transferrin
• 3 parts Dulbecco’s Modified Eagle’s
Medium
• 1 part Ham’s F12 Medium
• Adenine
• Penicillin
• Streptomycin
• Fetal Bovine Serum
• Hydrocortisone
• Cholera enterotoxin
• Epidermal Growth Factor
• Insulin
Presence of serum

5. Applications in KGM Lonza Culture and Complete FAD Medium.
KGM LONZA Medium : Cells grow mostly as undifferentiated
adherent monolayer.
Complete FAD Medium : Cells grow and differentiate into an
organotypic culture with different cell
types
(Ambler and Lamb 2012)
Applications
• Cells grown using FAD medium display better in vivo characteristics due to similar
morphology to the human skin (Skin-equivalent model).
• Thus, cells grown using FAD medium are ideal for use in Anatomical and
Physiological studies.

6. Applications of Monolayer Adherent Cultures
• Pathogenicity and Disease Progression
• The study of pathogenicity and disease progression may lead to the discovery of new
vaccines and drugs that combat illness!
KGM Lonza Medium: The “Gold Standard”
Figure _ : Growth Rate of Cells cultured in KGM Gold vs other
brands (Lonza 2017)
Figure _ : Confluency of Cells grown in KGM Gold vs other
Brands (Lonza n.d)
Why KGM Gold?
• High Yield.
• Faster Growth
Rate.
Why CFM?
• Higher similarity to
in-vivo models.
• More suited for
functional tests
such as toxicity
tests.

7. Discussion
Figure 2: Average depth of the epithelium layer in different type of media. (Lamb and
Ambler, 2013)
Figure 1: Comparison of Normal Human Epithelial Keratinocytes growth in KGM-Gold serum-free media and in complete
FAD medium with feeders. (Lamb and Ambler, 2013)
• Test for effect of different concentration of Calcium and addition of 10% serum on the
growth of epithelium layer.
• Based on the result, it is proven that higher growth of epithelium layer depends on
high concentration of calcium and addition of 10% serum.
• A-D : Test for cell motility. The cells grown in KGM-Gold are more motile and form temporary cell
attachment because the absence of calcium prevents the formation of desmosomes and cell junctions.
• E-F: Actin staining with phalloidin. Polymerised actin was detected at the leading edge of cell grown in
KGM-Gold while it is uniform in cell grown in complete FAD.

8. Discussion
Figure 4: The effect of heat-treated serum on the average depth of epithelium layer. (Lamb and Ambler, 2013)
Figure 3: The growth of epithelial layer in EpiLife serum-free medium with variation in
presence of Calcium and Serum. (Lamb and Ambler, 2013)
• Test for effect of second serum-free medium, EpiLife on the epithelial growth under
various conditions. Based on the result, EpiLife also requires both high concentration
of calcium and addition of 10% serum for the best growth result.
• Test for effect of heat-treated serum and it was found that heated serum would
increase the epidermal thickness and organisation.

9. Challenges
• Lonza KGM Gold Bullet kit
• Inability to form 3D structure (Lamb & Ambler 2013)
• Fixed through re-additon of serum
• Rheinwald & Green complete FAD medium
• Serum (Freshney 2007)
• Contamination & immunoglobulin
• Filtration (0.1 μm)
• Heat inactivation (56oC, 30 min)
• Inconsistent batches (eg: bovine) -> lasts 6 months
• Climate, animal, storage, sterilization

10. Current Developments
• Efficient Keratinocyte Differentiation Strictly Depends on JNK-Induced
Soluble Factors in Fibroblasts (Schumacher et al. 2014)
• Alternatives to the use of fetal bovine serum: serum-free cell culture
(Gstraunthaler 2003)
• Stable chemically defined surface for the culture of human
keratinocytes (Higham et al. 2003)
• A Defined, Feeder-Free, Serum-Free System to Generate In Vitro
Hematopoietic Progenitors and Differentiated Blood Cells from hESCs
and hiPSCs (Salvagiotto et al. 2011)

11. Conclusion
• Serum contains many essential growth factors that mimics inVivo
growth conditions for animal cells
• There is presence of serum in Lonza KGM-Gold™ Keratinocyte Growth
Medium while FAD formula does not have any.
• Cells grown in FAD formula shows more differentiated structures
compared to cells grown in Lonza KGM-Gold™ Keratinocyte Growth
Medium.
• Thus, cells grown in FAD formula can be used to study cell physiology
• However, Lonza KGM-Gold™ Keratinocyte Growth Medium provides
higher yield in a shorter amount of time.

12. Reference
• Schumacher, M., Schuster, C., Rogon, Z., Bauer, T., Caushaj, N., Baars, S., Szabowski, S., Bauer, C., Schorpp-Kistner, M., Hess, J., Holland-Cunz, S., Wagner, E., Eils, R.,
Angel, P. and Hartenstein, B. (2014). Efficient Keratinocyte Differentiation Strictly Depends on JNK-Induced Soluble Factors in Fibroblasts. [online]
http://www.sciencedirect.com. Available at: http://www.sciencedirect.com/science/article/pii/S0022202X15367555 [Accessed 20 Sep. 2017].
• G, G. (2003). Alternatives to the use of fetal bovine serum: serum-free cell culture. - PubMed - NCBI. [online] Ncbi.nlm.nih.gov. Available at:
https://www.ncbi.nlm.nih.gov/pubmed/14671707/ [Accessed 20 Sep. 2017].
• Higham MC, e. (2003). Development of a stable chemically defined surface for the culture of human keratinocytes under serum-free conditions for clinical use. -
PubMed - NCBI. [online] Ncbi.nlm.nih.gov. Available at: https://www.ncbi.nlm.nih.gov/pubmed/14633376 [Accessed 20 Sep. 2017].
• Salvagiotto, G., Burton, S., Daigh, C., Rajesh, D., Slukvin, I. and Seay, N. (2011). A Defined, Feeder-Free, Serum-Free System to Generate In Vitro Hematopoietic
Progenitors and Differentiated Blood Cells from hESCs and hiPSCs. [online] https://www.ncbi.nlm.nih.gov. Available at:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3060827/ [Accessed 20 Sep. 2017].
• http://bio.lonza.com. (n.d.). Clonetics™ Keratinocyte Media Products. [online] Available at:
http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_Clonetics_Keratinocyte_Media_Products.pdf [Accessed 20 Sep.
2017].http://booksc.org/book/14056580/7d6182
• Lindberg, K. and Badylak, S. (2001). Porcine small intestinal submucosa (SIS): a bioscaffold supporting in vitro primary human epidermal cell differentiation and
synthesis of basement membrane proteins. [online] http://www.sciencedirect.com. Available at:
http://www.sciencedirect.com/science/article/pii/S0305417900001133 [Accessed 20 Sep. 2017].
• Kollisch, G., Kalali, B., Voelcker, V., Wallich, R., Behrendt, H., Ring, J., Bauer, S., Jakob, T., Mempel, M. and Ollert, M. (2005). Various members of the Toll-like receptor
family contribute to the innate immune response of human epidermal keratinocytes. [online] http://onlinelibrary.wiley.com. Available at:
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2567.2005.02122.x/full [Accessed 20 Sep. 2017].
• Lamb, R. and Ambler, C. (2013). Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin
Model. [online] NCBI. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3543440/ [Accessed 20 Sep. 2017].
• Lonza.com. (n.d.). KGM-Gold™ Keratinocyte Growth Medium. [online] Available at: http://www.lonza.com/products-services/bio-research/primary-cells/human-
cells-and-media/keratinocytes-and-media/kgm-gold-keratinocyte-growth-medium.aspx [Accessed 20 Sep. 2017].
• Lamb, R. and Ambler, C. (2013). Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin
Model. PLoS ONE, [online] 8(1), pp.1-8. Available at: http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0052494&type=printable [Accessed 19
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