To investigate the effect of Lonza KGM Gold bullet kit and Rheinwald and Green complete FAD medium on the construction of reconstituting skin equivalent model.

1. To compare between Lonza KGM Gold BulletKit and
Rheinwald & Green Complete FAD medium in the
construction of reconstituted skin equivalent model
Alyaa Abdul
Cheong Kar Lok
Chong Ee Seng
Nageeswary Marimuthu
Vennishalini Maniam
GENE TISSUE & CULTURE TECHNOLOGY

2. Basic Concept
SKIN EQUIVALENT MODEL
Human skin equivalents (HSE) are three-
dimensional systems that are engineered by
seeding fibroblasts into a three-dimensional
dermal matrix. (El Ghalbzouri ,2018)
à Study cell-cell, cell-matrix, dermal-
epidermal interactions and epidermal
morphogenesis
à Mimic diseased skin disorders in vitro
à Suitable alternative for animal testing

3. - BPE (bovine pituitary extract)
- hEGF Insulin (recombinant human)
- Hydrocortisone GA-1000 (gentamicin, amphotericin-B)
- Epinephrine Transferrin - Contains non-serum derived growth factors
and antibiotics.
1. Expansion cell for the proliferation of keratinocytes serum-free
environment.
2. Support both clonal growth and high density keratinocyte
proliferation
3. The medium support superior growth and morphology of
keratinocyte (minimum presence of enlargement and vacuolated
cells)
4. Higher seedling efficiency ( more ≥15 Population Doublings)
Lonza KGM-Gold™ BulletKit
FUNCTION
CONTENT
SERUM FREE MEDIA

4. Rheinwald and Green complete FAD medium
SERUM CONTAINING MEDIA
CONTENT
- Serum (10% fetal-bovine serum)
- EGF (epidermal growth factor) (supplement)
- Trypsin
- Adenine (supplement)
- Insulin (supplement)
- Hydrocortisone (supplement)
- Cholera enterotoxin (supplement)
- Glutamine (supplement)
- Penicilin (supplement)
- Developed by James Rheinwald and Howard Green in 1970’s
- It culture the cells in the air-liquid interface
- It is a serum-containing medium supplements with the growth factors
(supplements) and antibiotics
- culturing the HEKs on a mitotically inactivated feeder layer (J2-3T3 cells) in
complete FAD media to support HEKs under control

5. Application
( KGM-Gold™ Keratinocyte Growth Medium, with low Ca2+ )
- Keratinocytes were distributed
throught out the culture as a single cell
or localised, in small losely-connected
groups.
- Cells are highly motile, with ruffling
lamellipodia. This is due to the low
availability of Calcium in the media.
“Calcium is responsible for the
formation of cell-to-cell junction. Low
amount of Calcium causes the cell to
have less anchouring.”
- Formed temporary attachment.
(Lamb & Ambler, 2013)
Figure below shows : Keratinocyte behaviour of monolayer cultures in KGM Gold media. ( Lamb& Ambler, 2013)
(Lamb & Ambler, 2013)

6. Application
( Rheinwald and Green complete FAD medium with high Ca2+)
- Cells formed tight clusters.
- They remained uniform and
undifferentiated within colonies.
Figure below shows : Keratinocyte behaviour of monolayer cultures in FAD media. ( Lamb & Ambler, 2013)
(Lamb & Ambler, 2013)

7. KGM-Gold™ BulletKit
Application
Rheinwald and Green complete FAD
medium
Effect of both medium on the thickness/depth of the epidermis
- T h i c k , m u l t i - l a y e r e d e p i d e r m i s
comprimised : basal, suprabasal (spindous &
granuar) and cornified layers are grown.
- No epidermis formed, only a small number
of nucleated cells were detected.
- Further investigation on KGM-Gold :
They supplied the KGM-Gold media with other variables
and observed that there's a layer of epidermis formed.
However, the performance for FAD media for the growth
of the cell outcompete KGM-Gold media.
Figure below shows : Differences of the depth between epidermis between FAD medium and KGM Gold medium. (Lamb & Ambler, 2013)
(Lamb & Ambler, 2013)

8. Current Issue and developments
Very few oppurtunities on the research value about culture media, as well
as their shortcomings and limitation. (Yao & Asamaya, 2017)
Morever, culture media and their equipments are now supplied in a
commercial basis, and it become possible for anyone to work easily with
culture cell.
The possible interaction among the component in the media are
sometimes overlooked & it can bring an effect on the performance of the
cell.

9. Conclusion
KGM-Gold™ BulletKit Rheinwald and Green complete
FAD medium
Advantage Disadvantage Advantage Disadvantage
- Undifferentiated
- Inability to form
stratified keratinocyte
- More than one type
of media obtain.
- Increased growth and
productivity.
- More consistent
performance.
- Low risk of
contamination.
- Time consuming batch
test.
- The Fetal Bovine serum
(growth factors), helps to
protect cells from adverse
condition and able to
stimulate cell growth and
function.
- Acts as a natural buffering
agent.
- Protease inhibitor (trypsin),
able to protect the cell from
proteolysis
- High risk
contamination. Partly
due to its nutrient-rich
medium.
- Batch to batch
variation in
composition.
- Has a high protein
content, that hinders
product purification.
-High cost

10. Conclusion
Rheinwald and Green complete FAD medium is
better option as it contains serum derived growth
factors and antibiotics and able to form a stratified epidermis
in a reconstituted skin model.

11. References
1. Advantages of Serum Free Cell Culture, PAN Biotech UK Ltd, <http://www.pan-biotech.co.uk/advantages-of-
serum-free-cell-culture.html>, Accessed date 19 April 2018.
2. El Ghalbzouri et al., Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for
human skin equivalent products. Biomaterials. 30(1):71-8. 2009
3. KGM-Gold™ Keratinocyte Growth Medium, Lonza.com, <https://www.lonza.com/products-services/bio-
research/primary-cells/human-cells-and-media/keratinocytes-and-media/kgm-gold-keratinocyte-growth-
medium.aspx>, Accessed date 19 April 2018.
4. Lamb, R. and Ambler, C.A., 2013. Keratinocytes propagated in serum-free, feeder-free culture conditions fail to
form stratified epidermis in a reconstituted skin model. PloS one, 8(1), p.e52494, Accessed date 19 April 2018.
5. Moore, J.C., Atze, K., Toro-Ramos, A., Ricupero, C., Hart, R.P. and Cohen, R.I., high-efficiency, high-throughput
Transfection of Pluripotent human Embryonic Stem Cells. Reliable, Cost-effective Transfection at the Touch of a
Finger, p.3, Accessed date 19 April 2018.

12. References
6. Serum-Free Media (SFM) Thermo Fisher Scientific, Thermofisher.com,
<https://www.thermofisher.com/my/en/home/life-science/cell-culture/mammalian-cell-culture/serum-
free-media.html>, Accessed date 19 April 2018.
7. Tatsa, Y., & Yuta, A., 2017, Animal cell culture media : History, characteristics and Current issue, Journal
of Reproductive medicine and biology, pp 101-117