Foreskin-derived skin equivalent in exchange for animal testing*

* dibuat sebagai essay persyaratan mengikuti seleksi Novartis International Biocamp 2015

The European union completely ban the use of animal testing for cosmetics development in 2013. The ban was a critical milestones in the effort to phase out experimentation using animals. The medical research community were currently guided by principles that whenever possible, to replace animal with other reliable methods, reduce the number of animal and refine the techniques used to minimize potential pain and distress and promote animal welfare. These trends leads to the development of alternatives to animal testing. One such alternatives is the use of cell culture and tissue engineering.

                The Fraunhofer institute in 2010 launch the “skin from factory” project that essentially produced skin equivalents in a fully automated process. The institute recognized that skin equivalents as in vitro test system for chemicals, cosmetics and drugs are rare. Production of an in vitro test system by manual cell culture and tissue engineering is time consuming and required specifically trained personnel. Most interesting was that the skin source for such system were foreskins which was an abundant by product/medical waste of compulsory practice of khitan in Indonesia.

                Rumah Sunatan a leading company focused in khitan services claimed to reach more than 15.000 patient a year. This is an untapped resource unique of Indonesia home to millions of observant Muslims. This point of view is, for sure, not without challenges. Among others is the ethical and religious issues that centered on the commercial use of human tissues. However research on the potential cultivation of foreskins could result benefit for many field such as stem cells, regenerative medicine, cell therapy, cryopreservation.

                Recently, the discovery of the non-tumorigenic multilineage-stress enduring cells (MUSE) from dermal fibroblast potentially resolve the concern on the use of iPS cells for therapy. Dermal fibroblast contains a pluripotent subpopulation of mesenchymal stem cells that evidently does not form tumour. This findings extends further the importance of foreskin as skin sample source in regenerative medicine. Cryopreservation of cells isolated from so many samples a year will also stimulate research in the field of cryobiology and bio banking.

                In conclusion, the production of foreskin-derived skin equivalent as an in vitro skin testing system on a large scale will largely reduce the demand on animal testing.  Indonesia has a unique potential to solve other problems in medical research. Communication with appropriate religious body and government agency should be commenced to resolve issues that aroused on the collection, cultivation and storage of foreskins for medical research.

Geraniin supplementation increases human keratinocyte proliferation in serum-free culture

Indra Kusuma¹ and Restu Syamsul Hadi^2
1Physiology Department, Faculty of Medicine, YARSI University ²Anatomy Department, Faculty of Medicine, YARSI University

Abstract 

Background

Various products used in cellular therapy utilize tissue culture techniques requiring keratinocyte culture. An efficient and clinically acceptable  keratinocyte culture system requires supplements with mitogenic activity. Geraniin is a phytochemical with the potential as a supplement for expansion culture of keratinocytes. The objective of the present study was to verify the mitogenic activity of geraniin on human keratinocytes.

Methods

This was an experimental study using two samples of human foreskin  obtained by circumcision  of a male child. Epidermal keratinocytes were isolated from the foreskin samples and cultured with the addition of epidermal growth factor and bovine pitutary extract. The keratinocytes were divided into paired groups, comprising intervention and control groups.  The intervention groups were cultured with geraniin supplementation, whereas the control groups were cultured with standard supplements, without the addition of geraniin. Mitochondrial activity of the cells was evaluated by means of the MTT proliferation assay. Absorbance values in each of the groups was measured at 450 nm. Data analysis was performed with the  paired t-test.

Results

Geraniin supplementation significantly increased the keratinocyte proliferation rates at dosages of  0.8 to 3.1 µM. An increase of 57% in the proliferation rate was obtained at a dosage of 1.6 µM, while at a dosage of 12.5 µM toxic effects were starting to appear. Geraniin presumably causes increased cellular energy status, resulting in increased proliferation rates.

Conclusion

The findings in this study provide evidence in support of the utilization of geraniin as a supplement for expansion culture of keratinocytes. Further studies may presumably identify the molecules acting as geraniin receptors and the intracellular mechanisms underlying the increase in proliferation rates.

Key words: Keratinocyte, geraniin, foreskin, proliferation

Keratinocytes, Cobble-stone Appearance >90% Confluence

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Cerita dari NUS : Laboratorium In Vitro Elektrofisiologi

Patch Clamp Rig

Saya suka travelling, terutama yang mengandung unsur petualangan. Petualangan tidak berarti harus rimba dan gunung, tapi juga tempat asing seperti Singapura. Saya datang kesini untuk belajar, gratis, dimungkinkan atas nama solidaritas sesama kolega. Saya tidak akan menceritakan keajaiban singapura dengan "pristine district-nya", tapi saya akan sebut satu dua hal yang cukup membuat saya terheran.

Pertama adalah tombol untuk penyebrang jalan yang ada di tiang lampu lalu lintas, waktu kecil tentu kita pernah lihat juga di Jakarta, saya agak takjub aja di sini teryata berfungsi :) ironis ya...Kedua yang tak kalah ironis adalah jadwal bus dan papan nomor bus yang terpampang di halte, ini juga berfungsi dengan baik :)...bagi saya sisanya adalah cerita keseimbangan...modernitas dan keteraturan di satu sisi dan hilangnya budaya malu dan kehangatan.

Resep Selapis Keratinosit

Reagen	Larutan alikuot	Konsentrasi tujuan	50 ml	100 ml
DMEM/F12 3:1	1x	1x	Add 50 ml	Add 100ml
FBS ( di Inaktivasi)	1x	5% total	2,5 ml	5 ml
Hidrokortison	50 ug/ml	10 ug/ml	10 ml	20 ml
ITS	(1 mg/ml)	10 ug/ ml	0,5 ml	1 ml
Amphotericin B	250 ug/ml	2,5 ug/ml	0,5 ml	1 ml
Pen-strep	5000 U pen-5 mg strep /ml	100 U P- 100 ug/ ml	1 ml	2 ml
L-glutamin	200mM	2 mM	0,5 ml	1 ml
EGF	50 ug/ml	10 ng/ml	10 ul	20 ul
KGF/FGF-7	500 ng/ml	1 ng/ml	100 ul	200 ul
BPE	1 mg/ml	60 ug/ml	3 ml