Download PRIMARY CULTURE OF HUMAN AMNIOTIC FLUID CELLS

Regenerative Research 3(2) 2014 120-121
PRIMARY CULTURE OF HUMAN AMNIOTIC FLUID CELLS HARVESTED FROM FULLTERM PREGNANCIES
Muhammad K J1,2, Thilakavathy K1,2, Zulida R1, Yazid M N1 and Norshariza N1,2*
1
2
Department of Obstetrics & Gynaecology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia
Genetic and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia
ARTICLE INFO
Published: 1st December,
2014
*Corresponding author
email:
[email protected]
KEYWORDS
Amniotic Fluid,
Stem cells,
Primary culture
ABSTRACT
Heterogeneous cells contained in human amniotic fluid (AF) are believed to hold therapeutic
potential. Mid-term AF has been discovered to harbor high potency stem cell population.
However, it is not clear whether AF of full-term pregnancy contains this type of cells. In an
effort to explore this possibility, primary culture of AF harvested from full-term pregnancies
or during deliveries needs to be established. Here, we aimed to culture and propagate AF
cells of 38-40 weeks gestation collected during normal (ARM) and cesarean section (C-Sect)
deliveries. The time taken for both cells to reach confluency and the cell morphology were
compared and evaluated. We found C-Sect samples took shorter time to confluent compared
to ARM samples in Amniomax with both exhibiting similar cell morphology. These results
prove the presence of viability cells residing in full-term AF, thus, giving us high hope for the
existence of stem cell population in the fluid, which is normally discarded.
1.0 Introduction
2.0 Materials and Methods
Human amniotic fluid has been used for prenatal diagnosis
since many years (1). More studies have discovered the
presence of stem cell population in AF (1-4). However, most
stem cells were isolated from AF of mid-term gestation
through an invasive procedure, amniocentesis. There are
concerns on the possible risks pose by the invasive procedure
in collecting mid-term AF samples, such as infection of the
amnion sac from the needle, leakage of the sac and in more
serious note; miscarriage (5,6). Alternatively, stem cells
could be isolated from AF of full-term pregnancies,
specifically during delivery. Thus, we are now trying to
culture and establish amniotic fluid stem cells from full-term
pregnancies. Here, we aimed to see whether AF cells can be
cultured from full-term pregnancies.
Human amniotic fluid was collected from normal, through
artificial rupture membrane (ARM, n=2), and cesarean section
(C-Sect, n=2) deliveries. Samples were cautiously collected
by O&G specialists at Medical Putra Medical Centre and
Serdang Hospital upon approval by the appropriate Ethics
Committee into a sterile container. The collected samples
were immediately processed. The AF was first washed with
PBS to get rid of blood contamination or other unwanted
debris, centrifuged and grown in Amniomax until attached
and confluent in a 370C incubator. Cells were observed daily
to see first day of attachment and also the cell morphology.
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Fig 1: Culturing of ARM sample from day 1 to day 24, showing the stages of
culturing and the cell morphology.
3.0 Results
Full-term AF cells from both the ARM and C-Sect samples
can grow in Amniomax. C-Sect samples took only 14 days to
confluent as compared to 24 days for the ARM samples. All
samples exhibited heterogeneous cells population with mostly
fibroblast- and epithelial-like morphology (Figure 1, 2).
4.0 Discussion and Conclusion
We managed to grow amniotic fluid cells from full-term
pregnancies indicating the presence of viable cells at this
stage of gestation.
Fig 2: Culturing of C-Sect sample from day 1 to day 14, showing the stages
of culturing and the cell morphology.
2. De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos CC,
Perin L, Mostoslavsky G, Serre AC, Snyder EY, Yoo JJ,
Furth ME, Sooker S, Atala A. Isolation of amniotic stem cell
lines with potential for therapy. Nat Biotechnol. 2007;
25:100-106.
3. Tsai MS, Lee JL, Chang YJ, Hwang SM. Isolation of
human multipotent mesenchymal stem cells from secondtrimester amniotic fluid using a novel two-stage culture
protocol. Hum Reprod. 2004; 19(6):1450-1456.
4. In`t Anker PS, Scherjon SA, Kleijburg-van der Keur C.
Amniotic fluid as a novel source of mesenchymal stem cells
for therapeutic transplantation. Blood. 2003; 102:1548-1549.
Acknowledgement
This research was funded by Ministry of Science, Technology
and Innovation (Mosti) and Graduate Research Fellow (GRF),
UPM; E-Science fund project no 02-10-04SF1651.
5. Nelson MM. Amniotic fluid cell culture and chromosome
studies. In: Emery AEH, eds. Antenatal Diagnosis of Genetic
Disease. Edinburgh: Churchill Livingstone. 1973; 69-81.
References
6. You Q, Tong X, Guan Y et al. The biological
characteristics of human third trimester amniotic fluid stem
cells. J INT MED RES 2009; 37(1):105-112
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derived pluripotent amniotic fluid stem (AFS) cells. Med J
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