CHAPTER I INTRODUCTION The use of amniotic fluid to assess the ...

CHAPTER I
INTRODUCTION
The use of amniotic fluid to assess the fetal condition has a history of only three
decades; within this short span of years it has become evidence that sampling of
amniotic fluid offer excellent opportunities for prenatal diagnosis (PND). Amniotic
cells are fetal cells found suspended within the amniotic fluid bathing around the fetus.
Suspended in the fluid are a number of intact cells together with cell debris and vernix
(Fuchs and Cederqvist, 1973). The fluid was obtained by ultrasonography-guide
amniocentesis from pregnant women with a gestational age ranging from 15 to 19
weeks (Kaviani et al., 2003). The cells originally ingress from several exposed
embryonic tissue, such as skin epithelium, gastro-intestinal tract, urethra and
respiratory tract. The primary cells were used in clinical diagnosis for the fetal
chromosomes abnormality such as; Patau’s syndrome, Turner’s syndrome and Down’s
syndrome. The risk of the chromosome abnormality increase with the advancement of
maternal age (Mevatee, 1998).
Two amniotic cell lines, AMC-K46 and AC-F2 have been developed from the
primary amniocentetic cells in laboratory at the Human and Animal Cell Technologies
Research Unit, Chiang Mai University (Wisedkaew, 2004 and Dasa, 2005). These cell
lines expressed heterogeneity with at least three cell types of morphology.
The proportions of cell number of each cell types were changed on microscopic
appearance through generations (Sangngam, 2005). The biology and basic
characteristics of the two cell lines are necessary in order to get them on any scientific
applications. With the heterogeneitic features of these cell lines, their application in
biological assay may be limited and misleading by the different response of each cell
types (Doyle and Griffiths, 1998). The traditional microbiological approach to the
problem of culture heterogeneity is to isolate pure cell strains by cloning. The cell
cloning is the procedure by which a cell type has been isolated as from initiated single
cell and subculture as a continuous cell line. This procedure of homogeneity is more
useful in the continuous cell lines than the primary cells (Freshney, 2000). The cloning
culture derived from normal tissue or finite cells may survive only for a limited number
of generations. By the time that a clone has produced a usable number of cells it may

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already be near to senescence. This research effort isolates cells by cloning for
homogeneity to retrieves the more unique and stable continuous amniotic cell lines.
When a new cell line has derived, it is necessary to evaluate details of its origin
and expressed features. This aspect of cell culture is a crucial important with the
widespread dissemination of a cell line through research laboratories and cell banks. In
particular if a cell line becomes incorporated into a research that requires validation of
its components, then the authentications of the cell line get necessary. It is, therefore,
vital that detailing the origin and handling of the cell lines are kept from the time of
isolation and periodically during use. The purpose of a cell line to be characterized are
depending on the situation of use such as; to confirm the species of origin, to correlate
with the tissue origin, to determine the cell line transformation, to demonstrate the
cross-contamination, to indicate the prone of genetic instability and phenotypic
variation, and to identify a specific cell line with in a group from the same origin
(Butler and Dowson, 1992; Freshney, 2000). Several detailed protocols to success of
the above purpose are widely available (Kuchler, 1977; Masters, 2000; Freshney,
2000). However, some basic characteristics are studied in this research for the newly
form cell lines and their sub-clones including; morphology (type of cells in cultures),
plating efficiency (the percentage of cells seeded at subculture that gives rise to
colony), seeding efficiency (the percentage of the inoculums that attaches to the
substrate within a stated period of time), growth pattern or growth curve (a
semilogarithmic plot of the cell number on a logarithmic scale against time on a linear
scale for a proliferating cell culture), viability (survival of the cells) and the
karyotyping (the distinctive chromosomal complement of cells). These characteristics
will be very useful for any future research with the continuous cell lines.
Amniotic cells containing many of cell types present in the original tissue.
A wide variety of investigations performed since the 1980s have provided evidences
that cells of all three germ layers (ectoderm, mesoderm and endoderm) can be
detected in human amniotic fluid (Hoenh and Salk, 1982 and Gosden, 1983). Despite,
the widespread and well-established use of amniotic cells in routine prenatal genetic
testing, current knowledge about the origin and properties of these cells is
limited (Prusa and Hengstschläger, 2002). Recently, some reports mentioned that the
amniotic fluid contained a variety of stem cells, which were shed from embryonic and

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extra-embryonic tissues during the process of fetal development and growth
(Prusa et al., 2003; Fauza, 2004; Tsai et al., 2004). Amniotic cells could transform into
several cells and may be the source of powerful therapy for human congenital disorder
and as a potential source of pluripotent stem cells (Kakishita, 2003 and Tsai et al.,
2005). In addition, Delo et al. (2006) mentioned about number of promising novel
therapeutic concepts using these cells in tissue engineering, cell transplantation and
gene therapy. Current stem cell based approaches could be employed in cellreplacement
therapy or drug treatments. Thus, amniotic cells are intriguing as a
possible source of pluripotent stem cells for cell-based therapeutics, and do not raise
the ethical concerns associated with use of embryonic stem cells (Kim et al., 2007).
Furthermore, many experiments carried out in vitro are for the sole purpose of
determining the potential cytotoxicity of the compounds being studied. Either because
the compounds are being used as pharmaceuticals or cosmetics or must be shown to be
nontoxic or designed as anticancer agents and cytotoxicity may be crucial to their
action (Freshney, 2000). In this study, the two new amniotic cell lines are studied for
their potential use in cytotoxic assay. The response of these cells to a standard
component of pharmaceutical and cosmetic compound will be provided with the
effective inhibitory concentrations.
OBJECTIVES
This study was initiated with the aims to;
(i) Establish the homogenous amniotic cell lines from amniotic fluid of
Thais by cell cloning.
(ii) Study some characteristics and examine the Stemness of the developed
cell lines.
(iii) Study the toxicity response potential of the cell lines in cytotoxicity
assay.