Vol.3, No.4, 299-304 (2013) Open Journal of Animal Sciences
Development of in vitro culture of rat Leydig cells
after purification with Nycodenz gradient
Ekayanti Mulyawati Kaiin1*, Ita Djuwita2, Tuty Laswardi Yusuf3, Mohamad Agus Setiadi3
1Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia;
*Corresponding Author: ekayantikaiin@yahoo.com
2Department of Anatomy, Physiology and Pharmacology, Faculty of Veterinary Medicine, Bogor Agriculture University (IPB),
Jl. Agatis Kampus Darmaga, Bogor, Indonesia
3Department of Veterinary Clinic, Reproduction and Pathology, Faculty of Veterinary Medicine, Bogor Agriculture University (IPB),
Jl. Agatis Kampus Darmaga, Bogor, Indonesia
Received 11 July 2013; revised 23 August 2013; accepted 12 September 2013
Copyright © 2013 Ekayanti Mulyawati Kaiin et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The study examined the effect of human Chori-
onic Gonadotrophin (hCG) and/or Insulin Trans-
ferring Sodium Selenite (ITS) on the proliferation
and development of rat Leydig cells purified by
Nycodenz gradient. Leydig cells purity, viability
and proliferation after purification and 3 days of
cultured were evaluated. Leydig cells 1 × 106
cells/ml were cultured in DMEM containing 10%
Newborn Calf Serum (NBCS) and divided into
four kinds of treatments 1) as a control, 2) control
+ 2.5 IU/ml hCG, 3) control + ITS (5 μg/ml insulin,
10 μg/ml transferrin, 5 μg/ml Se), and 4) control +
hCG + ITS. Leydig cells purification results
showed 91.40% of purity, viability was 98.17%
and concentration 7.03 × 106 cells/ml. The addi-
tion of ITS and hCG + ITS in DMEM produced
Leydig cell proliferation by 88.35% and 90.64%
higher than in controls (86.82%) (p < 0.05). The
addition of hCG did not increase Leydig cell
proliferation (86.99%). In the primary culture of
Leydig cells, the population doubling time (PDT)
was 1.03 days, similar with the addition of hCG
treatment (1.02 day). Declined in value of PDT
significantly (p < 0.05) occurred in DMEM sup-
plemented with ITS (0.97 day) and hCG + ITS
(0.88 day). This result was also seen in the first
and second Leydig cell lines. DMEM added with
hCG or hCG + ITS resulted in a higher amount of
testosterone (5.06 ng/ml; 5.25 ng/ml)) than the
culture medium without hCG (2.46 ng/ml) (p <
0.05). It can be concluded that the combination
of hCG + ITS can increase the number of Leydig
cells and testosterone levels in the culture me-
Keywords: Leydig; In Vitro; Rat; Nycodenz; hCG;
ITS; Testosterone
Androgen hormonal therapy for hypogonadism in men
has been done to maintain testosterone level physiologi-
cally. Bhasin & Bremer stated that this therapy can in-
crease muscle strength, improve osteoporosis, stabilized
bone density and restore the secondary sexual characters
[1]. However, provision of these hormones in the long
term can cause an increase in blood viscosity, red blood
cell formation abnormalities, hypertension, stroke, bone
density changes and emotional changes [1-3]. Therefore,
it is necessary to attempt an alternative therapy such as
Leydig cells transplantation which naturally produced
testosterone that can be used to replace the use of syn-
thetic testosterone hormone [1]. This cellular therapy has
limitations in terms of availability of tissue and cells.
Therefore, Leydig cell culture is required to obtain a
source of cells that will be used in cellular therapy. Tes-
ticular tissue consists of somatic cells like Sertoli cells,
Leydig cells, fibroblasts, and spermatogenic cells (male
gamet cells) in various stages of development, and other
cells so it requires to be isolated and purified with ap-
propriated methods like Nycodenz gradient [4]. Ny-
codenz or Iohexol has several advantages compared to
Percoll including lower osmolality and viscosity but
higher density. It was able to separate macromolecules
and cells effectively with a broader range of sizes for
more varied cell types. Nycodenz are stable, soluble in
water and can be sterilized with a syringe filter that is
easy to use in the laboratory and non cytotoxic against
mammalian cells. Additionally, Nycodenz can be easily
Copyright © 2013 SciRes. Openly accessible at http://www.scirp. org/journal/o jas/
E. M. Kaiin et al. / Open Journal of Animal Sciences 3 (2013) 299-304
removed from the cell suspension by centrifugation com-
pared with gradients of other materials because the re-
sidual gradient can lead to cell death. Nycodenz can be
used easily by a simple method without any limitations
in temperature, pH and stability [5]. Nycodenz gradient
was used to separate blood cells, liver cells, the primor-
dial germ cell (PGC), spermatogonia and Sertoli cells
To obtain an optimum condition in supporting prolife-
ration of Leydig cell culture in vitro, medium culture
requires some bioactive materials such as human Chorio-
genic Gonadotrophin (hCG) and Insulin Transfferin So-
dium Selenite (ITS). Leydig cell may secrete several
bioactive materials such as peptide, growth hormone,
Interleukin-1 (IL-1) and Interleukin-6 (IL-6), testoster-
one and others to support their growth [9-11]. Luiteniz-
ing Hormone (LH) or hCG is necessary for the prolifera-
tion and differentiation of Leydig cells so that the cells
are able to produce testosterone [12]. The concentration
of testosterone in human Leydig cell culture medium has
increased after the addition of 1 IU hCG/ml [9] whereas
the addition of 5 IU hCG on Leydig cell culture medium
of young rabbit produce a large amount of testosterone
[13]. From several studies before, it seems that addition
of ITS is required as a bioactive material for the Leydig
cell proliferation. Chemes et al. [9] added transferrin 10
μg/ml and insulin that can enhance the production of
testosterone by the Leydig cells of fetal mice in the cul-
ture medium for 24 and 48 hours [14]. Bernier et al. [15]
added insulin and transferrin each 5 μg/ml and 1 IU
hCG/ml into the culture of Leydig cells piglets which
causes an increase in the synthesis of testosterone com-
pared to those without addition of hCG. The purpose of
this study was to test the influence of the addition of hCG,
ITS or in combination in DMEM medium on prolifera-
tion and development of Leydig cells and to get an opti-
mum condition of adult rat Leydig cells in vitro.
2.1. Isolation and Purification of Leydig
Testes were collected from the rats (Sprague Dawley)
adult males aged 8 - 10 weeks after anesthetized and
sacrificed by cervical dislocation. Testicular tissue was
placed in a petri dish contains Dulbecco’s Phosphate
Buffer Saline (DPBS) without Ca and Mg (Gibco, 21600
- 010, Invitrogen, NY, USA). The tissue was then washed
three times using medium DPBS supplemented with
0.1% Newborn Calf Serum (NBCS, Gibco, 16010 - 159,
Invitrogen, New Zealand) (DBPS). Testicular tissue were
then put into tubes containing one ml of 0.04% colla-
genase type I (Sigma, C0130, St. Louis, MO, USA) and
10 μg/ml trypsin inhibitor (Sigma, T9003, St. Louis, MO,
USA) in DPBS and incubated in a water bath at a tem-
perature of 34˚C for 40 min. Cell suspension diluted four
times to initial volume with DPBS then allowed to pre-
cipitate. Supernatant was collected and centrifuged at
200 g for three minutes. Cell pellet then was washed
twice by using DPBS and diluted with 0.5 ml DPBS.
Isolation and purification of Leydig cells were conducted
by using Nycodenz gradient. The cell suspension were
put into Nycodenz gradient 4%, 8%, 10%, 12%, 15% and
then were centrifuge using rotor swing (KOKUSAN
H-26F) at 1500 g for 10 min. Cell layer formed were
collected and washed four times with DPBS and DMEM
(Sigma, D5532, St. Louis, MO, USA) supplemented 10%
NBCS and centrifuged at 200 g for three minutes. Cell
pellet was diluted with 0.5 ml DMEM and the cell con-
centration was calculated using Neubauer haemocytom-
2.2. In Vitro Culture and the Production of
Leydig Cells
Leydig cells as 1 × 106 cells/ml were placed in a petri
dish (Corning, 430 165, NY USA) 35 × 10 mm contain-
ing DMEM medium supplemented with 10% NBCS as a
control (group 1), with 2.5 IU/ml hCG (Chorulon, In-
tervet, EU) (group 2), with 5 mg/ml insulin, 10 mg/ml
transferrin, 5 μg/ml Se (ITS, Sigma I3146, St Louis, MO,
USA) (group 3) and hCG + ITS (group 4). All petri dish
then cultured in a 5% CO2 incubator (Sanyo, MCO-95,
Japan) at 37˚C. After cultured for three days, Leydig
cells were calculated for their concentration, viability
and purity. Leydig cells were stained by Trypan Blue and
specific staining 3-β HSD. Primary cultures from the
each treatment were passage on day 3 and after washing
with DPBS medium then the cells were calculated using
a Neubauer haemositometer. Cell was then cultured
again until reaching a confluent stage. Passage was per-
formed twice and was counted Population Doubling
Time (PDT) with the formula:
PDT day
log final cellnumberlog initial cellnumber3.32
2.3. Testosterone Content from Leydig Cell
Culture Medium
Leydig cell culture medium from each treatment were
collected on day 3 and tested by using Testosterone
ELISA kit (DRG Diagnostics EIA 1559). Tests were car-
ried out at the Laboratory of Hormones, Reproduction
Rehabilitation Unit, Faculty of Veterinary Medicine, Bo-
gor Agriculture University (IPB).
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E. M. Kaiin et al. / Open Journal of Animal Sciences 3 (2013) 299-304 301
2.4. Experimental Design and Data Analysis
Leydig cell cultures performed three replications for
each treatment. The parameters measured were viability,
purity, PDT and number of Leydig cells alive. Leydig
cell lines made up to second passage and repeated three
times. The content of testosterone in the culture medium
were conducted three replications in each treatment. Data
were analyzed using one way ANOVA and if there is a
difference between treatments followed by Duncan’s. All
cases significance was set at p < 0.05.
The results showed that a percentage of Leydig cells
after purified with Nycodenz gradients were 91.40%,
while the viability was 98.17% and the cell concentration
was 6.30 × 106 cells/ml (Table 1).
The effect of hCG, ITS or in combination on Leydig
cell proliferation in vitro were presented at Table 2. Ley-
dig cells proliferation were cultured in DMEM + ITS and
DMEM + hCG + ITS significantly (p < 0.05) higher at
88.35% and 90.64% when compared to control (86.82%).
The addition of hCG to the Leydig cell culture medium
did not increase the proliferation of Leydig cells (86.99%).
The percentage of Leydig cells tend to decline at the end
of treatment by hCG, ITS and the combination.
The result of the passage of Leydig cells increased in
cell number at the end of the primary culture and also at
first and second cell lines (Table 3). In between treat-
ments shows that the role of hCG and ITS increased the
number of Leydig cells compared with the other treat-
Table 1. Purity, viability and concentration of Leydig cell after
purification with Nycodenz gradient.
Parameter Leydig Cells
Purity (%) 91.40 ± 5.02
Viability (%) 98.17 ± 0.51
Cell concentration (106 cells/ml) 7.03 ± 1.04
Number of living cells (106 cells/ml) 6.30
Table 2. Primary cultures of Leydig cells after purified with
Nycodenz gradient.
Medium treatment
Parameter DMEM
+ hCG
rate (%) 86.82a 86.99a 88.35b 90.64c
Leydig cells (%)
-Initial culture 90.75 90.75 90.75 90.75
-End of culture 88.75 88.25 88.00 88.50
Note: Different superscript letters indicate significant differences (p < 0.05)
by Duncan test on the same line.
Table 3. Number of cells and PDT from cell suspense in vitro.
Cell line in several
medium treatment
Initial cell
End of cell
PDT (day)
Primary cultures
DMEM 1 7.60aA 1.03a
DMEM + hCG 1 7.69a 1.02a
DMEM + ITS 1 8.63b 0.97b
DMEM + hCG + ITS1 10.69c 0.88c
1st cell line
DMEM 1 6.71aAB 1.09a
DMEM + hCG 1 7.60a 1.03a
DMEM + ITS 1 8.30a 0.99a
DMEM + hCG + ITS1 13.56b 0.82b
2nd cell line
DMEM 1 6.28aB 1.14a
DMEM + hCG 1 7.27ab 1.05a
DMEM + ITS 1 8.76b 0.96b
DMEM + hCG + ITS1 9.17c 0.94b
Note: Different small superscript letters indicate significant differences (p <
0.05) with Duncan test on the same cell lines. Different capital superscript
letters indicate significant differences (p < 0.05) with Duncan test in the
same treatment with the different cell lines.
The percentage of Leydig cells and the viability was
seen in all treatments (80% - 91%). The ability of Leydig
cells to proliferate seen from the highest number of Ley-
dig cells in the treatment DMEM + hCG + ITS in pri-
mary culture, first and second cell lines compared to
other treatments (Table 4).
The addition of ITS, hCG + ITS in DMEM increasing
the number of live Leydig cells (p < 0.05) in primary
cultures (6.68 × 106/ml and 8.31 × 106/ml cells). Number
of live Leydig cells in first and secondary cell lines on all
treatments were also increased (p < 0.05) compared with
controls. Treatment of the same medium in first and sec-
ond cell lines reduced the number of live Leydig cells (p
< 0.05) compared to primary culture except the treatment
of hCG + ITS on the first cell line, the Leydig cells alive
increased in number.
Table 5 shows the content of testosterone increased (p
< 0.05) in medium with hCG and hCG + ITS to 5.06
ng/ml and 5.25 ng/ml than without hCG (2.46 ng/ml).
Several different methods of purification of Leydig
cells have been reported by other researchers. The percen-
tage of Leydig cells purified by Nycodenz gradient in
this experiment was higher than Risbridger and Hedger
[16] which was 87%. The same result by Yang et al. (2003)
was 95% and also by Kaiin et al. [4] was 92.22% using
Percoll gradient. However, the concentration of Leydig
cells purified by Percoll was higher at 15.42 × 106 cells/ml
[4] A higher proliferation of Leydig cells was found when .
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Table 4. The purity, viability and number of living cells of Leydig cell lines.
Cell line in several
medium treatment Cell concentration
(×106/ml) Leydig cells (%) Number of Leydig
cells (×106/ml) Cell Viability (%) Number of live
cells (×106/ml)
Primary culture
DMEM 7.60aA 85.17 6.47aA 88.67A 5.74aA
DMEM + hCG 7.69a 85.50A 6.58a 89.00C 5.85aD
DMEM + ITS 8.63b 86.67 7.48b 89.33E 6.68bG
DMEM + hCG + ITS 10.69c 87.00 9.30c 89.33G 8.31cI
1st cell line
DMEM 6.71aAB 85.67 5.74aAB 86.33A 4.96aB
DMEM + hCG 7.60a 85.33A 6.49a 84.00D 5.45bE
DMEM + ITS 8.30a 86.67 7.19a 87.00E 6.26cH
DMEM + hCG + ITS 13.56b 87.33 11.84b 88.33G 10.46dJ
2nd cell line
DMEM 6.28aB 86.67 5.44aB 80.67B 4.39aC
DMEM + hCG 7.27ab 89.17B 6.49ab 81.67D 5.30bF
DMEM + ITS 8.76b 90.67 7.94b 82.67F 6.57cH
DMEM + hCG + ITS 9.17c 91.17 8.36c 83.33H 6.97cK
Note: Different small superscript letters indicate significant differences (p < 0.05) with Duncan test in the same cell line. Different capital superscript letters
indicate significant differences (p < 0.05) with Duncan test in the same treatment with different cell lines.
Table 5. Testosterone content in Leydig cells culture medium.
Treatment n Testosterone (ng/ml)
DMEM + 10% NBCS (DMEM) 2 1.29a
Leydig cells + DMEM 3 2.46ab
Leydig cells + DMEM + hCG 3 5.06c
Leydig cells + DMEM + ITS 3 3.19b
Leydig cells + DMEM + hCG + ITS 3 5.25d
Note: The different superscript letters indicate significant difference (p <
0.05) in column.
cultured in vitro with ITS. Insulin Transferrin Sodium
Selenite (ITS) is the supplement that used to increase cell
proliferation. Insulin is a polypeptide hormone that helps
the absorption of glucose and amino acids, whereas
transferrin is an iron-carrying protein aims to help cell
nutrient absorption. Selenium is an essential trace ele-
ment present in the serum.
Doubling time is the time period required by the cell
to make twice of size or the number from the original
[18]. The faster of proliferation of the cell made the
value of PDT lower. The value of PDT from primary cul-
tures of Leydig cells was 1.03 days and that was equiva-
lent to DMEM + hCG treatment (1.02 day). The value of
PDT was significantly (p < 0.05) lower in DMEM + ITS
treatment (0.97 day) and DMEM + hCG + ITS (0.88
day). Similar results occurred in the first and second cell
lines. The results showed that the addition of ITS, as well
as hCG + ITS makes Leydig cells require shorter time to
achieve a cell number twice. It supports the ITS func-
tions as a bioactive material that can increase cell prolif-
eration. Butler [19] states that the time required for the
process of cell division in vivo occurs approximately 18 -
24 hours. After passage, the cell is more homogeneous
and able to adapt to the environmental conditions in vitro
[20]. Leydig cells in primary culture have a fairly high
percentage of purity (>90%) and because those in culture
were homogeneous, they made the value of PDT near to
the time of proliferation in vivo. Leydig cell number on
treatment with DMEM decreased in the first and second
cell lines (p < 0.05), therefore, resulted in a higher PDT
from primary culture. Similarly occurred in the other
treatment, except in the first cell line, that an increase in
the number of cells was occurred in hCG + ITS. The
addition of ITS in DMEM led to an increased in the
number of Leydig cells at the second cell lines compared
to the first lines. In general, a decline in the ability of
Leydig cell proliferation occurred after passage twice.
According to Hebert et al. [21], the stimulation of pro-
liferation of rat Leydig cell precursors is influenced by
growth factors such as Transforming Growth Factor
(TGF-α) and Insulin-like Growth Factor (IGF-I). In addi-
tion, Platelet-derived Growth Factor (PDGF-A) which is
secreted by Sertoli cells is also required for proliferation
and differentiation of adult Leydig cells. The decrease in
the number of live Leydig cells in the first and second
cell line is probably influenced by the availability of
these growth factors in the culture medium.
Leydig cells in culture have the capacity to secrete
testosterone up to 72 hours [22]. Level of testosterone
E. M. Kaiin et al. / Open Journal of Animal Sciences 3 (2013) 299-304 303
produced by isolated adult rat Leydig cells in vitro varies
from 0.5 ng up to 150 ng/106 Leydig cells per hour under
LH/hCG—stimulated condition [16]. Similar results oc-
curred in Leydig cell piglets cultures [15]. In this ex-
periment, Leydig cells were cultured in DMEM + ITS
which had the testosterone levels of 3.19 ng/ml. Pointis
et al. [14] stated that the addition of insulin in the culture
medium can increase the accumulation of testosterone in
fetal mouse Leydig cell cultures. Hormone that stimu-
lates steroidogenesis in Leydig cells is Luiteinizing
Hormone (LH) which is secreted by the pituitary.
Changes in LH levels in vivo can reflect variations of
physiological conditions that can induce morphological
changes and the ability of Leydig cells to synthesize and
secrete testosterone [22]. The use of hCG which is an
analog of LH that can bind to the same receptor with LH
receptors on Leydig cells [23] resulted in the secretion of
testosterone. The addition of hCG to the DMEM in-
creased the secretion of testosterone, whereas ITS in-
creased the cell proliferation that can increase the content
of testosterone in the culture medium.
The addition of 10% NBCS hormone in DMEM me-
dium produced the testosterone content of 1.29 ng/ml.
Sedelaar and Isaacs [24] reported that hormone testos-
terone concentrations between 1.2 and 7.5 ng/ml were
found in bovine serum from newborn to one year old
assayed using the RIA method. Serum contains several
nutritional components such as fatty acids, cholesterol,
T3, insulin, IGF, EGF and androgen [25]. Moreover, the
addition of serum is made to provide the hormones that
stimulate cell growth and function. Serum provides bio-
matriks that help the process of attachment and spreading
of cells, as well as carrier transport protein hormones,
minerals and lipids [26].
It can be concluded that the addition of ITS to the cul-
ture medium increased Leydig cell proliferation, whereas
addition of hCG increased concentrations of testosterone
hormone in the Leydig cell culture medium. The combi-
nation of ITS and hCG increased cell proliferation and
the concentration of testosterone in Leydig cell culture
medium. Further research is needed to increase the num-
ber of living cells in the production of Leydig cell line.
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