ORIGINAL RESEARCH PAPER
Establishment and characterization of outer root sheath (ORS)cell line from Jining grey goat
Zhifeng Cui ?Yanxia Hu ?Hui Wang ?
Yongqing Zeng ?Bin Dong ?Houshun Zhu ?
Zhongdian Dong ?Zhiyuan Liu
Received:31August 2011/Accepted:11November 2011/Published online:22November 2011óSpringer Science+Business Media B.V.2011
Abstract A new line of outer root sheath (ORS)cells was established from hair follicles of Jining grey goat by using a mechanical separation combined with enzyme digestion.Cell morphology is described at different phases.The chromosome analysis of ORS cells,identi?cation of the ORS cells and morpholog-ical reversion test were detected at the 4th and 40th passages.The ORS cells were healthy and the growth characteristics were stable with a population doubling time of 52h.Chromosome analysis showed that [58%of cells were diploid.Test for ORS cell line CK19expression was positive.This newly established ORS cell line not only lays the foundation for further studying on the growth,regeneration,development law of goat hair follicle but also provides a mirror for the research of human hair in medical ?eld.Keywords Outer root sheath cell áCell line áJining grey goat áGiemsa staining áBiological characteristic
Introduction
The hair follicle is an important skin derivative in the mammalian organism and has the ability to cyclically regenerate itself during the lifetime (Paus and Foitzik 2004;Stenn and Paus 2001).Each hair follicle contains cells of different types that mainly include dermal sheath (DS)cell,dermal papilla (DP)cell and outer root sheath (ORS)cell (Karoline et al.2006;Limat et al.1986).Among them,the ORS cells that have high proliferation potential play the most important role in hair regeneration,wound healing and differentiation of primary and secondary follicle.Stem cells for various epithelial cell populations of the skin are located in the ORS tissues of hair follicles (Jahoda et al.2000).
To study the growth and development of hair follicle,culturing the ORS cells in vitro is essential.Weterings et al.(1981)were ?rst able to grow human ORS cells in vitro by explanting plucked scalp hair follicles on bovine eye lens capsules.Wells et al.(1982)succeeded in explanting plucked human hair follicles directly on tissue culture plastic.Sotaro et al.(1994),Park et al.(2007)cultured the human ORS cells on the collagen-coated dished and tissue-culture plastic respectively.All the ORS culturing methods reported so far have limitations that the cultured ORS cells have a low reproductivity and easily age during culture periods in vitro.
So far,all the reported cultured ORS cells in vitro are from human hair follicle,the establishment of the
Z.Cui áY.Hu áH.Wang (&)áY.Zeng áB.Dong áZ.Dong áZ.Liu
Shandong Agricultural University,Daizong Road No.61,Taian 271018,Shandong,People’s Republic of China e-mail:wanghui2328@https://www.doczj.com/doc/7519038271.html,
H.Zhu
Yangzhou University,88South University Ave,Jiangsu 225009,Yangzhou,People’s Republic of China
Biotechnol Lett (2012)34:433–440DOI 10.1007/s10529-011-0799-x
ORS cell line from other mammalian has not been documented.In the present study,we cultured ORS cells from hair follicles of Jining grey goat using mechanical separation combined with enzyme diges-tive technology,and?rst successfully established a new ORS cell line.We aimed to initiate primary cultures of disaggregated hair follicle cells in order to increase the proliferative capacity of the primary ORS cells and to get a more de?ned and standardized culture system.
Materials and methods
Materials
Dulbecco’s Modi?ed Eagle’s Medium/Ham’s Nutri-ent Mixture F12(DMEM/F12,1:1),Keratinocyte-Serum Free Medium(K-SFM),phosphate-buffered saline(PBS),penicillin,and streptomycin were pur-chased from Gibco,Inc.;fetal bovine serum(FBS) from MDgenics,Inc.(USA);trypsin,colchicine,insu-lin-like growth factor-I(IGF-I),hydrocortisone and epidermal growth factor(EGF)from Sigma,Co.; Giemsa stain system from Fluka,Inc.(Switzerland); and monoclonal antibody against cytokeratin-19(CK-19)from Acris,Inc.(Germany).
Culture media formulations
Different culture mediums were used to establish the ORS cell line from the Jining grey goat.These culture mediums were de?ned as following:
Medium A:DMEM/F12containing10ng EGF/ml, 10ng IGF-I/ml,0.4l g hydrocortisone/ml and10% (v/v)FBS,
Medium B:K-SFM containing10ng EGF/ml, 10ng IGF-I/ml,and0.4l g hydrocortisone/ml, Medium C:DMEM/F12containing10ng EGF/ml, 10ng IGF-I/ml,0.4l g hydrocortisone/ml and2% (v/v)FBS.
Establishment of primary cell culture and serial subcultures
Skin tissue was obtained from healthy1–3days grey goats by surgery.Hair follicles were separated using an operating microscope and microsurgical instruments under sterile conditions as described by Cui et al.(2008).After the sterile hair follicles were incubated in PBS containing0.25%trypsin at37°C, for10min,the trypsin was removed and these hair follicles were picked up by curved forceps individ-ually and seeded on the bottom of the6-well plates coated with the extracellular matrix(ECM)of ?broblast cells from Jining grey goat following the method described by Freshney(2005).Following1h incubation at37°C,3ml medium A was added to each well of6-well plates containing hair follicles. The hair follicles were incubated at37°C in a 5%(v/v)CO2atmosphere,with daily observation to monitor the growth of the cells.When a number of epithelial-like cells appeared from the vicinity of the hair follicles,the medium A was replaced with3ml of the medium B per well and the cells were incubated in the same conditions.The medium B was changed every3days.
The ORS cells were split to subcultures when the primary ORS cells were completely con?uent by trypsin digestion method.Brie?y the cells were incubated in a0.25%trypsin solution for10min. The ORS cells were suspended in the fresh culture medium and seeded in new culture?asks at a density of19104cells/ml.Medium B was changed every 3days.After eight passages,the medium B was replaced with the same volume of medium C.All cultures were carried out at37°C in humidi?ed atmosphere containing5%(v/v)CO2.
Cell morphology observation
ORS cell cultures were observed every2days under an inverted phase contrast microscope.The cells at the 40th passage were stained with Giemsa.
Growth curve of ORS cells
Cells were seeded on the24-well culture plates at 104cells/ml.Cell density and cell numbers of each well were counted and recorded daily until the plateau phase was reached.The cell numbers from three wells were counted at each time point use the average to make the cell growth curve.The cell growth curve was plotted using the cell number data and the population doubling time was calculated from the growth curve. The experiment was repeated three times.
Identi?cation of the ORS cells
Chromosome number determination
and the karyotype of the ORS cells
To verify the source of the ORS cells,the chromosome numbers and karyotype of the ORS cells were analyzed following the method described by Masover et al. (1998).The40th passage cells at the logarithmic phase were incubated for2h with0.08l g colchicine/ml.The chromosomes of60cells in the metaphase were counted in ORS cell line.The karyotype of peripheral blood lymphocytes from normal Jining grey goat(female)was analyzed as a control using the same method.
Identi?cation of the ORS cells by speci?c monoclonal antibody
The ORS cell cultures were analyzed using immunocy-tochemical method.Brie?y,the ORS cell cultures at the 4th passage incubated with ORS cell speci?c primary antibody(anti-CK19(1:200))overnight at4°C.Then, the samples were incubated with secondary antibody system,[biotin SP-HRP(Dingguo Biotechnology Co., Ltd]for2h at room temperature.The colorization was performed by DAB(Dingguo Biotechnology Co.,Ltd). The?broblast cells from Jining grey goat and the Swiss 3T3mouse?broblast cells(3T3cells)were used as negative controls in the analysis.
Morphological reversion test of ORS cell
To study the stability of the subcultural ORS cells,the cells were seeded in two new?asks when ORS cells at passage40were completely con?uent.Following the method of Freshney(2005),one added medium C and the other added medium B.All cultures were carried out at37°C in humidi?ed atmosphere containing5% (v/v)CO2.After that,we observed the phenotypic characteristics and morphology of the ORS cells (Freshney2005).
Results
Establishment and growth pattern of ORS cell line We established a new ORS cell line from Jining grey goat using the technique of the mechanical separation combined with enzyme digestion.Cell cultures were initiated from individual hair follicles on the bottom of the ECM of the?broblast cell coated6-well plates. The ORS cells and?broblast-like cells migrated from the different hair follicles and grew well in medium A. When medium A was replaced with the medium B, only ORS cells showed rapid growth and formed monolayer during the2nd week.The cells showed rapid growth until passage8under these culture conditions.The colony forming ability of the cells was similar from the1st to8th passages.When the cellular growth began to slow down at8th passage,the medium B was replaced with the medium C.ORS cells were subcultured to the40th generation,and the subcultural cells at40th passage still showed good growth and proliferation.The morphology of the cells started to change in the culturing with medium C.The most ORS cells became a long spindle shape,and cells showed rapid growth under these culture conditions of medium C.
Our results suggest that the technique of the mechanical separation combined with enzyme diges-tion enables the generation of a large amount of the pure ORS cells,and that the combination of the different medium formula and ECM of the?broblast cells substrate is suitable for the culture of ORS cells.
Morphology of ORS cells
The cultured ORS cell morphology was observed and monitored regularly under inverted microscopy.The hair follicles were cultured in medium A for approx. 4–5days.A few epithelial-like and?broblast-like cells grew from the different hair follicles(Fig.1a). When medium A was replaced with the medium B, ORS cells showed a typical cobble-stone shape (Fig.1b).After subculturing,ORS cell growth accel-erated and maintained the typical cobble-stone shape, but the?broblast-like cell growth ceased.All ORS cells were cobble-stone shaped on the bottom of culture?asks at the8th passage.Then,medium B was replaced with the medium C after8passages.Most ORS cells were a long spindle shape(Fig.1c).When the culture conditions were changed,drastic morpho-logic changes also occurred in the cells in the manner of other types of mammalian epithelial cells.These results demonstrated that the cells were healthy and the culture conditions were optimal(Fig.1d).
Growth curve and characteristics of sub-cultural ORS cells
The subculture cells could be completely con?uent in a week.The growth curve of the ORS cells appeared as a typical ‘‘S’’shape.The population doubling time calculated from the mid-growth curve data was approx.52h.Cell growth is given in Table 1and Fig.2.
Identi?cation of the ORS cells
The results of chromosomal analysis indicated that majority of the cells were diploid (2n =60)(Fig.3a,b),but the cell chromosome number changed over time,with an increasing frequency of hypodiploid and hyperdiploid occurrences.The diploid proportion was approx.58%at the 40th generation in culture (Fig.3c).The ORS cells in vitro maintain the
same
Fig.1Outer root sheath cells of grey goat in vitro .a The early stage of migrated ORS cells after culture initiation.The cells displayed circle and polygon morphology (scale bar 50l m).b Cells migrated from ORS about 15days after culture initiation.The cells had a typical cobble-stone shape (scale bar 50l m).c Subcultural ORS cells at passage 10.Most ORS cells were a long spindle shape (scale bar 50l m).d The mitotic cells were stained with Giemsa (scale bar 25l m)
karyotype as the tissue cells(peripheral blood lym-phocytes)from normal Jining grey goat(Fig.3d). Majority of cell karyotype was normal(Fig.3b,d). These results suggest that the cell line was indeed from the Jining grey goat.
In general,large-size colony of ORS cells is considered as epithelial stem/progenitor compartment with long-term self-replication activity in vitro culture, the identi?cation of the ORS cells was performed by immunocytochemical staining with stem/progenitor speci?c monoclonal antibody using?broblast cells from Jining grey goat and the?broblast cells from mice (3T3)as negative controls.Young(passage less than ?ve times)ORS cells were stained positive(pale yellow)with speci?c monoclonal antibody,which suggests that the cells strongly expressed CK19 (Fig.4a).While the?broblast cells from Jining grey goat and3T3cells showed negative results in the same immunostaining experiment(Fig.4b,c).These results suggest that the established cell line is an ORS cell line.
To study the stability of the subcultural ORS cells, the cell morphological reversion test was conducted. At the40th passage,cells that changed to long spindle shape(Fig.5b)in medium C could change their shape back to a polygonal shape as primary ORS cells (Fig.5c)when the culture medium was changed back to the medium B(Fig.5a).The results suggest that the subcultural cells maintained the same character as the epithelial cells.
Discussion
Cultivation of outer root sheath cells has been a useful method for researchers seeking to reveal the mecha-nism behind the epithelial-mesenchymal interaction that leads to regenerate itself during the lifetime.The ?rst such trial was reported by Weterings et al.(1981) for culturing human ORS cells on bovine eye lens capsules,in which morphologic characteristics of the ORS cells were described,but the major drawbacks were the limited number of the ORS cells and the low propagation.Several attempts have been made since then to characterize the reproductivity potential of cultured ORS cells on rat tail collagen,?bronectin, and other biologic substrates(Hoeller et al.2001;
Table1ORS cells cultured for7days
Data on cell density was monitored and recorded each day until the plateau phase was reached;three wells were counted at each time point Time Well1
(9104cells/ml)
Well2
(9104cells/ml)
Well3
(9104cells/ml)
Average density
(9104cells/ml) 0d11 1.011
1d0.920.920.930.92
2d 4.8 4.8 4.78 4.8
3d14.9814.9614.9715
4d54.0255.3456.8755.4
5d90.5690.2690.6190.5
6d969696.3296
7d95.4595.496.3295.7 0
10
20
30
40
50
60
70
80
90
100
0d1d2d3d4d5d6d7d C
e
l
l
C
o
n
c
e
n
t
r
a
t
i
o
n
(
×
1
4
c
e
l
l
s
·
m
l
-
1
)
Time
Fig.2Growth curve of ORS cells.The inoculum was1.029104cells/ml. The growth curve included a latency phase,main growth phase,and stationary phase. The population doubling time was approx.52h
Limat et al.1994a ,b ).Rheinwald et al.(1983)?rst demonstrated that the cultured ORS cells sustained their growth capacity on 3T3cells feeder,but the ORS cells in culture lost their activity in later passage.In this study,we have developed an improved method to culture Jining grey goat ORS cells and successfully established the Jining grey goat ORS cell line.We used different biologic substrates to
cultured
Chromosome number
C e l l n u m b e r
5μm
1
2
3
4
5
6
7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 23 24
25 26 27 28 29 X X
1
2
3
4
5
6
7 8 9 10 11 1213 14 15 16 17 1819 20 21 22 23 2425 26
27 28
29 X X
a c
b
d
Fig.3Chromosomal analysis of ORS cells at passage 40.a Metaphase chromosome of ORS cells (female)(scale bar 5l m).b Karyotype of ORS cells (female).The chromosome number of ORS cells in vitro is 2n =60in which six pairs are metacentric chromosomes,?ve pairs are submetacentric chro-mosomes and nineteen pairs are telocentric chromosomes.c Chromosome number of ORS cells at passage 40.The diploid
proportion was approx.58%.d Karyotype of tissue cells (peripheral blood lymphocytes)from normal Jining grey goat (female).The normal Jining grey goat 2n chromosome number is 60in which six pairs are metacentric chromosomes,?ve pairs are submetacentric chromosomes and 19pairs are telocentric
chromosomes
Fig.4Immunocytochemical staining of ORS cells,?broblast cells and 3T3-L1at fourth passage against CK19(scale bar 50l m).a The ORS cells from Jining grey goat at the 4th passage expressed CK19.The 4th ORS cells in vitro were stained pale yellow.b There was no expression of CK19in the ?broblast cells from Jining grey goat at the same passage.The cells were not stained.c The 3T3cells (Swiss 3T3mouse ?broblast cells)also did not express CK19.(scale bar 50l m)
ORS cells and ?rst found that ECM of the ?broblast cells could support the goat ORS cells growth in vitro without a living cell feeder layer.The ECM of the ?broblast cells could provide the extracellular envi-ronment for the primary ORS cells growth.
The 1:1mixture of DMEM and F12medium as the basis for their formulations combine the richness of F12and the higher nutrient concentration of DMEM (Barnes and Sato 1980;Dulbecco and Freeman 1959;Morton 1970).K-SFM medium was originally devel-oped for the culture of epidermal keratinocytes and recently the complete medium has become commer-cially available (Kurata et al.1994;Limat et al.1986;Tausche et al.2003).In the culturing,we selected the DMEM/F12medium and the K-SFM (purify the ORS cells),this combination has provided an empirical formula that is suitable as a basic medium for supplementation with special additives for many different cell types (Freshney 2005).In our study,medium A containing 10%FBS provides the abundant nutrition for the dissociation hair follicle growth in vitro,the cells well-adherent easily in the high concentration of FBS,consequently stimulate the cells grow from the edge of the hair follicle.K-SFM is abundant in copper,selenium,zinc and essential amino acids,is a selection medium for the ORS cells (Yang et al.2000;Yoshida et al.2005).
For the young ORS cells,growth factor and hormone were added to allow the ORS cells to accommodate to the cultural environment in vitro,and provide the abundant nutrition for the ORS cells,and lay the foundation for the subculture.In addition,K-SFM contains a low concentration of Ca 2?(0.03M)and ORS cells could retain their characteristics in this medium (Cotsarelis et al.1990;Taylor et al.2000).Because K-SFM contains few amino acid and mic-roelements in comparison with DMEM/F12,the cel-lular growth began to slow down at the passage 8.When the K-SFM was replaced with medium C containing low concentration FBS,this not only provide abundant amino acids and microelements,but also avoided inhibition from the high concentration of FBS for growth of ORS cells.We have established the ORS cell line using different media (medium A,B,C)given at different times.This method is simple in operation and can be used for serial passage.
This ORS cell line is genetically stable and rapidly proliferating,suggesting that it represents an effective new experiment resource for further studying on ORS cells.The establishment of this cell line will lay the foundation for studying on the growth,regeneration,development law of hair follicle from Jining grey goat.ORS cells can de-differentiate to hair follicle stem cells,hair follicle stem cells not only involved in the formation of the hair follicle but also in the renovation of the epidermis,hair follicle epithelial stem cells were shown to give rise to all the epithelia of the hair follicle as well as potentially contribute to the epidermis (Taylor et al.2000).The ORS cell line can also provide a new resource for studying the hair follicle stem cells and will provide a reference for grafting of chronic wounds or restoration of burned skin and reconstruction of skin.In addition,this cell line as a new cell bank will make a valuable contribution to the preservation of the genetic resources of the Jining grey goat and provides useful biomaterial for future studies in cell biology,genomics,post-genomics and genetic
engineering.
Fig.5ORS cell morphology reversion test (scale bar 50l m).a Morphology of ORS cells which cultured in medium B.Most of the cells became a polygonal shape .b Morphology of ORS cells which cultured in medium C.They were still a long spindle shape .c Morphology of ORS cells in primary culture (scale bar 50l m)
Acknowledgments This research was supported by the National Natural Science Foundation of China(30671498) and Science and Technology Program of Shandong Province (2006GG2209004).
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