BACKGROUND
chronic airway diseases like human bronchial asthma and chronic obstructive pulmonary disease  and the equine recurrent airway obstruction  principally characterized by bronchial hyperreactivity and airflow obstruction result from several factors including structural alterations of the airway wall and cell function.
such airway remodelling is a common feature of abnormal deposition of extracellular matrix  components in airway mesenchymal layer associated with airway wall thickness  <cit> .
airway fibroblasts and those cells differentiated into myofibroblasts contribute to sub-epithelial fibrosis linked to airway remodelling by producing ecm proteins such as collagen, fibronectin and proteoglycans  <cit> .
there are several well characterized human fetal lung fibroblast cell lines that are cultured in defined media and for use as in vitro cell model to study airway diseases and remodeling, for example, hfl- <dig>  <cit> , imr- <dig>  <cit> , hel <dig>  mcr- <dig>  <cit> , wi- <dig>  <cit>  and gm  <dig>  <cit> .
culture of primary bronchial fibroblasts exists for human  <cit> , mice  <cit>  and rats  <cit> .
however, there is currently neither description of equine adult primary airway fibroblast cultures nor there are such cell lines for this species.
not all cells have the same requirements for growth and survival.
with this regard, for successful growth, maintenance and expression of differentiated metabolic functions of human or animal cells in vitro, either primary cultures or continuous cell lines, appropriate culture conditions are required that mimic the physiological conditions in vivo and situ.
in fact, it is well known that serum represents a fundamental source of nutrients, cytokines and adhesive molecules necessary for in vitro cell growth, metabolism and to stimulate proliferation  <cit> .
in serum-free medium, only the addition of growth factors could initiate mouse lung fibroblast proliferation  <cit> .
on the other hand, proliferating primary cardiac and dermal fibroblasts as well as established cell lines have been synchronized into a non-dividing g0-phase to cell cycle by serum withdrawal  <cit>  which favour fibrotic processes or cause cell death  <cit> .
while significant advances are made to culture conditions of permanent cell lines, current research is lacking that compares cultures of primary airway fibroblasts in different serum origins added to a standard culture medium or serum depleted medium, and how this may affect their differentiation.
the sera that most widely used are bovine origin ; as well the horse serum has been seen as an alternative to provide growth factors and hormones in modern cell biology.
indeed, almost it is not known about the responses of primary airway fibroblast cultures to horse serum factors.
aim of the present study was to develop a primary bronchial fibroblast culture technique and investigate the influence of horse serum in comparison to fbs on cell viability, morphology and immunocytochemical characteristics, cell proliferation and α-smooth muscle actin expression  to further understand the mechanism of peribronchial fibrosis  in the equine rao.
RESULTS
effect of serum absence and serum withdrawal on primary ebf culture
primary ebf cultured without serum presence in basic dmem failed to attach and to proliferate and those attached, disappeared completely from the flask surfaces within 1-week of culture.
also, we tested whether confluent ebf cultured in dmem can be affected by serum withdrawal.
serum withdrawal led to modification of ebf number in culture; they exhibited detachment within three days of serum starvation .
even the addition of 10% fbs after 24 h did not significantly enhance ebf attachment and proliferation.
cell viability and morphology under fbs and hs influence
cell yield from digested bronchial tissue was consistent.
under both culture conditions, ebf were stained with trypan blue and the percentage of viable cells was similar, usually > 95%.
no significant evidence of cell necrosis or cell apoptosis was observed under inverted light microscopic analysis.
with regard to cell morphology, ebf cultured in dmem with 10% fbs appeared to be typically flattened and spindle-shaped  over several weeks of passages compared to ebf cultured in dmem with 10% hs .
in medium containing fbs, ebf were grown over the growth surface with only loose cell-cell-contact until reaching confluence and then formed tight parallel lines which remained as typical fibroblastic monolayer until passage  <dig>  ebf between passages  <dig> –  <dig> started to change their morphology: cells were large, flat and more polygonal shaped, with a large, heterogeneous nucleus.
at the same time, cell growth was rapidly reduced and cell viability was diminished.
in contrast, ebf cultured in the presence of hs showed altered morphological changes within 2 days of culture; cells were small and more compact in shape combined with granula-like dark structures in the cytoplasm .
moreover, cells grew in clusters and chains and failed to reach confluence within a week.
this morphological behaviour of ebf was seen under this culture conditions until passage  <dig>  but thereafter, ebf failed to proliferate regularly and decreased in their viability .
under both culture conditions, most ebf  were positive for vimentin, but with more characteristic filamentous structures within the cytoplasm in medium containing fbs  than in the presence of hs .
sera effects on cell population, proliferation and total protein
number of passages and proliferation differed significantly between the two culture conditions.
by routinely passaging primary ebf , ebf maintained their typical morphological fibroblastic properties up to passage  <dig> in culture medium containing fetal bovine serum, whereas ebf in horse serum did exhibit significantly limited proliferation rate merely up to passage  <dig> .
under both experimental conditions, growth curves of ebf showed similar initial 24 h lag-phase.
the population data began to exhibit significant differences between the two conditions until day  <dig> of ebf culture .
cell doubling time was slow in the presence of horse serum, and here, it took significantly longer than in culture medium containing fbs .
under culture conditions in the presence of fbs, primary ebf reached confluence on day  <dig> but not in the presence of hs.
indeed, significant differences between serum groups were not found on day  <dig> and  <dig>  total protein content of the cell monolayers did not reveal significant difference between the two experimental conditions until day five , while after day six the amount of total protein increased under hs condition and statistically differed from fbs condition, though cell number was the vice versa.
when two different batches of fbs and hs were compared on maximum passage, population doubling time and total protein content of ebf, there was no remarkable differences between them .
moreover, cell proliferation was compared between freshly isolated and cryopreserved ebf.
we did not observe any effect of cryopreservation on ebf proliferation ; so routinely, thawed cells could be used for experimental purposes.
furthermore, we assessed the effect of time-dependent withdrawal of fetal bovine serum on cell proliferation in sub-confluent primary equine bronchial fibroblasts by -thymidine incorporation assay .
10% fbs induced markedly ebf proliferation compared to ebf cultured under serum withdrawal for  <dig> and 48 h. even if this investigation was carried out in two to four cell isolates  in triplicates, the results indicate that after 48 h of fbs deprivation, cells almost stopped to proliferate in comparison to 24 hours serum withdrawal.
α-smooth muscle actin expression
to evaluate differentiation of fibroblasts into myofibroblasts, the expression of α-sma was assessed in cells under culture conditions in the presence of fbs and hs.
under both experimental conditions, α-sma was detectable in all cell lysates.
in the presence of hs, α-sma was significantly expressed in comparison to ebf cultured in medium containing fbs .
the expression of the control protein β-actin also differed between both culture conditions ; in the presence of hs, it tended to increase even if not statistically significant.
also, when two different batches of fbs and hs were used, and the effect on α-sma and β-actin expression was compared, no significant difference was observed .
discussion
the overall aim of the present investigation was to assess viability, morphological and proliferation differences of primary bronchial fibroblasts of the horse subjected to basal medium without serum, or first serum containing but later serum-depleted medium, in the presence of fetal bovine serum or horse serum.
the results highlight differences in examined parameters when primary ebf were cultured within these differing conditions.
in the absence of serum, basal medium dmem did not support ebf attachment and proliferation; serum withdrawal in sub-confluent ebf has also led to decreased cell proliferation and attachment.
the most important factors in significantly affecting cell growth and differentiation were the sera types added to basal dmem.
ebf cultures were established from freshly isolated ebf  or from frozen isolated ebf.
despite cryopreservation, the proliferation data generated from frozen cells were similar to those from freshly solated and further cultured ebf.
after short-term trypsin digestion of peeled and minced equine bronchial mucosa and from cultures of epithelial cell mixture in dmem containing fbs, we could observe a complete disappearance of epithelial cells within 1-week and instead five days later a complete coverage of culture flasks with viable primary equine bronchial fibroblasts.
thus, dmem does not support epithelial cell growth despite the presence of serum.
this isolation procedure is described, to our knowledge, the first time for these cells and resulted in reproducible large cell yields with typical mesenchymal cell properties.
ebf cultured in serum-free dmem were, however, not only unable to proliferate but also were non-adherent after seven days in culture.
from this result, indeed, it is difficult to point out the proteins which are present in serum and absent in the basal medium that are essential for primary ebf growth.
the reason why ebf disappeared seems to be, even if we did not proof, the occurrence of apoptosis and necrosis.
thus, it can be argued that under serum-free conditions in our cultures ebf-specific growth factors which are usually present in serum are missing.
but in many established permanent cell line culture under serum-free conditions  <cit>  the serum-free environment did support growth and differentiation with clonal growth as well as mass culture .
although recent efforts have shown that human mesenchymal stem cells and primary epithelial cells can be isolated and expanded long-term in serum-free medium  <cit> , no published work had shown whether human or animal primary airway fibroblasts are able to expand under serum-free conditions for long-term in culture.
the selective nature of serum-free media formulations could be used in primary cultures of tissue explants by allowing growth suppression of other cell types which might quantitatively influence experiments with primary cells obtained from tumor tissues.
presumably, in our case, the serum-free condition should be optimized for enhanced primary fibroblast expansion by adding growth factors which have to be further investigated.
in sum, understanding of these processes is essential in understanding the use of these cells as in vitro model and to study their role in airway remodelling during respiratory disease mechanisms.
the importance of serum supplementation for cell attachment, growth and passaging of in vitro proliferating cells  <cit>  as well as the influence of serum deprivation on permanent cell line proliferation and cycle  <cit>  are well known.
on the other hand, no data are available on the effect of time–dependent serum withdrawal on primary airway fibroblast proliferation and differentiation.
since serum components might affect cell stimulation, many studies in vitro are conducted in the absence of serum, and cells can be deprived of serum for  <dig> – 8 days  <cit> , and thus, the aim of the present study was to investigate the response to serum withdrawal for  <dig> to 48 hours in sub-confluent primary equine bronchial fibroblasts which were first cultured in dmem containing 10% fbs.
in our study, we have clearly demonstrated a marked time-dependent decrease in -thymidine incorporation in cells deprived of serum for  <dig> to 48 hours, suggesting discontinuation of proliferation in the absence of fbs.
after 72 hours of serum depletion many cells were floating in the medium and the number of attached cells was decreasing, indicating altering cell viability and morphology, in agreement with data found for cardiac fibroblasts  <cit> .
these findings show that ebf in primary cultures are not able to adapt to serum deprivation for a given time, presumably sufficient serum growth factors are not available.
moreover, we have tested the effects of sera types on proliferation, morphology, population doubling and viability of primary equine bronchial fibroblasts.
the results highlight striking differences in cell proliferation, morphology, passaging time and number as well as total protein amount and α-sma expression when ebf were cultured within these differing conditions.
both fbs and hs are natural products, presumably, with varying concentrations of growth factors within different batches; however, the effects measured in our study were not dependent on batch/lot number.
ebf grown in dmem containing 10% fbs continue to proliferate even after reaching confluence, whereas these same cells, when cultured in dmem containing 10% hs, had relatively limited proliferation rate, longer population doubling time and somehow quite different morphological features.
cells in hs were smaller, chain-forming and more compact in shape with dark cytoplasmic granules which can be related to the occurrence of abnormal protein accumulation than those in fbs, and they failed to reach confluence.
in concordance, sera types affect other various cell types in a similar way: bovine adipocytes and sheep skeletal muscle satellite cells proliferated rapidly, when fbs was supplemented to growth medium instead of hs; thus, fbs-containing medium is often used as growth medium in these cultures  <cit> .
on the contrary, equine chondrocytes could equally proliferate under both conditions  <cit> .
even if they are equine cells, it is noteworthy to find that ebf exhibit a preference for fetal bovine serum factors over the horse serum factors, whereas the vice versa has been seen for neural cells  <cit> .
merely, early studies have proposed that horse serum is appropriate for neuronal cell cultures  <cit> .
moreover, despite the short passaging time and limited proliferation and population doubling rate, increased α-sma expression was accompanied by higher protein amount in the presence of horse serum than in bovine serum, suggesting that hs might stimulate fibroblast differentiation into myofibroblasts.
it is well known that myofibroblasts are able to produce large amount of extracellular matrix  proteins, as well as growth factors and cytokines and express cytoplasmic contractile structures including α-sma.
thus, it seems that the horse serum increases the protein content in ebf by increasing synthesis of structural proteins, ecm and cytokines.
indeed, it is not yet known if differential expression of α-sma and protein is functionally relevant.
CONCLUSIONS
fetal bovine serum favors fibroblastic morphology with enhanced proliferation rate, population doubling, passage number and triggers cell differentiation , suggesting serum factors essential for the equine airway fibroblasts are available in the fetal bovine serum, whereas in the horse serum cells there were signs of degeneration or cell granularity.
moreover, we can conclude that serum withdrawal for 48 hours rather decrease ebf adaptation and enhance cell detachment than the 24 hour fbs depletion and in the latter case cell viability was though not altered, thus, ebf cultured in 10% fbs represent a good model allowing studying the response to drugs that influence cell proliferation and pathways of airway remodeling in airway diseases.
study limitation: animal serum is a complex mixture of a large number and variety of components; therefore, it is difficult to assess, at this stage, the significance of the observation that horse serum inhibits the programmed progress of equine airway cells through the lineage of differentiation in cultures.

 chronic airway diseases like human bronchial asthma and chronic obstructive pulmonary disease and the equine recurrent airway obstruction principally characterized by bronchial hyperreactivity and airflow obstruction result from several factors including structural alterations of the airway wall and cell function. such airway remodelling is a common feature of abnormal deposition of extracellular matrix components in airway mesenchymal layer associated with airway wall thickness. airway fibroblasts and those cells differentiated into myofibroblasts contribute to sub-epithelial fibrosis linked to airway remodelling by producing ecm proteins such as collagen, fibronectin and proteoglycans. there are several well characterized human fetal lung fibroblast cell lines that are cultured in defined media and for use as in vitro cell model to study airway diseases and remodeling, for example, hfl-, imr-, hel mcr-, wi- and gm. culture of primary bronchial fibroblasts exists for human, mice and rats. however, there is currently neither description of equine adult primary airway fibroblast cultures nor there are such cell lines for this species. not all cells have the same requirements for growth and survival. with this regard, for successful growth, maintenance and expression of differentiated metabolic functions of human or animal cells in vitro, either primary cultures or continuous cell lines, appropriate culture conditions are required that mimic the physiological conditions in vivo and situ. in fact, it is well known that serum represents a fundamental source of nutrients, cytokines and adhesive molecules necessary for in vitro cell growth, metabolism and to stimulate proliferation. in serum-free medium, only the addition of growth factors could initiate mouse lung fibroblast proliferation. on the other hand, proliferating primary cardiac and dermal fibroblasts as well as established cell lines have been synchronized into a non-dividing g0-phase to cell cycle by serum withdrawal which favour fibrotic processes or cause cell death. while significant advances are made to culture conditions of permanent cell lines, current research is lacking that compares cultures of primary airway fibroblasts in different serum origins added to a standard culture medium or serum depleted medium, and how this may affect their differentiation. the sera that most widely used are bovine origin; as well the horse serum has been seen as an alternative to provide growth factors and hormones in modern cell biology. indeed, almost it is not known about the responses of primary airway fibroblast cultures to horse serum factors. aim of the present study was to develop a primary bronchial fibroblast culture technique and investigate the influence of horse serum in comparison to fbs on cell viability, morphology and immunocytochemical characteristics, cell proliferation and α-smooth muscle actin expression to further understand the mechanism of peribronchial fibrosis in the equine rao.  effect of serum absence and serum withdrawal on primary ebf culture primary ebf cultured without serum presence in basic dmem failed to attach and to proliferate and those attached, disappeared completely from the flask surfaces within 1-week of culture. also, we tested whether confluent ebf cultured in dmem can be affected by serum withdrawal. serum withdrawal led to modification of ebf number in culture; they exhibited detachment within three days of serum starvation. even the addition of 10% fbs after 24 h did not significantly enhance ebf attachment and proliferation. cell viability and morphology under fbs and hs influence cell yield from digested bronchial tissue was consistent. under both culture conditions, ebf were stained with trypan blue and the percentage of viable cells was similar, usually > 95%. no significant evidence of cell necrosis or cell apoptosis was observed under inverted light microscopic analysis. with regard to cell morphology, ebf cultured in dmem with 10% fbs appeared to be typically flattened and spindle-shaped over several weeks of passages compared to ebf cultured in dmem with 10% hs. in medium containing fbs, ebf were grown over the growth surface with only loose cell-cell-contact until reaching confluence and then formed tight parallel lines which remained as typical fibroblastic monolayer until passage ebf between passages – started to change their morphology: cells were large, flat and more polygonal shaped, with a large, heterogeneous nucleus. at the same time, cell growth was rapidly reduced and cell viability was diminished. in contrast, ebf cultured in the presence of hs showed altered morphological changes within 2 days of culture; cells were small and more compact in shape combined with granula-like dark structures in the cytoplasm. moreover, cells grew in clusters and chains and failed to reach confluence within a week. this morphological behaviour of ebf was seen under this culture conditions until passage but thereafter, ebf failed to proliferate regularly and decreased in their viability. under both culture conditions, most ebf were positive for vimentin, but with more characteristic filamentous structures within the cytoplasm in medium containing fbs than in the presence of hs. sera effects on cell population, proliferation and total protein number of passages and proliferation differed significantly between the two culture conditions. by routinely passaging primary ebf, ebf maintained their typical morphological fibroblastic properties up to passage in culture medium containing fetal bovine serum, whereas ebf in horse serum did exhibit significantly limited proliferation rate merely up to passage. under both experimental conditions, growth curves of ebf showed similar initial 24 h lag-phase. the population data began to exhibit significant differences between the two conditions until day of ebf culture. cell doubling time was slow in the presence of horse serum, and here, it took significantly longer than in culture medium containing fbs. under culture conditions in the presence of fbs, primary ebf reached confluence on day but not in the presence of hs. indeed, significant differences between serum groups were not found on day and total protein content of the cell monolayers did not reveal significant difference between the two experimental conditions until day five, while after day six the amount of total protein increased under hs condition and statistically differed from fbs condition, though cell number was the vice versa. when two different batches of fbs and hs were compared on maximum passage, population doubling time and total protein content of ebf, there was no remarkable differences between them. moreover, cell proliferation was compared between freshly isolated and cryopreserved ebf. we did not observe any effect of cryopreservation on ebf proliferation; so routinely, thawed cells could be used for experimental purposes. furthermore, we assessed the effect of time-dependent withdrawal of fetal bovine serum on cell proliferation in sub-confluent primary equine bronchial fibroblasts by -thymidine incorporation assay. 10% fbs induced markedly ebf proliferation compared to ebf cultured under serum withdrawal for and 48 h. even if this investigation was carried out in two to four cell isolates in triplicates, the results indicate that after 48 h of fbs deprivation, cells almost stopped to proliferate in comparison to 24 hours serum withdrawal. α-smooth muscle actin expression to evaluate differentiation of fibroblasts into myofibroblasts, the expression of α-sma was assessed in cells under culture conditions in the presence of fbs and hs. under both experimental conditions, α-sma was detectable in all cell lysates. in the presence of hs, α-sma was significantly expressed in comparison to ebf cultured in medium containing fbs. the expression of the control protein β-actin also differed between both culture conditions; in the presence of hs, it tended to increase even if not statistically significant. also, when two different batches of fbs and hs were used, and the effect on α-sma and β-actin expression was compared, no significant difference was observed. discussion the overall aim of the present investigation was to assess viability, morphological and proliferation differences of primary bronchial fibroblasts of the horse subjected to basal medium without serum, or first serum containing but later serum-depleted medium, in the presence of fetal bovine serum or horse serum. the results highlight differences in examined parameters when primary ebf were cultured within these differing conditions. in the absence of serum, basal medium dmem did not support ebf attachment and proliferation; serum withdrawal in sub-confluent ebf has also led to decreased cell proliferation and attachment. the most important factors in significantly affecting cell growth and differentiation were the sera types added to basal dmem. ebf cultures were established from freshly isolated ebf or from frozen isolated ebf. despite cryopreservation, the proliferation data generated from frozen cells were similar to those from freshly solated and further cultured ebf. after short-term trypsin digestion of peeled and minced equine bronchial mucosa and from cultures of epithelial cell mixture in dmem containing fbs, we could observe a complete disappearance of epithelial cells within 1-week and instead five days later a complete coverage of culture flasks with viable primary equine bronchial fibroblasts. thus, dmem does not support epithelial cell growth despite the presence of serum. this isolation procedure is described, to our knowledge, the first time for these cells and resulted in reproducible large cell yields with typical mesenchymal cell properties. ebf cultured in serum-free dmem were, however, not only unable to proliferate but also were non-adherent after seven days in culture. from this result, indeed, it is difficult to point out the proteins which are present in serum and absent in the basal medium that are essential for primary ebf growth. the reason why ebf disappeared seems to be, even if we did not proof, the occurrence of apoptosis and necrosis. thus, it can be argued that under serum-free conditions in our cultures ebf-specific growth factors which are usually present in serum are missing. but in many established permanent cell line culture under serum-free conditions the serum-free environment did support growth and differentiation with clonal growth as well as mass culture. although recent efforts have shown that human mesenchymal stem cells and primary epithelial cells can be isolated and expanded long-term in serum-free medium, no published work had shown whether human or animal primary airway fibroblasts are able to expand under serum-free conditions for long-term in culture. the selective nature of serum-free media formulations could be used in primary cultures of tissue explants by allowing growth suppression of other cell types which might quantitatively influence experiments with primary cells obtained from tumor tissues. presumably, in our case, the serum-free condition should be optimized for enhanced primary fibroblast expansion by adding growth factors which have to be further investigated. in sum, understanding of these processes is essential in understanding the use of these cells as in vitro model and to study their role in airway remodelling during respiratory disease mechanisms. the importance of serum supplementation for cell attachment, growth and passaging of in vitro proliferating cells as well as the influence of serum deprivation on permanent cell line proliferation and cycle are well known. on the other hand, no data are available on the effect of time–dependent serum withdrawal on primary airway fibroblast proliferation and differentiation. since serum components might affect cell stimulation, many studies in vitro are conducted in the absence of serum, and cells can be deprived of serum for – 8 days, and thus, the aim of the present study was to investigate the response to serum withdrawal for to 48 hours in sub-confluent primary equine bronchial fibroblasts which were first cultured in dmem containing 10% fbs. in our study, we have clearly demonstrated a marked time-dependent decrease in -thymidine incorporation in cells deprived of serum for to 48 hours, suggesting discontinuation of proliferation in the absence of fbs. after 72 hours of serum depletion many cells were floating in the medium and the number of attached cells was decreasing, indicating altering cell viability and morphology, in agreement with data found for cardiac fibroblasts. these findings show that ebf in primary cultures are not able to adapt to serum deprivation for a given time, presumably sufficient serum growth factors are not available. moreover, we have tested the effects of sera types on proliferation, morphology, population doubling and viability of primary equine bronchial fibroblasts. the results highlight striking differences in cell proliferation, morphology, passaging time and number as well as total protein amount and α-sma expression when ebf were cultured within these differing conditions. both fbs and hs are natural products, presumably, with varying concentrations of growth factors within different batches; however, the effects measured in our study were not dependent on batch/lot number. ebf grown in dmem containing 10% fbs continue to proliferate even after reaching confluence, whereas these same cells, when cultured in dmem containing 10% hs, had relatively limited proliferation rate, longer population doubling time and somehow quite different morphological features. cells in hs were smaller, chain-forming and more compact in shape with dark cytoplasmic granules which can be related to the occurrence of abnormal protein accumulation than those in fbs, and they failed to reach confluence. in concordance, sera types affect other various cell types in a similar way: bovine adipocytes and sheep skeletal muscle satellite cells proliferated rapidly, when fbs was supplemented to growth medium instead of hs; thus, fbs-containing medium is often used as growth medium in these cultures. on the contrary, equine chondrocytes could equally proliferate under both conditions. even if they are equine cells, it is noteworthy to find that ebf exhibit a preference for fetal bovine serum factors over the horse serum factors, whereas the vice versa has been seen for neural cells. merely, early studies have proposed that horse serum is appropriate for neuronal cell cultures. moreover, despite the short passaging time and limited proliferation and population doubling rate, increased α-sma expression was accompanied by higher protein amount in the presence of horse serum than in bovine serum, suggesting that hs might stimulate fibroblast differentiation into myofibroblasts. it is well known that myofibroblasts are able to produce large amount of extracellular matrix proteins, as well as growth factors and cytokines and express cytoplasmic contractile structures including α-sma. thus, it seems that the horse serum increases the protein content in ebf by increasing synthesis of structural proteins, ecm and cytokines. indeed, it is not yet known if differential expression of α-sma and protein is functionally relevant.  fetal bovine serum favors fibroblastic morphology with enhanced proliferation rate, population doubling, passage number and triggers cell differentiation, suggesting serum factors essential for the equine airway fibroblasts are available in the fetal bovine serum, whereas in the horse serum cells there were signs of degeneration or cell granularity. moreover, we can conclude that serum withdrawal for 48 hours rather decrease ebf adaptation and enhance cell detachment than the 24 hour fbs depletion and in the latter case cell viability was though not altered, thus, ebf cultured in 10% fbs represent a good model allowing studying the response to drugs that influence cell proliferation and pathways of airway remodeling in airway diseases. study limitation: animal serum is a complex mixture of a large number and variety of components; therefore, it is difficult to assess, at this stage, the significance of the observation that horse serum inhibits the programmed progress of equine airway cells through the lineage of differentiation in cultures. 
the protein content of the cell pellets was higher in ebf cultured in medium containing hs than fbs. large variations were found in the ability of fbs and hs to support growth and differentiation of ebf in monolayer culture. indications of growth-promoting actions, increasing passage number as well as maintaining fibroblast morphology were found rather in fbs than in hs. primary ebf were cultured on non-collagen coated flasks without serum or in the presence of fetal bovine serum or horse serum or in serum depleted medium. airway fibroblasts have become a critical addition to all facets of structural lung tissue changes such as in human asthma and chronic obstructive pulmonary disease, but little is known about their role in the equine recurrent airway obstruction, a disease that resembles to the human asthma.
 airway fibroblasts have become a critical addition to all facets of structural lung tissue changes such as in human asthma and chronic obstructive pulmonary disease, but little is known about their role in the equine recurrent airway obstruction, a disease that resembles to the human asthma. since the equine bronchial fibroblasts have not been isolated and characterized yet, the use of defined medium was investigated.  primary ebf were cultured on non-collagen coated flasks without serum or in the presence of fetal bovine serum or horse serum or in serum depleted medium. ebf cultured in serum-free basal media and those serum deprived were not able to proliferate and even exhibited considerable cell death. in media containing fbs or hs, proliferation of the cells was reproducible between different primary cultures and cells demonstrated expression of vimentin. large variations were found in the ability of fbs and hs to support growth and differentiation of ebf in monolayer culture. indications of growth-promoting actions, increasing passage number as well as maintaining fibroblast morphology were found rather in fbs than in hs. ebf culturing in hs needed longer doubling and confluence time. the protein content of the cell pellets was higher in ebf cultured in medium containing hs than fbs. alpha-smooth muscle actin seemed to be less expressed in ebf cultured in medium containing fbs than those in hs.  in sum, serum addition to basal ebf medium enhanced ebf differentiation into myofibroblasts, and these findings are useful to develop in vitro fibroblast culture models that mimic in vivo physiological processes and to study airway disease mechanisms and remodeling. airwaysprimary bronchial fibroblastscell culturein vitroserum types 