**1. Introduction**

Goats are one of the oldest domesticated species. They are bred for milk and meat and play an important role in human nutrition, especially in developing countries. Their number is constantly increasing through the years and the population has been estimated to over a billion

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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(FAOSTAT, 2013). However, goats are not useful only for food production. Because of their anatomical and physiological characteristics, relatively short gestation period, early sexual maturation, and inexpensive and simple maintenance, they are valuable for basic research, for biotechnology applications, and as animal models in medical research. For example, goats are used to study heart and joint diseases [1] and are an excellent model species to study mammary development and lactation [2].

**2.1. Establishment and maintenance of the primary goat mammary epithelial cells** 

Primary cultures were established from mammary tissue of slaughter animals. Lactating goats of different age and nonlactating juvenile goats from approximately four to seven months of age were used for tissue collection. The whole mammary gland was removed immediately after slaughter, wiped with 70% ethanol, and processed under aseptic conditions. First, larger pieces of the glandular tissue were removed from the gland. Alternatively, tissue biopsates can be used instead of whole mammary gland. Different quantities of the tissue can be processed, depending on the desired amount of cells in the culture. In our case, approximately 100 g of the dissected tissue pieces were washed in Hank's balanced salt solution (HBSS), containing penicillin (200 μg/mL), streptomycin (200 μg/mL), gentamicin (200 μg/mL), ampicillin (200 μg/mL), and amphotericin B (10 μg/mL), and mechanically minced with scissors and scalpels. Minced tissue was digested in a 100-ml solution of collagenase and hyaluronidase (400 U/mL of each), prepared in HBSS with HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), also containing all of the above listed antibiotics in the same concentrations. The digestion was carried out at 37°C with gentle shaking. Fractions of dissociated cells were collected after 60, 120, and 180 min of incubation, by filtering the contents through a steel mesh and adding fresh solution of the enzymes to the leftovers of the minced tissue. The filtrates were put in 50-ml tubes, washed (diluted) with HBSS, and centrifuged at 1200 rpm for 5 min. The pellets can be resuspended in HBSS and centrifuged several times to remove cell debris. Finally, the resuspended cells were filtered through a 40-μm cell strainer, centrifuged at 1200 rpm for 5 min, resuspended in growth medium, and plated on tissue culture vessels or resuspended in freezing medium (90% FBS and 10% DMSO) for freezing in liquid nitrogen. Major steps of tissue processing

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An alternate to enzymatic digestion, explant culture method is possible. In this case, it is important to mechanically mince the extracted tissue to very small pieces and incubate the finely minced tissue in growth vessels (supplied with growth medium) for several days, using conditions as described hereinafter. After several days, cells will start to explant and attach to culture dishes. Afterward, the tissue pieces can be removed from the vessels and the attached cells passaged into a new dish. To our experience, explant culture will produce lower cell yields; however, the obtained culture might be more enriched in a desired (epithelial) cell type(s) as in the case of enzymatic digestion, where other cell types (e.g. fibroblasts) might be

The cells were grown in RPMI 1640 growth medium, supplemented with 0.1 mM l-methi-

bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. For simulation of lactogenic

, 1 mM Na-pyruvate, 2 mM l-glutamine, 10% fetal

**(pgMECs)**

*2.1.1. Tissue processing*

are depicted in **Figure 1**.

present in a significant amount.

*2.1.2. Maintaining pgMECs in cell culture*

onine, 0.4 mM l-lysine, 2 g/l NaHCO3

Rodent mammary gland is the most widely studied and has provided many biological insights, but its anatomy and physiology are not fully representative of human or ruminant mammary gland. Morphological development of mammary gland is much more alike between humans and ruminants [3]. Considering the size, arrangement of the mammary gland (two main glands), and mechanism of secretion, which is apocrine in goats and humans, whereas merocrine in bovine, goats seem to be a better choice for modeling human mammary gland, compared to cows or rodents. Goat mammary tissue/cell cultures can serve as valuable models to study lactation, mammary development, and pathology, including neoplasia, which is for unknown reasons extremely rare in ruminants, despite the anatomical and physiological similarities to humans [4]. Additionally, genetically modified ruminants (especially goats) have been used as "bioreactors" for production of recombinant proteins. Recombinant proteins can be controlled by inducible, mammary-specific promotors and expressed in mammary gland, from where they can be relatively easily isolated from milk. For example, the first marketed human recombinant protein produced in transgenic animals was produced and extracted from milk of transgenic goats [5]. Furthermore, transgenic dairy goats can be used for production of milk with special nutritional characteristics [6], which can be beneficial especially in developing countries.

Knowledge on goat mammary cells and goat mammary biology is beneficial to different fields of science, for example, agriculture (enhancing lactation yield and persistency, and producing milk with special characteristics), basic research (understanding mammary biology), medicine (model organisms), and biopharma (expression of recombinant proteins in goat's milk). In this chapter, we describe methods for establishment and characterization of primary goat mammary cell cultures (pgMECs) and possible applications for which the cell model can be used instead of the mammary tissue. The established cells can be grown *in vitro* for several passages and remain hormone responsive and capable of milk protein synthesis. The cells can be used for basic research of lactation biology, mammary gland immunity studies, mammary stem/progenitor cell identification/isolation, and further applications.

## **2. Materials and methods**

In this section, we briefly describe materials and methods used for establishment, growth, characterization, and procedures with the primary cultures, which apply to the results described in the successive sections.

#### **2.1. Establishment and maintenance of the primary goat mammary epithelial cells (pgMECs)**

#### *2.1.1. Tissue processing*

(FAOSTAT, 2013). However, goats are not useful only for food production. Because of their anatomical and physiological characteristics, relatively short gestation period, early sexual maturation, and inexpensive and simple maintenance, they are valuable for basic research, for biotechnology applications, and as animal models in medical research. For example, goats are used to study heart and joint diseases [1] and are an excellent model species to study mam-

Rodent mammary gland is the most widely studied and has provided many biological insights, but its anatomy and physiology are not fully representative of human or ruminant mammary gland. Morphological development of mammary gland is much more alike between humans and ruminants [3]. Considering the size, arrangement of the mammary gland (two main glands), and mechanism of secretion, which is apocrine in goats and humans, whereas merocrine in bovine, goats seem to be a better choice for modeling human mammary gland, compared to cows or rodents. Goat mammary tissue/cell cultures can serve as valuable models to study lactation, mammary development, and pathology, including neoplasia, which is for unknown reasons extremely rare in ruminants, despite the anatomical and physiological similarities to humans [4]. Additionally, genetically modified ruminants (especially goats) have been used as "bioreactors" for production of recombinant proteins. Recombinant proteins can be controlled by inducible, mammary-specific promotors and expressed in mammary gland, from where they can be relatively easily isolated from milk. For example, the first marketed human recombinant protein produced in transgenic animals was produced and extracted from milk of transgenic goats [5]. Furthermore, transgenic dairy goats can be used for production of milk with special nutritional characteristics [6], which can be beneficial

Knowledge on goat mammary cells and goat mammary biology is beneficial to different fields of science, for example, agriculture (enhancing lactation yield and persistency, and producing milk with special characteristics), basic research (understanding mammary biology), medicine (model organisms), and biopharma (expression of recombinant proteins in goat's milk). In this chapter, we describe methods for establishment and characterization of primary goat mammary cell cultures (pgMECs) and possible applications for which the cell model can be used instead of the mammary tissue. The established cells can be grown *in vitro* for several passages and remain hormone responsive and capable of milk protein synthesis. The cells can be used for basic research of lactation biology, mammary gland immunity studies, mammary stem/progenitor cell identification/isolation, and further

In this section, we briefly describe materials and methods used for establishment, growth, characterization, and procedures with the primary cultures, which apply to the results

mary development and lactation [2].

166 Goat Science

especially in developing countries.

**2. Materials and methods**

described in the successive sections.

applications.

Primary cultures were established from mammary tissue of slaughter animals. Lactating goats of different age and nonlactating juvenile goats from approximately four to seven months of age were used for tissue collection. The whole mammary gland was removed immediately after slaughter, wiped with 70% ethanol, and processed under aseptic conditions. First, larger pieces of the glandular tissue were removed from the gland. Alternatively, tissue biopsates can be used instead of whole mammary gland. Different quantities of the tissue can be processed, depending on the desired amount of cells in the culture. In our case, approximately 100 g of the dissected tissue pieces were washed in Hank's balanced salt solution (HBSS), containing penicillin (200 μg/mL), streptomycin (200 μg/mL), gentamicin (200 μg/mL), ampicillin (200 μg/mL), and amphotericin B (10 μg/mL), and mechanically minced with scissors and scalpels. Minced tissue was digested in a 100-ml solution of collagenase and hyaluronidase (400 U/mL of each), prepared in HBSS with HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), also containing all of the above listed antibiotics in the same concentrations. The digestion was carried out at 37°C with gentle shaking. Fractions of dissociated cells were collected after 60, 120, and 180 min of incubation, by filtering the contents through a steel mesh and adding fresh solution of the enzymes to the leftovers of the minced tissue. The filtrates were put in 50-ml tubes, washed (diluted) with HBSS, and centrifuged at 1200 rpm for 5 min. The pellets can be resuspended in HBSS and centrifuged several times to remove cell debris. Finally, the resuspended cells were filtered through a 40-μm cell strainer, centrifuged at 1200 rpm for 5 min, resuspended in growth medium, and plated on tissue culture vessels or resuspended in freezing medium (90% FBS and 10% DMSO) for freezing in liquid nitrogen. Major steps of tissue processing are depicted in **Figure 1**.

An alternate to enzymatic digestion, explant culture method is possible. In this case, it is important to mechanically mince the extracted tissue to very small pieces and incubate the finely minced tissue in growth vessels (supplied with growth medium) for several days, using conditions as described hereinafter. After several days, cells will start to explant and attach to culture dishes. Afterward, the tissue pieces can be removed from the vessels and the attached cells passaged into a new dish. To our experience, explant culture will produce lower cell yields; however, the obtained culture might be more enriched in a desired (epithelial) cell type(s) as in the case of enzymatic digestion, where other cell types (e.g. fibroblasts) might be present in a significant amount.

#### *2.1.2. Maintaining pgMECs in cell culture*

The cells were grown in RPMI 1640 growth medium, supplemented with 0.1 mM l-methionine, 0.4 mM l-lysine, 2 g/l NaHCO3 , 1 mM Na-pyruvate, 2 mM l-glutamine, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. For simulation of lactogenic

The established cells should be routinely screened for possible infections with different mycoplasma species. We suggest PCR-based detection of mycoplasma-specific DNA sequences,

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For transcription profiling, total RNA was isolated from the aqueous phase of lysed pgMECs and reverse transcribed into complementary DNA (cDNA), which was subjected to new generation sequencing (NGS) as described in Ref. [8]. Additionally, reverse transcription polymerase chain reactions (RT-PCR) were performed to monitor expression of the selected cell-type specific markers in the culture. Real-time quantitative polymerase chain reaction (RT-qPCR) method was used to determine relative quantities of markers and ratios between them (e.g. expression of caseins in basal and lactogenic medium). Due to poor annotation of the goat genome, PCR primers were designed against *Bos taurus* RefSeq (NCBI) mRNA sequences and cross-matched against the goat reference sequences, if available. We determined expression of markers on mRNA level first and proceeded to protein level (e.g. immu-

In order to detect beta-casein (CSN2) mRNA culprits, we performed reverse transcription polymerase chain reaction (RT-PCR) on pgMEC-derived transcriptome library (cDNA), using the following primer pair CSN2-F: 5′-ACAGCCTCCCACAAAACATC-3′, CSN2-R: 5′-AGGAAGGTGCAGCTTTTCAA-3′. The resulting 206 bp product was isolated from agarose gel, using gel extraction kit, and sequenced by Sanger sequencing to validate that the sequences correspond to the portion of the exon seven of the CSN2 gene (GenBank:

Real-time quantitative polymerase chain reactions (RT-qPCRs) consisted of 2× SYBR Green PCR master mix (Life Technologies), water, and 0.5 μM of each primer in a total volume of 20 μl. The cycles were as follows: 10 min at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Melting curve was determined at 15s for 95°C, 1min at 58°C, and 15s at 95°C.The following primers were used: estrogen receptor 1 (ESR1) forward: 5′-ACAGCATGAAGTGCAAGAACGTGG-3′ and reverse: 5′-TGCAAGGAATGCGATGAAGTGCAG-3′; progesterone receptor (PGR) forward: 5′-AAGCCAAGCCCTAAGCCAGAGAAT-3′ and reverse: 5′-AGCTGGAGGTAT-CAGGTTTGCTGT-3′; and CSN2 forward: 5′-ACAGCCTCCCACAAAACATC-3′ and reverse: 5′-AGGAAGGTGCAGCTTTTCAA-3′. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as an endogenous control, using the primer pair: forward: 5′-CATGTTTGTGATGGGCGTGAACCA-3′ and reverse: 5′-TAAGTCCCTCCA-

The immunostaining protocols often differ for different markers and every marker might require optimization of the protocol. Generally, the protocol consisted of cell fixation for

using 16S ribosomal RNA universal primers as described previously [7].

**2.2. Characterization of the pgMECs**

*2.2.1. mRNA expression of pgMEC-specific markers*

nostainings and western blotting) afterward.

AJ011019).

CGATGCCAAAGT-3′.

*2.2.2. Immunostainings*

**Figure 1.** Mechanical and enzymatic processing of the mammary tissue (photo: J. Ogorevc). (A) Removal of the skin covering the mammary tissue. (B) Tissue pieces excised from the gland. (C) Fine mechanical processing of the tissue. (D) Dissociation of the tissue in the cocktail of enzymes. Finally, dissociated cells were collect by centrifugation and seeded in cell culture flasks.

conditions, basic growth medium was supplemented with lactogenic hormones such as insulin (1 μg/mL), hydrocortisone (1 μg/mL), and prolactin (1 μg/mL), and the cells were grown on a commercially prepared basement membrane matrix-covered surface (e.g. Matrigel, Geltrex). The cells were allowed to overgrow the surface, differentiate, and establish cell-cell and cell-surface interactions.

Cells were grown in a 5% CO2 atmosphere at 37°C, 5% CO2 , and saturated humidity. Growth medium was changed every two to three days. When performing passaging, the cells were treated with 0.25% trypsin-EDTA and incubated at 37°C until the cells detached from the surface. The cells were centrifuged and resuspended in growth medium in a 1:5 ratio. In case of fibroblast contamination, different detachment times of fibroblasts and epithelial cells can be exploited for refining the culture. Fibroblasts form weaker cell-cell and cell-surface interactions and normally detach faster. This characteristic can be used for enrichment of epithelial cells in the culture, using principle of differential trypsinization. Trypsin-EDTA can be diluted to a lower concentration (e.g. 0.05%) to extend detachment times for better control of the procedure.

The established cells should be routinely screened for possible infections with different mycoplasma species. We suggest PCR-based detection of mycoplasma-specific DNA sequences, using 16S ribosomal RNA universal primers as described previously [7].

## **2.2. Characterization of the pgMECs**

## *2.2.1. mRNA expression of pgMEC-specific markers*

For transcription profiling, total RNA was isolated from the aqueous phase of lysed pgMECs and reverse transcribed into complementary DNA (cDNA), which was subjected to new generation sequencing (NGS) as described in Ref. [8]. Additionally, reverse transcription polymerase chain reactions (RT-PCR) were performed to monitor expression of the selected cell-type specific markers in the culture. Real-time quantitative polymerase chain reaction (RT-qPCR) method was used to determine relative quantities of markers and ratios between them (e.g. expression of caseins in basal and lactogenic medium). Due to poor annotation of the goat genome, PCR primers were designed against *Bos taurus* RefSeq (NCBI) mRNA sequences and cross-matched against the goat reference sequences, if available. We determined expression of markers on mRNA level first and proceeded to protein level (e.g. immunostainings and western blotting) afterward.

In order to detect beta-casein (CSN2) mRNA culprits, we performed reverse transcription polymerase chain reaction (RT-PCR) on pgMEC-derived transcriptome library (cDNA), using the following primer pair CSN2-F: 5′-ACAGCCTCCCACAAAACATC-3′, CSN2-R: 5′-AGGAAGGTGCAGCTTTTCAA-3′. The resulting 206 bp product was isolated from agarose gel, using gel extraction kit, and sequenced by Sanger sequencing to validate that the sequences correspond to the portion of the exon seven of the CSN2 gene (GenBank: AJ011019).

Real-time quantitative polymerase chain reactions (RT-qPCRs) consisted of 2× SYBR Green PCR master mix (Life Technologies), water, and 0.5 μM of each primer in a total volume of 20 μl. The cycles were as follows: 10 min at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Melting curve was determined at 15s for 95°C, 1min at 58°C, and 15s at 95°C.The following primers were used: estrogen receptor 1 (ESR1) forward: 5′-ACAGCATGAAGTGCAAGAACGTGG-3′ and reverse: 5′-TGCAAGGAATGCGATGAAGTGCAG-3′; progesterone receptor (PGR) forward: 5′-AAGCCAAGCCCTAAGCCAGAGAAT-3′ and reverse: 5′-AGCTGGAGGTAT-CAGGTTTGCTGT-3′; and CSN2 forward: 5′-ACAGCCTCCCACAAAACATC-3′ and reverse: 5′-AGGAAGGTGCAGCTTTTCAA-3′. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as an endogenous control, using the primer pair: forward: 5′-CATGTTTGTGATGGGCGTGAACCA-3′ and reverse: 5′-TAAGTCCCTCCA-CGATGCCAAAGT-3′.

#### *2.2.2. Immunostainings*

conditions, basic growth medium was supplemented with lactogenic hormones such as insulin (1 μg/mL), hydrocortisone (1 μg/mL), and prolactin (1 μg/mL), and the cells were grown on a commercially prepared basement membrane matrix-covered surface (e.g. Matrigel, Geltrex). The cells were allowed to overgrow the surface, differentiate, and establish cell-cell

**Figure 1.** Mechanical and enzymatic processing of the mammary tissue (photo: J. Ogorevc). (A) Removal of the skin covering the mammary tissue. (B) Tissue pieces excised from the gland. (C) Fine mechanical processing of the tissue. (D) Dissociation of the tissue in the cocktail of enzymes. Finally, dissociated cells were collect by centrifugation and seeded

atmosphere at 37°C, 5% CO2

medium was changed every two to three days. When performing passaging, the cells were treated with 0.25% trypsin-EDTA and incubated at 37°C until the cells detached from the surface. The cells were centrifuged and resuspended in growth medium in a 1:5 ratio. In case of fibroblast contamination, different detachment times of fibroblasts and epithelial cells can be exploited for refining the culture. Fibroblasts form weaker cell-cell and cell-surface interactions and normally detach faster. This characteristic can be used for enrichment of epithelial cells in the culture, using principle of differential trypsinization. Trypsin-EDTA can be diluted to a lower concentration (e.g. 0.05%) to extend detachment times for better control of the

, and saturated humidity. Growth

and cell-surface interactions.

in cell culture flasks.

168 Goat Science

Cells were grown in a 5% CO2

procedure.

The immunostaining protocols often differ for different markers and every marker might require optimization of the protocol. Generally, the protocol consisted of cell fixation for 30 s to 1 min in ice cold acetone/methanol (1:1) or for several minutes in 4% paraformaldehyde. Fixation was followed by permeabilization (not necessary when using acetone-methanol fixation or in case of membrane-bound markers) with 0.3% Triton X-100 for 10 min. After washing with phosphate buffered saline (PBS), cells were blocked with 5–10% fetal serum (it is recommended to use fetal serum from species in which secondary antibodies were produced) and 1–3% bovine serum albumin (BSA) for 60 min. Incubation with primary antibodies was performed overnight at 4°C. Next day, cells were washed with PBS several times and incubated with fluorescently labeled secondary antibodies at room temperature for 1 h. After washing with PBS, cell nuclei can be counterstained with 4′,6-diamidino-2-phenylindole (DAPI), washed, and visualized under microscope. In case of paraffinized tissue, sections were deparaffinized using xylene and rehydrated in decreasing concentrations of ethanol. Rehydrated tissue slices were washed in PBS, followed by performing heat-induced antigen retrieval in a microwave oven, using 10 mM sodium-citrate buffer (pH 6). Afterward, the same protocol was used for immunofluorescent staining as described previously for pgMECs. The more detailed protocols and the antibodies used were described in our previous publications [8–10].

glandular tissue, followed by regression of the tissue due to apoptosis and loss of glandular structure and function (involution). The functional part of mammary gland is glandular tissue connected by a branched system of secondary ducts, combining into larger primary ducts, which end in the gland cistern. The milk is synthesized by secretory luminal cells arranged in spherical structures called alveoli, which form larger structures called lobules. Alveoli and ducts of lactating mammary gland are composed of different types of epithelial cells important for milk synthesis and secretion, while connective and fat tissue surround and support the epithelial structures. An alveolus is comprised of a single layer of milk secreting luminal epithelium, surrounded by a single layer of contractile myoepithelial cells, which lie adjacent

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In agreement with the "Replacement" of the three Rs principle (3Rs: Replacement, Reduction, and Refinement) adequate *in vitro* model, mimicking the function of the mammary gland allows the study of physiological, biochemical, and immune functions of the mammary gland, substituting *in vivo* experiments. In addition to the ethical issues, cell lines enable use of many technical replicas, better control of the environment, and surmount the problem of variation introduced by animal's individuality [13] and the problem of systemic effects, which makes elucidation of a contribution of a particular cell type of interest difficult [14]. A main limitation of primary cell models is a finite life span, and a limiting number of available biological replicas, as a derived cell culture, represent a single genotype, while establishment and characterization of a large number of cell cultures/lines are quite a challenging and laborious process. Additionally, cell cultures do not always properly model *in vivo* conditions; there-

Several ruminant immortalized mammary epithelial cell lines as MAC-T [15] and BME-UV [16] were generated by genomic integration of Simian virus large T-antigen (SV40LTA). However, transformed mammary cells are genetically and (usually) phenotypically changed. Transient mammary lines show low responsiveness to lactogenic hormones [17] and are not proper approximation of *in vivo* lactation. Genetic modifications and adaptations to growth in cell cultures alter metabolic pathways in continuous cell lines; therefore, the use of primary

The derived primary cell culture consisted of a heterogeneous population of mostly epithelial and mesenchymal (fibroblast-like) cells. Epithelial cells grew in round-shaped densely packed islands of cells with multiple nucleoli and exhibited typical cobblestone morphology. Cells randomly spreading around these islands were larger, spindle-shaped cells, morpho-

Cell proliferation was slow for the first week after seeding dissociated cells in plastic dishes. After the first passage, the cells started to proliferate much faster and overgrew the surface

to the basal membrane, where mammary stem/progenitor cells also reside [10].

fore, limitations should be considered for each individual purpose.

cells is a much better approximation of the *in vivo* system [18].

**4. pgMEC characteristics**

**4.1. Morphology and growth**

logically resembling fibroblasts (**Figure 2**).

#### *2.2.3. Mammosphere formation assay*

When performing mammosphere formation assay, a single-cell suspension of the mammary cells was grown in DMEM/F12 medium, supplemented with EGF (20 ng/mL), bFGF (human, 20 ng/mL), heparin (4 μg/mL), cholera toxin (10 ng/mL), hydrocortisone (0.5 μg/mL), insulin (0.5 μg/mL), and B27 supplement (2%), and grown in 6-well ultralow-attachment plates with or without extracellular membrane matrix or in hanging drops, according to the described protocol [11].

#### *2.2.4. Oil Red O staining*

Growth medium was aspirated and the pgMECs were fixed in 4% paraformaldehyde for 15 min. Oil Red O (0.5 g) was dissolved in 50 ml of isopropanol and diluted with water (3:2) and then left for 10 min, and the solution filtered through a 20-μm filter. Cells were briefly washed with isopropanol (60%) and incubated with solution of Oil Red O for 15 min at room temperature. The cells were then rinsed with isopropanol and washed under tap water. The formation of lipid droplets (red stain) was observed under bright field microscope.
